Immunological and Physiological Differences Between Layer- and Broiler Chickens after Concurrent Intratracheal Administration of Lipopolysaccharide and Human Serum Albumin

Layers and broilers were concurrently intratracheally challenged with 0.5 mg Lipopolysaccharide (LPS) and 0.1 mg Human Serum Albumin (HuSA) at 3 weeks of age. Specific total and isotype-specific (IgM, IgG, IgA) Antibody (Ab) responses to HuSA during 3 weeks following immunization, cellular in vitro mitogen responses to Concanavalin A (Con A) and specific cellular responses in vitro to different dosages of HuSA, blood serotonin (5-HT) levels, plasma Corticosterone (CORT) levels at 6 weeks of age and ex vivo nitric oxide (NO) production in the presence of LPS, respectively, were measured in all birds. Higher in vitro cellular responses to HuSA, but not Con A, were found in the broilers than in the layers. Also higher total, IgM and IgG antibody responses to HuSA were found in the broilers. Higher ex vivo NO production was found in the layers. A heavier spleen weight was found in the broilers, but relative spleen weight was higher in the layers. The broilers grew much heavier and also maintained a higher growth during the first 24 and 48 h after i.t. challenge with LPS and HuSA. No breed effect was found for body temperature responses after i.t. challenge. Blood 5-HT levels and plasma CORT levels were significantly higher in the layers. Number and type of significant correlations between 5-HT levels, cachectin response to LPS, antibody levels and cellular immunity differed between breeds. Our data suggest comparable immune responses to i.t. HuSA challenge in broilers and layers of similar age and confirm the earlier reported higher humoral immune response in broilers. On the other hand, the cachectin response to LPS differed between broilers and layers. Our results do not confirm the earlier reported higher cellular immune response of layers. Different significant relationships between physiological parameters in broilers and layers were found. Our results suggest that selection for enhanced growth does not necessarily affect specific immune competence of poultr

Spatial and temporal cellular responses to single-strand breaks in human cells

DNA single-strand breaks (SSB) are one of the most frequent DNA lesions produced by reactive oxygen species and during DNA metabolism, but the analysis of cellular responses to SSB remains difficult due to the lack of an experimental method to produce SSB alone in cells. By using human cells expressing a foreign UV damage endonuclease (UVDE) and irradiating the cells with UV through tiny pores in membrane filters, we created SSB in restricted areas in the nucleus by the immediate action of UVDE on UV-induced DNA lesions. Cellular responses to the SSB were characterized by using antibodies and fluorescence microscopy. Upon UV irradiation, poly(ADP-ribose) synthesis occurred immediately in the irradiated area. Simultaneously, but dependent on poly(ADP-ribosyl)ation, XRCC1 was translocated from throughout the nucleus, including nucleoli, to the SSB. The BRCT1 domain of XRCC1 protein was indispensable for its poly(ADP-ribose)-dependent recruitment to the SSB. Proliferating cell nuclear antigen and the p150 subunit of chromatin assembly factor 1 also accumulated at the SSB in a detergent-resistant form, which was significantly reduced by inhibition of poly(ADP-ribose) synthesis. Our results show the importance of poly(ADP-ribosyl)ation in sequential cellular responses to SSB

Cellular-level versus receptor-level response threshold hierarchies in T-Cell activation

Peptide-MHC (pMHC) ligand engagement by T-cell receptors (TCRs) elicits a variety of cellular responses, some of which require substantially more TCR-mediated stimulation than others. This threshold hierarchy could reside at the receptor level, where different response pathways branch off at different stages of the TCR/CD3 triggering cascade, or at the cellular level, where the cumulative TCR signal registered by the T-cell is compared to different threshold values. Alternatively, dual-level thresholds could exist. In this study, we show that the cellular hypothesis provides the most parsimonious explanation consistent with data obtained from an in-depth analysis of distinct functional responses elicited in a clonal T-cell system by a spectrum of biophysically defined altered peptide ligands across a range of concentrations. Further, we derive a mathematical model that describes how ligand density, affinity, and off-rate all affect signaling in distinct ways. However, under the kinetic regime prevailing in the experiments reported here, the TCR/pMHC class I (pMHCI) dissociation rate was found to be the main governing factor. The CD8 coreceptor modulated the TCR/pMHCI interaction and altered peptide ligand potency. Collectively, these findings elucidate the relationship between TCR/pMHCI kinetics and cellular function, thereby providing an integrated mechanistic understanding of T-cell response profiles

Salt-inducible kinases (SIKs) regulate TGFβ-mediated transcriptional and apoptotic responses

The signalling pathways initiated by members of the transforming growth factor-β (TGFβ) family of cytokines control many metazoan cellular processes, including proliferation and differentiation, epithelial-mesenchymal transition (EMT) and apoptosis. TGFβ signalling is therefore strictly regulated to ensure appropriate context-dependent physiological responses. In an attempt to identify novel regulatory components of the TGFβ signalling pathway, we performed a pharmacological screen by using a cell line engineered to report the endogenous transcription of the TGFβ-responsive target gene PAI-1. The screen revealed that small molecule inhibitors of salt-inducible kinases (SIKs) attenuate TGFβ-mediated transcription of PAI-1 without affecting receptor-mediated SMAD phosphorylation, SMAD complex formation or nuclear translocation. We provide evidence that genetic inactivation of SIK isoforms also attenuates TGFβ-dependent transcriptional responses. Pharmacological inhibition of SIKs by using multiple small-molecule inhibitors potentiated apoptotic cell death induced by TGFβ stimulation. Our data therefore provide evidence for a novel function of SIKs in modulating TGFβ-mediated transcriptional and cellular responses.</p

A Novel Non-invasive Method to Detect RELM Beta Transcript in Gut Barrier Related Changes During a Gastrointestinal Nematode Infection

Currently, methods for monitoring changes of gut barrier integrity and the associated immune response via non-invasive means are limited. Therefore, we aimed to develop a novel non-invasive technique to investigate immunological host responses representing gut barrier changes in response to infection. We identified the mucous layer on feces from mice to be mainly composed of exfoliated intestinal epithelial cells. Expression of RELM-β, a gene prominently expressed in intestinal nematode infections, was used as an indicator of intestinal cellular barrier changes to infection. RELM-β was detected as early as 6 days post-infection (dpi) in exfoliated epithelial cells. Interestingly, RELM-β expression also mirrored the quality of the immune response, with higher amounts being detectable in a secondary infection and in high dose nematode infection in laboratory mice. This technique was also applicable to captured worm-infected wild house mice. We have therefore developed a novel non-invasive method reflecting gut barrier changes associated with alterations in cellular responses to a gastrointestinal nematode infection

Cellular Responses to Replication Problems

During every S-phase cells need to duplicate their genomes so that both daughter cells inherit complete copies of genetic information. It is a tremendous task, given the large sizes of mammalian genomes and the required precision of DNA replication. A major threat to the accuracy and efficiency of DNA synthesis is the presence of damaged DNA, e.g. abasic sites, single stranded DNA breaks, DNA crosslinks and adducts. This damage can be caused by exogenous age

Immune dysregulation as a cause of autoinflammation in fragile X premutation carriers: link between FMRI CGG repeat number and decreased cytokine responses.

BackgroundIncreased rates of autoinflammatory and autoimmune disorders have been observed in female premutation carriers of CGG repeat expansion alleles of between 55-200 repeats in the fragile X mental retardation 1 (FMR1) gene. To determine whether an abnormal immune profile was present at a cellular level that may predispose female carriers to autoinflammatory conditions, we investigated dynamic cytokine production following stimulation of blood cells. In addition, splenocyte responses were examined in an FMR1 CGG knock-in mouse model of the fragile X premutation.MethodsHuman monocyte and peripheral blood leukocytes (PBLs) were isolated from the blood of 36 female FMR1 premutation carriers and 15 age-matched controls. Cells were cultured with media alone, LPS or PHA. In the animal model, splenocytes were isolated from 32 CGG knock-in mice and 32 wild type littermates. Splenocytes were cultured with media alone or LPS or PMA/Ionomycin. Concentrations of cytokines (GM-CSF, IL-1β, IL-6, IL-10, IL-13, IL-17, IFNγ, TNFα, and MCP-1) were determined from the supernatants of cellular cultures via Luminex multiplex assay. Additionally, phenotypic cellular markers were assessed on cells isolated from human subjects via flow cytometry.ResultsWe found decreases in cytokine production in human premutation carriers as well as in the FMR1 knock-in mice when compared with controls. Levels of cytokines were found to be associated with CGG repeat length in both human and mouse. Furthermore, T cells from human premutation carriers showed decreases in cell surface markers of activation when compared with controls.ConclusionsIn this study, FMR1 CGG repeat expansions are associated with decreased immune responses and immune dysregulation in both humans and mice. Deficits in immune responses in female premutation carriers may lead to increased susceptibility to autoimmunity and further research is warranted to determine the link between FMR1 CGG repeat lengths and onset of autoinflammatory conditions

The transcription factor ATF5: role in cellular differentiation, stress responses, and cancer.

Activating transcription factor 5 (ATF5) is a cellular prosurvival transcription factor within the basic leucine zipper (bZip) family that is involved in cellular differentiation and promotes cellular adaptation to stress. Recent studies have characterized the oncogenic role of ATF5 in the development of several different types of cancer, notably glioblastoma. Preclinical assessment of a systemically deliverable dominant-negative ATF5 (dnATF5) biologic has found that targeting ATF5 results in tumor regression and tumor growth inhibition of glioblastoma xenografts in mouse models. In this review, we comprehensively and critically detail the current scientific literature on ATF5 in the context of cellular differentiation, survival, and response to stressors in normal tissues. Furthermore, we will discuss how the prosurvival role of ATF5 aides in cancer development, followed by current advances in targeting ATF5 using dominant-negative biologics, and perspectives on future research

VEGF guides angiogenic sprouting utilizing endothelial tip cell filopodia

Vascular endothelial growth factor (VEGF-A) is a major regulator of blood vessel formation and function. it controls several processes in endothelial cells, such as proliferation, survival, and migration, but it is not known how these are coordinately regulated to result in more complex morphogenetic events, such as tubular sprouting, fusion, and network formation. We show here that VEGF-A controls angiogenic sprouting in the early postnatal retina by guiding filopodial extension from specialized endothelial cells situated at the tips of the vascular sprouts. The tip cells respond to VEGF-A only by guided migration; the proliferative response to VEGF-A occurs in the sprout stalks. These two cellular responses are both mediated by agonistic activity of VEGF-A on VEGF receptor 2. Whereas tip cell migration depends on a gradient of VEGF-A, proliferation is regulated by its concentration. Thus, vessel patterning during retinal angiogenesis depends on the balance between two different qualities of the extracellular VEGF-A distribution, which regulate distinct cellular responses in defined populations of endothelial cells

Organ-specific features of natural killer cells.

Natural killer (NK) cells can be swiftly mobilized by danger signals and are among the earliest arrivals at target organs of disease. However, the role of NK cells in mounting inflammatory responses is often complex and sometimes paradoxical. Here, we examine the divergent phenotypic and functional features of NK cells, as deduced largely from experimental mouse models of pathophysiological responses in the liver, mucosal tissues, uterus, pancreas, joints and brain. Moreover, we discuss how organ-specific factors, the local microenvironment and unique cellular interactions may influence the organ-specific properties of NK cells