Generation of ribosome imprinted polymers for sensitive detection of translational responses

Whilst the profiling of the transcriptome and proteome even of single-cells becomes feasible, the analysis of the translatome, which refers to all messenger RNAs (mRNAs) engaged with ribosomes for protein synthesis, is still an elaborate procedure requiring millions of cells. Herein, we report the generation and use of “smart materials”, namely molecularly imprinted polymers (MIPs) to facilitate the isolation of ribosomes and translated mRNAs from merely 1,000 cells. In particular, we show that a hydrogel-based ribosome imprinted polymer could recover ribosomes and associated mRNAs from human, simian and mice cellular extracts, but did not selectively enrich yeast ribosomes, thereby demonstrating selectivity. Furthermore, ribosome imprinted polymers enabled the sensitive measurement of an mRNA translational regulatory event, requiring 1,000-fold less cells than current methodologies. These results provide first evidence for the suitability of MIPs to selectively recover ribonucleoprotein complexes such as ribosomes, founding a novel means for sensitive detection of gene regulation

Two Host Factors Regulate Persistence of H7a-Specific T Cells Injected in Tumor-Bearing Mice

BACKGROUND: Injection of CD8 T cells primed against immunodominant minor histocompatibility antigens (MiHA) such as H7(a) can eradicate leukemia and solid tumors. To understand why MiHA-targeted T cells have such a potent antitumor effect it is essential to evaluate their in vivo behavior. In the present work, we therefore addressed two specific questions: what is the proliferative dynamics of H7(a)-specifc T cells in tumors, and do H7(a)-specific T cells persist long-term after adoptive transfer? METHODOLOGY/PRINCIPAL FINDINGS: By day 3 after adoptive transfer, we observed a selective infiltration of melanomas by anti-H7(a) T cells. Over the next five days, anti-H7(a) T cells expanded massively in the tumor but not in the spleen. Thus, by day 8 after injection, anti-H7(a) T cells in the tumor had undergone more cell divisions than those in the spleen. These data strongly suggest that anti-H7(a) T cells proliferate preferentially and extensively in the tumors. We also found that two host factors regulated long-term persistence of anti-H7(a) memory T cells: thymic function and expression of H7(a) by host cells. On day 100, anti-H7(a) memory T cells were abundant in euthymic H7(a)-negative (B10.H7(b)) mice, present in low numbers in thymectomized H7(a)-positive (B10) hosts, and undetectable in euthymic H7(a)-positive recipients. CONCLUSIONS/SIGNIFICANCE: Although in general the tumor environment is not propitious to T-cell invasion and expansion, the present work shows that this limitation may be overcome by adoptive transfer of primed CD8 T cells targeted to an immunodominant MiHA (here H7(a)). At least in some cases, prolonged persistence of adoptively transferred T cells may be valuable for prevention of late cancer relapse in adoptive hosts. Our findings therefore suggest that it may be advantageous to target MiHAs with a restricted tissue distribution in order to promote persistence of memory T cells and thereby minimize the risk of cancer recurrence

The Role of the Medial Prefrontal Cortex in Regulating Social Familiarity-Induced Anxiolysis

Overcoming specific fears and subsequent anxiety can be greatly enhanced by the presence of familiar social partners, but the neural circuitry that controls this phenomenon remains unclear. To overcome this, the social interaction (SI) habituation test was developed in this lab to systematically investigate the effects of social familiarity on anxiety-like behavior in rats. Here, we show that social familiarity selectively reduced anxiety-like behaviors induced by an ethological anxiogenic stimulus. The anxiolytic effect of social familiarity could be elicited over multiple training sessions and was specific to both the presence of the anxiogenic stimulus and the familiar social partner. In addition, socially familiar conspecifics served as a safety signal, as anxiety-like responses returned in the absence of the familiar partner. The expression of the social familiarity-induced anxiolysis (SFiA) appears dependent on the prefrontal cortex (PFC), an area associated with cortical regulation of fear and anxiety behaviors. Inhibition of the PFC, with bilateral injections of the GABAA agonist muscimol, selectively blocked the expression of SFiA while having no effect on SI with a novel partner. Finally, the effect of D-cycloserine, a cognitive enhancer that clinically enhances behavioral treatments for anxiety, was investigated with SFiA. D-cycloserine, when paired with familiarity training sessions, selectively enhanced the rate at which SFiA was acquired. Collectively, these outcomes suggest that the PFC has a pivotal role in SFiA, a complex behavior involving the integration of social cues of familiarity with contextual and emotional information to regulate anxiety-like behavior

Differential sensitivity of T lymphocytes and hematopoietic precursor cells to photochemotherapy with 8-methoxypsoralen and ultraviolet A light.

International audienceThe combination of 8-methoxypsoralen (8-MOP) and long wave ultraviolet radiation (UV-A) has immunomodulatory effects and might abolish both graft-vs-host and host-vs-graft reactions after allogeneic hematopoietic stem cell transplantation. In the present study, we have confirmed the sensitivity of T lymphocytes to 8-MOP treatment plus UV-A exposure as evidenced by the abrogation of the alloreactivity in mixed lymphocyte cultures as well as the inhibition of the response to phytohemagglutinin A. However, the clonogenic capacity of the bone marrow hematopoietic progenitors was inhibited with UV-A doses lower than the doses needed to inhibit T-lymphocytes alloreactivity. Moreover, long-term bone marrow cultures showed that 8-MOP plus UV-A treatment had detrimental effects on the more immature bone marrow stem cells. These data were confirmed when murine bone marrow graft was treated with 8-MOP, exposed to UV-A, then transplanted into semiallogeneic recipient mice. The treated cells could not maintain their clonogenic capacity in vivo resulting in death of all animals. Taken together, these data show that ex vivo 8-MOP plus UV-A treatment of the marrow graft cannot be used to prevent post-bone marrow transplantation alloreactivity