TRAF6-IRF5 kinetics, TRIF, and biophysical factors drive synergistic innate responses to particle-mediated MPLA-CpG co-presentation.

Innate immune responses to pathogens are driven by co-presentation of multiple pathogen-associated molecular patterns (PAMPs). Combinations of PAMPs can trigger synergistic immune responses, but the underlying molecular mechanisms of synergy are poorly understood. Here, we used synthetic particulate carriers co-loaded with monophosphoryl lipid A (MPLA) and CpG as pathogen-like particles (PLPs) to dissect the signaling pathways responsible for dual adjuvant immune responses. PLP-based co-delivery of MPLA and CpG to GM-CSF-driven mouse bone marrow-derived antigen-presenting cells (BM-APCs) elicited synergistic interferon-ß (IFN-ß) and interleukin-12p70 (IL-12p70) responses, which were strongly influenced by the biophysical properties of PLPs. Mechanistically, we found that MyD88 and interferon regulatory factor 5 (IRF5) were necessary for IFN-ß and IL-12p70 production, while TRIF signaling was required for the synergistic response. Both the kinetics and magnitude of downstream TRAF6 and IRF5 signaling drove the synergy. These results identify the key mechanisms of synergistic Toll-like receptor 4 (TLR4)-TLR9 co-signaling in mouse BM-APCs and underscore the critical role of signaling kinetics and biophysical properties on the integrated response to combination adjuvants.

Early treatment of SIV+ macaques with an α4ß7 mAb alters virus distribution and preserves CD4+ T cells in later stages of infection.

Integrin α4ß7 mediates the trafficking of leukocytes, including CD4+ T cells, to lymphoid tissues in the gut. Virus mediated damage to the gut is implicated in HIV and SIV mediated chronic immune activation and leads to irreversible damage to the immune system. We employed an immuno-PET/CT imaging technique to evaluate the impact of an anti-integrin α4ß7 mAb alone or in combination with ART, on the distribution of both SIV infected cells and CD4+ cells in rhesus macaques infected with SIV. We determined that α4ß7 mAb reduced viral antigen in an array of tissues of the lung, spleen, axillary, and inguinal lymph nodes. These sites are not directly linked to α4ß7 mediated homing; however, the most pronounced reduction in viral load was observed in the colon. Despite this reduction, α4ß7 mAb treatment did not prevent an apparent depletion of CD4+ T cells in gut in the acute phase of infection that is characteristic of HIV/SIV infection. However, α4ß7 mAb appeared to facilitate the preservation or restoration of CD4+ T cells in gut tissues at later stages of infection. Since damage to the gut is believed to play a central role in HIV pathogenesis, these results support further evaluation of α4ß7 antagonists in the study and treatment of HIV disease.

A Novel Method to Quantify RNA-Protein Interactions In Situ Using FMTRIP and Proximity Ligation.

RNA binding proteins (RBP) and small RNAs regulate the editing, localization, stabilization, translation, and degradation of ribonucleic acids (RNAs) through their interactions with specific cis-acting elements within target RNAs. Here, we describe a novel method to detect protein-mRNA interactions, which combines FLAG-peptide modified, multiply-labeled tetravalent RNA imaging probes (FMTRIPs) with proximity ligation (PLA), and rolling circle amplification (RCA). This assay detects native RNA in a sequence specific and single RNA sensitive manner, and PLA allows for the quantification and localization of protein-mRNA interactions with single-interaction sensitivity.

Can we observe changes in mRNA "state"? Overview of methods to study mRNA interactions with regulatory proteins relevant in cancer related processes.

RNA binding proteins (RBP) regulate the editing, localization, stabilization, translation, and degradation of ribonucleic acids (RNA) through their interactions with specific cis-acting elements within target RNAs. Post-transcriptional regulatory mechanisms are directly involved in the control of the immune response and stress response and their alterations play a crucial role in cancer related processes. In this review, we discuss mRNAs and RNA binding proteins relevant to tumorigenesis, current methodologies for detecting RNA interactions, and last, we describe a novel method to detect such interactions, which combines peptide modified, RNA imaging probes (FMTRIPs) with proximity ligation (PLA) and rolling circle amplification (RCA). This assay detects native RNA in a sequence specific and single RNA sensitive manner, and PLA allows for the quantification and localization of protein-mRNA interactions with single-interaction sensitivity in situ.

Demonstration of droplet size and vaporization rate measurements in the near field of a two-phase jet with droplet lasing spectroscopy.

Droplet lasing spectroscopy has been applied to the measurement of droplet size and evaporation rate in a spray. A single droplet, doped with laser dye, was injected along the centerline of a liquid spray. Filters were used to block the strong elastic-scattering signal. The lasing emission from the doped droplet could be detected against the background with mass loadings of liquid in the spray as high as 20%. An analysis of the spectrum of droplet lasing was used to evaluate the droplet diameter. The evaporation rate of the droplet was obtained from consecutive lasing spectra that were obtained from the same droplet. An error analysis of the drop size and drop evaporation measurements was carried out and showed that accurate measurements of evaporation rates were feasible.

Focused-image holography as a dense-spray diagnostic.

The denser regions of sprays need to be probed for us to understand further the basic phenomena of the breakup of a bulk liquid into droplets and the subsequent drop dynamics. The instruments currently available to spray diagnosticians, the Malvern spray analyzer and the phase/Doppler particle analyzer, cannot be used in the denser regions of the spray and in regions where ligaments or nonspherical droplets exist. Holography, as applied in the past, has permitted the interrogation of nonspherical drops but in general has been applied to droplet-dominated dilute regions of sprays. Using a focused-image holographic system, with the advantages of an imaging lens and side lighting, we can probe highly complex regions of sprays, such as those that include bubble explosions and complex ligament formation at the nozzle exit. In this paper we present components of a focused-image holographic system for spray analysis, advantages and limitations of that system, and how the longitudinal magnification varies as a funct on of the length of the object along the optical path.