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Despite reports of striking outcomes, immunotherapy efficacy in melanoma is limited to subsets of patients 1, 2. Combining immunotherapies with other modalities has yielded limited improvements but also adverse events requiring cessation of treatment 1. In addition to ineffective patient stratification, efficacy can be impaired by paucity of tumor-infiltrating lymphocytes (TILs). Thus, effective strategies to safely increase TILs are urgently needed to improve immunotherapies 3. Here, we report that dietary administration of the sugar L-fucose triggers CD4+T cell-mediated increases in TILs, anti-tumor immunity, and enhanced immune checkpoint blockade responses. This is induced by the fucosylation and cell surface enrichment of the MHC-II protein HLA-DRB1 in melanoma. Single-cell immunofluorescent staining analysis of patient melanoma specimens demonstrates that fucosylation and fucosylated HLA-DRB1 is associated with intratumoral T cell abundance and anti-PD1 responder status. Our findings demonstrate that fucosylation is a key mediator of anti-tumor immunity, via regulation of melanoma cell surface HLA-DRB1 and induction of anti-tumor immunity, suggesting use of melanoma fucosylation as a novel strategy to stratify patients for immunotherapies. Importantly, our study suggests that L-fucose represents a powerful, non-toxic agent for safely increasing anti-tumor immunity and immunotherapy efficacy in melanoma.

The 2021 Association of Pathology Chairs Annual Meeting included a chairs' session and a premeeting discussion-group webinar sponsored by the Senior Fellows Group (former chairs of academic departments of pathology who have remained active in the Association of Pathology Chairs) focused on generating discretionary income for departments. Discretionary income was defined as revenue that can be used by the department with few, if any, restrictions. Such income is particularly desirable given limitations on departmental budgets. Four discussion-group panelists presented the funds-flow model in their respective institutions and how they derived and used discretionary income. Discretionary income was obtained from both external sources (eg, philanthropy, indirect cost recovery, partnerships with outside entities, medical education courses, research laboratory agreements, clinical trials) and internal sources (eg, core facilities, institutional programmatic support, institutional incentive programs). Significant departmental variations were associated with differences in institutional financial structure and policies, revenue-generating capabilities of the department and individual faculty, practice plan policies, donor intentions, and geographic market forces. Most finances were dependent upon a robust funds-flow model. Uses of discretionary funds included salary support, recruitment expenses (including start-up packages), research equipment, space renovation, social events, support of academic programs, and travel. Panelists also discussed particular challenges of discretionary-fund generation and use during the coronavirus disease 2019 pandemic. Notably, each institution had its own unique methodology for generating discretionary income, and no obvious standard approach was identified. The 2 moderators emphasized the importance of identifying and understanding opportunities, issues, and institutional culture surrounding generation and use of discretionary funds.

ImportanceBladder-preserving trimodality therapy can be an effective alternative to radical cystectomy for treatment of muscle-invasive bladder cancer (MIBC), but biomarkers are needed to guide optimal patient selection. The DNA repair protein MRE11 is a candidate response biomarker that has not been validated in prospective cohorts using standardized measurement approaches.ObjectiveTo evaluate MRE11 expression as a prognostic biomarker in MIBC patients receiving trimodality therapy using automated quantitative image analysis.Design, Setting, and ParticipantsThis prognostic study analyzed patients with MIBC pooled from 6 prospective phase I/II, II, or III trials of trimodality therapy (Radiation Therapy Oncology Group [RTOG] 8802, 8903, 9506, 9706, 9906, and 0233) across 37 participating institutions in North America from 1988 to 2007. Eligible patients had nonmetastatic MIBC and were enrolled in 1 of the 6 trimodality therapy clinical trials. Analyses were completed August 2020.ExposuresTrimodality therapy with transurethral bladder tumor resection and cisplatin-based chemoradiation therapy.Main Outcomes and MeasuresMRE11 expression and association with disease-specific (bladder cancer) mortality (DSM), defined as death from bladder cancer. Pretreatment tumor tissues were processed for immunofluorescence with anti-MRE11 antibody and analyzed using automated quantitative image analysis to calculate a normalized score for MRE11 based on nuclear-to-cytoplasmic (NC) signal ratio.ResultsOf 465 patients from 6 trials, 168 patients had available tissue, of which 135 were analyzable for MRE11 expression (median age of 65 years [minimum-maximum, 34-90 years]; 111 [82.2%] men). Median (minimum-maximum) follow-up for alive patients was 5.0 (0.6-11.7) years. Median (Q1-Q3) MRE11 NC signal ratio was 2.41 (1.49-3.34). Patients with an MRE11 NC ratio above 1.49 (ie, above first quartile) had a significantly lower DSM (HR, 0.50; 95% CI, 0.26-0.93; P = .03). The 4-year DSM was 41.0% (95% CI, 23.2%-58.0%) for patients with an MRE11 NC signal ratio of 1.49 or lower vs 21.0% (95% CI, 13.4%-29.8%) for a ratio above 1.49. MRE11 NC signal ratio was not significantly associated with overall survival (HR, 0.84; 95% CI, 0.49-1.44).Conclusions and RelevanceHigher MRE11 NC signal ratios were associated with better DSM after trimodality therapy. Lower MRE11 NC signal ratios identified a poor prognosis subgroup that may benefit from intensification of therapy.

Background Muscle invasive bladder (MIBC) cancer is an aggressive disease with high rates of local recurrence following radical cystectomy (RC). Currently, there are no clinically validated biomarkers to predict local only recurrence (LOR) and guide adjuvant treatment decisions. This pilot study evaluated the role of Ki-67, MRE11 and PD-L1 as predictive biomarkers for recurrence patterns in patients undergoing RC for MIBC. Methods: Our institutional cystectomy database containing cases of urothelial MIBC from 1992–2014 was queried for patients with LOR and case-matched with patients who had distant recurrence (DR) and no recurrence (NR). Clinical and histopathological data were collected from selected patients and a tissue microarray was built and analyzed for presence of Ki-67, MRE11 and PD-L1 using immunofluorescence (IF) and immunohistochemistry (IHC). Univariate, multivariate, and principal component analyses (PCA) were performed to evaluate association of these biomarkers with recurrence pattern. Results: Pathologic specimens from 42 patients (18 NR, 16 LOR and 8 DR) were reviewed. Compared to adjacent normal bladder tissue, tumors had increased expression of PD-L1 on both IHC and IF (p

Summary A clearer understanding of the tumor immune microenvironment (TIME) in metastatic clear cell renal cell carcinoma (ccRCC) may help to inform precision treatment strategies. We sought to identify clinically meaningful TIME signatures in ccRCC. We studied tumors from 39 patients with metastatic ccRCC using quantitative multiplexed immunofluorescence and relevant immune marker panels. Cell densities were analyzed in three regions of interest (ROIs): tumor core, tumor–stroma interface and stroma. Patients were stratified into low- and high-marker density groups using median values as thresholds. Log-rank and Cox regression analyses while controlling for clinical variables were used to compare survival outcomes to patterns of immune cell distributions. There were significant associations with increased macrophage (CD68+CD163+CD206+) density and poor outcomes across multiple ROIs in primary and metastatic tumors. In primary tumors, T-bet+ T helper type 1 (Th1) cell density was highest at the tumor–stromal interface (P = 0·0021), and increased co-expression of CD3 and T-bet was associated with improved overall survival (P = 0·015) and survival after immunotherapy (P = 0·014). In metastatic tumor samples, decreased forkhead box protein 3 (FoxP3)+ T regulatory cell density correlated with improved survival after immunotherapy (P = 0·016). Increased macrophage markers and decreased Th1 T cell markers within the TIME correlated with poor overall survival and treatment outcomes. Immune markers such as FoxP3 showed consistent levels across the TIME, whereas others, such as T-bet, demonstrated significant variance across the distinct ROIs. These findings suggest that TIME profiling outside the tumor core may identify clinically relevant associations for patients with metastatic ccRCC.

INTRODUCTION AND OBJECTIVE:The recent success of anti-PD1 therapeutics is known to be associated with an upregulated Th1 gene signature and an immunogenic tumor microenvironment (TME). There remain...

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INTRODUCTION AND OBJECTIVE:Tumor-associated macrophages (TAMs) have been associated with tumor progression and metastasis in breast, ovarian and gastric cancers. Based on response to microenvironme...

Responsiveness to immunotherapies is limited in melanoma to subsets of patients. Despite attempts to improve responsiveness to immunotherapies by testing combination therapies, patients often experience significant adverse events that require the termination of treatment. Although causes of nonresponsiveness remain unclear and the subject of active investigation, a commonality among refractory tumors is low pretreatment levels of tumor-infiltrating lymphocytes (TILs). Thus, studies aiming to elucidate the interactions between melanoma and immune cells and to increase TIL levels are anticipated to improve therapies. We previously reported that fucosylation (the post-translational modification of proteins with the dietary plant sugar L-fucose), is altered in melanoma. Increasing fucosylation levels in melanoma—genetically or with oral L-fucose—suppresses tumor growth, metastasis, and increases TILs in immune competent mouse models of melanoma (Lau et al., 2015). The mechanisms underlying how melanoma fucosylation appears to trigger antitumor immunity are unclear. Here, we identified TIL subpopulations triggered by fucosylation to mediate tumor suppression and a key molecular mechanism. In NRAS- and BRAF-mutant melanoma models, dietary L-fucose suppressed tumor growth by ~50-60% and increased TILs by ~10-50-fold. Of all TIL subpopulations examined, CD3+ T cells were most increased by L-fucose, doubling or increasing by 15-fold, in NRAS- or BRAF-mutant models, respectively. Depletion of CD4+ T cells abrogated L-fucose-triggered TIL increases and tumor suppression, indicating that CD4+ T cells are central for antitumor immune effects of L-fucose. We identified the Class II MHC protein HLA-DRB1 as expressed and fucosylated in melanoma cells and confirmed its requirement for L-fucose-triggered, CD4+ T cell-dependent tumor suppression in vivo. Roles of HLA-DRB1 fucosylation in melanoma, effects on CD4+ T cell biology, and how L-fucose can enhance immunotherapies will be discussed. Our studies highlight how L-fucose supplementation can render tumors “immune hot” and represents a potential therapeutic strategy for improving immunotherapies. Citation Format: Daniel K. Lester, Matt Mercurio, Pasquale Innamorato, Kodumudi Krithika, Williamson Danial, Watson Gregory, Pilon-Thomas Shari, Messina Jane, Kerri L. Thomas, Susan McCarthy, Joseph Markowitz, Robert Haltiwanger, Eric Lau. Using L-fucose to render melanomas immune hot: Roles of melanoma HLA-DRB1 and CD4+T cell-mediated antitumor immunity [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2019 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(3 Suppl):Abstract nr B24.

While immunotherapies have had a striking efficacy in melanoma patients, significant proportions of melanoma patients exhibit poor responsiveness to immunotherapies. Further understanding of immunoregulatory mechanisms is needed to improve immunotherapies. Previously, we discovered increasing tumor fucosylation in melanomas reduces tumor growth and metastasis (Lau et al., 2015). To identify tumor-infiltrating lymphocytes (TILs) affected by L-fucose, we profiled lymphocytes from syngeneic tumors of control- or fucose-fed mice tumors. Dietary supplementation of L-fucose increased TILs by ~10-50-fold. Of total TILs, CD3+ T cells (including CD4+ and CD8+ T cells) doubled. L-fucose did not trigger tumor suppression in immune-deficient mouse melanoma models, indicating the immune system’s requirement for L-fucose-triggered suppression. Immunodepletion of CD4+ or CD8+ T cells during L-fucose treatment revealed that CD4+ T cells are crucial for tumor suppression. CD4+ T-cell depletion abrogated infiltration of NK, dendritic, and CD8+ T cells in the tumors, implicating these populations as downstream effectors of fucosylation- and CD4+ T cell-triggered tumor suppression. To identify proteins on melanomas contributing to fucosylation-triggered immune responses, we performed mass spectrometric analysis of all fucosylated proteins in melanoma and identified 2 MHC proteins, HLA-A and HLA-DRB1, as fucosylated. Knockdown of HLA- DRB1 and HLA-A in vivo revealed HLA-DRB1 is required for L-fucose-mediated tumor suppression, and notably, for recruitment of CD4+ T cells. We further investigated how fucosylation affects CD4+ T-cell biology using CD4+ T cells isolated from healthy donors, which we pharmacologically modulated fucosylation. The roles of fucosylation on tumor vs. CD4+ T cells and clinical utility/implications for melanoma patients will be discussed. Our data show how increased fucosylation drives tumor suppression through CD4+ T cells, and support the potential of dietary L-fucose to boost immunity in melanomas. Citation Format: Daniel K. Lester, Pasquale Innamarato, Krithika Kodumudi, Gregory Watson, Matt Mercurio, Shari Pilon-Thomas, Jane Messina, Susan McCarthy, Eric Lau. Fucosylation in CD4+ T cell-mediated melanoma suppression [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2019 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(3 Suppl):Abstract nr A73.

To detect the molecular changes of malignancy in histologically normal breast (HNB) tissues, we recently developed a novel 117-gene-malignancy-signature. Here we report validation of our leading malignancy-risk-genes, topoisomerase-2-alpha (TOP2A), minichromosome-maintenance-protein-2 (MCM2) and “budding-uninhibited-by-benzimidazoles-1-homolog-beta” (BUB1B) at the protein level. Using our 117-gene malignancy-signature, we classified 18 fresh-frozen HNB tissues from 18 adult female breast cancer patients into HNB-tissues with low-grade (HNB-LGMA; ) and high-grade molecular abnormality (HNB-HGMA; ). Archival sections of additional HNB tissues from these patients, and invasive ductal carcinoma (IDC) tissues from six other patients were immunostained for these biomarkers. TOP2A/MCM2 expression was assessed as staining index (%) and BUB1B expression as H-scores (0–300). Increasing TOP2A, MCM2, and BUB1B protein expression from HNB-LGMA to HNB-HGMA tissues to IDCs validated our microarray-based molecular classification of HNB tissues by immunohistochemistry. We also demonstrated an increasing expression of TOP2A protein on an independent test set of HNB/benign/reductionmammoplasties, atypical-ductal-hyperplasia with and without synchronous breast cancer, DCIS and IDC tissues using a custom tissue microarray (TMA). In conclusion, TOP2A, MCM2, and BUB1B proteins are potential molecular biomarkers of malignancy in histologically normal and benign breast tissues. Larger-scale clinical validation studies are needed to further evaluate the clinical utility of these molecular biomarkers.

Historical data have indicated the potential for the histologically-normal breast to harbor pre-malignant changes at the molecular level. We postulated that a histologically-normal tissue with "tumor-like" gene expression pattern might harbor substantial risk for future cancer development. Genes associated with these high-risk tissues were considered to be "malignancy-risk genes". From a total of 90 breast cancer patients, we collected a set of 143 histologically-normal breast tissues derived from patients harboring breast cancer who underwent curative mastectomy, as well as a set of 42 invasive ductal carcinomas (IDC) of various histologic grades. All samples were assessed for global gene expression differences using microarray analysis. For the purpose of this study we defined normal breast tissue to include histologically normal and benign lesions. Here we report the discovery of a "malignancy-risk" gene signature that may portend risk of breast cancer development in benign, but molecularly-abnormal, breast tissue. Pathway analysis showed that the malignancy-risk signature had a dramatic enrichment for genes with proliferative function, but appears to be independent of ER, PR, and HER2 status. The signature was validated by RT-PCR, with a high correlation (Pearson correlation = 0.95 with P < 0.0001) with microarray data. These results suggest a predictive role for the malignancy-risk signature in normal breast tissue. Proliferative biology dominates the earliest stages of tumor development.

The VERB ™ campaign used a social marketing approach to deliver its message through the mass media, school and community promotions, and partnerships to encourage children aged 9–13 years (tweens) to be physically active every day. This paper presents the VERB campaign's community and national partnership strategy, highlights three successful partnerships, and discusses challenges associated with the efforts. The national advertising generated awareness of and affinity for the product's brand and motivated the primary audience to seek out the product. The campaign's national and community partners were engaged to facilitate a product-distribution channel. The campaign developed a three-pronged partnership strategy to integrate the promotion with the placement of the campaign's product (physical activity): (1) reframe the way physical activity is positioned and delivered; (2) connect the brand to the point-of-purchase; and (3) refer (or drive) the audience to the action outlets, opportunities, places, spaces and programs to purchase the product. The VERB campaign provided partners with marketing training and resources to assist them as they leveraged tweens' brand awareness and supported regular physical activity among tweens. The method of technical assistance and the types of marketing tools were provided in relationship to four characteristics of the partner: (1) partner's network, (2) leaders and champions in the network, (3) partner's financial resources for community campaigns; and (4) partner's understanding of the marketing mindset. Coordinated, collaborative, and strong mass-media and community-based interventions within a national social marketing campaign can sustain the immediate effects of such campaigns.

Insulin-induced gene 2 (Insig2) was recently identified as a putative positive prognostic biomarker for colon cancer prognosis. Insig2 has been previously reported to be an endoplasmic reticulum (ER) membrane protein, and a negative regulator of cholesterol synthesis. Here we report that Insig2 was validated as a gene with univariate negative prognostic capacity to discriminate human colon cancer survivorship. To investigate the functional roles it plays in tumor development and malignancy, Insig2 was over-expressed in colon cancer cells resulting in increased cellular proliferation, invasion, anchorage independent growth and inhibition of apoptosis. Over-expression of Insig2 appeared to suppress chemotherapeutic drug treatment-induced Bcl2 associated X protein (Bax) expression and activation. Insig2 was also found to localize to the mitochondria/heavy membrane fraction and associate with conformationally changed Bax. Moreover, Insig2 altered the expression of several additional apoptosis genes located in mitochondria, further supporting its new functional role in regulating mitochondrial mediated apoptosis. Our findings show that Insig2 is a novel colon cancer biomarker, and suggest, for the first time, a reasonable connection between Insig2 and Bax-mediated apoptosis through the mitochondrial pathway.

Following the introduction of a National Curriculum for Science, all secondary science teachers in England need to be prepared to teach all aspects of a broad and balanced science curriculum. This is the second paper in which we explore science student teachers’ subject knowledge development during a one‐year postgraduate teacher preparation course. In this qualitative study we explore the role of university tutors and school‐based subject mentors in science student teachers’ subject knowledge development as perceived by student teachers, school‐based subject mentors and university tutors. The findings reveal that student teachers are reluctant to use university tutors and school mentors for subject knowledge development because they are aware of their assessment roles. The role of the university tutor in subject knowledge development is perceived as one of facilitation and of developing student teachers’ sense of professionalism. School mentors perceive that they do provide support for subject knowledge ...

This paper proposes the use of a key biological concept - broad classification - to teach the provisional and contested nature of science in school biology curricula. It also examines existing curriculum-related factors which might pose obstacles to implementing such a change. An investigation in South Africa highlights the problems regarding biological classification at the kingdom level of 50 biology students entering one university. Using interviews, questionnaires and document analysis, factors thought to affect learners' ideas were investigated, including the understanding of the changing and contested nature of scientific knowledge of a range of biology educators (35 teachers, 33 university academics, five teacher educators, four curriculum developers and two textbook authors). The students lacked knowledge of the concept of a biological kingdom, and showed archaic thinking about the number of kingdoms. Many of the teachers held similar views, used outmoded classification systems in their teaching, ...

Pancreatic ductal adenocarcinoma (PDAC) is one of the deadliest forms of cancer and is very difficult to treat with conventional chemotherapeutic regimens. Gemcitabine and 5-fluorouracil are used in the management of PDAC and act by indirectly blocking replicative forks. However, these drugs are not highly effective at suppressing disease progression, indicating a need for the development of innovative therapeutic approaches. Recent studies indicate that suppression of the MCM helicase may provide a novel means to sensitize cancer cells to chemotherapeutic agents that inhibit replicative fork progression. Mammalian cells assemble more MCM complexes on DNA than are required to start S-phase. The excess MCM complexes function as backup initiation sites under conditions of replicative stress. The current study provides definitive evidence that cosuppression of the excess/backup MCM complexes sensitizes PDAC tumor lines to both gemcitabine and 5-FU, leading to increased loss of proliferative capacity compared with drugs alone. This occurs because reduced MCM levels prevent efficient recovery of DNA replication in tumor cells exposed to drug. PDAC tumor cells are more sensitive to MCM loss in the presence of gemcitabine than are nontumor, immortalized epithelial cells. Similarly, colon tumor cells are rendered less viable when cosuppression of MCM complexes occurs during exposure to the crosslinking agent oxaliplatin or topoisomerase inhibitor etoposide. Implications: These studies demonstrate that suppressing the backup complement of MCM complexes provides an effective sensitizing approach with the potential to increase the therapeutic index of drugs used in the clinical management of PDAC and other cancers. Mol Cancer Res; 13(9); 1296–305. ©2015 AACR.

Background Intraductal papillary mucinous neoplasms (IPMNs) are pancreatic ductal adenocarcinoma (PDAC) precursors. Differentiating between high-risk IPMNs that warrant surgical resection and low-risk IPMNs that can be monitored is a significant clinical problem, and we sought to discover a panel of mi(cro)RNAs that accurately classify IPMN risk status. Methodology/Principal Findings In a discovery phase, genome-wide miRNA expression profiling was performed on 28 surgically-resected, pathologically-confirmed IPMNs (19 high-risk, 9 low-risk) using Taqman MicroRNA Arrays. A validation phase was performed in 21 independent IPMNs (13 high-risk, 8 low-risk). We also explored associations between miRNA expression level and various clinical and pathological factors and examined genes and pathways regulated by the identified miRNAs by integrating data from bioinformatic analyses and microarray analysis of miRNA gene targets. Six miRNAs (miR-100, miR-99b, miR-99a, miR-342-3p, miR-126, miR-130a) were down-regulated in high-risk versus low-risk IPMNs and distinguished between groups (P

The demand on science teachers in England and Wales in terms of their own subject knowledge has increased significantly since the 1980s. This has created pressure on pre‐service educators to adequately prepare pre‐service teachers regarding their science knowledge, during a very intensive course. This qualitative study explored the perceptions of students about the range of strategies they use to facilitate their subject knowledge development during a one‐year postgraduate initial teacher education course, with a particular focus on their use of peers. The majority of the pre‐service teachers used their peers in a range of different ways to develop their own knowledge and understanding of science. They valued this support highly and believed that with enhanced tutor facilitation, peers could be used to greater effect. These findings are discussed in terms of the demands of a highly regulated, standards‐driven model of teacher education and the desire to encourage the development in our students of a sense...

The development of a successful radiation sensitivity predictive assay has been a major goal of radiation biology for several decades. We have developed a radiation classifier that predicts the inherent radiosensitivity of tumor cell lines as measured by survival fraction at 2 Gy (SF2), based on gene expression profiles obtained from the literature. Our classifier correctly predicts the SF2 value in 22 of 35 cell lines from the National Cancer Institute panel of 60, a result significantly different from chance (P = 0.0002). In our approach, we treat radiation sensitivity as a continuous variable, significance analysis of microarrays is used for gene selection, and a multivariate linear regression model is used for radiosensitivity prediction. The gene selection step identified three novel genes (RbAp48, RGS19, and R5PIA) of which expression values are correlated with radiation sensitivity. Gene expression was confirmed by quantitative real-time PCR. To biologically validate our classifier, we transfected RbAp48 into three cancer cell lines (HS-578T, MALME-3M, and MDA-MB-231). RbAp48 overexpression induced radiosensitization (1.5- to 2-fold) when compared with mock-transfected cell lines. Furthermore, we show that HS-578T-RbAp48 overexpressors have a higher proportion of cells in G2-M (27% versus 5%), the radiosensitive phase of the cell cycle. Finally, RbAp48 overexpression is correlated with dephosphorylation of Akt, suggesting that RbAp48 may be exerting its effect by antagonizing the Ras pathway. The implications of our findings are significant. We establish that radiation sensitivity can be predicted based on gene expression profiles and we introduce a genomic approach to the identification of novel molecular markers of radiation sensitivity.

Background Tumors evade T cell-mediated rejection despite the presence of tumor associated antigens (TAAs) and T cells specific for these TAAs in cancer patients. Therapeutic tumor vaccines are being developed to prevent this evasion. Previous reports revealed that anti-tumor T cell responses could be activated in mice when granulocyte macrophage-colony stimulating factor (GM-CSF) or CD40L are produced at tumor vaccine sites. We sought to test the hypothesis that production of GM-CSF and CD40L by a bystander cell line could induce an anti-tumor T cell response in an in vitro human model. Materials and methods The K562 cell line was stably transfected with the human GM-CSF and CD40L genes. The effect of this cell line on T cell responses was tested in a human autologous mixed tumor cell/lymph node cell model using tissue from a series of cancer patients. Results There was no significant anti-tumor T cell response when human lymphocytes derived from tumor-draining lymph nodes were stimulated with autologous tumor cells in vitro . However, significant anti-tumor T cell responses were observed when bystander cells transfected with CD40L and GM-CSF were added to the cultures. Conclusions A fully autologous human model consisting of tumor cells as stimulator cells and tumor-draining lymph nodes as responder cells can be used to test immunotherapeutic strategies. T cells in these lymph nodes are unresponsive to autologous tumor cells, but this lack of responsiveness can be reversed in the presence of GM-CSF and CD40L. These data provide a rationale for testing tumor cell vaccines incorporating GM-CSF- and CD40L-expressing bystanders in clinical trials.

Osteopontin (OPN) is a secreted phosphoglycoprotein known to interact with a number of integrin receptors. While increased OPN expression has been reported in a number of human cancers, and its cognate receptors (alphav-beta3, alphav-beta5, and alphav-beta1 integrins and CD44) have been identified, its role in colon cancer development and progression has not been extensively studied. We previously identified, using a combination of gene expression and tissue microarrays, that increased OPN expression is concordant with tumor stage. The current study examined the functional role of OPN in colon cancer progression and metastatic potential. The principal findings of this study were that both endogenous OPN expression (via stable transfection) as well as exogenous OPN (added to culture medium) enhanced the motility and invasive capacity of human colon cancer cells in vitro. OPN appeared to regulate motility though interaction with CD44. OPN expression also reduced intercellular (homotypic) adhesion, an important characteristic of metastatic cancer cells. Stable transfection of four poorly tumorigenic human colon cancer cell lines with OPN also resulted in enhanced tumorigenicity in vivo with increased proliferation and increased CD31 positive microvessel counts, concordant with the degree of OPN expression. Collectively, these results suggest that OPN may affect multiple functional components contributing to human colon cancer progression and solidifies its role in this process.

PURPOSE While much is known about the interactions between the pharmaceutical industry and physicians, very little is known about pharmaceutical marketing directed toward medical students. This study sought to characterize the extent and forms of medical students' exposure to pharmaceutical industry marketing. METHOD In 2001-02, an anonymous, 17-item questionnaire was distributed to 165 preclinical and 116 clinical students at the University of Minnesota Medical School-Twin Cities. The main outcome measures were the number and forms of exposures to pharmaceutical industry marketing reported by medical students and whether students had discussed these exposures with teachers or advisors. Preclinical and clinical students were compared using chi(2) analysis (p < .05). RESULTS One hundred fourteen (69.1%) preclinical students and 107 (92.2%) clinical students responded. Nearly all students reported at least one exposure to pharmaceutical industry marketing. Seventy-six (71.7%) clinical students compared to 38 (33.3%) preclinical students recalled over 20 exposures (p < .005). Clinical students were more likely to have received a free meal (p < .01), textbook (p < .005), pocket text (p < .005), or trinket (p < .005) than were their preclinical colleagues. Most students (68.2%) had not discussed pharmaceutical marketing with an instructor or advisor; 59 (55.7%) clinical students as compared to 87 (80.6%) preclinical students recalled no such discussion (p < .005). CONCLUSION Medical students have extensive exposure to pharmaceutical industry marketing during their early years of training. Given existing evidence that such exposure influences physicians' practice and prescribing patterns, the authors propose that medical school curricula include formal instruction to prepare students to critically assess these contacts.

Clusterin is a widely expressed glycoprotein that has been paradoxically observed to have both pro- and antiapoptotic functions. Recent reports suggest this apparent dichotomy of function may be related to two different isoforms, one secreted and cytoplasmic, the other nuclear. To clarify the functional role of clusterin in regulating apoptosis, we examined its expression in human colon cancer tissues and in human colon cancer cell lines. We additionally explored its expression and activity using models of adenomatous polyposis coli (APC)- and chemotherapy-induced apoptosis. Clusterin RNA and protein levels were decreased in colon cancer tissues largely devoid of wild-type APC when compared with matched normal tissue controls, suggesting a means for invasive cancers to avoid apoptosis. Conversely, induction of apoptosis by expression of wild-type APC or by treatment with chemotherapy led to increased clusterin RNA and protein levels localizing to apoptotic nuclei. We found that transient transfection of clusterin to colon cancer cell lines directly enhanced basal and chemotherapy-induced apoptosis. Clusterin-induced apoptosis was inhibited by antisense clusterin and was found to be highly dependent on p21 but not p53 expression, yet a deficit in p21 can be subverted by clusterin transfection. Collectively, these data support the hypothesis that nuclear clusterin function is proapoptotic when induced by APC or chemotherapy in the context of p21 expression. Absent of p21, clusterin in not induced, and apoptosis is significantly inhibited. These data support a potential therapeutic role for clusterin in enhancing chemotherapy-induced apoptosis and in promoting apoptosis in cells deficient in p21.

SUMMARY The National Agricultural Library (NAL) of the United States of America and its national and international partners have developed a number of innovative services and collaborative programs in the last ten years. NAL's collaborative efforts have focused on providing access to agricultural information through preservation of publications, Internet access, electronic networks, and development of discipline-specific Web sites. This article describes four case studies demonstrating collaborative innovations in agricultural information services, developed through different partnership models: http://www.Invasivespecies.gov; http://www.Science.gov, AgNIC (Agriculture Network Information Center); and the National Preservation Program for Agricultural Literature.

Introduction : Dementia is a complex and chronic disease and is currently the second leading cause of death in Australia, three in ten people over the age of 85 and almost one in ten people over 65 have dementia. In Northern Sydney, there is projected growth of 23% in the population of people aged 75 years and older between 2011 and 2021, indicating a substantial increase in the number of people with dementia in the region over the next 6 years. There are a wide range of services available in Northern Sydney for people with dementia and their carers; however access and information about support services has been problematic. Information is often fragmented and difficult to find for consumers and Health Care Professionals. A partnership between Northern Sydney Local Health District (NSLHD), Sydney North Health Network, CCNB Ltd and Alzheimer’s Australia NSW was established to improve the journeys of people with dementia in Northern Sydney. Method : People with dementia (3) and many more carers (20) told us their stories, with key themes from these journeys identified. Over 40 health professionals from across sectors were separately engaged in mapping a number of typical journeys as well as an ideal journey. Gaps and issues were identified and prioritised, with a range of solutions then identified and prioritised. A project plan was implemented by the partnership, including strategies to address identified gaps in information, service access, referral pathways and education. Major project outputs include clarification of referral pathways for General Practitioners, a “Memory Problems?” booklet outlining the range of services available for people with dementia and their carers, distribution of the same across health sectors, government and non-government services throughout the region, a dementia hospital discharge follow–up service commissioned by Sydney North Health Network (SNHN), and a number of education events targeting different groups across the region. Other initiatives in progress being led by SNHN are the development of national quality indicators for dementia care in general practice and a localised dementia pathway for health professionals. Results : A number of typical journeys for people with dementia and their carers have been mapped, highlighting the range of complex interactions experienced and many opportunities to enable better journeys. The impact of the resulting initiatives are being evaluated through a range of indicators including the timing of referrals to memory clinics in patient’s journeys, unplanned hospital admissions for people with dementia and provider satisfaction. Results are expected to be available by the time of the conference. The NSW Agency for Clinical Innovation is undertaking evaluation of the partnership and it's benefits as part of a statewide program for integrating care for older people with complex health needs. Conclusions : This partnership has resulted in implementation of a plan which will have a substantially greater impact than if one organisation had undertaken this work alone. The project outputs are practical, sustainable and are expected to improve the journeys of people with dementia and their carers in Northern Sydney.

Background The epithelial-mesenchymal transition (EMT) is a key developmental program that is often activated during cancer progression and may promote resistance to therapy. An analysis of patients (n = 71) profiled with both gene expression and a global microRNA assessment (~415 miRs) identified miR-147 as highly anti-correlated with an EMT gene expression signature score and postulated to reverse EMT (MET). Methods and Findings miR-147 was transfected into colon cancer cells (HCT116, SW480) as well as lung cancer cells (A-549). The cells were assessed for morphological changes, and evaluated for effects on invasion, motility, and the expression of key EMT markers. Resistance to chemotherapy was evaluated by treating cells with gefitinib, an EGFR inhibitor. The downstream genes regulated by miR-147 were assayed using the Affymetrix GeneChip U133 Plus2.0 platform. miR-147 was identified to: 1. cause MET primarily by increasing the expression of CDH1 and decreasing that of ZEB1; 2. inhibit the invasion and motility of cells; 3. cause G1 arrest by up-regulating p27 and down-regulating cyclin D1. miR-147 also dramatically reversed the native drug resistance of the colon cancer cell line HCT116 to gefitinib. miR-147 significantly repressed Akt phosphorylation, and knockdown of Akt with siRNA induced MET. The morphologic effects of miR-147 on cells appear to be attenuated by TGF-B1, promoting a plastic and reversible transition between MET and EMT. Conclusion miR-147 induced cancer cells to undergo MET and induced cell cycle arrest, suggesting a potential tumor suppressor role. miR-147 strikingly increased the sensitivity to EGFR inhibitor, gefitinib in cell with native resistance. We conclude that miR-147 might have therapeutic potential given its ability to inhibit proliferation, induce MET, as well as reverse drug sensitivity.

BACKGROUND AND AIMS: Altered expression of specific microRNAs (miRNA) is known to occur during colorectal carcinogenesis. However, little is known about the genome-wide alterations in miRNA expression during the neoplastic progression of primary colorectal cancers. METHODS: Using a miRNA array platform, we simultaneously evaluated the expression of 668 miRNA in primary colonic adenocarcinomas. Prediction analysis for microarrays (PAM) was used to identify differentially expressed miRNA. Biological functions of selected miRNA were evaluated with in vitro invasion assays. RESULTS: Primary fresh frozen tissues from 65 patients (40 male and 25 female) with a mean age 65 +/− 13 years and with AJCC Stages I (n=7), II (n=22), III (n=18) and IV (n=18) colon cancers, underwent RNA extraction and miRNA array analysis. We identified a seven-miRNA expression signature that differentiated Stage I and Stage IV primary colon cancers. We then demonstrated this signature was able to discriminate between Stage II and III primary colon cancers. Interestingly 6 of the 7 differentially expressed miRNA were downregulated in association with the development of metastases, and all 7 miRNA were complementary strand miRNA*. We transfected HCT-116, a highly invasive colon cancer cell line, with corresponding downregulated miRNA* and demonstrated that overexpression of 3 of the 6 miRNA* (miR200c*, miR143*, and miR424*) significantly abrogated invasive potential. CONCLUSION: We have identified a seven-miRNA* signature that is associated with metastatic potential in the primary tumor. Forced over-expression of 3 of the 6 identified miRNA* resulted in an attenuation of in vitro invasion, suggesting direct tumor suppressive function and further supporting the biological importance of complementary strand miRNA*.

This article reports on 18 months of action research that monitored British pupils' learning about the nature of science, using some aspects of history of science for the purpose. The action research took place within five classrooms and involved practicing teachers who used a set of historical materials specially written for this study. Preliminary findings about the common perceptions of the nature of science held by middle school pupils (age 11–14 years) guided the work, which was carried out using a variety of methodologies. The results obtained show some areas of substantial progress in the pupils' understanding of the nature of science, and others where little change seems to have been effected.

Green tea catechins (GTCs) exert chemopreventive effects in many cancer models. Several studies implicate the DNA synthesis marker minichromosome maintenance protein 7 (MCM7) in prostate cancer progression, growth and invasion; representing a novel therapeutic target. In this study, we investigated the effect of GTCs on MCM7 expression in the transgenic adenocarcinoma mouse prostate model (TRAMP). DNA microarray, immunohistochemistry and western blot analysis showed that GTCs significantly suppressed MCM7 in the TRAMP mice treated with GTCs. Our study indicates that the cellular DNA replication factor MCM7 is involved in prostate cancer (CaP) and MCM7 gene expression was reduced by GTCs. Together, these results suggest a possible role of GTCs in CaP chemoprevention in which MCM7 plays a critical role.

The VERB campaign used a social marketing approach to deliver its message through the mass media, school and community promotions, and partnerships to encourage children aged 9-13 years (tweens) to be physically active every day. This paper presents the VERB campaign's community and national partnership strategy, highlights three successful partnerships, and discusses challenges associated with the efforts. The national advertising generated awareness of and affinity for the product's brand and motivated the primary audience to seek out the product. The campaign's national and community partners were engaged to facilitate a product-distribution channel. The campaign developed a three-pronged partnership strategy to integrate the promotion with the placement of the campaign's product (physical activity): (1) reframe the way physical activity is positioned and delivered; (2) connect the brand to the point-of-purchase; and (3) refer (or drive) the audience to the action outlets, opportunities, places, spaces and programs to purchase the product. The VERB campaign provided partners with marketing training and resources to assist them as they leveraged tweens' brand awareness and supported regular physical activity among tweens. The method of technical assistance and the types of marketing tools were provided in relationship to four characteristics of the partner: (1) partner's network, (2) leaders and champions in the network, (3) partner's financial resources for community campaigns; and (4) partner's understanding of the marketing mindset. Coordinated, collaborative, and strong mass-media and community-based interventions within a national social marketing campaign can sustain the immediate effects of such campaigns.

Background: Microbial infections are associated with the initial susceptibility to and flares of asthma. However, immunologic mechanisms whereby infections might alter the asthmatic phenotype are lacking. Objective: To test the hypothesis that memory T cells specific both for a viral antigen and an allergen could influence the pathogenesis of allergic disease in vivo. Methods: We developed a system in which 2 distinct T-cell receptors coexist on the T-cell surface, 1 specific for a virus and the other for an inhaled antigen. Results: We show that a population of dual-receptor T cells, polarized through a virus-specific T-cell receptor to contain TH 1o r TH2 cells, can be reactivated through an unrelated T-cell receptor in recall responses in vivo. Quiescent memory cells derived from a TH1-polarized effector population blocked the development of airway hyperreactivity in a model of allergic lung disease, in association with decreased induction of chemokines and eosinophil recruitment. Conversely, reactivation of quiescent TH2 cells after inhalation of antigen or virus infection was sufficient to lead to the development of airway hyperresponsiveness and allergic pulmonary inflammation in mice whose lungs were previously normal. Conclusion: These data provide evidence that dual-receptor memory T cells can regulate allergic disease susceptibility and suggest that they may play a role in mediating the influence of microbes on asthma pathogenesis. (J Allergy Clin Immunol 2004;114:1441-8.)

NF-kappaB/Rel transcription factors are linked to innate immune responses and APC activation. Whether and how the induction of NF-kappaB signaling in normal CD4(+) T cells regulates effector function are not well-understood. The liberation of NF-kappaB dimers from inhibitors of kappaB (IkappaBs) constitutes a central checkpoint for physiologic regulation of most forms of NF-kappaB. To investigate the role of NF-kappaB induction in effector T cell responses, we targeted inhibition of the NF-kappaB/Rel pathway specifically to T cells. The Th1 response in vivo is dramatically weakened when T cells defective in their NF-kappaB induction (referred to as IkappaBalpha(DeltaN) transgenic cells) are activated by a normal APC population. Analyses in vivo, and IL-12-supplemented T cell cultures in vitro, reveal that the mechanism underlying this T cell-intrinsic requirement for NF-kappaB involves activation of the IFN-gamma gene in addition to clonal expansion efficiency. The role of NF-kappaB in IFN-gamma gene expression includes a modest decrease in Stat4 activation, T box expressed in T cell levels, and differentiation efficiency along with a more prominent postdifferentiation step. Further, induced expression of Bcl-3, a trans-activating IkappaB-like protein, is decreased in T cells as a consequence of NF-kappaB inhibition. Together, these findings indicate that NF-kappaB induction in T cells regulates efficient clonal expansion, Th1 differentiation, and IFN-gamma production by Th1 lymphocytes at a control point downstream from differentiation.