Human γδ T cells expressing Vγ2Vδ2 TCRs monitor foreign- and self-prenyl pyrophosphate metabolites in isoprenoid biosynthesis to mediate immunity to microbes and tumors. Vγ2Vδ2 cells have been used for adoptive cancer immunotherapy with some partial and complete remissions. Most trials have used continuous zoledronate exposure to expand Vγ2Vδ2 cells. Zoledronate inhibits farnesyl pyrophosphate synthase causing isopentenyl pyrophosphate to accumulate that then stimulates Vγ2Vδ2 cells. Because zoledronate exposure is toxic, we hypothesized that a short period of exposure would reduce T cell toxicity but still be sufficient for monocytes uptake. Supporting this hypothesis, pulse zoledronate exposure with IL-2 resulted in more uniform expansion of Vγ2Vδ2 cells with higher purity and cell numbers as compared with continuous exposure. These Vγ2Vδ2 cells also had higher levels of CD107a and perforin and slightly increased tumor cytotoxicity. Importantly, adoptive immunotherapy with Vγ2Vδ2 cells derived by pulse stimulation controlled human PC-3 prostate cancer cells in immunodeficient NSG mice significantly better than those derived by continuous stimulation. Pulse zoledronate stimulation of Vγ2Vδ2 cells with IL-15 also resulted in higher purity and cell numbers. Like with CD8 αβ T cells, IL-15 preserved early memory Vγ2Vδ2 T cell subsets better than IL-2. However, despite this fact, adoptive immunotherapy with Vγ2Vδ2 cells derived with IL-15 showed similar inhibition of PC-3 tumor growth as those derived with IL-2. Thus, pulse zoledronate stimulation maximizes the purity, quantity, and quality of expanded Vγ2Vδ2 cells. This simple modification to existing protocols would likely enhance the effectiveness of adoptively transferred Vγ2Vδ2 T cells.

No description available.

Tumor-necrosis factor (TNF)-receptor (R) associated factor 3 (TRAF3) is an important adaptor protein that plays a variety of context-dependent regulatory roles in all types of immune cells. In B cells, TRAF3 mediates signaling downstream of CD40, B cell activating factor (BAFF)-R, and toll-like receptors (TLR)s to restrain B cell survival and function. Downstream of CD40 and BAFF-R, TRAF3 negatively regulates NF-κB2 activation through NF-κB inducing kinase (NIK) stabilization. NF-κB2 activation is important for B cell-homeostatic survival. However, the constitutively active NF-κB2 in other TRAF3 deficient immune cell types does not lead to increased cell survival. More importantly, loss-of-function mutations of the TRAF3 gene are found at relatively high frequencies in B cell malignancies such as multiple myeloma and B cell lymphoma. Therefore, TRAF3 plays a critical and unique role in B cells to restrain cell survival and differentiation that contributes to B cell malignancies. In this study, we aim to identify TRAF3 modulated survival pathways that contribute to homeostatic B-cell survival and B-cell differentiation. We found that TRAF3 degradation was not sufficient or necessary to induce NF-κB2 activation. We also showed that TRAF3 degradation is dependent on association with TRAF2 and cytoplasmic tail of CD40 or BAFF-R. TRAF3 regulation of NIK is important for mature B cell development; however, NIK only partially contributes to TRAF3-mediated B cell survival. TRAF3 also regulates the protein level of proviral integrations of Moloney virus (Pim2), a pro-survival serine/threonine protein kinase encoded by the Pim2 gene, to restrain B cell survival; this regulation can operate independently of the NF-κB2 pathway. Furthermore, we showed that TRAF3 negatively regulates IL-6R signaling, a pathway that contributes to expansion of the plasma cell compartment and to the pathogenesis of multiple myeloma, a plasma cell malignancy. We found that TRAF3 facilitates recruitment of PTPN22, a tyrosine phosphatase, to associate with Jak1 following IL-6 binding to the IL-6R complex. This regulation by TRAF3 restrains plasma cell differentiation, and also provides the first demonstration that PTPN22 regulates cytokine receptor signaling. Collectively, these findings highlight the importance of TRAF3 in the regulation of B cell-specific survival and differentiation pathways. This information could be exploited for more precise and effective therapeutic choices in treatment of B cell malignancies with TRAF3 deficiencies.

Segerstrom et al. (2017) found that more repetitive thought (RT) was related to lower interleukin-6 (IL-6), in older adults at average IQ. This study aimed to replicate and extend this finding in midlife adults, with a daily measure of RT, and additional inflammatory biomarkers. 153 participants were drawn from the Midlife in the United States (MIDUS) Refresher project; ages 25-70 (M = 45.07, SD = 10.96), 50.3% female, and 83% Caucasian. Cognition was assessed via the Brief Test of Adult Cognition by Telephone, biological data via fasted blood draw, and RT data were collected as part of the National Study of Daily Experiences daily diary. Total RT (amount one engages in RT) and RT valence (positive vs. negative thought content) were analyzed. As IQ increased, more positive RT was associated with lover levels of IL-6 and CRP after adjusting for age, BMI, and statin use (β = -.161, p = .029; β = -.240, p = .002). Results did not replicate Segerstrom et al. (2017) but suggested that crystallized intelligence and RT total reflect a cognitive system different than that of fluid intelligence, executive functioning, and RT valence. Future studies should continue to investigate effects of RT on health outcomes.

Melanoma differentiation associated gene 7/Interleukin-24 (MDA-7/IL-24) is a secreted cytokine which acts as a tumor suppressor. It is capable of selectively killing cancer cells, regardless of anatomic origin, while sparing normal cells. miRNAs are master regulators of gene expression that can play two roles in cancer: tumor-suppression and oncogenesis. We identified a number of miRNAs that are regulated by MDA-7/IL-24 using a PCR plate array containing probes for miRNAs known to play a role in prostate cancer. We independently validated the array with qRT-PCR to identify three miRNAs which are downregulated by MDA-7/IL-24 treatment in DU145, PC3, and PC3ML prostate cancer lines. These miRNAs were miR-125a, miR-145, and miR-23b. Their gene targets were identified using TargetScan and confirmed to be regulated in our prostate cancer model. NLRC5, KLF4, and KLF15, respectively, were upregulated after treatment with MDA-7/IL-24. We focused on NLRC5 as a novel target of MDA-7/IL-24, which plays a role in immune evasion by cancer cells. NLRC5 is upregulated following inhibition of miR-125a. It is not downregulated by overexpression of miR-125a which suggests that more than one miRNA may be acting to regulate its expression. Finally, we determined that miR-125a is downregulated by MDA-7 through DICER, an important processing enzyme for miRNA biogenesis.

Mast cells are tissue-resident immune cells known as effector cells for the innate and adaptive immune systems. Mast cells contribute to host defenses against parasites such as large roundworm parasites, bacterial pathogens, and toxins, and participate in wound healing, but they are mostly known for their role in allergic diseases. It has been well established that during allergic diseases, mast cells are stimulated by IgE cross-linkage to release proinflammatory mediators. However, a newly discovered cytokine, IL-33 has also been implicated in allergic disease. Recently, IL-33 has been implicated as a driver of several Type I sensitivities and previous studies have shown that IL-33 can stimulate mast cells in atopic inflammation. Although the importance of IL-33 has been established, there are still several things unknown about IL-33 signaling regulation or treatment. This dissertation will present two separate studies involving the modulation of IL-33-mediated mast cells function In the first study, the effects of fluvastatin are explored. In a previous study, fluvastatin was shown to inhibit proinflammatory functions of IgE crosslinked mast cells. Contrasting to IgE stimulation, fluvastatin augments IL-6 and TNF production in IL-33 stimulated mast cells, but suppressed MCP-1. This phenomenon was seen in mouse and human mast cells in vitro and replicated in a mast cell-dependent murine model of IL-33-induced inflammation in vivo. In the second study, IL-33 was found to induce miR-146a expression in mouse mast cells and mast cell-derived exosomes in vitro, and in plasma exosomes in vivo. IL-33 induced miR-146a was of interest because miR-146a is a known negative regulator of TLR signaling, which shares the MyD88 signaling pathway with IL-33. We found that miR-146a KO mast cells are hyperresponsive to IL-33 stimulation, data that were replicated by suppressing miR-146a-5p in WT mast cells. In an acute mast cell repopulation model, kitW-sh/W-sh mice containing miR-146a KO BMMC had increased IL-33 induced neutrophilia in comparison to their controls. Collectively, these data reveal new IL-33 signaling pathways and means of altering its inflammatory effects on mast cells. Because IL-33 has important roles in allergy and other Th2-mediated diseases, these results advance clinically relevant areas of immunology.

No description available.

Histatins, a group of proteins produced by human salivary glands, have a variety of innate immune functions including the ability to: kill oral microorganisms, neutralize toxins, inactivate protease/collagenase activities, inhibit co-aggregation of oral bacteria, and inhibit lipopolysaccharide mediated activities. Hemagglutinin B (HagB), a virulence factor of the periodontal pathogen Porphyromonas gingivalis, induces a robust cytokine and chemokine response in human myeloid dendritic cells. In this study, I hypothesize that histatin 5 can attenuate a HagB-induced chemokine response. Objectives: To characterize an expanded cytokine and chemokine response induced in human myeloid dendritic cells by HagB, and to determine if prior incubation of HagB with histatin 5 attenuates these responses. Methods: In my first experiment, 0.040 M HagB was mixed with dilutions of histatin 5 and histatin 8 (Sigma, 0.04 to 40.0 M), incubated at 37C for 30 minutes, and added to 2 x 104 human myeloid dendritic cells (Lonza, Walkersville, MD). At 24 hours, culture media was removed, and 6 cytokines and chemokines (pg/ml) were determined in cell-free supernatants (Millipore, Billerica, MA) using the Luminex 100 IS instrument (Luminex, Austin, TX). In my second experiment, 0.040 M HagB was mixed with 40.0 M histatin 5 only (e.g., 1:1000), incubated at 37C for 30 minutes, and added to 2 x 104 human myeloid dendritic cells. At 0, 1, 2, 4, 8, 16, and 24 hours post-inoculation, culture media was removed, and 26 cytokines and chemokines (pg/ml) were determined in cell-free supernatants. Results: In both experiments, human myeloid dendritic cells incubated with HagB produced Th1, Th2, and Th17 cytokines (IL-2, IL-12(p70), IFN-, IL-3, IL-4, IL-5, , IL-15, IL-17); pro-inflammatory cytokines (IL-1, IL-1, IL-6, TNF-, IL-12(p40); anti-inflammatory v cytokines (IL-10, IL-13, IFN2); chemokines (CXCL8/IL-8, CXCL10/IP-10, CCL2/MCP-1, CCL3/MIP-1, MIP-1b, CCL11/eotaxin); and colony stimulating factors (IL-7, G-CSF, GM-CSF). Histatin 5 significantly attenuated (p < 0.05) the IL-8 response induced by HagB at 8 - 16 hours and to a lesser extent, the IL-6, GM-CSF, MCP-1, MIP-1α, MIP-1β, and TNF-α response. Conclusion: Histatin 5 is an important salivary component capable of attenuating an IL-8 response. Together with human beta defensin 3, another peptide previously shown to attenuate pro-inflammatory cytokines, histatin 5 may help control and contain oral infection and inflammation by down regulating IL-8 chemotactic response.

Pseudomonas aeruginosa est une bactérie pathogène opportuniste responsable de nombreuses maladies nosocomiales ainsi que d'infections graves chez les patients atteints de mucoviscidose ou chez immunodéprimés. La résistance aux antibiotiques observée chez de nombreuses souches fait de cette bactérie un pathogène difficile à éradiquer. Sa capacité à former un biofilm renforce en particulier cette résistance. Au cours de son processus infectieux, la bactérie utilise des lectines qui lui permettent de reconnaitre et de fixer spécifiquement les oligosaccharides présents en particulier sur les cellules hôte. Parmi elles, sont retrouvées la lectine soluble PA-IL (lectine à galactose) ainsi qu'une lectine fimbriale nouvellement identifiée, CupB6. Ces protéines seraient impliquées dans l'adhésion aux tissus de l'hôte et dans l'élaboration du biofilm. Nos travaux portent sur l'étude biochimique et structurale de ces deux lectines. Par des techniques de microcalorimétrie, de résonance plasmonique de surface et de cristallographie aux rayons X, le site de liaison du sucre de PA-IL a été parfaitement caractérisé, et de nombreux inhibiteurs dérivés du galactose ont pu être testés. D'autre part, nous avons exprimé Cup6 sous forme recombinante et nous avons étudié sa spécificité. Nos études ont montré que le sucre reconnu pourrait être le Lewis b et des études structurales sont en cours. Ces travaux s'inscrivent dans l'optique d'élaborer des glycomimétiques qui pourraient apporter une alternative aux antibiotiques.

<p>El presente estudio versa sobre el análisis de errores de un grupo de suecos aprendientes de español como segunda lengua. El análisis trata de explorar si el tiempo de estadía en el país de la lengua meta, juega un papel significativo en la frecuencia de errores lingüísticos. Para este efecto, se analizaron las transcripciones de un ejercicio narrativo hecho por los aprendientes, para luego determinar los errores gramaticales y léxicos más frecuentes. Además de los aprendientes también se constó de un grupo de nativos monolingües de español quienes también hicieron el mismo ejercicio narrativo. El método de análisis en este trabajo es de tipo cuantitativo y como herramienta de análisis se utilizaron el t-test y el test correlacional Pearson. El t-test ayuda a determinar si la diferencia entre los dos grupos es suficientemente grande y significativa. El test correlacional Pearson ayuda a determinar la correlación entre el total de errores y el tiempo de estadía.</p><p>Las preguntas de investigación responden a dos preguntas: las diferencias entre nativos y aprendientes en lo que se refiere a los errores lingüísticos que comente y el papel que desempeña el tiempo de estadía en la frecuencia de errores cometidos. Seguido a estas preguntas se plantean dos hipótesis: los aprendientes cometen más errores que los nativos, y el tiempo de estadía juega un papel importante en este comportamiento en el sentido de que mientras más tiempo se lleva en el ámbito L2 menos errores lingüísticos se deben cometer.</p><p>Los resultados del análisis indican que el tiempo de estadía no presenta mayor efecto en la frecuencia de errores que los aprendientes cometen.</p>