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A small-molecule agonist of the Toll-like receptor 1/2 signaling pathway does it by stabilizing heterodimeric associations., Toll-like receptor (TLR) agonists activate both the innate and the adaptive immune systems. These TLR agonists have been exploited as potent vaccine adjuvants and antitumor agents. We describe the identification and characterization of a small molecule, N-methyl-4-nitro-2-(4-(4-(trifluoromethyl)phenyl)-1H-imidazol-1-yl)aniline (CU-T12-9), that directly targets TLR1/2 to initiate downstream signaling. CU-T12-9 specifically induces TLR1/2 activation, which can be blocked by either the anti-hTLR1 or the anti-hTLR2 antibody, but not the anti-hTLR6 antibody. Using a variety of different biophysical assays, we have demonstrated the binding mode of CU-T12-9. By binding to both TLR1 and TLR2, CU-T12-9 facilitates the TLR1/2 heterodimeric complex formation, which in turn activates the downstream signaling. Fluorescence anisotropy assays revealed competitive binding to the TLR1/2 complex between CU-T12-9 and Pam3CSK4 with a half-maximal inhibitory concentration (IC50) of 54.4 nM. Finally, we showed that CU-T12-9 signals through nuclear factor κB (NF-κB) and invokes an elevation of the downstream effectors tumor necrosis factor–α (TNF-α), interleukin-10 (IL-10), and inducible nitric oxide synthase (iNOS). Thus, our studies not only provide compelling new insights into the regulation of TLR1/2 signaling transduction but also may facilitate future therapeutic developments.

There is consensus that young patients with mantle cell lymphoma (MCL) should receive intensive immunochemotherapy regimens, but optimal treatment of elderly patients as well for as patients with limited or indolent disease is not defined. Our aim was to evaluate and compare outcome in relation to prognostic factors and first-line treatment in patients with MCL in a population-based data set. Data were collected from the Swedish and Danish Lymphoma Registries from the period of 2000 to 2011. A total of 1389 patients were diagnosed with MCL. During this period, age-standardized incidence MCL increased, most prominently among males. Furthermore, male gender was associated with inferior overall survival (OS) in multivariate analysis (hazard ratio [HR] = 1.36; P = .002). Forty-three (3.6%) patients with stage I-II disease received radiotherapy with curative intent, showing a 3-year OS of 93%. Twenty-nine (2.4%) patients followed a watch-and-wait approach and showed a 3-year OS of 79.8%. Among patients receiving systemic treatment, rituximab (n = 766; HR = 0.66; P = .001) and autologous stem cell transplant (n = 273; HR = 0.55; P = .004) were independently associated with improved OS in multivariate analysis. Hence, by a population-based approach, we were able to provide novel data on prognostic factors and primary treatment of MCL, applicable to routine clinical practice.

Exosomes, biologically active nanoparticles (40-100 nm) released by hematopoietic and non-hematopoietic cells, contain a variety of proteins and small, non-coding RNA known as microRNA (miRNA). Exposure to various pathogens and disease states modifies the composition and function of exosomes, but there are no studies examining in vivo exosomal changes evoked by the acute stress response. The present study reveals that exposing male Fisher 344 rats to an acute stressor modulates the protein and miRNA profile of circulating plasma exosomes, specifically increasing surface heat shock protein 72 (Hsp72) and decreasing miR-142-5p and -203. The selected miRNAs and Hsp72 are associated with immunomodulatory functions and are likely a critical component of stress-evoked modulation of immunity. Further, we demonstrate that some of these stress-induced modifications in plasma exosomes are mediated by sympathetic nervous system (SNS) activation of alpha-1 adrenergic receptors (ADRs), since drug-mediated blockade of the receptors significantly attenuates the stress-induced modifications of exosomal Hsp72 and miR-142-5p. Together, these findings demonstrate that activation of the acute stress response modifies the proteomic and miRNA profile of exosomes released into the circulation.

Introduction: Prothrombinase is an essential enzyme of the coagulation cascade that is made up of a heterodimer of coagulation factor Xa and Va. The complex most readily forms in the presence of lipid membranes containing the anionic lipid phosphatidylserine. The phosphatidylserine lipid head groups provide a surface for the prothrombinase complex to assemble and significantly increase the enzyme's turnover number. Here, we investigate how truncation and modification of the naturally occurring myristoylated alanine-rich C-kinase substrate (MARCKS) protein into small peptide and peptidomimetic fragments (MARCKS ED) forms lipid probes that bind to phosphatidylserine, and block the assembly and enzymatic activity of the prothrombinase complex. Compared to known phosphatidylserine binding proteins such as annexin V or lactadherin, the MARCKS ED probes are less than a tenth of the molecular weight and do not require calcium to bind to phosphatidylserine. Methods: Using biophysical assays, we examine how the MARCKS ED probes assemble on lipid membranes containing phosphatidylserine and how this assembly alters the binding of factor Xa to these surfaces. Effects on the enzymatic activity of the prothrombinase complex in the presence of activated platelets and other phosphatidylserine exposing membranes are explored using a modified prothrombinase assay. The binding and anticoagulant activity of the MARCKS ED probes during whole blood coagulation are examined by quantifying fluorescently labeled MARCKS ED probe localization, platelet accumulation, and fibrin formation in microfluidic flow assays. Results: We find that the MARCKS ED probes can antagonize factor Xa from binding phosphatidylserine by measuring reduced association of factor Xa to lipid membranes treated with the MARCKS ED probes. Pre-treatment of activated platelets with the MARCKS ED probes significantly reduced the assembly and enzymatic activity of prothrombinase (100% activity with vehicle control vs. 51% with 1 μM MARCKS ED peptide treatment, P < 0.05). Similar inhibitory activity was also observed when activated platelets were replaced with other phosphatidylserine exposing lipid membranes, such as synthetic liposomes or biologic microparticles. In the whole blood microfluidic flow assay, the MARCKS ED probes colocalized with adherent platelets and inhibited fibrin formation (100% peak fibrin intensity with vehicle control vs. 17% peak fibrin intensity with 1 μM MARCKS ED peptide treatment, P < 0.05). The MARCKS ED probes did not alter platelet surface area coverage. Electron microscopy images of the final clots formed in the microfluidic flow assay show an absence of fibrin formation with MARCKS ED probe treatment. Conclusions: We demonstrate that the MARCKS ED probes can inhibit the assembly and enzymatic activity of prothrombinase, and that this activity translates to significantly reduced fibrin formation in whole blood. The MARCKS ED peptide and peptidomimetic probes may provide a new therapeutic tool for inhibiting the assembly of phosphatidylserine dependent coagulation factors, as well as a research tool for identifying platelet phosphatidylserine exposure. Disclosures Kastelowitz: University of Colorado Boulder: Patents & Royalties. Yin:University of Colorado Boulder: Patents & Royalties.

Exosomes, biologically active nanoparticles (40–100 nm) released by hematopoietic and non-hematopoietic cells, contain a variety of proteins and small, non-coding RNA known as microRNA (miRNA). Exposure to various pathogens and disease states modifies the composition and function of exosomes, but recent studies reveal that the acute stress response can also evoke in vivo exosomal changes in the absence of disease. The present study demonstrates that exposing male Fisher 344 rats to an acute stressor modulates proteins and miRNA in their circulating plasma exosomes, specifically up-regulating heat shock protein 72 (Hsp72) and decreasing miR-142-5p and −203 expression. The selected miRNAs and Hsp72 are associated with immunomodulatory functions and are likely a critical component of stress-enhanced immunity. Further, we demonstrate that some of these stress-induced modifications in plasma exosomes are mediated by sympathetic nervous system (SNS) activation of alpha-1 adrenergic receptors (ADRs), since drug-mediated blockade of the receptors significantly attenuates the stress-induced modifications of exosomal Hsp72 and miR-142-5p. Moreover, in vivo analysis reveals that administering rats stress modified plasma exosomes improves their inflammatory response to subcutaneous injections of Escherichia coli (E. coli) compared to rats administered non-stressed plasma exosomes or saline. Together, these findings suggest that activation of the acute stress response potentiates innate immunity by modifying the proteomic and miRNA profile of exosomes released into the circulation. Supported by NSF IOS 1022451.