Ann E. Casey

Platelets and Megakaryocytes Contain Functional Nuclear Factor-κB

Objective—To investigate the presence and role of NF-&kgr;B proteins in megakaryocytes and platelets. The nuclear factor-&kgr;B (NF-&kgr;B) transcription factor family is well known for its role in eliciting inflammation and promoting cell survival. We discovered that human megakaryocytes and platelets express the majority of NF-&kgr;B family members, including the regulatory inhibitor-&kgr;B (I-&kgr;B) and I-&kgr; kinase (IKK) molecules. Methods and Results—Anucleate platelets exposed to NF-&kgr;B inhibitors demonstrated impaired fundamental functions involved in repairing vascular injury and thrombus formation. Specifically, NF-&kgr;B inhibition diminished lamellapodia formation, decreased clot retraction times, and reduced thrombus stability. Moreover, inhibition of I-&kgr;B-&agr; phosphorylation (BAY-11-7082) reverted fully spread platelets back to a spheroid morphology. Addition of recombinant IKK-&bgr; or I-&kgr;B-&agr; protein to BAY inhibitor–treated platelets partially restored platelet spreading in I-&kgr;B-&agr; inhibited platelets, and addition of active IKK-&bgr; increased endogenous I-&kgr;B-&agr; phosphorylation levels. Conclusion—These novel findings support a crucial and nonclassical role for the NF-&kgr;B family in modulating platelet function and reveal that platelets are sensitive to NF-&kgr;B inhibitors. As NF-&kgr;B inhibitors are being developed as antiinflammatory and anticancer agents, they may have unintended effects on platelets. On the basis of these data, NF-&kgr;B is also identified as a new target to dampen unwanted platelet activation.

HSV amplicon-mediated Aβ vaccination in Tg2576 mice: differential antigen-specific immune responses

Given the participation of amyloid beta (Abeta) in Alzheimers disease (AD) pathogenesis the derivation of experimental therapeutics to prevent Abeta fibrillogenesis and/or enhance removal of parenchymal amyloid deposits represent viable disease-modifying approaches. Active Abeta-based immunotherapies have shown promise in mouse AD models, but application in human trials was accompanied by moderate brain inflammation in a subset of patients. Immune-shaping vaccine platforms may mitigate adverse effects. Herein, we describe the use of herpes simplex virus (HSV)-derived amplicons to elicit distinctive immune responses against Abeta. Two vaccine vectors were constructed: one expressing Abeta1-42 alone (HSVAbeta), and a second expressing Abeta1-42 fused with the molecular adjuvant tetanus toxin Fragment C (HSVAbeta/TtxFC). Peripheral administration of these vaccines augmented humoral responses to Abeta and reduced CNS Abeta deposition in Tg2576 AD mice. Interestingly and unexpectedly, HSVAbeta vaccination was uniquely toxic and incited the expression of pro-inflammatory molecule transcripts within the hippocampi of Tg2576 mice, suggesting that this paradigm may serve as a relevant model to study Abeta vaccine-elicited CNS inflammatory syndromes.

Isoprostane and isofuran lipid mediators accumulate in stored red blood cells and influence platelet function in vitro

Stored red blood cells (RBCs) release hemoglobin (Hb) that leads to oxidative damage, which may contribute to thrombosis in susceptible transfusion recipients. Oxidative stress stimulates the generation of a new class of lipid mediators called F2‐isoprostanes (F2‐IsoPs) and isofurans (IsoFs) that influence cellular behavior. This study investigated RBC‐derived F2‐IsoPs and IsoFs during storage and their influence on human platelets (PLTs).

Alterations of platelet function and clot formation kinetics after in vitro exposure to anti‐A and ‐B

BACKGROUND: ABO‐mismatched platelets (PLTs) are commonly transfused despite reported complications. We hypothesized that because PLTs possess A and B antigens on their surface, ABO‐mismatched transfused or recipient PLTs could become activated and/or dysfunctional after exposure to anti‐A or ‐B in the transfused or recipient plasma. We present here in vitro modeling data on the functional effects of exposure of PLTs to ABO antibodies.

Hexamethylene bisacetamide leads to reduced helper virus-free HSV-1 amplicon expression titers via suppression of ICP0

The herpes simplex virus (HSV)‐derived amplicon vector has evolved into a promising gene transfer platform for widespread DNA delivery in gene replacement strategies and vaccine development given its ease of molecular manipulation, large transgene capacity, and transduction efficiencies of numerous cell types in vivo. The recent development of helper virus‐free packaging methodologies bodes well for this vector system in its eventual implementation as a clinically viable therapeutic modality. For realization of clinical application, efforts have been made to enhance yields and quality of helper‐free amplicon stocks. Hexamethylene bisacetamide (HMBA), a hybrid polar compound that exhibits stimulatory activity of HSV‐1 immediate‐early gene expression, has been employed as a standard reagent in helper virus‐free packaging given its purported mode of action on virus gene expression kinetics. Unexpectedly, we have found that HMBA exhibits no titer‐enhancing activity; in contrast, the compound enhances the proportion of amplicon virions that are non‐expressive. Omission of HMBA during vector packaging led to a marked reduction in the ratios of vector genome‐transducing to transgene‐expressing virions. This effect was neither packaging‐cell‐specific nor amplicon‐promoter‐dependent. Analysis of resultant vector stocks indicated amplicon genome replication/concatenation was unaffected, but the level of particle‐associated ICP0 was reduced in stocks packaged in the presence of HMBA. Inclusion of a co‐transfected, ICP0‐expressing plasmid into the packaging process led to significant rescue of amplicon expression titers, indicating that regulation of ICP0 concentrations is critical for maintenance of the amplicon genome expressive state. Copyright

Alterations of platelet function and clot formation kinetics following in vitro exposure to anti-A and -B antibodies

BACKGROUND: ABO‐mismatched platelets (PLTs) are commonly transfused despite reported complications. We hypothesized that because PLTs possess A and B antigens on their surface, ABO‐mismatched transfused or recipient PLTs could become activated and/or dysfunctional after exposure to anti‐A or ‐B in the transfused or recipient plasma. We present here in vitro modeling data on the functional effects of exposure of PLTs to ABO antibodies.

Alterations of platelet function and clot formation kinetics after in vitro exposure to anti-A and -B: PLT EXPOSURE TO ANTI-ABO

BACKGROUND: ABO‐mismatched platelets (PLTs) are commonly transfused despite reported complications. We hypothesized that because PLTs possess A and B antigens on their surface, ABO‐mismatched transfused or recipient PLTs could become activated and/or dysfunctional after exposure to anti‐A or ‐B in the transfused or recipient plasma. We present here in vitro modeling data on the functional effects of exposure of PLTs to ABO antibodies.