Single-shot characterization of multi-film structures based on combined spectral interferometry and spatially recorded spectroscopic ellipsometry.

In this investigation, we propose an improved combined scheme with spectral interferometry and spatially recorded spectroscopic ellipsometry to measure surface height and film thicknesses at once in real time. Instead of rotating polarizing optical components, a spatial phase retarder, which consists of spatially rotating liquid crystal arrays, is used to obtain the ellipsometric spectral data from a single image. In addition, interferometric configuration is combined to collect the surface height information in the same image. The spatial phase retarder can be characterized by the phase retardation and the rotation angle of the liquid crystal during the calibration procedure. In the experiments, single-layered and multi-layered film specimens were measured to verify the measurement capability of the proposed system, and it was confirmed that the measurement results were in good agreement with the provided reference values.

Mediators of Inflammation-Driven Expansion, Trafficking, and Function of Tumor-Infiltrating MDSCs.

Myeloid-derived suppressor cells (MDSC) are induced by and accumulate within many histologically distinct solid tumors, where they promote disease by secreting angiogenic and immunosuppressive molecules. Although IL1ß can drive the generation, accumulation, and functional capacity of MDSCs, the specific IL1ß-induced inflammatory mediators contributing to these activities remain incompletely defined. Here, we identified IL1ß-induced molecules that expand, mobilize, and modulate the accumulation and angiogenic and immunosuppressive potencies of polymorphonuclear (PMN)-MDSCs. Unlike parental CT26 tumors, which recruited primarily monocytic (M)-MDSCs by constitutively expressing GM-CSF- and CCR2-directed chemokines, IL1ß-transfected CT26 produced higher G-CSF, multiple CXC chemokines, and vascular adhesion molecules required for mediating infiltration of PMN-MDSCs with increased angiogenic and immunosuppressive properties. Conversely, CT26 tumors transfected with IL1ß-inducible molecules could mobilize PMN-MDSCs, but because they lacked the ability to upregulate IL1ß-inducible CXCR2-directed chemokines or vascular adhesion molecules, additional PMN-MDSCs could not infiltrate tumors. IL1ß-expressing CT26 increased angiogenic and immunosuppressive factors of tumor-infiltrating MDSCs, as did CT26 tumors individually transfected with G-CSF, Bv8, CXCL1, or CXCL5, demonstrating that mediators downstream of IL1ß could also modulate MDSC functional activity. Translational relevance was indicated by the finding that the same growth factors, cytokines, chemokines, and adhesion molecules responsible for the mobilization and recruitment of PMN-MDSCs into inflammatory CT26 murine tumors were also coordinately upregulated with increasing IL1ß expression in human renal cell carcinoma tumors. These studies demonstrated that IL1ß stimulated the components of a multifaceted inflammatory program that produces, mobilizes, chemoattracts, activates, and mediates the infiltration of PMN-MDSCs into inflammatory tumors to promote tumor progression.

Rap1A regulates generation of T regulatory cells via LFA-1-dependent and LFA-1-independent mechanisms.

The small GTPase Rap1A has a critical role in regulating cell-matrix and cell-cell adhesion. In T lymphocytes, Rap1A mediates LFA-1 activation and LFA-1-mediated adhesion. LFA-1 reduces the threshold of TCR signals for low affinity ligands. Previously, we determined that mice expressing constitutively active Rap1A on T cells have increased frequency of CD103(+) T regulatory cells (Treg). We hypothesized that Rap1A-GTP might affect the differentiation of Treg by regulating LFA-1 activation. Using Foxp3-GFP-KI, LFA-1-KO and Rap1A-GTP-Tg mice we determined that Rap1A has an active role in the development of thymic Treg but LFA-1 is not mandatory for this function. Rap1A is also involved in the generation of peripheral Treg and this effect is mediated via LFA-1-dependent and LFA-1-independent mechanisms. Identification of the signaling pathways via which Rap1-GTP contributes to the differentiation of Treg will provide new insights to the function of Rap1A and to designing targeted approaches for generation of Treg for therapeutic applications.

RIAM regulates the cytoskeletal distribution and activation of PLC-gamma1 in T cells.

Rap1-guanosine triphosphate (GTP)-interacting adaptor molecule (RIAM) plays a critical role in actin reorganization and inside-out activation of integrins in lymphocytes and platelets. We investigated the role of RIAM in T cell receptor (TCR)-mediated signaling. Although phosphorylation of the kinase ZAP-70 and formation of a signalosome recruited to the adaptor protein LAT were unaffected, elimination of endogenous RIAM by short hairpin RNA impaired generation of inositol 1,4,5-trisphosphate, mobilization of intracellular calcium ions (Ca(2+)), and translocation of the transcription factor NFAT to the nucleus. The activation of Ras guanine nucleotide-releasing protein 1 was also impaired, which led to the diminished expression of the gene encoding interleukin-2. These events were associated with the impaired translocation of phosphorylated phospholipase C-gamma1 (PLC-gamma1) to the actin cytoskeleton, which was required to bring PLC-gamma1 close to its substrate phosphatidylinositol 4,5-bisphosphate, and were reversed by reconstitution of cells with RIAM. Thus, by regulating the localization of PLC-gamma1, RIAM plays a central role in TCR signaling and the transcription of target genes.

The cyclin dependent kinase inhibitor (R)-roscovitine prevents alloreactive T cell clonal expansion and protects against acute GvHD.

Cell cycle re-entry of quiescent T lymphocytes regulated by cdk2 is required for antigen-specific clonal expansion and generation of productive T cell responses. Recently, we determined that induction of antigen-specific T cell tolerance results in impaired cdk2 activity leading to enhanced Smad3 transactivation, upregulation of p15 and blockade of cell cycle progression. Here we report that pharmacologic inhibition of cdk2 with (R)-roscovitine blocked expansion of alloreactive T cells in vitro and in vivo and protected from lethal acute GvHD. In addition to inhibiting alloreactive T cell responses, (R)-roscovitine prevented TNF-alpha-mediated activation of NF-kappa B pathway, which is involved in the inflammatory process leading to the development of GvHD. The combined anti-proliferative and anti-inflammatory properties of (R)-roscovitine make it an attractive treatment modality toward control of GvHD.

Human anisakiasis: Diversity in antibody response profiles to the changing antigens in larval excretions/secretions.

Anisakiasis is an infectious parasitic disease contracted by eating third stage larvae of Anisakis simplex (L3) carried by marine fishes. Human anisakiasis was researched for specific IgG with L3 excretory secretory products (ESP). L3ESP were prepared by daily harvesting of culture supernatant from day 2 to day 5, using two kinds culture media of RPMI-1640 and phosphate buffered saline (PBS). When the sera from persons diagnosed with anisakiasis by means of endoscopy were analyzed using indirect ELISA and Western blot, the sera was classified into four groups depending on specific antigen recognition patterns. In addition, the presence of a new antigen for L3, located at less than 13 kDa (AgLT13) was demonstrated in L3ESP with a modified Western blot condition. The production of AgLT13 was mainly found in L3ESP harvested both on day 2 and day 3, and that in PBS was predominant over that in RPMI-1640. Among those sera, the high reactive sera to L3ESP-day two prepared with phosphate buffer in indirect ELISA recognized AgLT13, and 33% of the low reactive sera did so. These results indicate that the binding pattern of human L3 specific antibody is diverse depending on L3ESP preparations and that AgLT13 demonstrated with a Western blot condition is a specific antigen for L3.