20 research outputs found

    Restriction landmark genomic scanning (RLGS) spot identification by second generation virtual RLGS in multiple genomes with multiple enzyme combinations.

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    BackgroundRestriction landmark genomic scanning (RLGS) is one of the most successfully applied methods for the identification of aberrant CpG island hypermethylation in cancer, as well as the identification of tissue specific methylation of CpG islands. However, a limitation to the utility of this method has been the ability to assign specific genomic sequences to RLGS spots, a process commonly referred to as "RLGS spot cloning."ResultsWe report the development of a virtual RLGS method (vRLGS) that allows for RLGS spot identification in any sequenced genome and with any enzyme combination. We report significant improvements in predicting DNA fragment migration patterns by incorporating sequence information into the migration models, and demonstrate a median Euclidian distance between actual and predicted spot migration of 0.18 centimeters for the most complex human RLGS pattern. We report the confirmed identification of 795 human and 530 mouse RLGS spots for the most commonly used enzyme combinations. We also developed a method to filter the virtual spots to reduce the number of extra spots seen on a virtual profile for both the mouse and human genomes. We demonstrate use of this filter to simplify spot cloning and to assist in the identification of spots exhibiting tissue-specific methylation.ConclusionThe new vRLGS system reported here is highly robust for the identification of novel RLGS spots. The migration models developed are not specific to the genome being studied or the enzyme combination being used, making this tool broadly applicable. The identification of hundreds of mouse and human RLGS spot loci confirms the strong bias of RLGS studies to focus on CpG islands and provides a valuable resource to rapidly study their methylation

    Downregulation and/or Release of NKG2D Ligands as Immune Evasion Strategy of Human Neuroblastoma

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    Neuroblastoma (NB) is a pediatric extracranial tumor characterized by downregulation of human leukocyte antigen class I and defects of the antigen processing machinery, two features that make it an appropriate target for natural killer (NK)-mediated lysis. NKG2D is an activating immunoreceptor expressed by cytotoxic T lymphocytes and NK cells. The ligands for NKG2D are the major histocompatibility complex class I-related chain (MIC)A and MICB glycoproteins, and the UL-16-binding proteins (ULBPs). Here, the expression of NKG2D ligands was investigated in human primary NB tumors and cell lines because scanty information is available on this issue. MICA, MICB, and ULBP transcripts were found in most tumors and cell lines. MICA protein was detected in some NB cell lines but not in primary tumors. A soluble form of MICA (sMICA) was identified in most patient sera and in some cell line supernatants. sMICA downregulated surface NKG2D in normal peripheral blood CD8+ cells and decreased NK-mediated killing of MICA+ NB cells. MICB was detected exclusively in the cytosol of primary tumors and cell lines. Approximately 50% of primary tumors expressed ULBP-2, but not ULBP-1 or -3. ULBP-3 was expressed in 5 of 9 cell lines, ULBP-2 in 2 of 9, whereas ULBP-1 was never detected. These studies delineate novel potential pathways of tumor escape and immunodeficiency in NB

    Pre-resectional RFA improves local CT26 tumor control and survival.

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    <p><b>(A)</b> Time schedule outlining the different treatments used. BALB/c mice (10–12 mice/group) were injected s.c. with 10<sup>6</sup> CT26 cells into the left flank on day 0. Treatments were performed 10 days after tumor innoculation. Primary tumors were treated as indicated with sham surgery, resection (Rxn), or RFA on days 10 or 17. <b>(B)</b> Schematic showing sequence of administration of RFA (90°C for 1 minute) 7 days prior to surgical resection. Tumor growth curves <b>(C)</b> and survival curves <b>(D)</b> of mice bearing CT26 tumors in different treatment groups. In <b>(C)</b>, the number of long-term survivors without tumor recurrence at 150 days is indicated for each experimental group; for <b>(D)</b> *<i>P</i> < 0.001 for pre-resectional RFA group compared to all other groups. <b>(E)</b> Surviving mice (n = 9) in the pre-resectional RFA group were rechallenged with 10<sup>6</sup> CT26 cells into the contralateral (right) flank on day 150. Tumor growth curves are depicted in which <i>T</i> = 0 corresponds to the time of injection of secondary tumors. As a control, tumor growth was monitored following inoculation of the same tumor cell dose into non-tumor (NT)-experienced naive BALB/c mice (n = 10); *<i>P</i> < 0.005 as determined by Kaplan-Meier analysis. One representative experiment of ≥ three independent experiments is shown.</p

    Survival benefit of pre-resectional RFA depends on adaptive immunity.

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    <p><b>(A)</b> Time schedule outlining treatment groups. SCID mice (5 mice/group) were implanted s.c. with 10<sup>6</sup> CT26 cells into the left flank on day 0. Treatment groups incuded sham surgery, resection (Rxn), or RFA on days 10 or 17. <b>(B)</b> Survival curves are representative of two independent experiments; n.s., not significant as determined by Kaplan-Meier analysis.</p

    Pre-resectional RFA elicits systemic antitumor effects in distant tumors.

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    <p><b>(A)</b> Time schedule outlining different treatments used in the experiements. BALB/c mice (5 mice/group) were injected s.c. with 10<sup>6</sup> CT26 cells into the left flank on day 0 to establish primary tumor, followed by s.c. injection of 10<sup>6</sup> CT26 cells in the right flank on day 7 to establish secondary tumor. Primary tumors or skin (at region outside of primary tumor; upper back) were treated as indicated with sham surgery, resection (Rxn), or RFA on days 10 or 17. <b>(B)</b> Growth curves of secondary CT26 tumors implanted s.c. on contralateral flank. *<i>P</i> <0.05. <b>(C)</b> Representative photomicrographs and quantification of immunostained endogenous CD8<sup>+</sup> T cells in distant secondary s.c. CT26 tumors. *<i>P</i> < 0.001; scale bar, 100 μm.</p
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