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It is commonly observed that plant species' range margins are enriched for increased selfing rates and, in otherwise self-incompatible species, for self-compatibility (SC). This has often been attributed to a response to selection under mate and/or pollinator limitation. However, range expansion can also cause reduced inbreeding depression, and this could facilitate the evolution of selfing in the absence of mate or pollinator limitation. Here, we explore this idea using spatially explicit individual-based simulations of a range expansion, in which inbreeding depression, variation in self-incompatibility (SI), and mate availability evolve. Under a wide range of conditions, the simulated range expansion brought about the evolution of selfing after the loss of SI in range-marginal populations. Under conditions of high recombination between the self-incompatibility locus (S-locus) and viability loci, SC remained marginal in the expanded metapopulation and could not invade the range core, which remained self-incompatible. In contrast, under low recombination and migration rates, SC was frequently able to displace SI in the range core by maintaining its association with a genomic background with purged genetic load. We conclude that the evolution of inbreeding depression during a range expansion promotes the evolution of SC at range margins, especially under high rates of recombination.‬.

Nipah virus (NiV) and respiratory syncytial virus (RSV) possess two surface glycoproteins involved in cellular attachment and membrane fusion, both of which are potential targets for vaccines. The majority of vaccine development is focused on the attachment (G) protein of NiV, which is the immunodominant target. In contrast, the fusion (F) protein of RSV is the main target in vaccine development. Despite this, neutralising epitopes have been described in NiV F and RSV G, making them alternate targets for vaccine design. Through rational design, we have developed a vaccine strategy applicable to phylogenetically divergent NiV and RSV that comprises both the F and G proteins (FxG). In a mouse immunization model, we found that NiV FxG elicited an improved immune response capable of neutralising pseudotyped NiV and a NiV mutant that is able to escape neutralisation by two known F-specific antibodies. RSV FxG elicited an immune response against both F and G and was able to neutralise RSV, however, this was inferior to the immune response of F alone. Despite this, RSV FxG elicited a response against a known protective epitope within G that is conserved across RSV A and B subgroups, which may provide additional protection in vivo. We conclude that inclusion of F and G antigens within a single design provides a streamlined subunit vaccine strategy against both emerging and established pathogens, with the potential for broader protection against NiV.

Efforts to develop and deploy effective vaccines against Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) continue at pace with more than 30 candidate vaccines now in clinical evaluation. Here we describe the preclinical development of an adjuvanted, prefusion-stabilised Spike (S) protein “Sclamp” subunit vaccine, from rational antigen design through to assessing manufacturability and vaccine efficacy. In mice, the vaccine candidate elicits high levels of neutralising antibodies to epitopes both within and outside the receptor binding domain (RBD) of S, as well as broadly reactive and polyfunctional S-specific CD4+ and cytotoxic CD8+ T cells. We also show protection in Syrian hamsters, which has emerged as a robust animal model for pulmonary SARS-CoV-2 infection. No evidence of vaccine enhanced disease was observed in animal challenge studies and pre-clinical safety was further demonstrated in a GLP toxicology study in rats. The Sclamp vaccine candidate is currently progressing rapidly through clinical evaluation in parallel with large-scale manufacture for pivotal efficacy trials and potential widespread distribution.

Aims To investigate the immunoglobulin (Ig) G response after being fed upon by Cimex lectularius L. Methods and results Participants were fed upon by three male C lectularius insects weekly for a month. Blood was obtained before the feeding and at the last feeding, which was used for immunoblots against bed bug salivary gland extract, with antihuman Immunoglobulin G (IgG) secondary antibodies. No consistent IgG changes developed in 11 humans serially fed upon by C lectularius. Two participants had new IgG responses to proteins at molecular weights of approximately 12-13 kDa, and one had an IgG response to a protein at approximately 40 kDa. At the last study visit, more intense IgG bands to proteins at molecular weights of 12-13 kDa had developed in 55% of participants (6/11) and at molecular weights of ≈30, ≈40 and ≈70 kDa in 45% (5/11) compared with the first study visit. Nitrophorin and apyrase were the most common C lectularius proteins identified with liquid chromatography-tandem mass spectrometry in both crushed bed bug salivary gland extract and post-bed bug feeding extract. Conclusions Human participants did not have consistent IgG responses to crushed C lectularius salivary gland extract.

The brain is a site of relative immune privilege. Although CD4 T cells have been reported in the central nervous system, their presence in the healthy brain remains controversial, and their function remains largely unknown. We used a combination of imaging, single cell, and surgical approaches to identify a CD69+ CD4 T cell population in both the mouse and human brain, distinct from circulating CD4 T cells. The brain-resident population was derived through in situ differentiation from activated circulatory cells and was shaped by self-antigen and the peripheral microbiome. Single-cell sequencing revealed that in the absence of murine CD4 T cells, resident microglia remained suspended between the fetal and adult states. This maturation defect resulted in excess immature neuronal synapses and behavioral abnormalities. These results illuminate a role for CD4 T cells in brain development and a potential interconnected dynamic between the evolution of the immunological and neurological systems. VIDEO ABSTRACT. ispartof: CELL vol:182 issue:3 pages:625-+ ispartof: location:United States status: published

We previously identified a cell wall-associated protein from Fusobacterium nucleatum , a Gram-negative bacterium of the oral cavity, that induces human beta defensin 2 (hBD-2) in primary human oral epithelial cells (HOECs) and designated it FAD-I ( F usobacterium - a ssociated d efensin i nducer). Here, we report differential induction of hBD-2 by different strains of F. nucleatum ; ATCC 25586 and ATCC 23726 induce significantly more hBD-2 mRNA than ATCC 10953. Heterologous expression of plasmid-borne fadI from the highly hBD-2-inducing strains in a Δ fadI mutant of ATCC 10953 resulted in hBD-2 induction to levels comparable to those of the highly inducing strains, indicating that FAD-I is the principal F. nucleatum agent for hBD-2 induction in HOECs. Moreover, anti-FAD-I antibodies blocked F. nucleatum induction of hBD-2 by more than 80%. Recombinant FAD-I (rFAD-I) expressed in Escherichia coli triggered levels of hBD-2 transcription and peptide release in HOECs similar to those of native FAD-I (nFAD-I) isolated from F. nucleatum ATCC 25586. Tandem mass spectrometry revealed a diacylglycerol modification at the cysteine residue in position 16 for both nFAD-I and rFAD-I. Cysteine-to-alanine substitution abrogated FAD-I's ability to induce hBD-2. Finally, FAD-I activation of hBD-2 expression was mediated via both Toll-like receptor-1/2 (TLR-1/2) and TLR-2/6 heterodimerization. Microbial molecules like FAD-I may be utilized in novel therapeutic ways to bolster the host innate immune response at mucosal surfaces.

The clinical extent of psoriasis pathology is regulated in part by defects in immune networks, including a defect in the suppressive actions of regulatory T cells. Recently, CD14(+) HLA-DR(-/low) monocytic myeloid-derived suppressor cells (Mo-MDSCs) have been shown to suppress T-cell activation as one of their suppressive mechanisms. However, little is known about the role of Mo-MDSCs and their functional relationship to T-cell suppression in relation to human chronic immune-mediated inflammatory diseases, including psoriasis. Despite psoriasis being a hyperinflammatory condition, Mo-MDSCs were elevated in psoriatic patient peripheral blood mononuclear cells compared to nonpsoriatic healthy controls (2.6% vs. 0.9%, P0.002). Freshly isolated psoriatic Mo-MDSCs directly suppressed CD8 T-cell proliferation less efficiently than healthy control Mo-MDSCs. In addition, psoriatic Mo-MDSCs expressed reduced surface expression of programmed cell death protein 1 compared to healthy controls. Additional in vitro assays also demonstrated that psoriatic and control Mo-MDSCs both induce regulatory T-cell conversion from naïve T effector cells, but, importantly, the regulatory T cells induced by psoriatic Mo-MDSCs displayed decreased suppressive functionality. These results suggest that aberrations in psoriatic Mo-MDSCs prevent proper suppression of effector T-cell expansion and hamper the immune system's ability to correctly self-regulate.

Cathepsins S (CatS) has been implicated in numerous tumourigenic processes and here we document for the first time its involvement in CCL2 regulation within the tumour microenvironment. Analysis of syngeneic tumours highlighted reduced infiltrating macrophages in CatS depleted tumours. Interrogation of tumours and serum revealed genetic ablation of CatS leads to the depletion of several pro-inflammatory chemokines, most notably, CCL2. This observation was validated in vitro, where shRNA depletion of CatS resulted in reduced CCL2 expression. This regulation is transcriptionally mediated, as evident from RT-PCR analysis and CCL2 promoter studies. We revealed that CatS regulation of CCL2 is modulated through CD74 (also known as the invariant chain), a known substrate of CatS and a mediator of NFkB activity. Furthermore, CatS and CCL2 show a strong clinical correlation in brain, breast and colon tumours. In summary, these results highlight a novel mechanism by which CatS controls CCL2, which may present a useful pharmacodynamic marker for CatS inhibition.

This Letter describes the further development and SAR exploration of a novel series of Legumain inhibitors. Based upon a previously identified Legumain inhibitor from our group, we explored the SAR of the carbamate phenyl ring system to probe the P3 pocket of the enzyme. This led to the identification of a sub-nanomolar inhibitor of Legumain.

Optogenetic inhibition of the electrical activity of neurons enables the causal assessment of their contributions to brain functions. Red light penetrates deeper into tissue than other visible wavelengths. We present a red-shifted cruxhalorhodopsin, Jaws, derived from Haloarcula (Halobacterium) salinarum (strain Shark) and engineered to result in red light–induced photocurrents three times those of earlier silencers. Jaws exhibits robust inhibition of sensory-evoked neural activity in the cortex and results in strong light responses when used in retinas of retinitis pigmentosa model mice. We also demonstrate that Jaws can noninvasively mediate transcranial optical inhibition of neurons deep in the brains of awake mice. The noninvasive optogenetic inhibition opened up by Jaws enables a variety of important neuroscience experiments and offers a powerful general-use chloride pump for basic and applied neuroscience.

IL-6 inhibition has been unsuccessful in treating psoriasis, despite high levels of tissue and serum IL-6 in patients. In addition, de novo psoriasis onset has been reported after IL-6 blockade in patients with rheumatoid arthritis. To explore mechanisms underlying these clinical observations, we backcrossed an established psoriasiform mouse model (IL-17C+ mice) with IL-6-deficient mice (IL-17C+KO) and examined the cutaneous phenotype. IL-17C+KO mice initially exhibited decreased skin inflammation; however, this decrease was transient and reversed rapidly, concomitant with increases in skin Tnf, Il36α/β/γ, Il24, Epgn, and S100a8/a9 to levels higher than those found in IL-17C+ mice. A comparison of IL-17C+ and IL-17C+KO mouse skin transcriptomes with that of human psoriasis skin revealed significant correlation among transcripts of skin of patients with psoriasis and IL-17C+KO mouse skin, and confirmed an exacerbation of the inflammatory signature in IL-17C+KO mice that aligns closely with human psoriasis. Transcriptional analyses of IL-17C+ and IL-17C+KO primary keratinocytes confirmed increased expression of proinflammatory molecules, suggesting that in the absence of IL-6, keratinocytes increase production of numerous additional proinflammatory cytokines. These preclinical findings may provide insight into why patients with arthritis being treated with IL-6 inhibitors develop new onset psoriasis and why IL-6 blockade for the treatment of psoriasis has not been clinically effective.

GM-CSF is a potent proinflammatory cytokine that plays a pathogenic role in the CNS inflammatory disease experimental autoimmune encephalomyelitis. As IL-27 alleviates experimental autoimmune encephalomyelitis, we hypothesized that IL-27 suppresses GM-CSF expression by T cells. We found that IL-27 suppressed GM-CSF expression in CD4+ and CD8+ T cells in splenocyte and purified T cell cultures. IL-27 suppressed GM-CSF in Th1, but not Th17, cells. IL-27 also suppressed GM-CSF expression by human T cells in nonpolarized and Th1- but not Th17-polarized PBMC cultures. In vivo, IL-27p28 deficiency resulted in increased GM-CSF expression by CNS-infiltrating T cells during Toxoplasma gondii infection. Although in vitro suppression of GM-CSF by IL-27 was independent of IL-2 suppression, IL-10 upregulation, or SOCS3 signaling, we observed that IL-27–driven suppression of GM-CSF was STAT1 dependent. Our findings demonstrate that IL-27 is a robust negative regulator of GM-CSF expression in T cells, which likely inhibits T cell pathogenicity in CNS inflammation.

Dynasore, a small molecule inhibitor of dynamin, was used to probe the role of dynamin in the endocytosis of wild-type and mutant CFTR (cystic fibrosis transmembrane conductance regulator). Internalization of both wild-type and ‘temperature-corrected’ ΔF508 CFTR was markedly inhibited by a short exposure to dynasore, implicating dynamin as a key element in the endocytic internalization of both wild-type and mutant CFTR. The inhibitory effect of dynasore was readily reversible upon washout of dynasore from the growth media. Corr-4 ({2-(5-chloro-2-methoxy-phenylamino)-4′-methyl-[4,5′]-bithiazolyl-2′-yl}-phenyl-methanonone), a pharmacological corrector of ΔF508 CFTR biosynthesis, caused a marked increase in the cell surface expression of mutant CFTR. Co-incubation of ΔF508 CFTR expressing cells with Corr-4 and dynasore caused a significantly greater level of cell surface CFTR than that observed in the presence of Corr-4 alone. These results argue that inhibiting the endocytic internalization of mutant CFTR provides a novel therapeutic target for augmenting the benefits of small molecule correctors of mutant CFTR biosynthesis.

Gamma-aminobutyric acid (GABA)-releasing inhibitory interneurons, a critical component of cortical circuitry, are involved in myriad known functional roles. However, information regarding the cytoarchitectural, physiological, and molecular properties of interneurons in posterior piriform cortex (PPC) is sparse. Taking advantage of the glutamic acid decarboxylase (GAD)67-enhanced green fluorescent protein (EGFP) mouse, we used in vitro whole-cell patch-clamp techniques to record from GABAergic interneurons across all 3 layers of PPC and, subsequently, to reconstruct their morphology. For the first time, 5 groups of interneurons are identified, whose firing types are defined based on those described within neocortex. Interestingly, each interneuron group with a distinct firing type also exhibits unique morphological properties, laminar distributions, and excitatory synaptic properties. The dendritic and axonal processes demonstrate subtype-specific orientations and a differential expression of spines and boutons, respectively. In addition, the active and passive electrophysiological properties of these cells show marked intergroup differences. Immunohistochemical techniques revealed a laminar-specific distribution of calcium-binding proteins and vasoactive intestinal peptide (VIP) expression. Surprisingly, excitatory synaptic properties in several groups lack target-specific differences seen in neocortical circuits, reflecting a circuit arranged with less complexity. These data aid in the identification of PPC interneurons and allow us to make well-supported postulations about their functional properties.

Understanding how the brain captures transient experience and converts it into long lasting changes in neural circuits requires the identification and investigation of the specific ensembles of neurons that are responsible for the encoding of each experience. We have developed a Robust Activity Marking (RAM) system that allows for the identification and interrogation of ensembles of neurons. The RAM system provides unprecedented high sensitivity and selectivity through the use of an optimized synthetic activity-regulated promoter that is strongly induced by neuronal activity and a modified Tet-Off system that achieves improved temporal control. Due to its compact design, RAM can be packaged into a single adeno-associated virus (AAV), providing great versatility and ease of use, including application to mice, rats, flies, and potentially many other species. Cre-dependent RAM, CRAM, allows for the study of active ensembles of a specific cell type and anatomical connectivity, further expanding the RAM system’s versatility., Swedish Brain Foundation (Research Fellowship), National Institutes of Health (U.S.). Ruth L. Kirschstein National Research Service Award (Predoctoral Fellowship), National Institutes of Health (U.S.) (Grants MH091220), National Institutes of Health (U.S.) (Grant NS088421), National Institutes of Health (U.S.) (Grant MH106817), MIT Simons Initiatives on Autism and the Brain (Postdoctoral Fellowship), John Merck Fund (Scholars Program)

This Letter describes the continued SAR exploration of small molecule Legumain inhibitors with the aim of developing a potent and selective in vitro tool compound. Work continued in this Letter explores the use of alternative P2-P3 linker units and the P3 group SAR which led to the identification of 10t, a potent, selective and cellularly active Legumain inhibitor. We also demonstrate that 10t has activity in both cancer cell viability and colony formation assays.

GATA-3 is a T cell-specific transcription factor and is essential for the development of the T cell lineage. The transcriptional regulation of GATA-3, however, remains elusive. In this study, we report the identification of a regulatory region located within the first intron of the murine GATA-3 gene. The intronic regulatory region contains both a positive and a negative cis-acting element but, as a whole, serves as a potent T cell-specific enhancer and is essential for the promoter activity in vitro. By using yeast one-hybrid screening, we discovered that fetal liver zinc finger protein 1 (Fliz1) could bind specifically to the negative cis-acting element, the sequence of which is conserved between the mouse and human GATA-3 genes. More importantly, overexpression of Fliz1 repressed the expression of GATA-3 in vivo and in vitro. Our data suggest that the expression of GATA-3 might be partly regulated by the intronic regulatory region and Fliz1 in a developmental stage-specific fashion.

Little is known about cimicosis, the resultant dermal reaction from feeding activity by the common bedbug, Cimex lectularius L. We fed C. lectularius on human study subjects four times over four weeks and measured serum cytokine and antibody levels, and subjects recorded any cimicosis. The average time for subjects to develop cimicosis decreased with each feeding from 8.4, to 2.1, 1.5 and 1.3 days, respectively. There were no significant changes in total IgG, IgG1, IgG2, IgG4 or IgE levels between the first and fourth bedbug feedings, but there was a significant decrease in total IgG3 levels (P

IL-13, a Th2 cell-specific cytokine, is a major effector molecule mediating several pathological features of allergic asthma. However, the transcriptional regulation of the IL-13 gene remains unclear. Here we demonstrate, by using intracellular cytokine staining, that IL-13 is not always coexpressed with other Th2 cytokines in normal Th cells on a single cell basis. In addition, we identified and cloned a minimal inducible and cell type-specific promoter of the murine IL-13 gene. The cell type specificity of the minimal IL-13 promoter is mediated by a functionally critical GATA-3 site that binds endogenous GATA-3 proteins, whereas the induction by PMA/ionomycin is mediated by distinct cis-acting elements. Furthermore, by expressing GATA-3 in wild-type and c-maf transgenic Th1 cells, we demonstrate that the expression of IL-13 is regulated by a mechanism distinct from that regulating the expression of IL-4, and that the expression of Th1 and Th2 cytokine genes does not have to be mutually exclusive in effector Th cells.

We report a novel phospholipase A(2) (PLA(2)), group XII (GXII) PLA(2), distinct from other cysteine-rich groups with a catalytic histidine motif, by its 20-kDa size and distribution of the 14 cysteine residues within the protein. Alternative spliced forms with distinct subcellular localization, designated GXII-1 and GXII-2, were identified by reverse transcription-polymerase chain reaction. Importantly, GXII PLA(2)s, in particular GXII-2 PLA(2), and group V PLA(2), but not group X PLA(2), were selectively expressed in murine type 2 helper T (Th2) clones and in vitro differentiated mouse CD4 Th2 cells as compared with type 1 helper T clones and cells. Stimulation with anti-CD3 appreciably up-regulated expression of GXII PLA(2)s and group V PLA(2) by steady state analysis of the Th2 cells as compared with type 1 helper T cells. These results suggest that group XII and group V PLA(2)s might participate in helper T cell immune response through release of immediate second signals and generation of downstream eicosanoids.

Mating system parameters and genetic diversity were examined for five populations of the endangered shrub Grevillea iaspicula (Proteaceae). Controlled pollinations show that G. iaspicula has an effective self-incompatibility system and little potential for agamospermy. This is reflected in uniformly high multilocus outcrossing rates (tm=0.96-1.00). However, average paternal diversity within open-pollinated sibships is low (rp=0.31-0.54), suggesting that mating within populations is quite restricted. Despite the small size of most populations (four of the five populations studied have fewer than 20 reproductive individuals) the species still possesses moderate to high allelic richness (A=1.6-2.5). Interpopulation genetic differentiation is high (D=0.04-0.32), suggesting that gene flow is limited, even among populations separated by only a few kilometres.

CCR5 expression on CD4+CD25highFoxp3+ regulatory T cells (Tregs) has been reported to be crucial for limiting Th1 inflammation associated with autoimmunity and bacterial infections. We inquired whether abnormalities in chemokine receptors expressed on Tregs might be involved in the psoriatic pathogenesis. Indeed, the proportion of CCR5+ Treg was 58.8% in healthy individuals (n = 9), whereas only half as many CCR5+ Treg cells were found in psoriatic individuals (29.1%, n = 8, p

Human autoimmune diseases are characterized by systemic T cell dysfunction, resulting in chronically activated Th1 and Th17 cells that are inadequately suppressed by regulatory T cells (Tregs). IL-6, which is overexpressed in tissue and serum of patients with autoimmune diseases, inhibits human Treg function. We sought to determine the mechanism for the antitolerogenic properties of IL-6 by examining the signaling pathways downstream of IL-6R in primary human T cells. Inhibition of Stat3 signaling in MLCs containing IL-6 restores Treg-mediated suppression, demonstrating that IL-6–mediated loss of Treg suppression requires phosphorylation of Stat3. Cultures in which either effector T cells (Teffs) or Tregs were pretreated with Stat3 inhibitors indicate that phosphorylated (p)Stat3 is required in both T cell populations for IL-6–mediated reversal of Treg function. IL-21, which signals preferentially through pStat3, also reverses Treg suppression, in contrast to IL-27 and IFN-γ, which signal preferentially through Stat1 and do not inhibit Treg function. Interestingly, both Teffs and Tregs respond to IL-6 stimulation through strong Stat3 phosphorylation with minimal MAPK/Erk activation and moderate Stat1 phosphorylation. Finally, Teffs stimulated strongly through the TCR are also resistant to suppression by Tregs and show concurrent Stat3 phosphorylation. In these cultures, inhibition of pStat3 restores functional suppression by Tregs. Taken together, our findings suggest that an early dominance of Stat3 signaling, prior to subsequent T cell activation, is required for the loss of functional Treg suppression and that kinase-specific inhibitors may hold therapeutic promise in the treatment of autoimmune and chronic inflammatory diseases.

Background Elevated numbers of regulatory T cells (Tregs) have been implicated in certain cancers. Depletion of Tregs has been shown to increase anti-tumor immunity. Tregs also play a critical role in the suppression of autoimmune responses. The study of Tregs has been hampered by a lack of adequate surface markers. Leucine Rich Repeat Containing 32 (LRRC32), also known as Glycoprotein A Repetitions Predominant (GARP), has been postulated as a novel surface marker of activated Tregs. However, there is limited information regarding the processing of LRRC32 or the regulatory phenotype and functional activity of Tregs expressing LRRC32. Results Using naturally-occurring freshly isolated Tregs, we demonstrate that low levels of LRRC32 are present intracellularly prior to activation and that freshly isolated LRRC32+ Tregs are distinct from LRRC32- Tregs with respect to the expression of surface CD62L. Using LRRC32 transfectants of HEK cells, we demonstrate that the N-terminus of LRRC32 is cleaved prior to expression of the protein at the cell surface. Furthermore, we demonstrate using a construct containing a deleted putative signal peptide region that the presence of a signal peptide region is critical to cell surface expression of LRRC32. Finally, mixed lymphocyte assays demonstrate that LRRC32+ Tregs are more potent suppressors than LRRC32- Tregs. Conclusions A cleaved signal peptide site in LRRC32 is necessary for surface localization of native LRRC32 following activation of naturally-occurring freshly-isolated regulatory T cells. LRRC32 expression appears to alter the surface expression of activation markers of T cells such as CD62L. LRRC32 surface expression may be useful as a marker that selects for more potent Treg populations. In summary, understanding the processing and expression of LRRC32 may provide insight into the mechanism of action of Tregs and the refinement of immunotherapeutic strategies aimed at targeting these cells.

The unique nature of extraocular muscle (EOM) and its differential disease susceptibility have been extensively established by anatomic, histologic, physiological, and genomic characterizations.1,2 However, studies to date have focused on the most prominent cell component of EOM—the muscle fibers—and have ignored the contribution of other cells, such as fibroblasts or endothelial cells, to the properties of the muscle as a whole. Dissection of the functions of each cell type in the context of the tissue is not trivial. Ultimately, investigations will necessitate application of multiple methods to gain understanding of the integration of each cell type in a cellular and chemical milieu and their individual contributions to a distinct tissue phenotype. There is a growing appreciation that fibroblasts do not simply function in formation of extracellular matrix, but also provide critical signaling molecules for the cell migration, proliferation, and adherence involved in a broad array of physiological functions.3,4 Fibroblasts possess extensive phenotypic heterogeneity and plasticity across tissues5 with respect to extracellular matrix production, immune response, metabolism, and proliferative capabilities, but even within a given tissue there is heterogeneity based on expression of cell surface markers6,7 and response to growth factors and cytokines.5,8,9 Human orbital fibroblasts and the fibroblasts that reside within the EOM have been a focus of intense study in the context of the pathology of Graves' ophthalmopathy.10,11 The thyrotropin receptor and the insulin-like growth factor receptor, which have been proposed to be autoantigens, are expressed by both the orbital fibroblasts and the perimysial fibroblasts of the EOMs.12,13 Evaluation of synovial, dermal, and orbital fibroblasts has demonstrated their differential response to inflammatory mediators in a disease-specific manner for rheumatoid arthritis, Graves' dermopathy, and Graves' orbitopathy (see Smith10 for a comprehensive review). Therefore, based on investigations of Graves' disease, it is clear that fibroblasts possess tissue-specific properties. Because of the phenotypic differences between EOM and limb skeletal muscles, we hypothesized that fibroblasts derived from EOM (designated, EOM-Fibro) possess properties that differentiate them from fibroblasts of limb muscle (designated LM-Fibro). In vitro investigations indicate that fibroblasts support the differentiation and survival of skeletal myoblasts, providing a substrate for contraction and trophic support.14 Therefore, we further hypothesized that EOM-Fibro would preferentially support the in vitro maturation of myotubes from EOM cell lines (CL-EOM) compared with LM-Fibro. To evaluate these hypotheses, we first performed comprehensive assessment of EOM-Fibro morphology, expression of Thy-1, a standard marker of fibroblast subpopulations, and broad-based characterization of gene expression. The results supported our hypothesis that EOM-Fibro possess distinct properties compared with LM-Fibro and encouraged further functional experiments, which demonstrated that EOM-Fibro sustain greater in vitro maturation of EOM myotubes than LM-Fibro. The data suggest that the perimysial fibroblasts of EOMs contribute to the unique phenotype of EOMs and therefore, may serve as a target for manipulation to modify the EOM phenotype, especially for therapeutic purposes.

We examined the population dynamics and seasonal change in body size of a population of Daphnia galeata mendotae experiencing strong vertebrate predation. The intensity of food limitation was quantified seasonally by in situ food addition. We also collected live animals early and late in the summer for laboratory studies designed to estimate genetic variation and seasonal change in life-history traits for the population. The population was characterized by nearly equal short-term birth and death rates that were strongly correlated with population density, suggesting density dependence. The population generally exhibited very high birth and death rates and no significant response to experimental food addition. The one exception was June 1988, when the population achieved high densities and exhibited strong food limitation. Throughout both summers, minimal and average body sizes of adults decreased. Laboratory studies document substantial genetic variation for body size and other life-history traits among animals collected early in summer. By late summer, however, there was little genetic variation for these same traits. Furthermore, clones collected early in summer were genetically larger in body size at birth and maturity than clones collected in late summer. We interpret these results as indicating natural selection for body size.

Putative living entities called nanobacteria (NB) are unusual for their small sizes (50–500 nm), pleomorphic nature, and accumulation of hydroxyapatite (HAP), and have been implicated in numerous diseases involving extraskeletal calcification. By adding precipitating ions to cell culture medium containing serum, mineral nanoparticles are generated that are morphologically and chemically identical to the so-called NB. These nanoparticles are shown here to be formed of amorphous mineral complexes containing calcium as well as other ions like carbonate, which then rapidly acquire phosphate, forming HAP. The main constituent proteins of serum-derived NB are albumin, fetuin-A, and apolipoprotein A1, but their involvement appears circumstantial since so-called NB from different body fluids harbor other proteins. Accordingly, by passage through various culture media, the protein composition of these particles can be modulated. Immunoblotting experiments reveal that antibodies deemed specific for NB react in fact with either albumin, fetuin-A, or both, indicating that previous studies using these reagents may have detected these serum proteins from the same as well as different species, with human tissue nanoparticles presumably absorbing bovine serum antigens from the culture medium. Both fetal bovine serum and human serum, used earlier by other investigators as sources of NB, paradoxically inhibit the formation of these entities, and this inhibition is trypsin-sensitive, indicating a role for proteins in this inhibitory process. Fetuin-A, and to a lesser degree albumin, inhibit nanoparticle formation, an inhibition that is overcome with time, ending with formation of the so-called NB. Together, these data demonstrate that NB are most likely formed by calcium or apatite crystallization inhibitors that are somehow overwhelmed by excess calcium or calcium phosphate found in culture medium or in body fluids, thereby becoming seeds for calcification. The structures described earlier as NB may thus represent remnants and by-products of physiological mechanisms used for calcium homeostasis, a concept which explains the vast body of NB literature as well as explains the true origin of NB as lifeless protein-mineralo entities with questionable role in pathogenesis.

Myasthenia gravis (MG) is a neuromuscular transmission disease caused primarily by acetylcholine receptor (AChR) autoantibodies,1,2 and several lines of evidence indicate that the fixation of complement at the neuromuscular junction (NMJ) is an important factor in determining disease severity.3 C3 activation fragments and the membrane attack complex are detected at the NMJ of patients with MG, depletion of serum C3 and C4 is appreciated in some patients, and terminal complement components are found in patient sera.4 – 6 In humans, antigenic modulation and direct blockade of the AChR also likely contribute to MG pathology, but their relative significance is not known.7,8 Complement is paramount in driving disease pathology in both models of the experimentally acquired myasthenia gravis (EAMG), whether produced by administration of AChR antibodies or through immunization with purified AChR. With rare exception,9 passive transfer EAMG is induced only by complement-fixing antibodies, and complement depletion by cobra venom factor eliminates NMJ injury and weakness.10 Antibodies directed toward C511 and soluble complement receptor (sCR1)12 are protective in EAMG produced by AChR antibody administration. C5-deficient mice13 or anti-C6 Fab antibody–treated EAMG rats14 are more resistant and develop less severe disease, whereas an absence of cell surface regulators of complement leads to significantly greater disease with EAMG induced by AChR antibodies.15–17 The inhibition of complement as a therapeutic approach is beginning to be applied to human disorders. Pexelizumab, a monoclonal antibody directed against C5, has demonstrated short-term safety and efficacy in humans in myocardial infarction,18 coronary artery bypass graft surgery,19 and lung transplantation.20 A phase 3 trial of eculizumab, a single-chain fragment of anti-C5, in treatment of paroxysmal nocturnal hemoglobinuria21 demonstrated significant clinical benefits with minimal adverse effects. MG would be expected to respond well to complement inhibition especially in acute exacerbations when complement-mediated NMJ injury is expected to be maximal. rEV576 is a novel complement inhibitor, which appears ideally suited for consideration as a therapeutic agent. It is a 17kDa protein22 identified from the saliva of the tick Ornithodoros moubata and belongs to the lipocalin family of proteins, which inhibit inflammatory mediators.23 Because the agent is derived from ticks that apply themselves to mammalian species for weeks without inducing a reaction, there is an expectation that rEV576 will have limited adverse effects in humans. The compound is not known to cause allergic reactions or to have effects beyond those related to complement inactivation.24 rEV576 specifically targets the C5 activation step and has been shown to inhibit both the classic and the alternative pathways, and act specifically at the C5 step of complement activation.25 The simplest explanation for the activity of rEV576 is direct binding to C5 that prevents interaction with the C5 convertase rather than by blocking the C5a cleavage site.26 In this study, the active and passive models of EAMG were used to investigate the therapeutic activity of rEV576. Rats treated with rEV576 demonstrated dramatic improvements in weakness, recovery from weight loss, and reduced C9 deposition at the NMJ. The findings suggest that this novel complement inhibitor could prove to be an effective treatment in human MG.

Afferent olfactory information, both in vivo and in vitro, can be rapidly adapted to through an mGluR mediated attenuation of synaptic strength. Specific cellular and synaptic mechanisms underlying olfactory learning and habituation at the cortical level are not entirely clear. Using whole-cell recording methods, both spontaneous and evoked excitatory post-synaptic currents (EPSCs) were recorded from principal cells of the piriform cortex (PC). Taking advantage of a coincidental, pre- and post-synaptic stimulation protocol, long term depression (LTD) in synaptic strength was induced at associative, excitatory synapses onto layer II pyramidal neurons of the PC in mice (P15–27). The LTD was mimicked and occluded by metabotropic glutamate receptor (mGluR) agonists and blocked by the nonselective mGluR antagonist (RS)-α-Methyl-4-sulfonophenylglycine (MSPG), but not by the NMDA receptor antagonist 2-amino-5-phosphonovaleric acid (APV). Analysis of the paired-pulse ratio, the AMPA/NMDA current ratio, and spontaneous excitatory postsynaptic currents (sEPSCs) indicates that the electrically induced LTD was mediated predominantly by postsynaptic mechanisms. In addition, presynaptic mGluRs were also involved in agonist-mediated synaptic depression. Immunohistochemical analysis supports the presence of multiple subclasses of mGluRs throughout the PC, with large concentrations of several receptors present in layer II. These observations provide further evidence of activity-dependent, long-term modification of associative inputs and its underlying mechanisms. Cortical adaptation at associative synapses provides a further link between cortical olfactory processing and subcortical centers that influence behavior.

The diseases of cystic fibrosis, chronic obstructive pulmonary disease (COPD), and chronic bronchitis are characterized by mucus-congested and inflamed airways. Anti-inflammatory agents that can simultaneously restore or enhance mucociliary clearance through cystic fibrosis transmembrane conductance regulator (CFTR) activation may represent new therapeutics in their treatment. Herein, we report the activation of CFTR-mediated chloride secretion by phosphodiesterase (PDE) 4 inhibitors in T84 monolayer using (125)I anion as tracer. In the absence of forskolin, the iodide secretion was insensitive to PDE4 inhibitor L-826,141 [4-[2-(3,4-bis-difluoromethoxyphenyl)-2-[4-(1,1,1,3,3,3-hexafluoro-2-hydroxypropan-2-yl)phenyl]-ethyl]-3-methylpyridine-1-oxide], roflumilast, or to PDE3 inhibitor trequinsin. However, these inhibitors potently augmented iodide secretion after forskolin stimulation, with efficacy coupled to the activation states of adenylyl cyclase. The iodide secretion from PDE3 or PDE4 inhibition was characterized at first by a prolonged efflux duration, followed by progressively elevated peak efflux rates at higher inhibitor concentrations. Paralleled with an increased phosphor-cAMP response element-binding protein formation, the CFTR activation dissociated from a global cAMP elevation and was blocked by H89 [N-[2-((p-bromocinnamyl)amino)ethyl]-5-isoquinolinesulfonamide]. 2-(4-Fluorophenoxy)-N-[(1S)-1-(4-methoxyphenyl)ethyl]nicotinamide, a stereoselective PDE4D inhibitor, augmented iodide efflux more efficiently than its less potent (R)-isomer. The peak efflux from maximal PDE4 and PDE3 inhibition matched that from full adenylyl cyclase activation. These data suggest that PDE3 and PDE4 (mainly PDE4D) form the major cAMP diffusion barrier in T84 cells to ensure a compartmentalized CFTR signaling. Together with their potent anti-inflammatory properties, the potentially enhanced airway mucociliary clearance from CFTR activation may have contributed to the efficacy of PDE4 inhibitors in COPD and asthmatic patients. PDE4 inhibitors may represent new opportunities to combat cystic fibrosis and other respiratory diseases in future.

The psbC gene encodes CP43, a component of Photosystem II (PSII) in higher plants, algae, and cyanobacteria. Previous work demonstrated that alteration of an arginine residue occurring at position 305 to serine produced a strain (R305S) with altered PSII activity (Knoepfle, N., Bricker, T. M., and Putnam-Evans, C. (1999) Biochemistry 38, 1582-1588). This strain grew at wild-type rates in complete BG-11 media (480 microM chloride) and evolved oxygen at rates that were 60-70% of the observed wild-type rates. The R305S strain assembled approximately 70-80% of the functional PSII centers contained in the control strain, and these PSII centers were very sensitive to photoinactivation at high light intensities. We recently observed that the R305S mutant exhibited a pronounced chloride effect. When this mutant was grown in media depleted of chloride (30 microM chloride), it exhibited a severely reduced photoautotrophic growth rate. The effect of chloride depletion on the growth rate of the mutant was reversed by the addition of 480 microM bromide to the chloride-depleted BG-11 media. Oxygen evolution rates for the mutant were further depressed to about 22% of that observed in control cells under chloride-limiting conditions. Addition of bromide restored these rates to those observed under chloride-sufficient conditions. The mutant exhibited a significantly lower relative quantum yield for oxygen evolution than did the control strain, and this was exacerbated under chloride-limiting conditions. Fluorescence yield measurements indicated that both the mutant and the control strains assembled fewer PSII reaction centers under chloride-limiting conditions. The reaction centers assembled by the mutant exhibited an enhanced sensitivity to photoinactivation under chloride-limiting conditions, with a t(1/2) of photoinactivation of 2.6 min under chloride-limiting conditions as compared to a t(1/2) of 4.7 min under normal growth conditions. The mutant also exhibited an enhanced stability of its S(2) state and increased number of centers in the S(1) state following dark incubation. These results indicate that the mutant R305S exhibits a defect in its ability to utilize chloride in support of efficient oxygen evolution in PSII. This is the first mutant of this type described in the CP43 protein.

Ethanol (EtOH) administration to rats for 4 wk markedly decreased Mg2+content in several tissues, including liver. Total cellular Mg2+accounted for 26.8 ± 2.4 vs. 36.0 ± 1.4 nmol Mg2+/mg protein in hepatocytes from EtOH-fed and control rats, respectively, and paralleled a 13% decrease in cellular ATP content. Stimulation of α1- or β-adrenergic receptor or acute EtOH administration did not elicit an extrusion of Mg2+from liver cells of EtOH-fed rats while releasing 5% of total tissue Mg2+content from hepatocytes of control rats. Despite the 25% decrease in Mg2+content, hepatocytes from EtOH-fed rats did not accumulate Mg2+following stimulation of protein kinase C signaling pathway, whereas control hepatocytes accumulated ∼2 nmol Mg2+· mg protein−1· 4 min−1. Together, these data indicate that Mg2+homeostasis and transport are markedly impaired in liver cells after prolonged exposure to alcohol. The inability of liver cells, and possibly other tissues, to accumulate Mg2+can help explain the reduction in tissue Mg2+content following chronic alcohol consumption.

To the Editor: As data accumulate on the recently identified genetic variant in the 3′-untranslated region of the prothrombin (factor II) gene (G20210A), it is apparent that this mutation confers a moderate risk for venous thrombosis.[1-3][1] Data from several abstracts presented at the 1997 ISTH

Therapy-related acute myeloid leukemia (t-AML) and therapy-related myelodysplastic syndrome (t-MDS) are well-recognized complications of cytotoxic chemotherapy and/or radiotherapy. Compared to de novo AML, there is a higher incidence of TP53 mutations, abnormalities of chromosomes 5 or 7, and complex cytogenetics in t-AML. The response to chemotherapy is also reduced compared to de novo AML, and long-term remissions are rare. From a pathogenesis perspective, the key difference between de novo AML and t-AML is the prior exposure of t-AML patients to cytotoxic therapy during treatment of their primary cancer. We previously reported the initial findings of our study to sequence the genome of 22 cases of t-AML (Ramsingh et al, Abstract #784, 2012). These data were compared to the genomic sequence of 49 cases of de novo AML (TCGA, NEJM, 2013). Surprisingly, the data showed that the total number of somatic single nucleotide variants (SNVs) and the percentage of transversions (which are associated with chemotherapy-induced DNA damage) were similar in t-AML and de novo AML. Thus, there is no evidence that chemotherapy induces genome-wide DNA damage in t-AML. We extended this data set by sequencing 149 genes of interest in an additional 89 patients with t-AML/t-MDS. Mutations of TP53 were the most frequent (31.5%), and TP53 was the only gene mutated at a higher frequency in t-AML or t-MDS compared with de novo AML or MDS respectively. The mechanism by which TP53 mutations are selectively enriched in t-AML/t-MDS is unclear. Since t-AML/t-MDS has a similar mutation burden compared to de novo AML, it is not likely that chemotherapy directly induces TP53 mutations. We recently reported that individual HSCs accumulate somatic mutations as a function of age. By age 50, there are on average 5 coding gene mutations per HSC clone (Welch et al, Cell, 2012). As it is estimated that there are 10,000 HSCs in humans, it is possible that HSC clones harboring aging-related TP53 mutations are present in a subset of healthy individuals. Based on these observations, we propose a model in which rare TP53 mutant-bearing HSC clones have a selective growth advantage in patients undergoing chemotherapy. This results in their clonal expansion, with additional mutations in the HSC clone undoubtedly required for full leukemic transformation. This model predicts that TP53 mutations are present in many patients years prior to the development of t-AML/t-MDS. To test this prediction, we developed a next generation sequencing technique able to detect rare subclones harboring TP53 mutations to as low as 1 in 1,000 cells. We identified 7 cases of t-AML/t-MDS with specific TP53 mutations for whom we had blood or bone marrow banked 3-10 years prior to the development of t-AML/t-MDS. The specific TP53 mutations were detected in two cases. In patient UPN 530447, bi-allelic mutations of TP53 were identified in mobilized peripheral blood leukocytes 6 years prior to the development of t-AML at frequencies of 0.5 and 0.7%. In patient UPN 341666, a heterozygous TP53 mutation was identified in mobilized peripheral blood leukocytes 3 years prior to t-MDS diagnosis at a frequency of 0.1%. In at least some cases, we have clear evidence that the TP53 mutations occurred prior to the acquisition of other driver mutations or the development of cytogenetic abnormalities. To directly test the hypothesis that TP53 mutations confer a clonal advantage after chemotherapy, we generated mixed bone marrow chimeras containing wild type and Tp53+/- cells. No clonal advantage of Tp53+/- cells was identified in untreated chimeras. However, upon treatment with N-ethyl-N-nitrosourea, Tp53+/- HSCs had a significant growth advantage. Collectively, these data suggest that HSCs that acquire heterozygous TP53 mutations as a function of normal aging may be selected for in the presence of cytotoxic therapy. This provides a potential mechanism for the high incidence of TP53 mutations in t-AML/t-MDS. The early acquisition of TP53 mutations in the founding clone likely contributes to the frequent cytogenetic abnormalities and poor response to chemotherapy that are typical of t-AML/t-MDS. Disclosures: Welch: Eisai: Research Funding.

Accumulating evidence from studies of a variety of morphological, aliozyme and DNA markers indicates that plants generally exhibit high levels of intraspecific genetic variation (see Venable, 1984; Hamrick and Godt, 1989; Clegg, 1990 for reviews). Furthermore, this variation is often not distributed randomly among individuals but, for species with a variety of ecologies, there is evidence of significant genetic structure at various spatial and organizational scales, for example among individuals within populations (e.g. Schmitt and Gamble, 1990), among subpopulations within a region (e.g. Linhart et al, 1981) and among populations across a species’ range (e.g. Li and Adams, 1989). Several studies have pointed to correlations between genetic variation and environmental patterns (e.g. Snaydon and Davies, 1976; Warwick and Black, 1986; Xie and Knowles, 1992). Despite this, it is only quite recently that investigations of genetic variation and genetic processes have been more formally incorporated into the synthetic discipline of landscape ecology (e.g. Manicacci et al., 1992; Fore, Hickey and Vankat, 1992). This chapter deals with the empirical evidence for the influence of landscape structure on genetic variation in plants and the distribution of this variation: genetic structure. Underlying genetic processes are also discussed.

The occurrence of intersex fish is widespread in the rivers of England and Wales. The extent of intersex in fish populations is believed to be strongly linked to their exposure to steroid estrogens. The present study presents, to our knowledge, the first national, catchment-based risk assessment for steroid estrogens in the world. A graphical information system-based model predicted the concentrations of estradiol (E2), estrone, and ethinylestradiol, which were combined and compared with known biological effect levels to predict the risk of endocrine disruption for 10,313 individual river reaches (21,452 km) receiving effluent from more than 2000 sewage treatment plants serving more than 29 million people. The large scale of this assessment underlines the usefulness of computer-based risk assessment methods. Overall, 61% [corrected] of the modeled reaches (all percentages are in terms of the total river length modeled) in England and Wales were predicted to be not at risk from endocrine disruption (mean concentrations,1 ng/L E2 equivalents). A large range existed in the percentage of river reaches at risk in the various regions, from 5% in Wales to 67% in the Thames catchment. Important factors influencing this proportion are the population density, particularly their location, and the available dilution. A very small proportion of reaches (approximately 1-3%) were predicted to be at high risk (10 ng/L E2 equivalents). Many of these high-risk reaches, however, were ditches, which were composed almost entirely of sewage effluent. The model could be applied equally well to any other chemical of concern emanating from the human population that would be impractical to assess by measurement.

GATA-3 is a T cell–specific transcription factor and is essential for the development of the T cell lineage. Recently, it was shown that the expression of GATA-3 is further induced in CD4 + helper T cells upon differentiation into type 2 but not type 1 effector cells. Here, we report the molecular cloning of a GATA-3 interacting protein, repressor of GATA (ROG). ROG is a lymphoid-specific gene and is rapidly induced in Th cells upon stimulation with anti-CD3. In in vitro assays, ROG represses the GATA-3-induced transactivation. Furthermore, overexpression of ROG in Th clones inhibits the production of Th cytokines. Taken together, our results suggest that ROG might play a critical role in regulating the differentiation and activation of Th cells.

Mr Young has here taken up the subject of the salmon's growth where it was necessarily left off by Mr Shaw. So far as the earliest or fresh-water state of the fish is concerned, he entirely agrees with the observer just named. He then states the various opinions which prevail regarding the more or less rapid growth of smolts and grilse, and shews by tabular lists (the result of frequently repeated experiments), that the increase in their dimensions is extraordinary so soon as they descend into the salt water.

Background Experimental autoimmune encephalomyelitis (EAE) is an animal model of autoimmune inflammatory demyelination that is mediated by Th1 and Th17 cells. The transcription factor interferon regulatory factor 3 (IRF3) is activated by pathogen recognition receptors and induces interferon-β production. Methods To determine the role of IRF3 in autoimmune inflammation, we immunised wild-type (WT) and irf3−/− mice to induce EAE. Splenocytes from WT and irf3−/− mice were also activated in vitro in Th17-polarising conditions. Results Clinical signs of disease were significantly lower in mice lacking IRF3, with reduced Th1 and Th17 cells in the central nervous system. Peripheral T-cell responses were also diminished, including impaired proliferation and Th17 development in irf3−/− mice. Myelin-reactive CD4+ cells lacking IRF3 completely failed to transfer EAE in Th17-polarised models as did WT cells transferred into irf3−/− recipients. Furthermore, IRF3 deficiency in non-CD4+ cells conferred impairment of Th17 development in antigen-activated cultures. Conclusion These data show that IRF3 plays a crucial role in development of Th17 responses and EAE and warrants investigation in human multiple sclerosis.