High-level expression of epithelial cell adhesion molecule (EpCAM) is associated with unfavorable prognosis in breast cancer. This study was designed to investigate two doses of the fully human IgG1 ...anti-EpCAM antibody adecatumumab (MT201) in patients with metastatic breast cancer (MBC).
A total of 109 patients were stratified into high- and low-level EpCAM expression by immunohistochemical staining of primary tumors and subsequently randomly assigned to receive monotherapy with either high- (6 mg/kg every two weeks (q2w)) or low-dose adecatumumab (2 mg/kg/ q2w) until disease progression.
No complete or partial tumor responses could be confirmed by central RECIST assessment. The probability for tumor progression was significantly lower in patients receiving high-dose adecatumumab and expressing high levels of EpCAM (hazard ratio 0.43; P=0.0057 versus low dose and low EpCAM). Three of 18 patients with highest EpCAM expression treated with adecatumumab developed new metastases up to week 6, compared with 14 of 29 patients with low EpCAM. Most frequent treatment-related adverse events (high dose/low dose) were chills (59%/20%), nausea (55%/18%), fatigue (39%/23%) and diarrhea (43%/7%).
Single-agent adecatumumab shows dose- and target-dependent clinical activity in EpCAM-positive MBC, albeit no objective tumor regression. Further investigation of adecatumumab in patients with EpCAM-overexpressing tumors and lower tumor burden is warranted.
Objective We evaluated the expression of epithelial cell adhesion molecule (EpCAM) and the potential of MT201 (adecatumumab), a human-monoclonal-antibody that targets EpCAM against ...chemotherapy-resistant ovarian disease. Study Design EpCAM expression was evaluated by real-time polymerase chain reaction and flow cytometry. Sensitivity to MT201 antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity was tested in 4-hour chromium-release assays. The effect of interleukin-2 on MT201 ADCC was also studied. Results High messenger RNA expression by real-time polymerase chain reaction and high EpCAM surface expression by flow cytometry was detected in 71% of ovarian cancers (5 of 7 cell lines). Although these cell lines were highly resistant to complement-dependent cytotoxicity and natural killer-dependent cytotoxicity in vitro (range of killing, 0–7%), EpCAM-positive cell lines showed high sensitivity to MT201 ADCC (range of killing, 27–66%). Incubation with interleukin-2 further increased the cytotoxic activity against EpCAM-positive ovarian cancer cell lines. Conclusion MT201 may represent a novel, potentially highly effective treatment option for patients with ovarian carcinoma whose body is harboring disease refractory to chemotherapy.
The predominant testicular gap junctional protein connexin43 (cx43) is located between neighboring Sertoli cells (SCs) and between SCs and germ cells. It is assumed to be involved in testicular ...development, cell differentiation, initiation, and maintenance of spermatogenesis with alterations of its expression being correlated with various testicular disorders. Because total disruption of the cx43 gene leads to perinatal death, we generated a conditional cx43 knockout (KO) mouse using the Cre/loxP recombination system, which lacks the cx43 gene solely in SCs (SCCx43KO), to evaluate the SC-specific functions of cx43 on spermatogenesis in vivo . Adult SCCx43KO−/− mice showed normal testis descent and development of the urogenital tract, but testis size and weight were drastically lower compared with heterozygous and wild-type littermates. Histological analysis and quantitation of mRNA expression of germ cell-specific marker genes revealed a significant reduction in the number of spermatogonia but increased SC numbers/tubule with only a few tubules left showing normal spermatogenesis. Thus, SC-specific deletion of cx43 mostly resulted in an arrest of spermatogenesis at the level of spermatogonia or SC-only syndrome and in intratubular SC clusters. Our data demonstrate for the first time that cx43 expression in SCs is an absolute requirement for normal testicular development and spermatogenesis.
Quantitative distance measurements are difficult to obtain in spite of the strong distance dependency of the energy transfer efficiency. One problem for the interpretation of the Forster resonant ...energy transfer (FRET) efficiency is the so-called zero-efficiency peak caused by FRET pairs with missing or nonfluorescent acceptors. Other problems occurring are direct excitation of the acceptor, spectral crosstalk, and the determination of the quantum efficiency of the dyes as well as the detector sensitivity. Our approach to overcome these limitations is based on the pulsed-interleaved excitation (PIE) of both the acceptor and the donor molecule. PIE is used to excite the acceptor dye independently of the FRET process and to prove its existence via fluorescence. This technique enables us to differentiate a FRET molecule, even with a very low FRET efficiency, from a molecule with an absent or non-fluorescent acceptor. Crosstalk, direct acceptor excitation, and molecular brightness of acceptor and donor molecules are determined by analyzing the data with fluorescence correlation spectroscopy (FCS). FRET efficiencies of the same data set are also determined by analyzing the lifetimes of the donor fluorophores. The advantages of the PIE-FRET approach are demonstrated on a polyproline assay labeled with Alexa-555 and Alexa-647 as donor and acceptor, respectively.
Recent developments in immunology have provided new strategies to induce and augment the immune response to cancer. Nonetheless, objective clinical responses after vaccination are rare and even when ...high frequencies of tumor-specific T cells are achieved after adoptive immunotherapy, tumor cells continue to evade the immune response. We hypothesize that 1 mechanism of resistance of tumor cells to destruction by T cells is an elevated threshold for the induction of apoptosis. Inhibitor of apoptosis proteins (IAPs) are overexpressed in various tumors and have been associated with treatment failure and poor prognosis. As the mitochondrial peptide second mitochondria-derived activator of caspase (Smac) can antagonize IAPs, we designed a GFP-Smac fusion protein with a granzyme B (GrB) cleavage site. This fusion protein should be cleaved when tumor-specific cytolytic T cells recognize the tumor and, using the pore-forming protein perforin, insert GrB into the target. Here we report that transfer of a construct encoding a novel eGFP-Smac fusion protein (pro-Smac) containing a specific cleavage site for GrB, into the poorly immunogenic mouse melanoma cell line, B16BL6-D5 (D5), sensitizes tumor cells for killing by tumor-specific wild type, but not perforin-deficient (perforin-knockout), effector T cells in vitro and in vivo. These results describe the first example of a tumor-specific, T-cell-mediated approach to amplify the GrB-mediated cytotoxicity pathway with a pro-Smac fusion protein and provide an innovative approach to overcome IAPs and improve the efficacy of immunotherapy.
From our way of thinking the problem facing vaccine strategies for cancer is not that we do not have "enough" tumour antigens. The problem is we cannot induce an immune response that is sufficient to ...mediate tumour regression. The normal "checks and balances" found in the body prevent the sustained expansion and subsequent persistence of immune killer cells. If vaccine strategies are going to become effective treatments for cancer patients, they will need to overcome this substantial roadblock. Recent developments in immunology have provided insights into the mechanisms that regulate the expansion and persistence of T cells. This has allowed investigators to reinterpret decades-old observations suggesting that chemotherapy administered before vaccination often led to a stronger immune response. This manuscript will review experiments that offer an explanation for these observations and present pre-clinical data from our laboratory that describes an innovative new approach to combining chemotherapy and vaccination. This approach is readily translatable to the clinic and is broadly applicable to any vaccine strategy for advanced cancer.
How can multi-stakeholder dialogue be used to assess and address the roots of environmental resource conflict in complex commons characterized by multiple levels of governance? This paper presents ...the results of a collaborative initiative aimed at strengthening aquatic resources governance in three ecoregions-Lake Victoria (Uganda), Lake Kariba (Zambia), and Tonle Sap Lake (Cambodia)- implemented from mid-2010 through early 2013. Analysis drawn from multi-stakeholder dialogue workshops and follow-on activities in each of the lake systems characterizes the sources of resource conflict, associated governance challenges, and opportunities for institutional innovation to address these. These innovations include attempts to increase community voices in private sector investment decisions and secure access rights for marginalized households in the face of competition, as well as strengthen community-based co-management, resource protection, public health, and gender equity. For each case, we consider interactions across scales with regard to ecosystem services, livelihood opportunities, and institutions of government, civil society, and the private sector. We then compare the process of participatory dialogue, action, and learning in each of the sites, noting the core set of principles employed, the diverse approaches to applying these in different socio-cultural and political contexts, and the outcomes as measured through evaluations led by local actors. The paper concludes with a synthesis of lessons regarding the conditions under which such an approach to multi-stakeholder dialogue is appropriate, the contributions this approach can make to improve multilevel governance as well as its limitations, and strategies for improving this domain of practice in the future.
Increased cardiovascular morbidity and mortality among cigarette smokers may be mediated in part by enhanced platelet function. Previous data showed that cigarette smoking--induced lowering of the ...platelet aggregate ratio of normal individuals was prevented by taking aspirin before smoking. Our study was undertaken to determine whether similar results would occur in men with coronary artery disease and whether platelet factor 4 would be released. A random-order, double-blind crossover study comparing the effects of placebo, 0.15 gm aspirin, and 0.30 gm aspirin was done in 30 male habitual smokers with coronary artery disease. Each man took a tablet containing placebo or aspirin and then abstained from smoking for 12 hours before each of three 20-minute periods of smoking two tobacco cigarettes. Immediately before and after smoking, the platelet aggregate ratio and the concentration of platelet factor 4 in platelet-poor plasma were determined from antecubital venous blood. Twelve hours after placebo, the geometric mean concentration of platelet factor 4 was 13.6 ng/ml before and 19.7 ng/ml after smoking (P = 0.0006). The mean platelet aggregate ratio was 0.77 and 0.72, respectively (P less than 0.00001). Neither dose of aspirin affected the presmoking value of or the smoking-induced change in either variable. The data indicate that smoking stimulated platelet aggregate formation and release of the contents of platelet alpha-granules, which were unaffected by preadministration of aspirin. This contrasts with the previous study of normal habitual smokers whose ingestion of 0.32 gm aspirin prevented a smoking-induced decrease in the platelet aggregate ratio.
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