Identification of Replication Competent Murine Gammaretroviruses in Commonly Used Prostate Cancer Cell Lines

A newly discovered gammaretrovirus, termed XMRV, was recently reported to be present in the prostate cancer cell line CWR22Rv1. Using a combination of both immunohistochemistry with broadly-reactive murine leukemia virus (MLV) anti-sera and PCR, we determined if additional prostate cancer or other cell lines contain XMRV or MLV-related viruses. Our study included a total of 72 cell lines, which included 58 of the 60 human cancer cell lines used in anticancer drug screens and maintained at the NCI-Frederick (NCI-60). We have identified gammaretroviruses in two additional prostate cancer cell lines: LAPC4 and VCaP, and show that these viruses are replication competent. Viral genome sequencing identified the virus in LAPC4 and VCaP as nearly identical to another known xenotropic MLV, Bxv-1. We also identified a gammaretrovirus in the non-small-cell lung carcinoma cell line EKVX. Prostate cancer cell lines appear to have a propensity for infection with murine gammaretroviruses, and we propose that this may be in part due to cell line establishment by xenograft passage in immunocompromised mice. It is unclear if infection with these viruses is necessary for cell line establishment, or what confounding role they may play in experiments performed with these commonly used lines. Importantly, our results suggest a need for regular screening of cancer cell lines for retroviral “contamination”, much like routine mycoplasma testing

Cytotoxic T lymphocytes that recognize decameric peptide sequences of retinoblastoma binding protein 1 (RBP-1) associated with human breast cancer

Retinoblastoma binding protein 1 (RBP-1) is a 143-kDa nuclear phosphoprotein that promotes cell growth by inhibiting the product of retinoblastoma tumour suppressor gene (pRB). We recently found that RBP-1 contains KASIFLK, a heptameric peptide (250–256) recognized by human antibodies and overexpressed by breast cancer cells. In the present study, we demonstrate that human T-cells stimulated with RBP-1 decameric peptides containing KASIFLK can kill human breast cancer cells. These decamers, GLQKASIFLK (247–256) and KASIFLKTRV (250–259), have anchor motifs for both HLA-A2 and HLA-A3. Peripheral blood lymphocytes from 41 normal donors were stimulated by these peptides in culture media containing 15 IU ml−1 interleukin-2, 25 IU ml−1 interleukin-7 and 500 IU ml−1 granulocyte–macrophage colony-stimulating factor. Cytotoxic activity of the T-cells was assessed against autologous B lymphoblastoid cells pulsed with each peptide. Stimulation by GLQKASIFLK generated specific cytotoxic T lymphocyte (CTL) lines from HLA-A2, A3 donors, HLA-A2 donors and HLA-A3 donors. Stimulation with KASIFLKTRV generated specific CTL lines from HLA-A2 donors. No HLA-A2−, A3− CTL line showed specific cytotoxicity against these target cells. These CTL lines were also cytotoxic against HLA-A2 and HLA-A3 breast cancer cells but not against normal fibroblastoid cell lines, normal epidermal cell lines, or a melanoma cell line. RBP-1 peptide antigens may be of clinical significance as a potential peptide vaccine against human breast cancer. © 1999 Cancer Research Campaig

An Efficient Strategy to Induce and Maintain In Vitro Human T Cells Specific for Autologous Non-Small Cell Lung Carcinoma

BACKGROUND: The efficient expansion in vitro of cytolytic CD8+ T cells (CTLs) specific for autologous tumors is crucial both for basic and translational aspects of tumor immunology. We investigated strategies to generate CTLs specific for autologous Non-Small Cell Lung Carcinoma (NSCLC), the most frequent tumor in mankind, using circulating lymphocytes. PRINCIPAL FINDINGS: Classic Mixed Lymphocyte Tumor Cultures with NSCLC cells consistently failed to induce tumor-specific CTLs. Cross-presentation in vitro of irradiated NSCLC cells by autologous dendritic cells, by contrast, induced specific CTL lines from which we obtained a high number of tumor-specific T cell clones (TCCs). The TCCs displayed a limited TCR diversity, suggesting an origin from few tumor-specific T cell precursors, while their TCR molecular fingerprints were detected in the patient's tumor infiltrating lymphocytes, implying a role in the spontaneous anti-tumor response. Grafting NSCLC-specific TCR into primary allogeneic T cells by lentiviral vectors expressing human V-mouse C chimeric TCRalpha/beta chains overcame the growth limits of these TCCs. The resulting, rapidly expanding CD4+ and CD8+ T cell lines stably expressed the grafted chimeric TCR and specifically recognized the original NSCLC. CONCLUSIONS: This study defines a strategy to efficiently induce and propagate in vitro T cells specific for NSCLC starting from autologous peripheral blood lymphocytes

Photodynamic Therapy of Tumors Can Lead to Development of Systemic Antigen-Specific Immune Response

Background: The mechanism by which the immune system can effectively recognize and destroy tumors is dependent on recognition of tumor antigens. The molecular identity of a number of these antigens has recently been identified and several immunotherapies have explored them as targets. Photodynamic therapy (PDT) is an anti-cancer modality that uses a non-toxic photosensitizer and visible light to produce cytotoxic reactive oxygen species that destroy tumors. PDT has been shown to lead to local destruction of tumors as well as to induction of anti-tumor immune response. Methodology/Principal Findings: We used a pair of equally lethal BALB/c colon adenocarcinomas, CT26 wild-type (CT26WT) and CT26.CL25 that expressed a tumor antigen, β-galactosidase (β-gal), and we treated them with vascular PDT. All mice bearing antigen-positive, but not antigen-negative tumors were cured and resistant to rechallenge. T lymphocytes isolated from cured mice were able to specifically lyse antigen positive cells and recognize the epitope derived from beta-galactosidase antigen. PDT was capable of destroying distant, untreated, established, antigen-expressing tumors in 70% of the mice. The remaining 30% escaped destruction due to loss of expression of tumor antigen. The PDT anti-tumor effects were completely abrogated in the absence of the adaptive immune response. Conclusion: Understanding the role of antigen-expression in PDT immune response may allow application of PDT in metastatic as well as localized disease. To the best of our knowledge, this is the first time that PDT has been shown to lead to systemic, antigen- specific anti-tumor immunity.United States. National Cancer Institute (grant RO1CA/AI838801)United States. National Cancer Institute (grant R01AI050875

Het verdiepen en professionaliseren van het toegepaste onderzoek binnen het Lectoraat ZorgGericht Bouwen

Vanuit het lectoraat ZorgGericht Bouwen dat wordt aangestuurd door Tom Vroon en Jarno Nillesen heeft men de visie dat het onderzoek de sleutel is voor succes op de langere termijn. Vanuit dit onderzoek wordt nieuwe kennis ontwikkeld en worden inzichten vergaard die als basis dienen voor de positie attractiviteit en verdere ontwikkeling van het Lectorraad

Process simulation during the design process makes the difference: process simulations applied to a traditional design

Objective: The objective is evaluation of a traditionally designed operating room using simulation of various surgical workflows.Background: A literature search showed that there is no evidence for an optimal operating room layout regarding the position and size of an ultraclean ventilation (UCV) canopy with a separate preparation room for laying out instruments and in which patients are induced in the operating room itself. Neither was literature found reporting on process simulation being used for this application. Many technical guidelines and designs have mainly evolved over time, and there is no evidence on whether the proposed measures are also effective for the optimization of the layout for workflows.Methods: The study was conducted by applying observational techniques to simulated typical surgical procedures. Process simulations which included complete surgical teams and equipment required for the intervention were carried out for four typical interventions. Four observers used a form to record conflicts with the clean area boundaries and the height of the supply bridge. Preferences for particular layouts were discussed with the surgical team after each simulated procedure.Results: We established that a clean area measuring 3 × 3 m and a supply bridge height of 2.05 m was satisfactory for most situations, provided a movable operation table is used. The only cases in which conflicts with the supply bridge were observed were during the use of a surgical robot (Da Vinci) and a surgical microscope. During multiple trauma interventions, bottlenecks regarding the dimensions of the clean area will probably arise.Conclusions: The process simulation of four typical interventions has led to significantly different operating room layouts than were arrived at through the traditional design process.Keywords: Evidence-based design, human factors, work environment, operating room, traditional design, process simulation, surgical workflows

Relationships between antral follicle count, blood serum concentration of anti-Müllerian hormone and fertility in mares

The anti-Müllerian hormone (AMH) plays an inhibitory role during folliculogenesis by regulating the number of follicles entering the growing pool. Antral follicle counts (AFC) are highly correlated with serum AMH concentrations and both appear to be related to the ovarian reserve in several species. Few data on AMH and AFC in mares exist, especially with regard to fertility. Therefore, the objective of the current study was to investigate the interrelationship between antral follicle count, serum AMH concentrations and fertility outcome in mares. One hundred and twenty-seven mares were enrolled in the study and grouped according to their reproductive status. Around time of estrus, serum AMH concentrations and AFC before and after ovulation were determined. Mares were artificially inseminated and pregnancy diagnosis was performed 14 to 18 days later. A high inter-individual variability in AFC and AMH concentration and a positive relationship between AMH and AFC for follicles ≤ 30 mm in diameter were observed, with a stronger correlation in mares older than 18 years. A high correlation between AFC measurements before and after ovulation was identified. The AFC after ovulation was higher than AFC before ovulation. AMH concentrations were neither related to the mares' reproductive status nor to age, number of cycles needed for pregnancy and pregnancy outcome. Excepted for a higher AFC in the middle-aged mares (9-18 years) compared to the younger mares (< 9 years), no associations between AFC and age, reproductive status as well as fertility of mares were found. This study confirms the relationship between AFC and AMH and a high degree of reproducibility of AFC measurements. However, based on our findings, neither AFC nor AMH are useful predictors of fertility in mares

Virtual Prototyping technique applied to the design of a crankmechanism of a process reciprocating compressor

Virtual Prototyping (VP) is a novel computational approach that reproduces a complete mechanism to test it several times, as a scale 1:1 laboratory prototype. VP utilizes various CAE tools, such as 3D modelling, Structural FEA, Multibody Dynamic Analysis (MDA), Multiaxial fatigue analysis, in an integrated way. The VP technique allows considering a realistic stepless loading pattern throughout the complete revolution and determining automatically the fatigue safety factors within the whole machine assembly. This new approach was used to review the design of the crankmechanism of an existing reciprocating compressor. The loads (including inertia forces) were applied to the gudgeon pin and, by means of the MDA, to all the other component