Aberrant Development of Thymocytes in Mice Lacking Laminin-2

In previous in vitro studies, we proposed a role for the extracellular matrix component, laminin- 2, and its integrin receptor, VLA-6, in thymocyte development. The characterization of two dystrophic mouse strains with different defects in laminin-2 allowed us to examine this proposal in vivo. Mice deficient in laminin-2, dy/dy, show a significant reduction in thymus size and number of thymocytes compared to normal littermates. These mice also exhibited apparent alterations of thymic architecture. Examination of the CD4/CD8 populations in dy/dy thymi showed large relative increases in the DN (CD4-CD8-) and SP (CD4+CD8-, CD4-CD8+) populations and a significant decrease in the DP (CD4+CD8+) population. Further examination of the DN population for CD44 and CD25 expression showed a remarkable decrease in the more mature pre-T cell populations. Analysis of apoptosis in situ, and by flow cytometry, in dy/dy thymi revealed a significant increase in apoptotic DN thymocytes in the capsule and subcapsular regions. Interestingly, thymocyte development appeared to proceed normally in dystrophic mice expressing a mutant form of laminin-2, dy2J, as well as, in fetal and neonatal dy/dy mice. We propose that laminin-2 plays an active role in thymocyte development by delivering cell survival and differentiation signals at specific stages of development in young adult mice

Finishing the euchromatic sequence of the human genome

The sequence of the human genome encodes the genetic instructions for human physiology, as well as rich information about human evolution. In 2001, the International Human Genome Sequencing Consortium reported a draft sequence of the euchromatic portion of the human genome. Since then, the international collaboration has worked to convert this draft into a genome sequence with high accuracy and nearly complete coverage. Here, we report the result of this finishing process. The current genome sequence (Build 35) contains 2.85 billion nucleotides interrupted by only 341 gaps. It covers ∼99% of the euchromatic genome and is accurate to an error rate of ∼1 event per 100,000 bases. Many of the remaining euchromatic gaps are associated with segmental duplications and will require focused work with new methods. The near-complete sequence, the first for a vertebrate, greatly improves the precision of biological analyses of the human genome including studies of gene number, birth and death. Notably, the human enome seems to encode only 20,000-25,000 protein-coding genes. The genome sequence reported here should serve as a firm foundation for biomedical research in the decades ahead

An epigenetic vaccine model active in the prevention and treatment of melanoma

Abstract Background Numerous immune genes are epigenetically silenced in tumor cells and agents such as histone deacetylase inhibitors (HDACi), which reverse these effects, could potentially be used to develop therapeutic vaccines. The conversion of cancer cells to antigen presenting cells (APCs) by HDACi treatment could potentially provide an additional pathway, together with cross-presentation of tumor antigens by host APCs, to establish tumor immunity. Methods HDACi-treated B16 melanoma cells were used in a murine vaccine model, lymphocyte subset depletion, ELISpot and Cytotoxicity assays were employed to evaluate immunity. Antigen presentation assays, vaccination with isolated apoptotic preparations and tumorigenesis in MHC-deficient mice and radiation chimeras were performed to elucidate the mechanisms of vaccine-induced immunity. Results HDACi treatment enhanced the expression of MHC class II, CD40 and B7-1/2 on B16 cells and vaccination with HDACi-treated melanoma cells elicited tumor specific immunity in both prevention and treatment models. Cytotoxic and IFN-γ-producing cells were identified in splenocytes and CD4+, CD8+ T cells and NK cells were all involved in the induction of immunity. Apoptotic cells derived from HDACi treatments, but not H2O2, significantly enhanced the effectiveness of the vaccine. HDACi-treated B16 cells become APCs in vitro and studies in chimeras defective in cross presentation demonstrate direct presentation in vivo and short-term but not memory responses and long-term immunity. Conclusion The efficacy of this vaccine derives mainly from cross-presentation which is enhanced by HDACi-induced apoptosis. Additionally, epigenetic activation of immune genes may contribute to direct antigen presentation by tumor cells. Epigenetically altered cancer cells should be further explored as a vaccine strategy.</p

The epigenetic regulation of Dicer and microRNA biogenesis by Panobinostat

<p>microRNAs (miRs) are small noncoding RNAs that regulate/fine tune many cellular protein networks by targeting mRNAs for either degradation or translational inhibition. Dicer, a type III endoribonuclease, is a critical component in miR biogenesis and is required for mature microRNA production. Abnormal Dicer expression occurs in numerous cancer types and correlates with poor patient prognosis. Recent reports have demonstrated that epigenetic agents, including histone deacetylase inhibitors (HDACi), may regulate Dicer and miR expression. HDACi are a class of epigenetic agents used to treat cancer, viral infections, and inflammatory disorders. However, little is known regarding the epigenetic regulation of miR biogenesis and function. We therefore investigated whether clinically successful HDACi modulated Dicer expression and found that Panobinostat, a clinically approved HDACi, enhanced Dicer expression via posttranscriptional mechanisms. Studies using proteasome inhibitors suggested that Panobinostat regulated the proteasomal degradation of Dicer. Further studies demonstrated that Panobinostat, despite increasing Dicer protein expression, decreased Dicer activity. This suggests that Dicer protein levels do not necessarily correlate with Dicer activity and mature miR levels. Taken together, we present evidence here that Panobinostat posttranscriptionally regulates Dicer/miR biogenesis and suggest Dicer as a potential therapeutic target in cancer.</p

TSA treatment enhances apoptosis and immune gene expression on melanoma cells and TSA-treated vaccines generate immunity

<p><b>Copyright information:</b></p><p>Taken from "An epigenetic vaccine model active in the prevention and treatment of melanoma"</p><p>http://www.translational-medicine.com/content/5/1/64</p><p>Journal of Translational Medicine 2007;5():64-64.</p><p>Published online 10 Dec 2007</p><p>PMCID:PMC2231344.</p><p></p> ) B16 cells were stained with mAb, isotype controls and annexinV after treatment with TSA and analyzed by flow cytometry for the expression of MHC class I, class II, CD80, CD86 and CD40. Isotype controls are shown as shaded peaks and heavy lines represent expression determined by specific mAb staining. Values indicated in the histograms are the percent of cells positive for the respective mAb relative to the isotype staining. The data presented here are representative of more than three independent experiments. ) Kaplan-Meier plot of B6 mice (10 in each group) inoculated with TSA-treated (500 nM for 48 h) or untreated B16 cells in the trunk. ) Durable immunity in 100% of the immune animals. Tumor-free mice, 40 days after vaccination with TSA-treated B16, were re-challenged with untreated B16 cells and observed for another 60 days. The number of tumor-free mice compared to total numbers used in each treatment group is shown in parentheses