Necroptosis promotes autophagy-dependent upregulation of DAMP and results in immunosurveillance.

Programmed necrosis, necroptosis, is considered to be a highly immunogenic activity, often mediated via the release of damage-associated molecular patterns (DAMPs). Interestingly, enhanced macroautophagic/autophagic activity is often found to be accompanied by necroptosis. However, the possible role of autophagy in the immunogenicity of necroptotic death remains largely obscure. In this study, we investigated the possible mechanistic correlation between phytochemical shikonin-induced autophagy and the shikonin-induced necroptosis for tumor immunogenicity. We show that shikonin can instigate RIPK1 (receptor [TNFRSF]-interacting serine-threonine kinase 1)- and RIPK3 (receptor-interacting serine-threonine kinase 3)-dependent necroptosis that is accompanied by enhanced autophagy. Shikonin-induced autophagy can directly contribute to DAMP upregulation. Counterintuitively, among the released and ectoDAMPs, only the latter were shown to be able to activate the cocultured dendritic cells (DCs). Interruption of autophagic flux via chloroquine further upregulated ectoDAMP activity and resultant DC activation. For potential clinical application, DC vaccine preparations treated with tumor cells that were already pretreated with chloroquine and shikonin further enhanced the antimetastatic activity of 4T1 tumors and reduced the effective dosage of doxorubicin. The enhanced immunogenicity and vaccine efficacy obtained via shikonin and chloroquine cotreatment of tumor cells may thus constitute a compelling strategy for developing cancer vaccines via the use of a combinational drug treatment.

EBV oncogene N-LMP1 induces CD4 T cell-mediated angiogenic blockade in the murine tumor model.

Antivascular immunity may provide long-term protection by preventing neovascularization that precedes tumor progression. Although the tumorigenesis promoted by EBV-encoded oncogene latent membrane protein 1 derived from Taiwanese nasopharyngeal carcinoma (N-LMP1) has been demonstrated, the potential of N-LMP1 for inducing immune surveillance remains elusive. In this article, we describe the immunogenicity of N-LMP1 (1510) and its induction of antivascular immunity in a transplantable tumor model in immunocompetent BALB/c mice. The immunogenicity of N-LMP1 was evaluated on the basis of tumor rejection following immunization. The impact of the immunization on the dynamics of tumor angiogenesis was assessed by temporal noninvasive dynamic contrast-enhanced magnetic resonance imaging and was further confirmed by histologic study and vascular count. Through the experiments of in vivo depletion and adoptive transfer, CD4 T cells were identified as effectors that depend on IFN-γ for tumor prevention. The response was further verified by the identification of an MHC H-2 I-E(d)-restricted peptide derived from N-LMP1 and by the immunization of mice with N-LMP1 peptide-loaded dendritic cells. These studies provide insight into N-LMP1-specific immunity in vivo, which suggests that CD4 T cells may play an important role in angiogenic surveillance against LMP1-associated cancer via tumor stroma targeting.

Magnetic-composite-modified polycrystalline silicon nanowire field-effect transistor for vascular endothelial growth factor detection and cancer diagnosis.

This study proposes a vascular endothelial growth factor (VEGF) biosensor for diagnosing various stages of cervical carcinoma. In addition, VEGF concentrations at various stages of cancer therapy are determined and compared to data obtained by computed tomography (CT) and cancer antigen 125 (CA-125). The increase in VEGF concentrations during operations offers useful insight into dosage timing during cancer therapy. This biosensor uses Avastin as the biorecognition element for the potential cancer biomarker VEGF and is based on a n-type polycrystalline silicon nanowire field-effect transistor (poly-SiNW-FET). Magnetic nanoparticles with poly[aniline-co-N-(1-one-butyric acid) aniline]-Fe3O4 (SPAnH-Fe3O4) shell-core structures are used as carriers for Avastin loading and provide rapid purification due to their magnetic properties, which prevent the loss of bioactivity; furthermore, the high surface area of these structures increases the quantity of Avastin immobilized. Average concentrations in human blood for species that interfere with detection specificity are also evaluated. The detection range of the biosensor for serum samples covers the results expected from both healthy individuals and cancer patients.

High-resolution melting molecular signatures for rapid identification of human papillomavirus genotypes.

BACKGROUND: Genotyping of human papillomarvirus (HPV) is crucial for patient management in a clinical setting. This study accesses the combined use of broad-range real-time PCR and high-resolution melting (HRM) analysis for rapid identification of HPV genotypes. METHODS: Genomic DNA sequences of 8 high-risk genotypes (HPV16/18/39/45/52/56/58/68) were subject to bioinformatic analysis to select for appropriate PCR amplicon. Asymmetric broad-range real-time PCR in the presence of HRM dye and two unlabeled probes specific to HPV16 and 18 was employed to generate HRM molecular signatures for HPV genotyping. The method was validated via assessment of 119 clinical HPV isolates. RESULTS: A DNA fragment within the L1 region was selected as the PCR amplicon ranging from 215-221 bp for different HPV genotypes. Each genotype displayed a distinct HRM molecular signature with minimal inter-assay variability. According to the HRM molecular signatures, HPV genotypes can be determined with one PCR within 3 h from the time of viral DNA isolation. In the validation assay, a 91% accuracy rate was achieved when the genotypes were in the database. Concomitantly, the HRM molecular signatures for additional 6 low-risk genotypes were established. CONCLUSIONS: This assay provides a novel approach for HPV genotyping in a rapid and cost-effective manner.

Selective reduction of post-selection CD8 thymocyte proliferation in IL-15Rα deficient mice.

Peripheral CD8(+) T cells are defective in both IL-15 and IL-15Rα knock-out (KO) mice; however, whether IL-15/IL-15Rα deficiency has a similar effect on CD8 single-positive (SP) thymocytes remains unclear. In this study, we investigated whether the absence of IL-15 transpresentation in IL-15Rα KO mice results in a defect in thymic CD8 single positive (SP) TCR(hi) thymocytes. Comparison of CD8SP TCR(hi) thymocytes from IL-15Rα KO mice with their wild type (WT) counterparts by flow cytometry showed a significant reduction in the percentage of CD69(-) CD8SP TCR(hi) thymocytes, which represent thymic premigrants. In addition, analysis of in vivo 5-bromo-2-deoxyuridine (BrdU) incorporation demonstrated that premigrant expansion of CD8SP TCR(hi) thymocytes was reduced in IL-15Rα KO mice. The presence of IL-15 transpresentation-dependent expansion in CD8SP TCR(hi) thymocytes was assessed by culturing total thymocytes in IL-15Rα-Fc fusion protein-pre-bound plates that were pre-incubated with IL-15 to mimic IL-15 transpresentation in vitro. The results demonstrated that CD8SP thymocytes selectively outgrew other thymic subsets. The contribution of the newly divided CD8SP thymocytes to the peripheral CD8(+) T cell pool was examined using double labeling with intrathymically injected FITC and intravenously injected BrdU. A marked decrease in FITC(+) BrdU(+) CD8(+) T cells was observed in the IL-15Rα KO lymph nodes. Through these experiments, we identified an IL-15 transpresentation-dependent proliferation process selective for the mature CD8SP premigrant subpopulation. Importantly, this process may contribute to the maintenance of the normal peripheral CD8(+) T cell pool.

Enhanced IL-10 production by CD4+ T cells primed in IL-15Rα-deficient mice.

In this study, we investigated the functional outcomes of CD4(+) T cells primed in the absence of IL-15 transpresentation. Compared with their WT counterparts primed in WT mice, IL-15Rα KO CD4(+) T cells primed in KO mice were found to exclusively overproduce IL-10 upon in vitro restimulation(.) The comparable expression of IL-4 and Foxp3 in CD4(+) T cells primed in the WT and IL-15Rα KO mice indicated that this was neither due to T(H) 2- nor Treg cell-differentiation. IL-10 overproduction was also observed when OVA-specific TCR transgenic CD4(+) T (OT-II) cells were primed in KO mice, excluding an intrinsic deficiency of KO CD4(+) T cells. To investigate the WT and KO microenvironment, DCs from both WT and IL-15Rα KO mice were compared. DCs from both backgrounds were indistinguishable in their steady-state survival and in their expression of MHC class II and costimulatory molecules CD80, CD86, and CD40. However, IL-15Rα KO DCs primed OT-II cells in vitro to produce higher levels of IL-10 upon their restimulation. Additionally, IL-15Rα KO DCs produced significantly more IL-10 upon activation, and IL-10 neutralization during DC-mediated in vitro priming abolished IL-10 overproduction by CD4(+) T cells. Thus, IL-15Rα KO DCs provide an IL-10-enriched environment that preferentially primes CD4(+) T cells for more IL-10 production, highlighting a regulatory role for IL-15 transpresentation in CD4(+) T-cell priming.

Mutation of mouse Cyp11a1 promoter caused tissue-specific reduction of gene expression and blunted stress response without affecting reproduction.

Steroids are synthesized mainly from the adrenal glands catalyzed by steroidogenic enzymes; the expression of these enzymes is controlled by transcription factor steroidogenic factor-1 (SF-1; NR5A1). To understand the physiological effect of genetic changes on steroid secretion, we used Cre-LoxP and gene targeting technology to mutate the binding sequence for SF-1 (SF-1 response element) on the promoter of the mouse Cyp11a1 gene, which encodes a critical enzyme for steroid biosynthesis. The resulting Cyp11a1 L/L mice expressed about 7-fold less cytochrome P450 side-chain cleavage enzyme (CYP11A1) in the adrenal and testis but expressed normal amounts of CYP11A1 in the placenta and ovary. This tissue-specific reduction of gene expression did not affect basal steroid secretion but attenuated the circadian rhythm of glucocorticoid secretion. These mice also failed to induce glucocorticoid secretion in response to stress, leading to retention of CD4+CD8+ double-positive thymocytes. Unlike complete Cyp11a1 disruption, which causes neonatal death, promoter mutation did not decrease life span and caused no defect in reproduction. Thus, CYP11A1 appears in normal mice to be expressed above the minimal required level, providing a large capacity for use in response to stress. Mutation of the SF-1 response element of Cyp11a1 results in reduced stress response due to decreased adrenal CYP11A1 expression and insufficient stress-induced glucocorticoids secretion.

Reduced expression of Bcl-2 in CD8+ T cells deficient in the IL-15 receptor alpha-chain.

Mice that lack IL-15 or the IL-15R alpha-chain (IL-15Ralpha) are deficient in peripheral CD8(+), but not in CD4(+), T cells. This CD8(+) T cell-specific deficiency has now been investigated further by characterization of a new strain of IL-15Ralpha(-/-) mice. The adult mutant mice exhibited a specific reduction in the percentage of CD8-single positive TCR(high) thymocytes. The expression of Bcl-2 was reduced in both CD8(+) thymocytes and naive T cells of the mutant animals, and the susceptibility of these cells to death was increased. Memory CD8(+) cells were profoundly deficient in IL-15Ralpha(-/-)mice, and the residual memory-like CD8(+) cells contained a high percentage of dead cells and failed to up-regulate Bcl-2 expression compared with naive CD8(+) cells. Moreover, exogenous IL-15 both up-regulated the level of Bcl-2 in and reduced the death rate of wild-type and mutant CD8(+) T cells activated in vitro. These results indicate that IL-15 and IL-15Ralpha regulate the expression of Bcl-2 in CD8(+) T cells at all developmental stages. The reduced Bcl-2 content in CD8(+) cells might result in survival defect and contribute to the reduction of CD8(+) cells in IL-15Ralpha(-/-)mice.