Characterization of Protective Human CD4+CD25+ FOXP3+ Regulatory T Cells Generated with IL-2, TGF-β and Retinoic Acid

BACKGROUND: Protective CD4+CD25+ regulatory T cells bearing the Forkhead Foxp3 transcription factor can now be divided into three subsets: Endogenous thymus-derived cells, those induced in the periphery, and another subset induced ex-vivo with pharmacological amounts of IL-2 and TGF-β. Unfortunately, endogenous CD4+CD25+ regulatory T cells are unstable and can be converted to effector cells by pro-inflammatory cytokines. Although protective Foxp3+CD4+CD25+ cells resistant to proinflammatory cytokines have been generated in mice, in humans this result has been elusive. Our objective, therefore, was to induce human naïve CD4+ cells to become stable, functional CD25+ Foxp3+ regulatory cells that were also resistant to the inhibitory effects of proinflammatory cytokines. METHODOLOGY/PRINCIPAL FINDINGS: The addition of the vitamin A metabolite, all-trans retinoic acid (atRA) to human naïve CD4+ cells suboptimally activated with IL-2 and TGF-β enhanced and stabilized FOXP3 expression, and accelerated their maturation to protective regulatory T cells. AtRA, by itself, accelerated conversion of naïve to mature cells but did not induce FOXP3 or suppressive activity. The combination of atRA and TGF-β enabled CD4+CD45RA+ cells to express a phenotype and trafficking receptors similar to natural Tregs. AtRA/TGF-β-induced CD4+ regs were anergic and low producers of IL-2. They had potent in vitro suppressive activity and protected immunodeficient mice from a human-anti-mouse GVHD as well as expanded endogenous Tregs. However, treatment of endogenous Tregs with IL-1β and IL-6 decreased FOXP3 expression and diminished their protective effects in vivo while atRA-induced iTregs were resistant to these inhibitory effects. CONCLUSIONS/SIGNIFICANCE: We have developed a methodology that induces human CD4(+) cells to rapidly become stable, fully functional suppressor cells that are also resistant to proinflammatory cytokines. This methodology offers a practical novel strategy to treat human autoimmune diseases and prevent allograft rejection without the use of agents that kill cells or interfere with signaling pathways

A polymeric immunoglobulin-antigen fusion protein strategy for enhancing vaccine immunogenicity.

In this study, a strategy based on polymeric immunoglobulin G scaffolds (PIGS) was used to produce a vaccine candidate for Mycobacterium tuberculosis. A genetic fusion construct comprising genes encoding the mycobacterial Ag85B antigen, an immunoglobulin γ‐chain fragment and the tailpiece from immunoglobulin μ chain was engineered. Expression was attempted in Chinese Hamster Ovary (CHO) cells and in Nicotiana benthamiana. The recombinant protein assembled into polymeric structures (TB‐PIGS) in N. benthamiana, similar in size to polymeric IgM. These complexes were subsequently shown to bind to the complement protein C1q and FcγRs with increased affinity. Modification of the N‐glycans linked to TB‐PIGS by removal of xylose and fucose residues that are normally found in plant glycosylated proteins also resulted in increased affinity for low‐affinity FcγRs. Immunization studies in mice indicated that TB‐PIGS are highly immunogenic with and without adjuvant. However, they did not improve protective efficacy in mice against challenge with M. tuberculosis compared to conventional vaccination with BCG, suggesting that additional or alternative antigens may be needed to protect against this disease. Nevertheless, these results establish a novel platform for producing polymeric antigen‐IgG γ‐chain molecules with inherent functional characteristics that are desirable in vaccines

An essential function for the ATR-Activation-Domain (AAD) of TopBP1 in mouse development and cellular senescence

ATR activation is dependent on temporal and spatial interactions with partner proteins. In the budding yeast model, three proteins – Dpb11TopBP1, Ddc1Rad9 and Dna2 - all interact with and activate Mec1ATR. Each contains an ATR activation domain (ADD) that interacts directly with the Mec1ATR:Ddc2ATRIP complex. Any of the Dpb11TopBP1, Ddc1Rad9 or Dna2 ADDs is sufficient to activate Mec1ATR in vitro. All three can also independently activate Mec1ATR in vivo: the checkpoint is lost only when all three AADs are absent. In metazoans, only TopBP1 has been identified as a direct ATR activator. Depletion-replacement approaches suggest the TopBP1-AAD is both sufficient and necessary for ATR activation. The physiological function of the TopBP1 AAD is, however, unknown. We created a knock-in point mutation (W1147R) that ablates mouse TopBP1-AAD function. TopBP1-W1147R is early embryonic lethal. To analyse TopBP1-W1147R cellular function in vivo, we silenced the wild type TopBP1 allele in heterozygous MEFs. AAD inactivation impaired cell proliferation, promoted premature senescence and compromised Chk1 signalling following UV irradiation. We also show enforced TopBP1 dimerization promotes ATR-dependent Chk1 phosphorylation. Our data suggest that, unlike the yeast models, the TopBP1-AAD is the major activator of ATR, sustaining cell proliferation and embryonic development

Women and Autoimmune Diseases1

Recent evidence indicates that sex hormones may exacerbate autoimmune diseases, particularly in women, by increasing the adjuvant effect of infections

The role of cranial CT in the investigation of meningitis

More patients with meningitis are undergoing CT and the number of inappropriate requests are increasing. There are few abnormal CT scans presenting a contraindication for lumbar puncture and the majority of these patients usually have clinical signs to suggest raised intracranial pressure

Increased CD45RA+FoxP3low Regulatory T Cells with Impaired Suppressive Function in Patients with Systemic Lupus Erythematosus

BACKGROUND: The role of naturally occurring regulatory T cells (Treg) in the control of the development of systemic lupus erythematosus (SLE) has not been well defined. Therefore, we dissect the phenotypically heterogeneous CD4(+)FoxP3(+) T cells into subpopulations during the dynamic SLE development. METHODLOGY/PRINCIPAL FINDINGS: To evaluate the proliferative and suppressive capacities of different CD4(+) T cell subgroups between active SLE patients and healthy donors, we employed CD45RA and CD25 as surface markers and carboxyfluorescein diacetatesuccinimidyl ester (CFSE) dilution assay. In addition, multiplex cytokines expression in active SLE patients was assessed using Luminex assay. Here, we showed a significant increase in the frequency of CD45RA(+)FoxP3(low) naive Treg cells (nTreg cells) and CD45RA(-)FoxP3(low) (non-Treg) cells in patients with active SLE. In active SLE patients, the increased proportions of CD45RA(+)FoxP3(low) nTreg cells were positively correlated with the disease based on SLE disease activity index (SLEDAI) and the status of serum anti-dsDNA antibodies. We found that the surface marker combination of CD25(+)CD45RA(+) can be used to defined CD45RA(+)FoxP3(low) nTreg cells for functional assays, wherein nTreg cells from active SLE patients demonstrated defective suppression function. A significant correlation was observed between inflammatory cytokines, such as IL-6, IL-12 and TNFα, and the frequency of nTreg cells. Furthermore, the CD45RA(+)FoxP3(low) nTreg cell subset increased when cultured with SLE serum compared to healthy donor serum, suggesting that the elevated inflammatory cytokines of SLE serum may promote nTreg cell proliferation/expansion. CONCLUSIONS/SIGNIFICANCE: Our results indicate that impaired numbers of functional CD45RA(+)FoxP3(low) naive Treg cell and CD45RA(-)FoxP3(low) non-suppressive T cell subsets in inflammatory conditions may contribute to SLE development. Therefore, analysis of subsets of FoxP3(+) T cells, using a combination of FoxP3, CD25 and CD45RA, rather than whole FoxP3(+) T cells, will help us to better understand the pathogenesis of SLE and may lead to the development of new therapeutic strategies

A proposal for a coordinated effort for the determination of brainwide neuroanatomical connectivity in model organisms at a mesoscopic scale

In this era of complete genomes, our knowledge of neuroanatomical circuitry remains surprisingly sparse. Such knowledge is however critical both for basic and clinical research into brain function. Here we advocate for a concerted effort to fill this gap, through systematic, experimental mapping of neural circuits at a mesoscopic scale of resolution suitable for comprehensive, brain-wide coverage, using injections of tracers or viral vectors. We detail the scientific and medical rationale and briefly review existing knowledge and experimental techniques. We define a set of desiderata, including brain-wide coverage; validated and extensible experimental techniques suitable for standardization and automation; centralized, open access data repository; compatibility with existing resources, and tractability with current informatics technology. We discuss a hypothetical but tractable plan for mouse, additional efforts for the macaque, and technique development for human. We estimate that the mouse connectivity project could be completed within five years with a comparatively modest budget.Comment: 41 page

Small but crucial : the novel small heat shock protein Hsp21 mediates stress adaptation and virulence in Candida albicans

Peer reviewedPublisher PD

Mycoplasma Contamination Revisited: Mesenchymal Stromal Cells Harboring Mycoplasma hyorhinis Potently Inhibit Lymphocyte Proliferation In Vitro

Mesenchymal stromal cells (MSC) have important immunomodulatory effects that can be exploited in the clinical setting, e.g. in patients suffering from graft-versus-host disease after allogeneic stem cell transplantation. In an experimental animal model, cultures of rat T lymphocytes were stimulated in vitro either with the mitogen Concanavalin A or with irradiated allogeneic cells in mixed lymphocyte reactions, the latter to simulate allo-immunogenic activation of transplanted T cells in vivo. This study investigated the inhibitory effects of rat bone marrow-derived MSC subsequently found to be infected with a common mycoplasma species (Mycoplasma hyorhinis) on T cell activation in vitro and experimental graft-versus-host disease in vivo.We found that M. hyorhinis infection increased the anti-proliferative effect of MSC dramatically, as measured by both radiometric and fluorimetric methods. Inhibition could not be explained solely by the well-known ability of mycoplasmas to degrade tritiated thymidine, but likely was the result of rapid dissemination of M. hyorhinis in the lymphocyte culture.This study demonstrates the potent inhibitory effect exerted by M. hyorhinis in standard lymphocyte proliferation assays in vitro. MSC are efficient vectors of mycoplasma infection, emphasizing the importance of monitoring cell cultures for contamination