Gendler, Sandra

Abstract Chronic inflammation associated with aging is termed inflamm-aging. It manifests as a rise in many proinflammatory mediators and contributes to a decline in immune function. Unregulated inflammasome activation initiates an inappropriate inflammatory response. It is particularly imperative to understand inflammasome dysregulation in the elderly (>65 yrs), since it is a likely basis of immune dysfunction. Analyses of freshly isolated memory CD4+ and CD8+ splenic T cells demonstrated that even without further activation, inflammasome genes were already significantly up-regulated in old mice compared to young, including factors critical to myeloid-derived suppressor cell (MDSC) expansion including S100a8, S100a9, IL-1β, IL-13 and IL-6. A corresponding 2-3 fold increase in CD11b+Gr1+cells was observed in the spleens of old naïve mice compared to young. Importantly, cells with the CD11b+Gr1+ phenotype from old mice, unlike young, were functionally MDSCs and suppressed T cell proliferation. Therefore, the immunosuppressive environment of the tumor-bearing host also appears to be operative in aging, even in the absence of tumor. We hypothesize that constituitive activation of inflammasome complex results in a chronic inflammatory state, altering immune responses in the elderly similarly to the tumor-bearing state. Preemptive reversal of inflammasome-induced MDSCs may enhance T cell immunosurveillance in the elderly, a population with a heightened susceptibility to malignancy.

Abstract A C57BL/6 mouse transgenic for human MUC1 (MUC1.Tg) was developed to evaluate MUC1-specific tumor immunity in an animal that expresses MUC1 as a normal self protein. Previous studies showed that MUC1.Tg mice, challenged with syngeneic tumors expressing MUC1 (B16.MUC1), developed progressively growing MUC1-positive tumors, whereas wild-type C57BL/6 (wt) mice developed MUC1-negative tumors at a significantly slower rate. The results of a limiting dilution CTL frequency assay were not informative, in that similar numbers of MUC1-specific CTL precursors (CTL) were detected in MUC1.Tg and wt mice. Tumor immunity in vivo was characterized by an adoptive transfer method to evaluate the degree of MUC1 or non-MUC1 tumor immunity in wt or MUC1.Tg mice. The results revealed that wt mice developed protective tumor immunity mediated by MUC1-specific CD4+ lymphocytes, while MUC1.Tg mice were functionally tolerant to MUC1 in vivo. The potential of adoptive immunotherapy to provide immunity to tumors expressing MUC1 and to produce undesirable autoimmunity in recipient MUC1.Tg mice expressing MUC1 as a self Ag was evaluated. Adoptive transfer of immune cells from wt mice primed in vivo with B16.MUC1 tumor cells into MUC1.Tg recipients resulted in significant increases in the survival of MUC1.Tg recipients compared with unmanipulated control MUC1.Tg mice challenged with B16.MUC1 tumor cells. This response was specific for MUC1 since control tumors developed at equivalent rates in recipient or control MUC1.Tg mice. No gross or histologic evidence of autoimmunity was observed in recipient MUC1.Tg mice, indicating that tumor immune responses mediated by MUC1-specific CD4+ lymphocytes spare nontransformed epithelia-expressing MUC1.

AbstractThe MUC1 mucin (CD227) is a cell surface mucin originally thought to be restricted to epithelial tissues. We report that CD227 is expressed on human blood dendritic cells (DC) and monocyte-derived DC following in vitro activation. Freshly isolated murine splenic DC had very low levels of CD227; however, all DC expressed CD227 following in vitro culture. In the mouse spleen, CD227 was seen on clusters within the red pulp and surrounding the marginal zone in the white pulp. Additionally, we confirm CD227 expression by activated human T cells and show for the first time that the CD227 cytoplasmic domain is tyrosine-phosphorylated in activated T cells and DC and is associated with other phosphoproteins, indicating a role in signaling. The function of CD227 on DC and T cells requires further elucidation.

Abstract Cyclooxygenase-2 (COX-2) inhibitors are rapidly emerging as a new generation of therapeutic drug in combination with chemotherapy or radiation therapy for the treatment of cancer. The mechanisms underlying its antitumor effects are not fully understood and more thorough preclinical trials are needed to determine if COX-2 inhibition represents a useful approach for prevention and/or treatment of breast cancer. The purpose of this study was to evaluate the growth inhibitory mechanism of a highly selective COX-2 inhibitor, celecoxib, in an in vivo oncogenic mouse model of spontaneous breast cancer that resembles human disease. The oncogenic mice carry the polyoma middle T antigen driven by the mouse mammary tumor virus promoter and develop primary adenocarcinomas of the breast. Results show that oral administration of celecoxib caused significant reduction in mammary tumor burden associated with increased tumor cell apoptosis and decreased proliferation in vivo. In vivo apoptosis correlated with significant decrease in activation of protein kinase B/Akt, a cell survival signaling kinase, with increased expression of the proapoptotic protein Bax and decreased expression of the antiapoptotic protein Bcl-2. In addition, celecoxib treatment reduced levels of proangiogenic factor (vascular endothelial growth factor), suggesting a role of celecoxib in suppression of angiogenesis in this model. Results from these preclinical studies will form the basis for assessing the feasibility of celecoxib therapy alone or in combination with conventional therapies for treatment and/or prevention of breast cancer.

Inositol hexaphosphate (IP6 or phytic acid) is thought to be the active ingredient in legumes and cereals that provides anti‐cancer activity. Our goal is to understand the influence of IP6 on mucin expression (Muc1 and Muc2, specifically) in the mouse intestine. C57BL/6 mice were randomized into 3 groups of 6 mice. Mice were orally dosed with IP6 (0, 1, 2 g/kg BW) for 5 days and sacrificed on the sixth day. Sections of the small intestine were removed, fixed in methacarn, sectioned and stained with Alcian blue‐PAS to visualize mucins. Mucosa from the jejunum and colon were removed and total RNA prepared using Trizol®. cDNA was prepared using random hexamers and TaqMan minor groove binding probes were used for real time PCR amplification of message for Muc1 and Muc2. Expression of Muc1 in the jejunum was minimal whereas that of Muc2 increased 1.4‐fold at the higher IP6 dose rate. In the colon, expression of both Muc1 and Muc2 increased in mice dosed with IP6. Expression of Muc1 increased 1.6‐fold and 3.8‐fold in mice dosed with 1 and 2 g/kg IP6, respectively. Expression of Muc2 increased 2.0‐fold and 1.7‐fold in mice dosed with 1 and 2 g/kg IP6, respectively. Alcian blue‐PAS stained sections of the colon showed an increase in mucus. Thus, IP6 seems to increase the expression of Muc1 and Muc2 in the mucosa lining of the small intestine, particularly in the colon. Research was supported by East Tennessee State University and Mayo Clinic.

Background Though the importance of the transmembrane mucin MUC1 in mammary oncogenesis has long been recognized, the relative contributions of the cytoplasmic tail and tandem repeat domains are poorly understood. Methods To address this, mouse models of mammary carcinogenesis were created expressing full-length cytoplasmic tail-deleted, or tandem repeat-deleted MUC1 constructs. Results Overexpression of full-length MUC1 resulted in tumor formation in young mice (≤ 12 months); however, loss of either the cytoplasmic tail or the tandem repeat domain abrogated this oncogenic capacity. Aged mice in all strains developed late-onset mammary tumors similar to those previously described for the FVB background. Conclusions This study is the first spontaneous cancer model to address the relative importance of the cytoplasmic tail and tandem repeat domains to MUC1-driven mammary oncogenesis, and suggests that both of these domains are essential for tumor formation.

AbstractA fully synthetic MUC1‐based cancer vaccine was designed and chemically synthesized containing an endogenous helper T‐epitope (MHC class II epitope). The vaccine elicited robust IgG titers that could neutralize cancer cells by antibody‐dependent cell‐mediated cytotoxicity (ADCC). It also activated cytotoxic T‐lymphocytes. Collectively, the immunological data demonstrate engagement of helper T‐cells in immune activation. A synthetic methodology was developed for a penta‐glycosylated MUC1 glycopeptide, and antisera of mice immunized by the new vaccine recognized such a structure. Previously reported fully synthetic MUC1‐based cancer vaccines that elicited potent immune responses employed exogenous helper T‐epitopes derived from microbes. It is the expectation that the use of the newly identified endogenous helper T‐epitope will be more attractive, because it will activate cognate CD4+ T‐cells that will provide critical tumor‐specific help intratumorally during the effector stage of tumor rejection and will aid in the generation of sustained immunological memory.