Immunological unresponsiveness established by the elimination or anergy of self-reactive lymphocyte clones is of importance to immunization against tumorassociated antigens. In this study, we have investigated induction of immunity against the human MUC1 carcinomaassociated antigen in MUC1 transgenic mice unresponsive to The human DF3͞MUC1 glycoprotein is overexpressed and aberrantly glycosylated in breast and other carcinomas (1-4). The finding that lymphocytes from certain patients with carcinomas recognize and lyse MUC1-positive tumor cells (5, 6) has suggested that this antigen is a potential target for anticancer vaccines. Whereas MUC1 is expressed on the apical borders of normal epithelium (1-3) and unresponsiveness to self-antigens is an obstacle to the development of antitumor immunity, MUC1 transgenic (MUC1.Tg) mice provide a potential model to assess the induction of anti-MUC1 immune responses. In this context, MUC1.Tg C57BL6 mice express MUC1 in a pattern and at a level similar to that found in humans (7). Significantly, the MUC1.Tg mice are tolerant to stimulation by MUC1 antigen (7).Dendritic cells (DC) are potent antigen-presenting cells (8) that sensitize CD4 ϩ T cells to specific antigens in a major histocompatibility complex-restricted manner (9, 10) and generate antigen-specific cytotoxic T lymphocytes (CTLs) from naive T cells in vitro (11,12). Moreover, DCs are the only antigen-presenting cells known to prime naive CTLs and to induce antigen-specific CTLs in vivo (13). DCs pulsed with tumor antigens or synthetic peptides derived from such antigens have been effective as vaccines in the induction of CTL responses and antitumor activity (14)(15)(16)(17). Other studies have demonstrated that transduction of DC with recombinant viral vectors expressing tumor antigens generates vaccines that induce antigen-specific antitumor immune responses (18)(19)(20). Fusions resulting in heterokaryons of DC and carcinoma cells as vaccines have provided an alternative strategy for inducing immunity against both known and unidentified tumor antigens (21).The present studies demonstrate that MUC1.Tg mice respond to fusions of DC and MUC1-positive MC-38 carcinoma cells with induction of anti-MUC1 immunity. The findings demonstrate that a DC fusion cell vaccine can reverse unresponsiveness to a tumor-associated antigen and induce the rejection of established metastases. MATERIALS AND METHODS MUC1 Transgenic Mice.A C57BL͞6 mouse strain transgenic for human MUC1 was established as described (7). Tail DNA (500 ng) was subjected to PCR amplification by using MUC1 primers (bp 745-765 and bp 1,086-1,065) to confirm the presence of MUC1 sequences. The PCR product was detected by electrophoresis in a 1% agarose gel (7).Cell Culture and Fusion. Murine (C57BL͞6) MC-38 and MB49 carcinoma cells were stably transfected with a MUC1 cDNA (22-24). Cells were maintained in DMEM supplemented with 10% heat-inactivated fetal calf serum, 2 mM

The mucin gene, Muc-1, encodes a high molecular weight integral membrane glycoprotein that is present on the apical surface of most simple secretory epithelial cells. Muc-1 is highly expressed and aberrantly glycosylated by most carcinomas and metastatic lesions. Numerous functions have been proposed for this molecule, including protection of the epithelial cell surface, an involvement in epithelial organogenesis, and a role in tumor progression. Mice deficient in Muc-1 were generated using homologous recombination in embryonic stem cells. These mice appeared to develop normally and were healthy and fertile. However, the growth rate of primary breast tumors induced by polyoma middle T antigen was found to be significantly slower in Muc-1 deficient mice. This suggests that Muc-1 plays an important role in the progression of mammary carcinoma.

BackgroundDroxidopa, a prodrug of norepinephrine, was approved for treatment of neurogenic orthostatic hypotension (nOH) due to primary autonomic disorders based on 3 randomized double-blind studies. We performed safety and efficacy analyses of this pooled dataset (n = 460).MethodsEfficacy was assessed using Orthostatic Hypotension Questionnaire (OHQ) scores (composite and individual items). Safety and tolerability were also examined.ResultsDroxidopa improved virtually all nOH symptom scores compared with placebo, significantly reducing OHQ composite score (−2.68 ± 2.20 vs −1.82 ± 2.34 units; P < 0.001), dizziness/lightheadedness score (−3.0 ± 2.9 vs −1.8 ± 3.1 units; P < 0.001), and 3 of 5 other symptom assessments (visual disturbances, weakness, and fatigue [P ≤ 0.010]). Droxidopa significantly improved 3 of 4 measures of activities of daily living (standing a long time, walking a short time, and walking a long time [P ≤ 0.003]) and significantly increased upright systolic blood pressure (11.5 ± 20.5 vs 4.8 ± 21.0 mmHg for placebo; P < 0.001). Droxidopa was effective in patients using inhibitors of dopa decarboxylase (DDCI; the enzyme that converts droxidopa to norepinephrine), but its efficacy was numerically greater in non-DDCI users. Droxidopa was well-tolerated. Rates of most adverse events were similar between groups. Supine hypertension rates were low, but slightly higher in patients receiving droxidopa (≤7.9% vs ≤4.6% for placebo); patients with severe hypertension at screening were excluded from these studies.ConclusionsDroxidopa is effective for the treatment of nOH in patients with primary autonomic disorders and is generally well-tolerated. A longer trial is underway to confirm efficacy beyond the ≤2 to 10 - week period assessed in the current trials.Trial registration ClinicalTrials.gov identifiers: NCT00782340, first received October 29, 2008; NCT00633880, first received March 5, 2008; and NCT01176240, first received July 30, 2010.