Effective low-dimensional dynamics of a mean-field coupled network of slow-fast spiking lasers.

Low-dimensional dynamics of large networks is the focus of many theoretical works, but controlled laboratory experiments are comparatively very few. Here, we discuss experimental observations on a mean-field coupled network of hundreds of semiconductor lasers, which collectively display effectively low-dimensional mixed mode oscillations and chaotic spiking typical of slow-fast systems. We demonstrate that such a reduced dimensionality originates from the slow-fast nature of the system and of the existence of a critical manifold of the network where most of the dynamics takes place. Experimental measurement of the bifurcation parameter for different network sizes corroborates the theory.

Effect of siltuximab, omalizumab, infliximab, pembrolizumab and vedolizumab on selected haematological and biochemical parameters in a pig model.

From the regulatory point of view a strong link between an animal model and human pharmacodynamics of biological drugs is very important to qualify the model as "relevant". Consistent changes in cell population between human physiology and animal model gain value of this model which then can be pharmacodynamically "relevant" from the regulatory point of view. Consequently, the aim of this study was to determine how similar to human observations is the effect of selected biological drugs on blood cells in a pig model. The study was to carry out a comparative analysis of the variability of selected biochemical and hematological parameters of the blood after administration of five different human therapeutic monoclonal antibodies (mAbs) after a single subcutaneous (SC) dose in breeding pigs. The tested drugs were siltuximab (Syl- vant®), omalizumab (Xolair®), infliximab (Inflectra®), pembrolizumab (Keytruda®), and vedoli- zumab (Entyvio®) given in a single 1 mg/kg SC injection. Each of the tested drugs exerted a sig- nificant effect on at least two of the tested parameters three weeks after the administration. Siltuximab significantly influenced 9 of the analyzed parameters. Vedolizumab significantly influenced 8 of the analyzed parameters. Infliximab had the lowest impact of all the tested drugs, as it significantly influenced only two of the analyzed parameters. The study has proved that the impact of mAbs on the analyzed parameters can be significantly extended over time. This requires the monitoring of hematological parameters in the pig model even many weeks after administration of a drug in a relatively small dose.

Phorbol ester-mediated re-expression of endogenous LAT adapter in J.CaM2 cells: a model for dissecting drivers and blockers of LAT transcription.

Linker for activation of T cells (LAT) is a raft-associated, transmembrane adapter protein critical for T-cell development and function. LAT expression is transiently upregulated upon T-cell receptor (TCR) engagement, but molecular mechanisms conveying TCR signaling to enhanced LAT transcription are not fully understood. Here we found that a Jurkat subline J.CaM2, initially characterized as LAT deficient, conditionally re-expressed LAT upon the treatment with a protein kinase C activator, phorbol 12-myristate 13-acetate (PMA). We took advantage of the above observation for studying cis-elements and trans-acting factors contributing to the activation-induced expression of LAT. We identified a LAT gene region spanning nucleotide position -14 to +357 relative to the ATG start codon as containing novel cis-regulatory elements that were able to promote PMA-induced reporter transcription in the absence of the core LAT promoter. Interestingly, a point mutation in LAT intron 1, identified in J.CaM2 cells, downmodulated LAT promoter activity by 50%. Mithramycin A, a selective Sp1 DNA-binding inhibitor, abolished LAT expression upon PMA treatment as did calcium ionophore ionomycin (Iono) and valproic acid (VPA), widely used as an anti-epileptic drug. Our data introduce J.CaM2 cells as a model for dissecting drivers and blockers of activation induced expression of LAT.

Identification of functional, short-lived isoform of linker for activation of T cells (LAT).

Linker for activation of T cells (LAT) is a transmembrane adaptor protein playing a key role in the development, activation and maintenance of peripheral homeostasis of T cells. In this study we identified a functional isoform of LAT. It originates from an intron 6 retention event generating an in-frame splice variant of LAT mRNA denoted as LATi6. Comparison of LATi6 expression in peripheral blood leukocytes of human and several other mammalian species revealed that it varied from being virtually absent in the mouse to being predominant in the cow. Analysis of LAT isoform frequency expressed from minigene splicing reporters carrying loss- or gain-of-function point mutations within intronic polyguanine sequences showed that these elements are critical for controlling the intron 6 removal. The protein product of LATi6 isoform (LATi6) ectopically expressed in LAT-deficient JCam 2.5 cell line localized correctly to subcellular compartments and supported T-cell receptor signaling but differed from the canonical LAT protein by displaying a shorter half-life and mediating an increased interleukin-2 secretion upon prolonged CD3/CD28 crosslinking. Altogether, our data suggest that the appearance of LATi6 isoform is an evolutionary innovation that may contribute to a more efficient proofreading control of effector T-cell response.

NWC, a new gene within RAG locus: could it keep GOD under control?

NWC, newly discovered, evolutionarily conserved gene within recombination activating gene (RAG) locus is constitutively expressed in all cells except lymphocytes, in which it is developmentally regulated by RAG1 promoter. In lymphocytes, NWC promoter, which is located within RAG2 intron and drives expression of NWC in non-lymphocytes, is inactive. Here, a hypothesis on the role of transcription of NWC in lymphocyte-specific regulation of RAG expression and their suppression in all other cells is presented. It is proposed that during development, inactivation of NWC promoter and the placement of NWC under the control of RAG1 promoter releases RAG genes from permanent suppression and allows their lymphocyte specific expression but at the same time subjects them to transcriptional feedback inhibition type of suppression which could permit for a stringent control over their threat to genome stability and oncogenic potential.

The chemokine TECK is expressed by thymic and intestinal epithelial cells and attracts double- and single-positive thymocytes expressing the TECK receptor CCR9.

Chemokines are key regulators of migration in lymphoid tissues. In the thymus, maturing thymocytes move from the outer capsule to the inner medulla and thereby interact with different types of stromal cells that control their maturation and selection. In the process of searching for molecules specifically expressed at different stages of mouse thymic differentiation, we have characterized the cDNA coding for the thymus-expressed chemokine (TECK) and its receptor CCR9. The TECK receptor gene was isolated and shown to be localized on the mouse chromosome 9F1-F4. Thymic dendritic cells have been initially thought to be a prevalent source of TECK. In contrast, our results indicate that thymic epithelial cells constitute the predominant source of TECK. Consistent with the latter distribution, the TECK receptor is highly expressed by double-positive thymocytes, and TECK can chemoattract both double-positive and single-positive thymocytes. The TECK transcript is also abundantly expressed in the epithelial cells lining the small intestine. In conclusion, the interplay of TECK and its receptor CCR9 is likely to have a significant role in the recruitment of developing thymocytes to discrete compartments of the thymus.

Differential gene expression in CD3epsilon- and RAG1-deficient thymuses: definition of a set of genes potentially involved in thymocyte maturation.

A set of 3000 mouse thymus cDNAs was analyzed by extensive measurement of expression using complex-probe hybridization of DNA arrays ("quantitative differential screening"). The complex probes were initially prepared using total thymus RNA isolated from C57BL/6 wild-type (WT), CD3epsilon- and RAG1-deficient mice. Over 100 clones displaying over- or under-expression by at least a factor of two between WT and knockout (KO) thymuses were further analyzed by measuring hybridization signatures with probes from a wide range of KO thymuses, cell types, organs, and embryonic thymuses. A restricted set of clones was selected by virtue of their expression spectra (modulation in KO thymuses and thymocytes, lymphoid cell specificity, and differential expression during embryonic thymus development), sequenced at one extremity, and compared to sequences in databases. Clones corresponding to previously identified genes (e.g., Tcrbeta, Tcf1 or CD25) showed expression patterns that were consistent with existing data. Ten distinct clones corresponding to new genes were subjected to further study: Northern blot hybridization, in situ hybridization on thymus sections, and partial or complete mRNA sequence determination. Among these genes, we report a new serine peptidase highly expressed in cortical epithelial cells that we have named thymus-specific serine peptidase (TSSP), and an acidic protein expressed in thymocytes and of unknown function that we have named thymus-expressed acidic protein (TEAP). This approach identifies new molecules likely to be involved in thymocyte differentiation and function.

Role of thymic selection in the development of thymic lymphomas in TCR transgenic mice.

To investigate the role of antigen receptor-mediated interactions in lymphomagenesis we have analyzed the influence of alpha beta TCR-mediated selection on the development of spontaneous thymic lymphomas, which appear with a high (up to 50%) frequency in mice expressing a transgenic TCR specific for the male antigen (HY) in the context of H-2Db molecules. To this end we compared the kinetics and the incidence of thymic lymphomas developing in females and males with selecting (H-2b) and non-selecting (H-2k) MHC molecules. The kinetics of development of thymic lymphomas was similar in positively selecting (H-2b females) and non-selecting (H-2k females and males) environments but significantly slower (P < 0.01) in the negatively selecting environment (H-2b male). Injection of lymphoma cells derived from a H-2b female into the thymus of a H-2b male resulted in strong, antigen-specific inhibition of growth, indicating that the slower kinetics of lymphomagenesis in H-2b males could be due, at least partially, to the sensitivity of oncogenically transformed thymocytes to TCR-mediated negative selection. Phenotypic and functional analysis of lymphoma cells indicated that they originated from the stage of pre-TCR-dependent transition of immature CD4-CD8- to CD4+ CD8+ thymocytes, which in H-2b females and males developed into tumors under different environmental pressures. These results failed to provide convincing evidence for the role of positive selection but provided a strong indication that self antigen-induced negative selection, in addition to its well established role in self tolerance, can occasionally act as a tumor surveillance mechanism by eliminating or suppressing growth of thymocytes undergoing oncogenic transformation.

Intrathymic education of alpha beta and gamma delta T cells is accompanied by cell surface expression of RNA/DNA helicase [corrected].

Despite ubiquitous expression of the gene, RNA/DNA helicase protein was found to be expressed specifically in all cells of the T cell lineage. Interestingly, immature thymocytes that are rearranging T cell receptor (TCR) genes express the helicase strongly on the cell surface and the surface expression is terminated upon engagement of functional TCR by positively selecting ligands. This provides the first evidence that a protein that binds nucleic acids can directly contact the extracellular environment in a developmentally controlled manner. Our discovery of a novel molecular link between the cell surface and nuclear events specific for thymocytes suggests that thymic education is supervised by a previously unknown molecular mechanism, which can now be experimentally explored.

Positive selection of T cells: rescue from programmed cell death and differentiation require continual engagement of the T cell receptor.

Positive selection of T cells is a complex developmental process generating long-lived, functionally mature CD4+CD8- and CD4-CD8+ cells from short-lived, immature CD4+CD8+ precursors. The process is initiated in the thymus by interaction of the alpha beta TCR with molecules encoded by the MHC, occurs without cell division, and involves rescue from programmed cell death (PCD), as well as induction of differentiation and maturation of selected precursors. It is unclear whether development of small, positively selected CD4+CD8+ thymocytes (characterized by up-regulated levels of TCR and CD69 molecules) depends on further interactions with MHC molecules and, if so, whether such interactions are required for survival, for maturation, or for both. The involvement of the TCR and/or CD4/CD8 coreceptors in transmitting additional signals is also unknown. We have examined these questions by analyzing survival and differentiation of early (CD4+CD8+TCRhi) and later (CD4-CD8+TCRhi) postselection stages of thymocytes from normal and bcl-2 transgenic mice expressing transgenic, class I MHC-restricted TCR, upon intrathymic transfer into recipients that lacked ligands either for both the TCR and CD8 coreceptor, or for the TCR only. The results provide direct evidence that induction of differentiation of CD4+CD8+ thymocytes by recognition of MHC molecules does not rescue them from PCD and is insufficient to activate the entire maturation program. Both processes require continual engagement of the TCR by positively selecting MHC molecules that, at least in the case of class I MHC-restricted CD4-CD8+ T cells, cannot be substituted by the engagement of coreceptor alone.