Histochemical and molecular overview of the thymus as site for T-cells development

The thymus represents the primary site for T cell lymphopoiesis, providing a coordinated set for critical factors to induce and support lineage commitment, differentiation and survival of thymus-seeding cells. One irrefutable fact is that the presence of non-lymphoid cells through the thymic parenchyma serves to provide coordinated migration and differentiation of T lymphocytes. Moreover, the link between foetal development and normal anatomy has been stressed in this review. Regarding thymic embryology, its epithelium is derived from the embryonic endodermal layer, with possible contributions from the ectoderm. A series of differentiating steps is essential, each of which must be completed in order to provide the optimum environment for thymic development and function. The second part of this article is focused on thymic T-cell development and differentiation, which is a stepwise process, mediated by a variety of stromal cells in different regions of the organ. It depends strongly on the thymic microenvironment, a cellular network formed by epithelial cells, macrophages, dendritic cells and fibroblasts, that provide the combination of cellular interactions, cytokines and chemokines to induce thymocyte precursors for the generation of functional T cells. The mediators of this process are not well defined but it has been demonstrated that some interactions are under neuroendocrine control. Moreover, some studies pointed out that reciprocal signals from developing T cells also are essential for establishment and maintenance of the thymic microenvironment. Finally, we have also highlighted the heterogeneity of the lymphoid, non-lymphoid components and the multi-phasic steps of thymic differentiation. In conclusion, this review contributes to an understanding of the complex mechanisms in which the foetal and postnatal thymus is involved. This could be a prerequisite for developing new therapies specifically aimed to overcome immunological defects, linked or not-linked to aging.     

Genomic deletion and promoter methylation status of Hypermethylated in Cancer 1 (HIC1) in mantle cell lymphoma

Mantle cell lymphomas (MCL), characterized by the t(11;14)(q13;q32), frequently carry secondary genetic alterations such as deletions in chromosome 17p involving the TP53 locus. Given that the association between TP53-deletions and concurrent mutations of the remaining allele is weak and based on our recent report that the Hypermethylated in Cancer 1 (HIC1) gene, that is located telomeric to the TP53 gene, may be targeted by deletions in 17p in diffuse large B-cell lymphoma (DLBCL), we investigated whether HIC1 inactivations might also occur in MCL. Monoallelic deletions of the TP53 locus were detected in 18 out of 59 MCL (31%), while overexpression of p53 protein occurred in only 8 out of 18 of these MCL (44%). In TP53-deleted MCL, the HIC1 gene locus was co-deleted in 11 out of 18 cases (61%). However, neither TP53 nor HIC1 deletions did affect survival of MCL patients. In most analyzed cases, no hypermethylation of the HIC1 exon 1A promoter was observed (17 out of 20, 85%). However, in MCL cell lines without HIC1-hypermethylation, the mRNA expression levels of HIC1 were nevertheless significantly reduced, when compared to reactive lymph node specimens, pointing to the occurrence of mechanisms other than epigenetic or genetic events for the inactivation of HIC1 in this entity.     

Biological effect of mono- and dinuclear alkylamine platinum(II) compounds on human lymphoma cells

The effects of the mononuclear chloro[meso-1,2-bis(4-fluorophenyl)ethylenediamine][hexylamine]platinum(II) chloride HACl and the dinuclear di[meso-1,2-bis(4-fluorophenyl)ethylenediamine]dichloro(mu-1,n-diaminoalkane-N:N')diplatinum(II)dichloride complexes DAHCl (alkane:hexane), DANCl (alkane:nonane) and DADCl (alkane:dodecane) with different alkyl chain length (n) were investigated on non-Hodgkin's lymphoma (NHL) and chronic myeloid leukemia (CML) cell lines. All compounds showed an antiproliferative effect on the NHL cell lines RAJI and U-937 accompanied in the case of DANCl, DAHCl, HACl and cisplatin by an increase in apoptosis. The growth of another NHL (JEKO-1) and one CML cell line (K-562) was decreased only by cisplatin. In contrast to HACl, DAHCl, DANCl and cisplatin, DADCl induced necrosis, suggesting toxicity because cell viability decreased. Similar effects were observed when bone marrow-derived lymphoma cells from a patient with high-grade B-NHL were incubated with the platinum complexes.     

Changes in the expression of genes related to apoptosis and fibrosis pathways in CCl4-treated rats

Chronic liver diseases are accompanied by changes in the biochemical pathways related to the regulation of apoptosis and extra-cellular matrix deposition. The present study was designed to investigate, using low density arrays, changes in the hepatic gene expression together with hepatic biochemical and histological alterations in rats that had liver impairment induced by chronic exposure to CCl₄. Further, we examined the possible recovery of genetic and pathological changes following the cessation of the hepatotoxic injury. Experimental fibrosis was induced in male Wistar rats by CCl₄ administration. Animals were subdivided into two groups. One group was given CCl₄ and animals were killed at 8 and 12 weeks of treatment. The other group was treated with CCl₄ for 6 weeks, the CCl₄ was then stopped and, subsequently, subgroups of animals were killed after 1 and 2 weeks of recovery. CCl₄ administration over 12 weeks was associated with significant changes in B-cell leukemia/lymphoma 2, procollagen type I α 2, matrix metalloproteinases 3 and 8, tissue inhibitors of metalloproteinases 1, 2, and 3 and the inhibitor of apoptosis 4 gene expressions. Recovery after CCl₄ cessation was associated with changes in procollagen type I α 2, matrix metalloproteinase 7, tissue inhibitors of metalloproteinases 1 and 2, inhibitor of apoptosis 4, and survivin gene expressions. This study shows an association between changes in the expression of several genes regulating hepatic cell apoptosis, the fibrosis process, and the recovery of the hepatic function after removal of the toxic injury.     

Two-hit wonders: The expanding universe of multitargeting epigenetic agents

Multitargeting involves the application of molecules that are deliberately intended to bind to two or more unrelated cellular targets with high affinity. In epigenetic chemical biology and drug discovery, the rational design of multitargeting agents has evolved to a sophisticated level, and there are now five examples that have reached clinical trials. This review covers recent developments in the field and future prospects.     

Constitutive autophagy contributes to resistance to TP53-mediated apoptosis in Epstein-Barr virus-positive latency III B-cell lymphoproliferations

The Epstein-Barr virus (EBV) is associated with various lymphoproliferative disorders and lymphomas. We have previously demonstrated that treating wild-type TP53-expressing B cell lines with the TP53 pathway activator nutlin-3 induced apoptosis in EBV-negative and EBV-positive latency I cells whereas EBV-positive latency III cells remained much more apoptosis-resistant. Here, we report a constitutively high level of autophagy in these resistant cells which express high levels of the proautophagic protein BECN1/Beclin 1 based, at least in part, on the activation of the NFKB signaling pathway by the viral protein LMP1. Following treatment with nutlin-3, several autophagy-stimulating genes were upregulated both in EBV-negative and EBV-positive latency III cells. However the process of autophagy was only triggered in the latter and was associated with an upregulation of SESN1/sestrin 1 and inhibition of MTOR more rapid than in EBV-negative cells. A treatment with chloroquine, an inhibitor of autophagy, potentiated the apoptotic effect of nutlin-3, particularly in those EBV-positive cells which were resistant to apoptosis induced by nutlin-3 alone, thereby showing that autophagy participates in this resistant phenotype. Finally, using immunohistochemical staining, clinical samples from various B cell lymphoproliferations with the EBV-positive latency II or III phenotype were found to harbor a constitutively active autophagy.     

Deciphering Spatial Protein–Protein Interactions in Brain Using Proximity Labeling

Cellular biomolecular complexes including protein–protein, protein–RNA, and protein–DNA interactions regulate and execute most biological functions. In particular in brain, protein–protein interactions (PPIs) mediate or regulate virtually all nerve cell functions, such as neurotransmission, cell–cell communication, neurogenesis, synaptogenesis, and synaptic plasticity. Perturbations of PPIs in specific subsets of neurons and glia are thought to underly a majority of neurobiological disorders. Therefore, understanding biological functions at a cellular level requires a reasonably complete catalog of all physical interactions between proteins. An enzyme-catalyzed method to biotinylate proximal interacting proteins within 10 to 300 nm of each other is being increasingly used to characterize the spatiotemporal features of complex PPIs in brain. Thus, proximity labeling has emerged recently as a powerful tool to identify proteomes in distinct cell types in brain as well as proteomes and PPIs in structures difficult to isolate, such as the synaptic cleft, axonal projections, or astrocyte–neuron junctions. In this review, we summarize recent advances in proximity labeling methods and their application to neurobiology.     

Immunohistochemical characterization of the thymic microenvironment

Summary The epithelial framework of the human thymus has been studied in parallel by immunohistochemical methods at the light- and electron-microscopic levels. Different monoclonal antibodies were used, reacting with components of the major histocompatibility complex, keratins, thymic hormones and other as yet antigenically undefined substances, which show specific immunoreactivities with human thymus epithelial cells.The electron-microscopic immunocytochemical observations clearly confirm microtopographical differences of epithelial cells not only between the thymic cortex and medulla, but also within the cortex itself. At least four subtypes of epithelial cells could be distinguished: 1) the cortical surface epithelium; 2) the main cortical epithelial cells and thymic nurse cells; 3) the medullary epithelial cells; and 4) the epithelial cells of Hassall's corpuscles.The various epithelial cell types of the thymus display several common features like tonofilaments, desmosomes and some surface antigens as demonstrated by anti-KiM3. In other respects, however, they differ from each other. The cortical subtype of thymic epithelial cells including the thymic nurse cells shows a distinct pattern of surface antigens reacting positively with antibodies against HLA-DR (anti-HLA-DR) and anti-21A62E. Electron-microscopic immunocytochemistry with these antibodies clearly reveals a surface labeling and a narrow contact to cortical thymocytes particularly in the peripheral cortical regions. An alternative staining pattern is realized by antibodies to some antigens associated with other subtypes of thymic epithelial cells. Medullary epithelial cells as well as the cortical surface epithelium react likewise positively with antibodies to special surface antigens (anti-Ep-1), to special epitopes of cytokeratin (anti-IV/82), and to thymic hormones (anti-FTS). The functional significance of distinct microenvironments within the thymus provided by different epithelial cells is discussed in view of the maturation of T-precursor cells.Glossary of Abbreviations Anti-X anti-X antibody - APUD-cells amine precursor uptake and decarboxylation (gastro-intestinal endocrine cells) - DAB diamino-benzidine - DMSO dimethyl sulfoxide - FTS facteur thymique sérique - HLA-A, B, C human leucocyte antigen, A, B, C-region related - HLA-DR human leucocyte antigen, D-region related - IDC interdigitating cell - MHC major histocompatibility gene complex - PBS phosphate-buffered saline - TNC thymic nurse cell This investigation was supported by grants from the Deutsche Forschungsgemeinschaft, and its Sonderforschungsbereich 111Fellow of the Alexander von Humbold-Stiftung, Institute of Pathology, University of Würzburg, Federal Republic of GermanyThe authors appreciate the contribution of human thymus tissue from Professor Alexander Bernhard, Abteilung kardiovasculäre Chirurgie der Universität Kiel; the gift of monoclonal antibodies from Dr. M.J.D. Anderson, Dr. M. Dardenne and Dr. H.J. Radzun; and the excellent technical assistence of Mrs. O.M. Bracker, Mrs. H. Hansen, Mrs. R. Köpke, Mrs. M. v. Kolszynski, Mrs. J. Quitzau, Mrs. H. Siebke, and Mrs. H. Waluk     

Continuous signaling of CD79b and CD19 is required for the fitness of Burkitt lymphoma B cells

Expression of the B‐cell antigen receptor (BCR) is essential not only for the development but also for the maintenance of mature B cells. Similarly, many B‐cell lymphomas, including Burkitt lymphoma (BL), require continuous BCR signaling for their tumor growth. This growth is driven by immunoreceptor tyrosine‐based activation motif (ITAM) and PI3 kinase (PI3K) signaling. Here, we employ CRISPR/Cas9 to delete BCR and B‐cell co‐receptor genes in the human BL cell line Ramos. We find that Ramos B cells require the expression of the BCR signaling component Igβ (CD79b), and the co‐receptor CD19, for their fitness and competitive growth in culture. Furthermore, we show that in the absence of any other BCR component, Igβ can be expressed on the B‐cell surface, where it is found in close proximity to CD19 and signals in an ITAM‐dependent manner. These data suggest that Igβ and CD19 are part of an alternative B‐cell signaling module that use continuous ITAM/PI3K signaling to promote the survival of B lymphoma and normal B cells.