RAGs and Regulation of Autoantibodies

Autoreactive antibodies are etiologic agents in a number of autoimmune diseases. Like all other antibodies these antibodies are produced in developing B cells by V(D)J recombination in the bone marrow. Three mechanisms regulate autoreactive B cells: deletion, receptor editing, and anergy. Here we review the prevalence of autoantibodies in the initial antibody repertoire, their regulation by receptor editing, and the role of the recombinase proteins (RAG l and RAG2) in this process

Generation and Evaluation of an IPTG-Regulated Version of Vav-Gene Promoter for Mouse Transgenesis

Different bacteria-derived systems for regulatable gene expression have been developed for the use in mammalian cells and some were also successfully adopted for in vivo use in vertebrate model organisms. However, certain limitations apply to most of these systems, including leakiness of transgene expression, inefficient transgene silencing or activation, as well as limited tissue accessibility of transgene-inducers or their unfavourable pharmacokinetics. In this study, we evaluated the suitability of the lac-operon/lac-repressor (lacO/lacI) system for the regulation of the well-established Vav-gene promoter that allows inducible transgene expression in different haematopoietic lineages in mice. Using the fluorescence marker protein Venus as a reporter, we observed that the lacO/lacI system could be amended to modulate transgene-expression in haematopoietic cells. However, reporter expression was not uniform and the lacO elements introduced into the Vav-gene promoter only conferred limited repression and reversion of lacI-mediated gene silencing after administration of IPTG. Although further optimization of the system is required, the lacO-modified version of the Vav-gene promoter may be adopted as a tool where low basal gene-expression and limited transient induction of protein expression are desired, e.g. for the activation of oncogenes or transgenes that act in a dominant-negative manner

Positive and Negative Selection in Transgenic Mice Expressing a T-Cell Receptor Specific for Influenza Nucleoprotein and Endogenous Superantigen

A transgenic mouse was generated expressing on most (>80%) of thymocytes and peripheral T cells a T-cell receptor isolated from a cytotoxic T-cell clone (F5). This clone is CD8+ and recognizes αα366-374 of the nucleoprotein (NP 366-374) of influenza virus (A/NT/60/68), in the context of Class ,MHC Db (Townsend et al., 1986). The receptor utilizes the Vβ11 and Vα4 gene segments for the β chain and α chain, respectively (Palmer et al., 1989). The usage of Vβ11 makes this TcR reactive to Class II IE molecules and an endogenous ligand recently identified as a product of the endogenous mammary tumour viruses (Mtv) 8, 9, and 11 (Dyson et al., 1991). Here we report the development of F5 transgenic T cells and their function in mice of the appropriate MHC (C57BL/10 H-2b, IE-) or in mice expressing Class II MHC IE (e.g., CBA/Ca H-2k and BALB/c H-2d) and the endogenous Mtv ligands. Positive selection of CD8+ T cells expressing the Vβ11 is seen in C57BL/10 transgenic mice (H-2b). Peripheral T cells from these mice are capable of killing target cells in an antigen-dependent manner after a period of in vitro culture with IL-2. In the presence of Class II MHC IE molecules and the endogenous Mtv ligand, most of the single-positive cells carrying the transgenic T-cell receptor are absent in the thymus. Unexpectedly, CD8+ peripheral T-cells in these (H-2k or H-2d) F5 mice are predominantly Vβ11 positive and also have the capacity to kill targets in an antigen-dependent manner. This is true even following backcrossing of the F5 TcR transgene to H-2d scid/scid mice, in which functional rearrangement of endogenous TcR alpha- and beta-chain genes is impaired

Lineage-specific compaction of Tcrb requires a chromatin barrier to protect the function of a long-range tethering element

Gene regulation relies on dynamic changes in three-dimensional chromatin conformation, which are shaped by composite regulatory and architectural elements. However, mechanisms that govern such conformational switches within chromosomal domains remain unknown. We identify a novel mechanism by which cis-elements promote long-range interactions, inducing conformational changes critical for diversification of the TCRβ antigen receptor locus (Tcrb). Association between distal Vβ gene segments and the highly expressed DβJβ clusters, termed the recombination center (RC), is independent of enhancer function and recruitment of V(D)J recombinase. Instead, we find that tissue-specific folding of Tcrb relies on two distinct architectural elements located upstream of the RC. The first, a CTCF-containing element, directly tethers distal portions of the Vβ array to the RC. The second element is a chromatin barrier that protects the tether from hyperactive RC chromatin. When the second element is removed, active RC chromatin spreads upstream, forcing the tether to serve as a new barrier. Acquisition of barrier function by the CTCF element disrupts contacts between distal Vβ gene segments and significantly alters Tcrb repertoires. Our findings reveal a separation of function for RC-flanking regions, in which anchors for long-range recombination must be cordoned off from hyperactive RC landscapes by chromatin barriers

Co-receptor choice by Vα14i NKT cells is driven by Th-POK expression rather than avoidance of CD8-mediated negative selection

Mouse natural killer T (NKT) cells with an invariant Vα14-Jα18 rearrangement (Vα14 invariant [Vα14i] NKT cells) are either CD4+CD8− or CD4−CD8−. Because transgenic mice with forced CD8 expression in all T cells exhibited a profound NKT cell deficit, the absence of CD8 has been attributed to negative selection. We now present evidence that CD8 does not serve as a coreceptor for CD1d recognition and that the defect in development in CD8 transgene homozygous mice is the result of a reduction in secondary T cell receptor α rearrangements. Thymocytes from mice hemizygous for the CD8 transgene have a less severe rearrangement defect and have functional CD8+ Vα14i NKT cells. Furthermore, we demonstrate that the transcription factor Th, Poxviruses and Zinc finger, and Krüppel family (Th-POK) is expressed by Vα14i NKT cells throughout their differentiation and is necessary both to silence CD8 expression and for the functional maturity of Vα14i NKT cells. We therefore suggest that Th-POK expression is required for the normal development of Vα14i NKT cells and that the absence of CD8 expression by these cells is a by-product of such expression, as opposed to the result of negative selection of CD8-expressing Vα14i NKT cells

Coherence and Coupling Functions Reveal Microvascular Impairment in Treated Hypertension

The complex interactions that give rise to heart rate variability (HRV) involve coupled physiological oscillators operating over a wide range of different frequencies and length-scales. Based on the premise that interactions are key to the functioning of complex systems, the time-dependent deterministic coupling parameters underlying cardiac, respiratory and vascular regulation have been investigated at both the central and microvascular levels. Hypertension was considered as an example of a globally altered state of the complex dynamics of the cardiovascular system. Its effects were established through analysis of simultaneous recordings of the electrocardiogram (ECG), respiratory effort, and microvascular blood flow [by laser Doppler flowmetry (LDF)]. The signals were analyzed by methods developed to capture time-dependent dynamics, including the wavelet transform, wavelet-based phase coherence, non-linear mode decomposition, and dynamical Bayesian inference, all of which can encompass the inherent frequency and coupling variability of living systems. Phases of oscillatory modes corresponding to the cardiac (around 1.0 Hz), respiratory (around 0.25 Hz), and vascular myogenic activities (around 0.1 Hz) were extracted and combined into two coupled networks describing the central and peripheral systems, respectively. The corresponding spectral powers and coupling functions were computed. The same measurements and analyses were performed for three groups of subjects: healthy young (Y group, 24.4 ± 3.4 y), healthy aged (A group, 71.1 ± 6.6 y), and aged treated hypertensive patients (ATH group, 70.3 ± 6.7 y). It was established that the degree of coherence between low-frequency oscillations near 0.1 Hz in blood flow and in HRV time series differs markedly between the groups, declining with age and nearly disappearing in treated hypertension. Comparing the two healthy groups it was found that the couplings to the cardiac rhythm from both respiration and vascular myogenic activity decrease significantly in aging. Comparing the data from A and ATH groups it was found that the coupling from the vascular myogenic activity is significantly weaker in treated hypertension subjects, implying that the mechanisms of microcirculation are not completely restored by current anti-hypertension medications

Recent advances in understanding hypertension development in sub-Saharan Africa

Consistent reports indicate that hypertension is a particularly common finding in black populations. Hypertension occurs at younger ages and is often more severe in terms of blood pressure levels and organ damage than in whites, resulting in a higher incidence of cardiovascular disease and mortality. This review provides an outline of recent advances in the pathophysiological understanding of blood pressure elevation and the consequences thereof in black populations in Africa. This is set against the backdrop of populations undergoing demanding and rapid demographic transition, where infection with the Human Immunodeficiency Virus predominates, and where under and over-nutrition coexist. Collectively, recent findings from Africa illustrate an increased lifetime risk to hypertension from foetal life onwards. From young ages black populations display early endothelial dysfunction, increased vascular tone and reactivity, microvascular structural adaptions, as well as increased aortic stiffness resulting in elevated central and brachial blood pressures during the day and night, when compared to whites. Together with knowledge on the contributions of sympathetic activation and abnormal renal sodium handling, these pathophysiological adaptations result in subclinical and clinical organ damage at younger ages. This overall enhanced understanding on the determinants of blood pressure elevation in blacks encourages (a) novel approaches to assess and manage hypertension in Africa better, (b) further scientific discovery to develop more effective prevention and treatment strategies, and (c) policymakers and health advocates to collectively contribute in creating health-promoting environments in Africa

T-cell identity and epigenetic memory

T-cell development endows cells with a flexible range of effector differentiation options, superimposed on a stable core of lineage-specific gene expression that is maintained while access to alternative hematopoietic lineages is permanently renounced. This combination of features could be explained by environmentally responsive transcription factor mobilization overlaying an epigenetically stabilized base gene expression state. For example, "poising" of promoters could offer preferential access to T-cell genes, while repressive histone modifications and DNA methylation of non-T regulatory genes could be responsible for keeping non-T developmental options closed. Here, we critically review the evidence for the actual deployment of epigenetic marking to support the stable aspects of T-cell identity. Much of epigenetic marking is dynamically maintained or subject to rapid modification by local action of transcription factors. Repressive histone marks are used in gene-specific ways that do not fit a simple, developmental lineage-exclusion hierarchy. We argue that epigenetic analysis may achieve its greatest impact for illuminating regulatory biology when it is used to locate cis-regulatory elements by catching them in the act of mediating regulatory change