Initiated by the finding that platelets express functional CD40 ligand (CD40L, CD154), many new roles for platelets have been discovered in unanticipated areas, including the immune response. When current literature is considered as a whole, the picture that is emerging begins to show that platelets are able to significantly affect, for better or worse, the overall health and condition of the mammalian host. Animal models have made significant contributions to our expanding knowledge of platelet function, much of which is anticipated to be clinically relevant. While still mostly circumstantial, the evidence supports a critical role for CD40L in many normal and disease processes.

Understanding the adaptive immune response is an area of research critically important in medicine. Several positive regulators of B- and T-cell activation exist to eliminate pathogens, in which CD40 ligand (CD154) plays a fundamental role. It is well documented that CD154 expressed by CD4 T helper cells can be critical in the proper activation of dendritic cells for the productive stimulation of CD8 T cells and is required for proper T-dependent B-cell immunity. However, platelets are an abundant and systemic source of CD154. While classically known to be important for hemostasis and inflammation, several lines of evidence suggest that platelet-derived ligands can modulate the adaptive immune compartment.

Platelets' primary role is hemostasis. However, a growing body of research has demonstrated that platelets are integral to the initiation of an inflammatory response and are potent effector cells of the innate immune response. Activated platelets express CD154, a molecule critical to adaptive immune responses, which has been implicated in plateletmediated modulation of innate immune responses and inflammation. Recent studies utilizing CD154 knockout mice extend the role of platelet-derived CD154 to the modulation of adaptive immune response by enhancing antigen presentation, improving CD8 + T cell responses, and playing a critical function in T-dependent humoral immunity under physiological conditions. Together these data provide a basis for the expansion of the current paradigm of B cell activation and germinal center formation to include a role for platelets.

The adjuvant therapy of choice for superficial bladder cancer is the intravesical instillation of live Mycobacterium bovis Bacillus Calmette-Guerin (BCG). In spite of the fact that this therapy is the most effective treatment for superficial bladder cancer, intravesical administration of BCG is associated with high local morbidity and the potential for systemic infection. Therefore, there is a need for the development of safer, less toxic approaches to fight this disease. Since fibronectin attachment protein (FAP) is a key element in BCG retention and targeting to cells, we hypothesize that this protein can be used as targeting agent to deliver cytotoxic cargo for the treatment of bladder tumors. Here we evaluated the ability of bladder tumor cells to bind and endocytose FAP via fibronectin-integrin complexes. We found that microaggregation induced by an anti-FAP polyclonal antibody accelerated FAP uptake by T24 bladder tumor cells. FAP was determined to be internalized via a clathrin-independent, caveolae-dependent mechanism. Further, once within the endosomal compartment, FAP was targeted to the lysosomal compartment with negligible recycling to the plasma membrane. Importantly, we demonstrated that FAP microaggregation and internalization could also be triggered by multivalent Ni2+NTA-bearing liposomes. Overall, our studies validate the use of FAP as a targeting vector and provide the foundation for the design of more effective, less toxic bladder cancer therapeutics.

We previously showed that degradation of cellular sphingomyelin (SM) by SMase C results in a greater stimulation of cholesterol translocation to endoplasmic reticulum, compared to its degradation by SMase D. Here we investigated the hypothesis that the effect of SMase C is partly due to the generation of ceramide, rather than due to depletion of SM alone. Inhibition of hydroxymethylglutaryl CoA reductase (HMGCR) activity was used as a measure of cholesterol translocation. Treatment of fibroblasts with SMase C resulted in a 90% inhibition of HMGCR, whereas SMase D treatment inhibited it by 29%. Treatment with exogenous ceramides, or increasing the endogenous ceramide levels also inhibited HMGCR by 60-80%. Phosphorylation of HMGCR was stimulated by SMase C or exogenous ceramide. The effects of ceramide and SMase D were additive, indicating the independent effects of SM depletion and ceramide generation. These results show that ceramide regulates sterol trafficking independent of cellular SM levels.

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The percentage of saturated cholesteryl esters (CEs) synthesized by human LCAT is several times higher than expected from the sn -2 acyl composition of plasma phosphatidylcholine (PC), whereas the synthesis of 20:4 CE and 22:6 CE is much lower than expected. To explain these discrepancies, we proposed that LCAT transfers some saturated fatty acids from the sn -1 position of PC species that contain 20:4 or 22:6 at sn -2. The present studies provide in vivo evidence for this hypothesis. We determined the composition and synthesis of CE species in plasma of volunteers before and after a 6 week dietary supplementation with docosahexaenoic acid (22:6; DHA). In addition to an increase in the DHA content of all plasma lipids, there was a significant ( ؉ 12%; P Ͻ 0.005) increase of 16:0 CE, although there was no increase in 16:0 at sn -2 of PC. The increase of DHA in CE was much lower than its increase at sn -2 of PC. Most of the cholesteryl esters (CEs) in the lipoproteins of human plasma are derived from the action of LCAT (1). Although this enzyme has been shown to be specific for the sn-2 position of phosphatidylcholine (PC), the composition of the CEs in human plasma does not match that of the sn-2 acyl group (1-4). For example, although 16:0 constitutes only ‫ف‬ 2-3% of the PC sn-2 acyl groups, it accounts for 10-12% of plasma CE. On the other hand, although 20:4 constitutes ‫ف‬ 16% of the sn-2 acyl groups of PC, only ‫ف‬ 5% of the plasma CE is 20:4. Similarly, the concentration of 22:6 at sn-2 of PC ( ‫ف‬ 5%) is much higher than that in CE ( ‫ف‬ 0.4%). Although these discrepancies have been attributed to the preference of the enzyme for the PC substrates containing 16:0 at sn-2 (5), in vitro studies with synthetic substrates and isolated enzyme did not support this mechanism (6). Our studies on the use of PC species by LCAT in native plasma also showed that 16:0-16:0 PC, the major PC with 16:0 at sn -2, is not preferred over "average" PC in plasma. Based on our studies with synthetic PC substrates and isolated LCAT (7, 8), we proposed that human LCAT transfers significant amounts of sn-1 acyl group from the PC species containing 20:4 or 22:6 at the sn -2 position. This mechanism not only explains the synthesis of higher than expected percentages of saturated CE species but also accounts for the formation of lower than expected percentages of 20:4 and 22:6 CE. This alteration in positional specificity appears to be a function of the architecture of the enzyme's active site, because rat and mouse enzyme are not substantially affected by the presence of sn-2-20:4 PC (8) and because the human LCAT behaves like the mouse enzyme if its active site domain is replaced by the corresponding domain from mouse enzyme (4, 9).Although we have provided multiple lines of evidence for the altered positional specificity of human LCAT in vitro, there is no direct evidence that this occurs in vivo. Previous studies of the effects of fish oil feeding in humans (10) and nonhuman primates (11,12) reported an increase in the ap...

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