MASALAH-MASALAH PEMBELAJARAN YANG DIHADAPI WIDYAISWARA : Studi Kasus Pada Lembaga Diktat Pemda Tk.I Propinsi Bengkulu

<div><p>Rat strains differ dramatically in their susceptibility to mammary carcinogenesis. On the assumption that susceptibility genes are conserved across mammalian species and hence inform human carcinogenesis, numerous investigators have used genetic linkage studies in rats to identify genes responsible for differential susceptibility to carcinogenesis. Using a genetic backcross between the resistant Copenhagen (Cop) and susceptible Fischer 344 (F344) strains, we mapped a novel mammary carcinoma susceptibility (<i>Mcs30</i>) locus to the centromeric region on chromosome 12 (LOD score of ∼8.6 at the D12Rat59 marker). The <i>Mcs30</i> locus comprises approximately 12 Mbp on the long arm of rat RNO12 whose synteny is conserved on human chromosome 13q12 to 13q13. After analyzing numerous genes comprising this locus, we identified <i>Fry</i>, the rat ortholog of the furry gene of <i>Drosophila melanogaster,</i> as a candidate <i>Mcs</i> gene. We cloned and determined the complete nucleotide sequence of the 13 kbp <i>Fry</i> mRNA. Sequence analysis indicated that the <i>Fry</i> gene was highly conserved across evolution, with 90% similarity of the predicted amino acid sequence among eutherian mammals. Comparison of the <i>Fry</i> sequence in the Cop and F344 strains identified two non-synonymous single nucleotide polymorphisms (SNPs), one of which creates a putative, de novo phosphorylation site. Further analysis showed that the expression of the <i>Fry</i> gene is reduced in a majority of rat mammary tumors. Our results also suggested that FRY activity was reduced in human breast carcinoma cell lines as a result of reduced levels or mutation. This study is the first to identify the <i>Fry</i> gene as a candidate <i>Mcs</i> gene. Our data suggest that the SNPs within the <i>Fry</i> gene contribute to the genetic susceptibility of the F344 rat strain to mammary carcinogenesis. These results provide the foundation for analyzing the role of the human <i>FRY</i> gene in cancer susceptibility and progression.</p></div

HSV-1 antigens recognized by human TG-derived CD4 T-cells.

<p>(A) Representative data from the TG-TCL of the donors TG2 and TG3 assayed for T-cell reactivity by a proliferation assay to proteins encoded by individual HSV-1 open reading frames (ORFs). Cell lysates of mock- and HSV-1 infected Cos-7 cells were used as negative and positive controls, respectively. Mean [³H]-thymidine incorporation by TG-TCL exposed in duplicate to γ-irradiated donor HLA-DQ/DR-matched allogeneic peripheral blood mononuclear cells (PBMC) pulsed with lysates generated from Cos-7 cells transfected with the individual HSV-1 ORFs arrayed in nominal genomic order on the x-axis. The names of the HSV-1 ORFs and corresponding proteins driving the positive responses are indicated by an arrow. ICP47, infected cell polypeptide 47 and VP16, virus protein 16. (B) Proliferation assay data of the TG-TCL of donor TG2 with γ-irradiated HLA-DQ/DR TG 2-matched allogeneic PBMC pulsed with whole HSV-1 ICP47 protein (gene US12) spanning synthetic peptides (15-meric peptides with 10 amino acid (aa) overlap) as antigen presenting cell (APC). (C; left panel) Proliferation assay data of the TG-TCL of donor TG3 with γ-irradiated HLA-DQ/DR-matched allogeneic PBMC pulsed with the indicated recombinant HSV-1 VP16 protein (gene UL48) fragments as APC. (C; right panel) Proliferation assay data of the TG-TCL of donor TG3 with γ-irradiated HLA-DQ/DR-matched allogeneic PBMC pulsed with HSV-1 VP16 protein fragment (aa151–240) spanning synthetic peptides (13-meric peptides with 8 aa overlap) as APC. Data are presented as mean counts per minute of triplicate experiments. Data presented in (B) and (C) are the means ± standard error of the mean.</p

Localization and phenotype of T-cells in HSV-1 latently infected human TG.

<p>(A) Representative image of an HSV-1 latently infected TG stained by immunohistochemistry (IHC) for CD3 (red). Inset: magnification of the TG tissue showing a cluster of CD3⁺ cells in panel A. (B; left panel) Double immunofluoresence staining for CD4 (red) and CD8 (green) combined with DNA counterstaining (DAPI; blue nuclei). The white arrows signify autofluorescent cytoplasmatic granules in neurons containing lipofuscin and neuron outlines are marked with white dotted lines. (B; right panel) Consecutive TG tissue sections stained for CD8 (brown) and granzyme B (brown), CD8 (brown) and TIA-1 (brown), and CD3 (red) and CD137 (red). Sections were developed with diaminobenzidine (brown staining pattern) or 3-amino-9-ethylcarbazole (red staining pattern) and counterstained with hematoxylin (blue nuclei). Magnifications were: (A) ×20 and inset ×200, (B; left panel) ×400 and (B; right panel) ×1000. Representative images from 10 HSV-1 latently infected TG donors analyzed.</p

HSV-1 antigens recognized by human TG-derived CD8 T-cells.

<p>Representative data from TG-TCL of 6 TG donors assayed for T-cell reactivity to proteins encoded by individual HSV-1 open reading frames (ORFs). Mean IFN-γ secretion levels, shown as arbitrary OD₄₅₀ values, by TG-TCL exposed in duplicate to Cos-7 cells that co-express the respective donor-specific HLA class I allele and the individual HSV-1 ORFs arrayed in nominal genomic order on the x-axis. The names of the HSV-1 ORFs and corresponding proteins specifically recognized are indicated by an arrow. The HSV-1 ORFs recognized by the TG-TCL of donors TG2 and TG7 are shown in colors of the respective TG donor-specific HLA class I allele. ICP, infected cell polypeptide; VP, virion protein and in case of a viral glycoprotein (e.g. gB, glycoprotein B). Currently, no proteins names are available for HSV-1 ORFs <i>UL6</i> and <i>UL25</i>.</p

Phenotype and HLA class I allele restriction of HSV-1 reactive T-cells recovered from human TG.

*<p>TG-derived T-cell lines were incubated with mock– and HSV-1–infected autologous B-cell lines and assayed by flow cytometry for intra-cellular interferon gamma (IFN-γ) expression.</p>#<p>The ratio of CD4 and CD8 T-cells of the respective TG-derived T-cell lines are indicated.</p>$<p>Patient HLA class I allele restricted HSV-1 reactive CD8 T-cell responses were defined using partially HLA class I matched BLCL. The values represent mean net percentages of live/CD3-gated IFN-γ⁺ T-cells (HSV-1 minus mock) of at least 2 separate experiments.</p><p>nd, not done.</p

HSV-1 antigens and epitopes recognized by CD8 T-cells recovered from human TG.

*<p>HSV-1 gene and protein names, and expression kinetic class, are from reference 1 and Genbank NC_001806. The amino acid (aa) location of CD8 T-cell epitopes identified are in parentheses. na, not applicable.</p>#<p>HLA allele by which the indicated proteins and peptides are recognized by the specific CD8 T-cells.</p

Comparison of T-cell cytolytic granule and cytokine transcripts to HSV-1 and VZV DNA load, and CD8β transcript levels, in human TG.

<p>(A) Scatter plot showing the mean HSV-1 and VZV genome equivalent copies (gec) per 10⁵ TG cells. (B) Comparison of HSV-1 and VZV DNA load in TG of individual donors. (C) Comparison between relative CD8β transcript levels and HSV-1 and VZV DNA load. (D) Comparison of HSV-1 DNA load with relative transcript levels of perforin, granzyme B (grB), interferon γ (IFN-γ) and tumor necrosis factor α (TNF-α). (E) Comparison of VZV DNA load with relative transcript levels of perforin, grB, IFN-γ and TNF-α. (F) Comparison between relative CD8β transcript levels and perforin, granzyme B, IFN-γ and TNF-α. (G) Comparison of the transcript levels of CD8β, perforin, grB, IFN-γ and TNF-α between paired left and right TG of individual donors. The paired <i>T</i>-test (A), Spearman correlation (B–F) and Wilcoxon matched pairs test (G) were used for statistical analysis. Data of 26 TG specimens analyzed.</p

HSV-1 epitope-specific CD8 T-cells are localized close to sensory neuron cell bodies in the contralateral human TG.

<p>Representative image of the TG tissue of donor TG2 stained with DAPI (blue), anti-CD8 (green) and tetramers (red) that consisted of both the synthetic HSV-1 peptides ICP0_642–651 and ICP8_1096–1105 conjugated to HLA-A*0201. Inserts, lower left and upper right corner, are enlargements of areas containing tetramer-positive CD8 T-cells. The white arrows and arrowheads signify autofluorescent granules containing lipofuscin and tetramer-positive CD8 T-cells, respectively. Neuron outlines are marked with a white dashed line. Magnification was ×400.</p

Interval mapping of putative quantitative trait loci (QTL) on rat chromosomes 12 that confer susceptibility to NMU-induced mammary carcinogenesis.

<p>The genetic map positions of polymorphic STR markers are indicated by hatch marks and are designated to the left. Genetic distances between markers in centiMorgans are drawn to scale within each panel. The calculated Likelihood Ration Statistic (LRS) values for linkage of the Quantitative trait (measured as log of tumor numbers) within chromosomal intervals are plotted as X to the right (black line). <b>A)</b> Linkage calculated on chromosome 12, using 99 animals genotyped at the markers indicated on the left. <b>B)</b> Linkage on chromosome 12, as calculated using 324 animals genotyped at the markers indicated on the left. The vertical green lines indicate the suggestive (LRS = 2.7 for Panels A; LRS = 1.9 for Panel B), significant (LRS = 8.0 for Panels A; LRS = 7.2 for Panels B), and highly significant (LRS = 15.0 for Panels A; LRS = 13.4 for Panels B) thresholds as calculated by permutation studies. The additive effect functions are represented as dotted red lines, with negative values to the left and positive values to the right.</p

Calculated LOD scores for markers on rat chromosome 12.

<p>Note: LOD, logarithm of the odds ratio.</p