Interrogating accessibility of telomeric sequences with FRET-PAINT: evidence for length-dependent telomere compaction

Single-stranded telomeric overhangs are ∼200 nucleotides long and can form tandem G-quadruplex (GQ) structures, which reduce their accessibility to nucleases and proteins that activate DNA damage response. Whether these tandem GQs further stack to form compact superstructures, which may provide better protection for longer telomeres, is not known. We report single-molecule measurements where the accessibility of 24–144 nucleotide long human telomeric DNA molecules is interrogated by a short PNA molecule that is complementary to a single GGGTTA repeat, as implemented in the FRET-PAINT method. Binding of the PNA strand to available GGGTTA sequences results in discrete FRET bursts which were analyzed in terms of their dwell times, binding frequencies, and topographic distributions. The binding frequencies were greater for binding to intermediate regions of telomeric DNA compared to 3′- or 5′-ends, suggesting these regions are more accessible. Significantly, the binding frequency per telomeric repeat monotonically decreased with increasing telomere length. These results are consistent with telomeres forming more compact structures at longer lengths, reducing accessibility of these critical genomic sites.     

Molecular aberrations in human systemic lupus erythematosus

Systemic lupus erythematosus is an autoimmune disorder that predominantly affects women during the childbearing years. Clinically, major organ systems are affected, including the skin, kidneys and nervous system. Genetic, hormonal, environmental and immunoregulatory factors contribute to the highly variable expression of the disease. Impaired cellular and humoral immune responses reflect disordered biochemical and molecular functions that might be determined genetically. Enhanced understanding of these molecular abnormalities should enable development of new, effective therapeutic agents in the near future.     

Regulatory T cells and control of the germinal centre response

Germinal centres (GCs) are specialised lymphoid microenvironments that form in secondary B-cell follicles upon exposure to T-dependent antigens. In the GC, clonal expansion, selection and differentiation of GC B cells result in the production of high-affinity plasma cells and memory B cells that provide protection against subsequent infection. The GC is carefully regulated to fulfil its critical role in defence against infection and to ensure that immunological tolerance is not broken in the process. The GC response can be controlled by a number of mechanisms, one of which is by forkhead box p3 expressing regulatory T (Treg) cells, a suppressive population of CD4⁺ T cells. A specialised subset of Treg cells – follicular regulatory T (Tfr) cells – form after immunisation and are able to access the GC, where they control the size and output of the response. Our knowledge of Treg cell control of the GC is expanding. In this review we will discuss recent advances in the field, with a particular emphasis on the differentiation and function of Tfr cells in the GC.     

Immunodeficiency and autoimmunity: lessons from systemic lupus erythematosus

Recent evidence suggests that systemic autoimmunity and immunodeficiency are not separate entities, but rather are interconnected processes. Immunodeficiency results from distinct defects of the immune response and primarily presents as infections but also frequently with autoimmune features. Systemic autoimmunity is the combined effect of multiple genetic variations and infectious and immunoregulatory factors that result in dominant autoimmune manifestations, in addition to frequent and opportunistic infections. The overlap in disease manifestations and symptoms suggests that immunodeficiency should be considered in the presence of autoimmunity, and vice versa. In this review, we present the shared or similar aspects of immunodeficiency and autoimmunity using systemic lupus erythematosus as a paradigm and discuss the implications for clinical care.     

Cholera toxin B accelerates disease progression in lupus-prone mice by promoting lipid raft aggregation

Infectious agents, including bacteria and viruses, are thought to provide triggers for the development or exacerbation of autoimmune diseases such as systemic lupus erythematosus in the genetically predisposed individual. Molecular mimicry and engagement of TLRs have been assigned limited roles that link infection to autoimmunity, but additional mechanisms are suspected to be involved. In this study we show that T cells from lupus-prone mice display aggregated lipid rafts that harbor signaling, costimulatory, inflammatory, adhesion, and TLR molecules. The percentage of T cells with clustered lipid rafts increases with age and peaks before the development of lupus pathology. We show that cholera toxin B, a component of Vibrio cholerae, promotes autoantibody production and glomerulonephritis in lupus-prone mice by enhancing lipid raft aggregation in T cells. In contrast, disruption of lipid raft aggregation results in delay of disease pathology. Our results demonstrate that lipid rafts contribute significantly to the pathogenesis of lupus and provide a novel mechanism whereby aggregated lipid rafts represent a potential link between infection and autoimmunity.     

Complement inhibitor, complement receptor 1-related gene/protein y-Ig attenuates intestinal damage after the onset of mesenteric ischemia/reperfusion injury in mice.

Complement receptor 1-related gene/protein y (Crry) is a murine membrane protein that regulates the activity of both classical and alternative complement pathways. We used a recombinant soluble form of Crry fused to the hinge, CH2, and CH3 domains of mouse IgG1 (Crry-Ig) to determine whether inhibition of complement activation prevents and/or reverses mesenteric ischemia/reperfusion-induced injury in mice. Mice were subjected to 30 min of ischemia, followed by 2 h of reperfusion. Crry-Ig was administered either 5 min before or 30 min after initiation of the reperfusion phase. Pretreatment with Crry-Ig reduced local intestinal mucosal injury and decreased generation of leukotriene B(4) (LTB(4)). When given 30 min after the beginning of the reperfusion phase, Crry-Ig resulted in a decrease in ischemia/reperfusion-induced intestinal mucosal injury comparable to that occurring when it was given 5 min before initiation of the reperfusion phase. The beneficial effect of Crry-Ig administered 30 min after the initiation of reperfusion coincided with a decrease in PGE(2) generation despite the fact that it did not prevent local infiltration of neutrophils and did not have a significant effect on LTB(4) production. These data suggest that complement inhibition protects animals from reperfusion-induced intestinal damage even if administered as late as 30 min into reperfusion and that the mechanism of protection is independent of neutrophil infiltration or LTB(4) inhibition.     

Respiratory and calcium transport properties of spiny lobster hepatopancreas mitochondria

Mitochondria were isolated from the hepatopancreas of the Florida spiny lobster Panulirus argus using a high osmolarity medium containing 600 mm mannitol, 83 mm sucrose, 5 mm 4-morpholinepropanesulfonic acid, pH 7.6, 0.5% bovine serum albumin (BSA), and 1 mm EDTA. O₂ uptake and Ca²⁺ transport were measured by electrode methods in similar media (plus 4 mm KP_i, 3.3 mm MgCl₂, and 0.67 mg/ml BSA, with 80 mm KCl replacing a portion of the osmotic support). Substrate-supported respiration was observed to be coupled to phosphorylation of ADP or uptake of Ca²⁺ ions. State 3 rates (nanogram atoms O × minute^?1 × milligram protein^?1 ± SEM (N)) were: 49.2 ± 3.9 (19), succinate; 30.9 ± 3.9 (6), dl-palmitoyl carnitine; 29.0 ± 2.7 (9), l-malate; 40.0 ± 2.3 (3), l-glutamate; 27.7 ± 2.2 (5), d-3-hydroxybutyrate; and 26.4 ± 2.4 (18), l-proline ± pyruvate. α-Glycerol phosphate was not oxidized. Ca²⁺ uptake driven by succinate oxidation proceeded with Ca:O ratios of 4.0 ± 0.2 (SEM). Hepatopancreas mitochondria were not uncoupled by Ca²⁺ uptake in excess of 1100 ng atoms × mg protein^?1. Ca²⁺ efflux could be induced by ruthenium red, indicating the presence of an active Ca²⁺ cycle. These mitochondria may provide a favorable model system in which to study regulation of the Ca²⁺ cycle.     

T cell receptor alpha/beta expressing double-negative (CD4-/CD8-) and CD4+ T helper cells in humans augment the production of pathogenic anti-DNA autoantibodies associated with lupus nephritis

It is generally accepted that human Th cells express the surface glycoproteins CD4 and alpha/beta-chain heterodimer of the TCR whereas cytotoxic/suppressor cells are usually CD8+ and alpha/beta TCR+. Another minor set of T cells found in the periphery are CD4-/CD8- (double negative) and express the gamma/delta TCR; these cells can manifest MHC-restricted or nonrestricted cytotoxicity but no helper function. Herein we describe the existence of an unusual Th population in the peripheral blood of humans that are CD4-/CD8- and alpha/beta TCR+. These double-negative Th were markedly expanded in patients with the autoimmune disease SLE and along with CD4+ Th, they induced production of the pathogenic variety of anti-DNA autoantibodies that are IgG in class and cationic in charge. The cationic anti-DNA antibodies induced by the Th were markedly restricted in spectrotype indicating that an oligoclonal population of B cells were committed to produce the pathogenic autoantibodies in active lupus. IL-2-dependent T cell lines were also derived from the patients with active lupus nephritis but the majority of those T cell lines lacked pathogenic autoantibody-inducing capability. Only 4 out of 42 T cell lines from a lupus patient could induce the production of cationic IgG class anti-DNA autoantibodies. The phenotypes of the pathogenic autoantibody-inducing Th lines were similar to the Th subsets: CD4+, alpha/beta TCR+ or CD4-/CD8-, alpha/beta TCR+. These studies suggest that production of pathogenic autoantibodies in human lupus is mediated by mechanisms that are distinct from the generalized, nonspecific polyclonal B cell hyperactivity that leads to excessive production of natural autoantibodies.