Fermented fish oil suppresses T helper 1/2 cell response in a mouse model of atopic dermatitis via generation of CD4+ CD25+ Foxp3+ T cells

Sang-Chul Han, Gyeoung-Jin Kang, Yeong-Jong Ko, Hee-Kyoung Kang, Sang-Wook Moon,Yong-Seok Ann and Eun-Sook Yoo

Abstract
Background: Allergic skin inflammation such as atopic dermatitis (AD), which is characterized by pruritus and inflammation, is regulated partly through the activity of regulatory T cells (Tregs). Tregs play key roles in the immune response by preventing or suppressing the differentiation, proliferation and function of various immune cells, including CD4+T cells. Recent studies report that fermentation has a tremendous capacity to transform chemical structures or create new substances, and the omega-3 polyunsaturated fatty acids (n-3 PUFAs) in fish oil can reduce inflammation in allergic patients. The beneficial effects of natural fish oil (NFO) have been described in many diseases, but the mechanism by which fermented fish oil (FFO) modulates the immune system and the allergic response is poorly understood. In this study, we produced FFO and tested its ability to suppress the allergic inflammatory response and to activate CD4+CD25+Foxp3 Tregs.

Results: The ability of FFO and NFO to modulate the immune system was investigated using a mouse model of AD. Administration of FFO or NFO in the drinking water alleviated the allergic inflammation in the skin, and FFO was more effective than NFO. FFO treatment did increase the expression of the immune-suppressive cytokines TGF-β and IL-10. In addition, ingestion of FFO increased Foxp3 expression and the number of CD4+CD25+Foxp3+ Tregs compared with NFO.

Conclusions: These results suggest that the anti-allergic effect of FFO is associated with enrichment of CD4+CD25+Foxp3+T cells at the inflamed sites and that FFO may be effective in treating the allergic symptoms of AD

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Immune responses in the lungs of patients with tuberculous pleural effusion without pulmonary tuberculosis

Diana Qama, Won-Il Choi  and Kun Young Kwon

Abstract
Background: Tuberculous pleural effusion (TPE) is one of the most common forms of extrapulmonary tuberculosis.Because most studies of TPE focused on the pleural space, little information regarding lung parenchyma is available. We therefore aimed to investigate immune responses in the lung parenchyma of TPE patients without pulmonary tuberculosis.

Methods: Patients with any evidence of pulmonary tuberculosis, either from radiologic or bacteriologic evaluation, were excluded. Bronchoalveolar lavage fluid (BALF) was collected from 10 newly diagnosed, untreated, HIV-negative TPE patients and 10 healthy controls. We analyzed T-lymphocyte subpopulations and measured 10 cytokines in BALF. Cytokine levels in BALF were standardised using urea.

Results: The concentrations of interferon-γ (IFN-γ), tumor necrosis factor-α (TNF-α), vascular endothelial growth factor (VEGF), and the CD4+/CD8+ ratio of T-lymphocytes were significantly higher in TPE patients without pulmonary tuberculosis than in the controls. Of the cytokines measured in BALF, VEGF showed the highest concentration. No difference was observed in T-helper type 2 cytokines between the 2 groups.

Conclusion: There were significant immune responses and increases in IFN-γ, TNF-α, and VEGF in the lung parenchyma of TPE patients without pulmonary tuberculosis. This result suggests that TPE may induce a significant immune response in lung parenchyma.

Keywords: Interferon-γ, Tuberculosis, Tumor necrosis factor-α, Vascular endothelial growth factor

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Regulation of in vitro human T cell development through interleukin-7 deprivation and anti-CD3 stimulation

Ekta S Patel, Starlyn Okada, Kevin Hachey, Li-jun Yang, Scott K Durum, Jan S Moreband Lung-Ji Chang

 

Abstract
Background: The role of IL-7 and pre-TCR signaling during T cell development has been well characterized in murine but not in human system. We and others have reported that human BM hematopoietic progenitor cells (HPCs) display poor proliferation, inefficient double negative (DN) to double positive (DP) transition and no
functional maturation in the in vitro OP9-Delta-like 1 (DL1) culture system.

Results: In this study, we investigated the importance of optimal IL-7 and pre-TCR signaling during adult human T cell development. Using a modified OP9-DL1 culture ectopically expressing IL-7 and Fms-like tyrosine kinase 3 ligand (Flt3L), we demonstrated enhanced T cell precursor expansion. IL-7 removal at various time points during T cell development promoted a slight increase of DP cells; however, these cells did not differentiate further and underwent cell death. As pre-TCR signaling rescues DN cells from programmed cell death, we treated the culture with anti-CD3 antibody. Upon pre-TCR stimulation, the IL-7 deprived T precursors differentiated into CD3 +TCRαβ+ DP cells and further matured into functional CD4 T cells, albeit displayed a skewed TCR Vβ repertoire.

Conclusions: Our study establishes for the first time a critical control for differentiation and maturation of adult human T cells from HPCs by concomitant regulation of IL-7 and pre-TCR signaling.

Keywords: T cell development, Interleukin-7, T cell receptor, Vbeta repertoire

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In-vitro inhibition of IFNγ + iTreg mediated by monoclonal antibodies against cell surface determinants essential for iTreg function

Volker Daniel, Mahmoud Sadeghi, Haihao Wang and Gerhard Opelz

Abstract
Background: IFNγ-producing CD4+CD25+Foxp3+PBL represent a subtype of iTreg that are associated with good long-term graft outcome in renal transplant recipients and suppress alloresponses in-vitro. To study the mechanism of immunosuppression, we attempted to block cell surface receptors and thereby inhibited the function of this iTreg subset in-vitro using monoclonal antibodies and recombinant proteins.

Methods: PBL of healthy control individuals were stimulated polyclonally in-vitro in the presence of monoclonal antibodies or recombinant proteins against/of CD178, CD152, CD279, CD28, CD95, and HLA-DR. Induction of IFNγ+iTreg and proliferation of effector cells was determined using four-color fluorescence flow cytometry.
Blockade of iTreg function was analyzed using polyclonally stimulated co-cultures with separated CD4+CD25+CD127-IFNγ+ PBL.

Results: High monoclonal antibody concentrations inhibited the induction of CD4+CD25+Foxp3+IFNγ+PBL(anti-CD152, anti-CD279, anti-CD95: p < 0.05) and CD4+
CD25+CD127-IFNγ+PBL (anti-CD178, anti-CD152, anti-CD279,anti-CD95: p < 0.05). Effector cell proliferation increased with increasing antibody concentrations in culture medium(anti-CD178 and anti-CD279: p < 0.05). Conversely, high concentrations of recombinant proteins induced formation of CD4+CD25+Foxp3+IFNγ+PBL (rCD152 and rCD95: p < 0.05) and decreased cell proliferation dose-dependently (rCD178 and rCD95: p < 0.05). Our data suggest an inverse association of iTreg induction with effector cell proliferation in cell culture which is dependent on the concentration of monoclonal antibodies against iTreg surface determinants. 3-day co-cultures of polyclonally stimulated PBL with separated CD4+ CD25+CD127-IFNγ+PBL showed lower cell proliferation than co-cultures with CD4+CD25+CD127-
IFNγ-PBL (p < 0.05). Cell proliferation increased strongly in CD4+CD25+CD127-IFNγ-PBL-containing co-cultures in the presence of monoclonal antibody (anti-CD28, anti-CD152, anti-CD279: p < 0.05) but remained low in co-cultures with CD4+ CD25+CD127-IFNγ+PBL (with the exception anti-CD28 monoclonal antibody: p < 0.05). Monoclonal antibodies prevent iTreg induction in co-cultures with CD4+CD25+CD127-IFNγ-PBL but do not efficiently block suppressive iTreg function in
co-cultures with CD4+CD25+CD12-IFNγ+PBL.

Conclusions: CD178, CD152, CD279, CD28, CD95, and HLA-DR determinants are important for induction and suppressive function of IFNγ +iTreg.

Keywords: IFNγ+iTreg, IFNγ+Foxp3+, IFNγ+CD127-, CD178, CD152, CD279, CD28, CD95, HLA-DR, Inhibition,Cell proliferation

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Serum activity of DPPIV and its expression on lymphocytes in patients with melanoma and in people with vitiligo

Ivana Z Matić, Marija Đorđić, Nađa Grozdanić, Ana Damjanović, Branka Kolundžija, Aleksandra Erić-Nikolić,Radan Džodić, Miomir Šašić, Srđan Nikolić, Danijela Dobrosavljević, Sanvila Rašković, Slađana Andrejević,Dušica Gavrilović, Oscar J Cordero and Zorica D Juranić

Abstract
Background: Dipeptidyl peptidase IV, a multifunctional serine protease, is implicated in regulation of malignant transformation, promotion and further progression of cancer, exerting tumor-suppressing or even completely opposite - tumor-promoting activities.The aim of present research was to determine the serum DPPIV activity, as well as the percentages of CD26+lymphocytes, CD26+ overall white blood cells and the mean fluorescence intensity of CD26 expression on
lymphocytes in patients with melanoma, people with vitiligo and in healthy controls.

Methods: The activity of DPPIV in serum was determined by colorimetric test. Expression of DPPIV (as CD26) on immunocompetent peripheral white blood cells was done using flow cytometry analysis.

Results: Data from our study show for the first time statistically significant decrease: in the serum DPPIV activity, in the percentage of CD26+ overall white blood cells and in the percentage of lymphocytes in patients with melanoma in comparison to healthy control people. In addition, significantly lower serum DPPIV activity was found in the group of patients with melanoma in relation to people with vitiligo too.

Conclusion: This study indicates the need for exploring the cause and the importance of the disturbances in the serum DPPIV activity and in the CD26 expression on immunocompetent cells in complex molecular mechanisms underlying the development and progression of melanoma.

Keywords: CD26 expression, DPPIV serum activity, Melanoma, Vitiligo

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Responses to Pandemic ASO3-adjuvanted A/California/07/09 H1N1 influenza vaccine in human immunodeficiency virus-infected individuals

Deborah Kelly, Kimberley Burt, Bayan Missaghi, Lisa Barrett, Yoav Keynan, Keith Fowke and Michael Grant

Abstract
Background: Influenza infection may be more serious in human immunodeficiency virus (HIV)-infected individuals, therefore, vaccination against seasonal and pandemic strains is highly advised. Seasonal influenza vaccines have had no significant negative effects in well controlled HIV infection, but the impact of adjuvanted pandemic A/California/07/2009 H1N1 influenza hemaglutinin (HA) vaccine, which was used for the first time in the Canadian population as an authorized vaccine in autumn 2009, has not been extensively studied.

Objective: Assess vaccine-related effects on CD4+ T cell counts and humoral responses to the vaccine in individuals attending the Newfoundland and Labrador Provincial HIV clinic.

Methods: A single dose of ArepanrixTM split vaccine including 3.75 μg A/California/07/2009 H1N1 HA antigen and ASO3 adjuvant was administered to 81 HIV-infected individuals by intramuscular injection. Plasma samples from shortly before, and 1–5 months after vaccination were collected from 80/81 individuals to assess humoral anti-H1N1 HA responses using a sensitive microbead-based array assay. Data on CD4+T cell counts, plasma viral load,antiretroviral therapy and patient age were collected from clinical records of 81 individuals.

Results: Overall, 36/80 responded to vaccination either by seroconversion to H1N1 HA or with a clear increase in anti-H1N1 HA antibody levels. Approximately 1/3 (28/80) had pre-existing anti-H1N1 HA antibodies and were more likely to respond to vaccination (22/28). Responders had higher baseline CD4+
T cell counts and responders without pre-existing antibodies against H1N1 HA were younger than either non-responders or responders with pre-existing antibodies. Compared to changes in their CD4+ T cell counts observed over a similar time period one year later, vaccine recipients displayed a minor, transient fall in CD4+
T cell numbers, which was greater amongst responders.

Conclusions: We observed low response rates to the 2009 pandemic influenza vaccine among HIV-infected individuals without pre-existing antibodies against H1N1 HA and a minor transient fall in CD4+ T cell numbers,which was accentuated in responders. A single injection of the ArepanrixTM pandemic A/California/07/2009 H1N1 HA split vaccine may be insufficient to induce protective immunity in HIV-infected individuals without pre-existing anti-H1N1 HA responses.

Keywords: HIV, influenza, pandemic, A/California/07/2009 H1N1 HA antigen, AS03 oil in water adjuvant,inflammation, CD4+T cells, age

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Residue analysis of a CTL epitope of SARS-CoV spike protein by IFN-gamma production and bioinformatics prediction

Jun Huang, Yingnan Cao, Xianzhang Bu and Changyou Wu

Abstract
Background: Severe acute respiratory syndrome (SARS) is an emerging infectious disease caused by the novel coronavirus SARS-CoV. The T cell epitopes of the SARS CoV spike protein are well known, but no systematic evaluation of the functional and structural roles of each residue has been reported for these antigenic epitopes.Analysis of the functional importance of side-chains by mutational study may exaggerate the effect by imposing a structural disturbance or an unusual steric, electrostatic or hydrophobic interaction.

Results: We demonstrated that N50 could induce significant IFN-gamma response from SARS-CoV S DNA immunized mice splenocytes by the means of ELISA, ELISPOT and FACS. Moreover, S366-374 was predicted to be an optimal epitope by bioinformatics tools: ANN, SMM, ARB and BIMAS, and confirmed by IFN-gamma response induced by a series of S358-374-derived peptides. Furthermore, each of S366-374 was replaced by alanine (A), lysine (K) or aspartic acid (D), respectively. ANN was used to estimate the binding affinity of single S366-374 mutants to H-2 Kd. Y367 and L374 were predicated to possess the most important role in peptide binding. Additionally, these one residue mutated peptides were synthesized, and IFN-gamma production induced by G368, V369, A371, T372
and K373 mutated S366-374 were decreased obviously.

Conclusions: We demonstrated that S366-374 is an optimal H-2 Kd CTL epitope in the SARS CoV S protein. Moreover, Y367, S370, and L374 are anchors in the epitope, while C366, G368, V369, A371, T372, and K373 may directly interact with TCR on the surface of CD8-T cells.

Keywords: SARS-CoV, CTL, Epitope, Residue

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Characterization of functional mannose receptor in a continuous hybridoma cell line

David J Vigerust, Sherell Vick and Virginia L Shepherd

Abstract
Background: The mannose receptor is the best described member of the type I transmembrane C-type lectins; however much remains unanswered about the biology of the receptor. One difficulty has been the inability to consistently express high levels of a functional full length mannose receptor cDNA in mammalian cells. Another difficulty has been the lack of a human macrophage cell line expressing a fully functional receptor. Commonly used human macrophage cell lines such as U937, THP-1, Mono-Mac and HL60 do not express the mannose receptor. We have developed a macrophage hybridoma cell line (43MR cells) created by fusion of U937 cells with primary human monocyte-derived macrophages, resulting in a non-adherent cell line expressing several properties of primary macrophages. The purpose of this study was to identify and select mannose receptor-expressing cells using fluorescence-activated cell sorting and to characterize the expression and function of the receptor.

Results: In the current study we show that the mannose receptor found on this novel cell has endocytic characteristics consistent with and similar to the mannose receptor found on the surface of monocyte-derived human macrophages and rat bone marrow-derived macrophages. In addition, we demonstrate that these cells
engage and internalize pathogen particles such as S. aureus and C. albicans. We further establish the transfectability of these cells via the introduction of a plasmid expressing influenza A hemagglutinin.

Conclusions: The 43MR cell line represents the first naturally expressed MR-positive cell line derived from a human macrophage background. This cell line provides an important cell model for other researchers for the study of human MR biology and host-pathogen interactions.

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Application of circular consensus sequencing and network analysis to characterize the bovine IgG repertoire

Peter A Larsen and Timothy P L Smith

Abstract
Background: Vertebrate immune systems generate diverse repertoires of antibodies capable of mediating response to a variety of antigens. Next generation sequencing methods provide unique approaches to a number of immuno-based research areas including antibody discovery and engineering, disease surveillance, and host immune response to vaccines. In particular, single-molecule circular consensus sequencing permits the sequencing of antibody repertoires at previously unattainable depths of coverage and accuracy. We approached the bovine immunoglobulin G (IgG) repertoire with the objective of characterizing diversity of expressed IgG transcripts. Here we present single-molecule real-time sequencing data of expressed IgG heavy-chain repertoires of four individual cattle. We describe the diversity observed within antigen binding regions and visualize this diversity using a network-based approach.

Results: We generated 49,945 high quality cDNA sequences, each spanning the entire IgG variable region from four Bos taurus calves. From these sequences we identified 49,521 antigen binding regions using the automated Paratome web server. Approximately 9% of all unique complementarity determining 2 (CDR2) sequences were of variable lengths. A bimodal distribution of unique CDR3 sequence lengths was observed, with common lengths of 5–6 and 21–25 amino acids. The average number of cysteine residues in CDR3s increased with CDR3 length and we observed that cysteine residues were centrally located in CDR3s. We identified 19 extremely long CDR3 sequences (up to 62 amino acids in length) within IgG transcripts. Network analyses revealed distinct patterns among the expressed IgG antigen binding repertoires of the examined individuals.

Conclusions: We utilized circular consensus sequencing technology to provide baseline data of the expressed bovine IgG repertoire that can be used for future studies important to livestock research. Somatic mutation resulting in base insertions and deletions in CDR2 further diversifies the bovine antibody repertoire. In contrast to previous studies, our data indicate that unusually long CDR3 sequences are not unique to IgM antibodies in cattle. Centrally located cysteine residues in bovine CDR3s provide further evidence that disulfide bond formation is likely of structural importance. We hypothesize that network or cluster-based analyses of expressed antibody repertoires from controlled challenge experiments will help identify novel natural antigen binding solutions to specific pathogens of interest.

Keywords: Antibody diversity, Bos taurus, SMRT sequencing, Immunoglobulin G

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Orally administered Lactobacillus rhamnosus modulates the respiratory immune response triggered by the viral pathogen-associated molecular pattern poly(I:C)

Julio Villena, Eriko Chiba, Yohsuke Tomosada, Susana Salva, Gabriela Marranzino, Haruki Kitazawa and Susana Alvarez

Abstract
Background: Some studies have shown that probiotics, including Lactobacillus rhamnosus CRL1505, had the potential to beneficially modulate the outcome of certain bacterial and viral respiratory infections. However, these studies did not determine the mechanism(s) by which probiotics contribute to host defense against respiratory viruses.

Results: In this work we demonstrated that orally administered Lactobacillus rhamnosus CRL1505 (Lr1505) was able to increase the levels of IFN-γ, IL-10 and IL-6 in the respiratory tract and the number of lung CD3+ CD4+ IFN-γ + T cells. To mimic the pro-inflammatory and physiopathological consecuences of RNA viral infections in the lung, we used an experimental model of lung inflammation based on the administration of the artificial viral pathogenassociated molecular pattern poly(I:C). Nasal administration of poly(I:C) to mice induced a marked impairment of lung function that was accompanied by the production of pro-inflammatory mediators and inflammatory cell recruitment into the airways. The preventive administration of Lr1505 reduced lung injuries and the production of TNF-α, IL-6, IL-8 and MCP-1 in the respiratory tract after the challenge with poly(I:C). Moreover, Lr1505 induced a significant increase in lung and serum IL-10. We also observed that Lr1505 was able to increase respiratory IFN-γ levels and the number of lung CD3+CD4+ IFN-γ + T cells after poly(I:C) challenge. Moreover, higher numbers of both
CD103+ and CD11bhigh dendritic cells and increased expression of MHC-II, IL-12 and IFN-γ in these cell populationswere found in lungs of Lr1505-treated mice. Therefore, Lr1505 treatment would beneficially regulate the balance between pro-inflammatory mediators and IL-10, allowing an effective inflammatory response against infection and avoiding tissue damage.

Conclusions: Results showed that Lr1505 would induce a mobilization of cells from intestine and changes in cytokine profile that would be able to beneficially modulate the respiratory mucosal immunity. Although deeper studies are needed using challenges with respiratory viruses, the results in this study suggest that Lr1505, a potent inducer of antiviral cytokines, may be useful as a prophylactic agent to control respiratory virus infection.

Keywords: L. rhamnosus CRL1505, Poly(I:C), Antiviral immunity, Respiratory tract

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Comparative immunological evaluation of recombinant Salmonella Typhimurium strains expressing model antigens as live oral vaccines

Song-yue Zheng, Bin Yu, Ke Zhang, Min Chen, Yan-Hong Hua, Shuofeng Yuan, Rory M Watt,Bo-Jian Zheng, Kwok-Yung Yuen  and Jian-Dong Huang

Abstract
Background: Despite the development of various systems to generate live recombinant Salmonella Typhimurium vaccine strains, little work has been performed to systematically evaluate and compare their relative immunogenicity. Such information would provide invaluable guidance for the future rational design of live
recombinant Salmonella oral vaccines.

Result: To compare vaccine strains encoded with different antigen delivery and expression strategies, a series of recombinant Salmonella Typhimurium strains were constructed that expressed either the enhanced green fluorescent protein (EGFP) or a fragment of the hemagglutinin (HA) protein from the H5N1 influenza virus, as model antigens. The antigens were expressed from the chromosome, from high or low-copy plasmids, or encoded on a eukaryotic expression plasmid. Antigens were targeted for expression in either the cytoplasm or the outer membrane. Combinations of strategies were employed to evaluate the efficacy of combined delivery/expression approaches. After investigating in vitro and in vivo antigen expression, growth and infection abilities; the immunogenicity of the constructed recombinant Salmonella strains was evaluated in mice. Using the soluble model antigen EGFP, our results indicated that vaccine strains with high and stable antigen expression exhibited high B cell responses, whilst eukaryotic expression or colonization with good construct stability was critical for T cell responses. For the insoluble model antigen HA, an outer membrane expression strategy induced better B cell and T cell responses than a cytoplasmic strategy. Most notably, the combination of two different expression strategies did not increase the immune response elicited.

Conclusion: Through systematically evaluating and comparing the immunogenicity of the constructed recombinant Salmonella strains in mice, we identified their respective advantages and deleterious or synergistic effects. Different construction strategies were optimally-required for soluble versus insoluble forms of the protein antigens. If an antigen, such as EGFP, is soluble and expressed at high levels, a low-copy plasmid-cytoplasmic expression strategy is recommended; since it provokes the highest B cell responses and also induces good T cell responses. If a T cell response is preferred, a eukaryotic expression plasmid or a chromosome-based, cytoplasmic-expression strategy is more effective. For insoluble antigens such as HA, an outer membrane expression strategy is recommended.

Keywords: Salmonella Typhimurium, Live oral vaccine, Soluble and insoluble antigens, Construction strategies, Immunological comparison

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Boosting immune response with the invariant chain segments via association with non-peptide binding region of major histocompatibility complex class II molecules

Fangfang Chen, Fantao Meng, Ling Pan, Fazhi Xu, Xuelan Liu and Weiyi Yu*

Abstract
Background: Based on binding of invariant chain (Ii) to major histocompatibility complex (MHC) class II molecules to form complexes, Ii-segment hybrids, Ii-key structure linking an epitope, or Ii class II-associated invariant chain peptide (CLIP) replaced with an epitope were used to increase immune response. It is currently unknown whether the Ii-segment cytosolic and transmembrane domains bind to the MHC non-peptide binding region (PBR) and consequently influence immune response. To investigate the potential role of Ii-segments in the immune response via MHC II/peptide complexes, a few hybrids containing Ii-segments and a multiepitope (F306) from Newcastle disease virus fusion protein (F) were constructed, and their binding effects on MHC II molecules and specific antibody production were compared using confocal microscopy, immunoprecipitation, western blotting and animal experiments.

Results: One of the Ii-segment/F306 hybrids, containing ND (Asn–Asp) outside the F306 in the Ii-key structure (Ii-key/F306/ND), neither co-localized with MHC II molecules on plasma membrane nor bound to MHC II molecules to form complexes. However, stimulation of mice with the structure produced 4-fold higher antibody titers compared with F306 alone. The two other Ii-segment/F306 hybrids, in which the transmembrane and cytosolic domains of Ii were linked to this structure (Cyt/TM/Ii-key/F306/ND), partially co-localized on plasma membrane with MHC class II molecules and weakly bound MHC II molecules to form complexes. They induced mice to produce approximately 9-fold higher antibody titers compared with F306 alone. Furthermore, an Ii/F306 hybrid (F306 substituting CLIP) co-localized well with MHC II molecules on the membrane to form complexes, although it increased antibody titer about 3-fold relative to F306 alone.

Conclusions: These results suggest that Ii-segments improve specific immune response by binding to the non-PBR on MHC class II molecules and enabling membrane co-localization with MHC II molecules, resulting in the formation of relatively stable MHC II/peptide complexes on the plasma membrane, and signal transduction.

Keywords: Li-segments, Epitope, Hybrid, MHC II, Antibody, Membrane co-localization

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Important role of CCR2 in a murine model of coronary vasculitis

Hernan G Martinez, Marlon P Quinones, Fabio Jimenez, Carlos Estrada, Kassandra M Clark, Kazuo Suzuki,Noriko Miura, Naohito Ohno, Sunil K Ahuja2,6 and Seema S Ahuj

Abstract
Background: Chemokines and their receptors play a role in the innate immune response as well as in the disruption of the balance between pro-inflammatory Th17 cells and regulatory T cells (Treg), underlying the pathogenesis of coronary vasculitis in Kawasaki disease (KD).

Results: Here we show that genetic inactivation of chemokine receptor (CCR)-2 is protective against the induction of aortic and coronary vasculitis following injection of Candida albicans water-soluble cell wall extracts (CAWS). Mechanistically, both T and B cells were required for the induction of vasculitis, a role that was directly modulated by CCR2. CAWS administration promoted mobilization of CCR2-dependent inflammatory monocytes (iMo) from the bone marrow (BM) to the periphery as well as production of IL-6. IL-6 was likely to contribute to the depletion of Treg and expansion of Th17 cells in CAWS-injected Ccr2+/+ mice, processes that were ameliorated following the genetic inactivation of CCR2.

Conclusion: Collectively, our findings provide novel insights into the role of CCR2 in the pathogenesis of vasculitis as seen in KD and highlight novel therapeutic targets, specifically for individuals resistant to first-line treatments.

Keywords: CCR2, Coronary vasculitis, Treg, Treg/Th17 imbalance

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Quantitative GPCR and ion channel transcriptomics in primary alveolar macrophages and macrophage surrogates

Paul J Groot-Kormelink, Lindsay Fawcett, Paul D Wright, Martin Gosling and Toby C Kent

Abstract
Background: Alveolar macrophages are one of the first lines of defence against invading pathogens and play a central role in modulating both the innate and acquired immune systems. By responding to endogenous stimuli within the lung, alveolar macrophages contribute towards the regulation of the local inflammatory
microenvironment, the initiation of wound healing and the pathogenesis of viral and bacterial infections. Despite the availability of protocols for isolating primary alveolar macrophages from the lung these cells remain recalcitrant to expansion in-vitro and therefore surrogate cell types, such as monocyte derived macrophages and phorbol ester-differentiated cell lines (e.g. U937, THP-1, HL60) are frequently used to model macrophage function.

Methods: The availability of high throughput gene expression technologies for accurate quantification of transcript levels enables the re-evaluation of these surrogate cell types for use as cellular models of the alveolar macrophage. Utilising high-throughput TaqMan arrays and focussing on dynamically regulated families of integral membrane proteins, we explore the similarities and differences in G-protein coupled receptor (GPCR) and ion channel expression in alveolar macrophages and their widely used surrogates.

Results: The complete non-sensory GPCR and ion channel transcriptome is described for primary alveolar macrophages and macrophage surrogates. The expression of numerous GPCRs and ion channels whose expression were hitherto not described in human alveolar macrophages are compared across primary macrophages and
commonly used macrophage cell models. Several membrane proteins known to have critical roles in regulating macrophage function, including CXCR6, CCR8 and TRPV4, were found to be highly expressed in macrophages but not expressed in PMA-differentiated surrogates.

Conclusions: The data described in this report provides insight into the appropriate choice of cell models for Investigating macrophage biology and highlights the importance of confirming experimental data in primary alveolar macrophages.

Keywords: COPD, Microfluidics, TaqMan, Arrays, High-throughput

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DNA methylation profile of Aire-deficient mouse medullary thymic epithelial cells

Guoying Wu, Keiji Hirabayashi, Shinya Sato, Nobuko Akiyama, Taishin Akiyama, Kunio Shiota and Shintaro Yag

Abstract
Background: Medullary thymic epithelial cells (mTECs) are characterized by ectopic expression of self-antigens during the establishment of central tolerance. The autoimmune regulator (Aire), which is specifically expressed in mTECs, is responsible for the expression of a large repertoire of tissue-restricted antigens (TRAs) and plays a role in the development of mTECs. However, Aire-deficient mTECs still express TRAs. Moreover, a subset of mTECs, which are considered to be at a stage of terminal differentiation, exists in the Aire-deficient thymus. The phenotype of a specific cell type in a multicellular organism is governed by the epigenetic regulation system. DNA methylation modification is an important component of this system. Every cell or tissue type displays a DNA methylation profile, consisting of tissue-dependent and differentially methylated regions (T-DMRs), and this profile is involved in cell-type-specific genome usage. The aim of this study was to examine the DNA methylation profile of mTECs by using Aire-deficient mTECs as a model.

Results: We identified the T-DMRs of mTECs (mTEC-T-DMRs) via genome-wide DNA methylation analysis of Aire−/− mTECs by comparison with the liver, brain, thymus, and embryonic stem cells. The hypomethylated mTEC-T-DMRs in Aire−/− mTECs were associated with mTEC-specific genes, including Aire, CD80, and Trp63, as well as other genes involved in the RANK signaling pathway. While these mTEC-T-DMRs were also hypomethylated in Aire+/+ mTECs, they were hypermethylated in control thymic stromal cells. We compared the pattern of DNA methylation levels at a total of 55 mTEC-T-DMRs and adjacent regions and found that the DNA methylation status was similar for Aire+/+ and Aire−/− mTECs but distinct from that of athymic cells and tissues.

Conclusions: These results indicate a unique DNA methylation profile that is independent of Aire in mTECs. This profile is distinct from other cell types in the thymic microenvironment and is indicated to be involved in the differentiation of the mTEC lineage.

Keywords: Medullary thymic epithelial cells, Aire, T-DMR

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DNA methylation profile of Aire-deficient mouse medullary thymic epithelial cells

Guoying Wu, Keiji Hirabayashi, Shinya Sato, Nobuko Akiyama, Taishin Akiyama, Kunio Shiota and Shintaro Yagi

 

Abstract
Background: Medullary thymic epithelial cells (mTECs) are characterized by ectopic expression of self-antigens during the establishment of central tolerance. The autoimmune regulator (Aire), which is specifically expressed in mTECs, is responsible for the expression of a large repertoire of tissue-restricted antigens (TRAs) and plays a role in the development of mTECs. However, Aire-deficient mTECs still express TRAs. Moreover, a subset of mTECs, which are considered to be at a stage of terminal differentiation, exists in the Aire-deficient thymus. The phenotype of a specific cell type in a multicellular organism is governed by the epigenetic regulation system. DNA methylation modification is an important component of this system. Every cell or tissue type displays a DNA methylation profile, consisting of tissue-dependent and differentially methylated regions (T-DMRs), and this profile is involved in cell-type-specific genome usage. The aim of this study was to examine the DNA methylation profile of mTECs by using Aire-deficient mTECs as a model.

Results: We identified the T-DMRs of mTECs (mTEC-T-DMRs) via genome-wide DNA methylation analysis of Aire−/− mTECs by comparison with the liver, brain, thymus, and embryonic stem cells. The hypomethylated mTEC-T-DMRs in Aire−/− mTECs were associated with mTEC-specific genes, including Aire, CD80, and Trp63, as well as other genes involved in the RANK signaling pathway. While these mTEC-T-DMRs were also hypomethylated in Aire+/+ mTECs, they were hypermethylated in control thymic stromal cells. We compared the pattern of DNA methylation levels at a total of 55 mTEC-T-DMRs and adjacent regions and found that the DNA methylation status was similar for Aire+/+ and Aire−/− mTECs but distinct from that of athymic cells and tissues.

Conclusions: These results indicate a unique DNA methylation profile that is independent of Aire in mTECs. This profile is distinct from other cell types in the thymic microenvironment and is indicated to be involved in the differentiation of the mTEC lineage.

Keywords: Medullary thymic epithelial cells, Aire, T-DMR

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Low-dose plasmid DNA treatment increases plasma vasopressin and regulates blood pressure in experimental endotoxemia

Thiago Malardo, Marcelo E Batalhão, Ademilson Panunto-Castelo, Luciana P Almeida, Everton Padilha,sabela C Fontoura, Célio L Silva , Evelin C Carnio and Arlete AM Coelho-Castelo

Abstract
Background: Although plasmid DNA encoding an antigen from pathogens or tumor cells has been widely studied as vaccine, the use of plasmid vector (without insert) as therapeutic agent requires further investigation.

Results: Here, we showed that plasmid DNA (pcDNA3) at low doses inhibits the production of IL-6 and TNF-α by lipopolysaccharide (LPS)-stimulated macrophage cell line J774. These findings led us to evaluate whether plasmid DNA could act as an anti-inflammatory agent in a Wistar rat endotoxemia model. Rats injected simultaneously with 1.5 mg/kg of LPS and 10 or 20 μg of plasmid DNA had a remarkable attenuation of mean arterial blood pressure (MAP) drop at 2 hours after treatment when compared with rats injected with LPS only. The beneficial effect of the plasmid DNA on MAP was associated with decreased expression of IL-6 in liver and increased concentration of plasma vasopressin (AVP), a known vasoconstrictor that has been investigated in hemorrhagic shock management.
No difference was observed in relation to nitric oxide (NO) production.

Conclusion: Our results demonstrate for the first time that plasmid DNA vector at low doses presents anti-inflammatory property and constitutes a novel approach with therapeutic potential in inflammatory diseases.

Keywords: Endotoxemic shock, Interleukin-6, Naked pcDNA3

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Zinc-finger nuclease mediated disruption of Rag1 in the LEW/Ztm rat

Nils-Holger Zschemisch, Silke Glage, Dirk Wedekind, Edward J Weinstein, Xiaoxia Cui, Martina Dorsch and Hans-Jürgen Hedrich

 

Abstract
Background: Engineered zinc-finger nucleases (ZFN) represented an innovative method for the genome manipulation in vertebrates. ZFN introduced targeted DNA double strand breaks (DSB) and initiated non-homologous end joining (NHEJ) after pronuclear or cytoplasmatic microinjection into zygotes. Resulting frame shift mutations led to functional gene ablations in zebra fish, mice, pigs and also in laboratory rats. Therefore, we targeted the rat Rag1 gene essential for the V(D)J recombination within the immunoglobulin production process and for the differentiation of mature B and T lymphocytes to generate an immunodeficient rat model in the LEW/Ztm strain.

Results: After microinjection of Rag1 specific ZFN mRNAs in 623 zygotes of inbred LEW/Ztm rats 59 offspring were born from which one carried a 4 bp deletion. This frame shift mutation led to a premature stop codon and a subsequently truncated Rag1 protein confirmed by the loss of the full-length protein in Western Blot analysis. Truncation of the Rag1 protein was characterized by the complete depletion of mature B cells. The remaining T cell population contained mature CD4+/CD3+/TCRαβ+as well as CD8+ /CD3+/TCRαβ+positive lymphocytes accompanied by a compensatory increase of natural killer cells in the peripheral blood. Reduction of T cell development in Rag1 mutant rats was associated with a hypoplastic thymus that lacked follicular structures.Histological evaluation also revealed the near-complete absence of lymphocytes in spleen and lymph nodes in the immunodeficient Rag1 mutant rat.

Conclusion: The Rag1 mutant rat will serve as an important model for transplantation studies. Furthermore, it may be used as a model for reconstitution experiments related to the immune system, particularly with respect to different populations of human lymphocytes, natural killer cells and autoimmune phenomena.

Keywords: Rag1, Zinc-finger nucleases, Rat, Lymphocytes, Natural killer cells, Hypoplastic thymus

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New perspectives for natural antimicrobial peptides: application as antinflammatory drugs in a murine model

Rosanna Capparelli, Francesco De Chiara, Nunzia Nocerino, Rosa Chiara Montella, Marco Iannaccone,Andrea Fulgione, Alessandra Romanelli, Concetta Avitabile, Giuseppe Blaiotta and Federico Capuano

Abstract
Background: Antimicrobial peptides (AMPs) are an ancient group of defense molecules. AMPs are widely distributed in nature (being present in mammals, birds, amphibians, insects, plants, and microorganisms). They display bactericidal as well as immunomodulatory properties. The aim of this study was to investigate the
antimicrobial and anti-inflammatory activities of a combination of two AMPs (temporin B and the royal jellein I) against Staphylococcus epidermidis.

Results: The temporin B (TB-KK) and the royal jelleins I, II, III chemically modified at the C terminal (RJI-C, RJII-C, RJIII-C), were tested for their activity against 10 different Staphylococcus epidermidis strains, alone and in combination. Of the three royal jelleins, RJI-C showed the highest activity. Moreover, the combination of RJI-C and TB-KK (MIX) displayed synergistic activity. In vitro, the MIX displayed low hemolytic activity, no NO2-production and the ability to curb the synthesis of the pro-inflammatory cytokines TNF-α and IFN-γ to the same extent as acetylsalicylic acid. In vivo, the MIX sterilized mice infected with Staphylococcus epidermidis in eleven days and inhibited the expression of genes encoding the prostaglandin-endoperoxide synthase 2 (COX-2) and CD64, two important parameters of inflammation.

Conclusion: The study shows that the MIX – a combination of two naturally occurring peptides - displays both antimicrobial and anti-inflammatory activities

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Elevation of soluble major histocompatibility complex class I related chain A protein in malignant and infectious diseases in Chinese patients

Xiaoxin Jiang, Ju-Fang Huang, Zhi Huo, Qiuqui Zhang, Yan Jiang, Xiaoping Wu, Yanwen Li,Guanmin Jiang, Leping Zeng, Xiao-Xin Yan, Ping Yuand Renxian Cao

Abstract
Background: Elevation of soluble major histocompatibility complex class I chain-related gene A (sMICA) products in serum has been linked to tissue/organ transplantation, autoimmune diseases and some malignant disorders. Cells infected by microbiological pathogens may release sMICA, whereas less is known whether and to what extent serum sMICA levels may change in infectious diseases.

Methods: The present study determined serum sMICA levels by enzyme-linked immunosorbent assay (ELISA) in a southern China population, including patients (n = 1041) suffering from several types of malignant and infectious diseases and healthy controls (n = 141).

Results: Relative to controls, serum sMICA elevation was significant in patients of hepatic cancer, and was approaching statistical significance in patients with lung, gastric and nasopharyngeal cancers. sMICA elevation was also associated with some bacterial (Enterobacteriaceae, Mycobacterium tuberculosis, non-fermenting Gramnegative bacteria and Gram-positive cocci), viral (hepatitis B and C) and the Microspironema pallidum infections.

Conclusion: Serum sMICA levels may be informative for the diagnosis of some malignant and infectious diseases. The results also indicate that microbiological infections should be considered as a potential confounding clinical condition causing serum sMICA elevation while using this test to evaluate the status of other disorders, such as cancers, host-graft response and autoimmune diseases.

Keywords: MHC, sMICA/B, NKG2D, Cancer diagnosis, Serum

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White-nose syndrome in bats: illuminating the darkness

Paul M Cryan, Carol Uphoff Meteyer, Justin G Boyles and David S Blehert

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Bacteria tracking by in vivo magnetic resonance imaging

Verena Hoerr, Lorena Tuchscherr, Jana Hüve, Nadine Nippe, Karin Loser, Nataliya Glyvuk,Yaroslav Tsytsyura, Michael Holtkamp, Cord Sunderkötter, Uwe Karst, Jürgen Klingauf, Georg Peters,Bettina Löffler and Cornelius Faber

 

Abstract
Background: Different non-invasive real-time imaging techniques have been developed over the last decades to study bacterial pathogenic mechanisms in mouse models by following infections over a time course. In vivo investigations of bacterial infections previously relied mostly on bioluminescence imaging (BLI), which is able to localize metabolically active bacteria, but provides no data on the status of the involved organs in the infected host organism. In this study we established an in vivo imaging platform by magnetic resonance imaging (MRI) for tracking bacteria in mouse models of infection to study infection biology of clinically relevant bacteria.

Results: We have developed a method to label Gram-positive and Gram-negative bacteria with iron oxide nano particles and detected and pursued these with MRI. The key step for successful labeling was to manipulate the bacterial surface charge by producing electro-competent cells enabling charge interactions between the iron particles and the cell wall. Different particle sizes and coatings were tested for their ability to attach to the cell wall and possible labeling mechanisms were elaborated by comparing Gram-positive and -negative bacterial characteristics. With 5-nm citrate-coated particles an iron load of 0.015 ± 0.002 pg Fe/bacterial cell was achieved for Staphylococcus aureus. In both a subcutaneous and a systemic infection model induced by iron-labeled S. aureus bacteria, high resolution MR images allowed for bacterial tracking and provided information on the morphology of organs and the inflammatory response.

Conclusion: Labeled with iron oxide particles, in vivo detection of small S. aureus colonies in infection models is feasible by MRI and provides a versatile tool to follow bacterial infections in vivo. The established cell labeling strategy can easily be transferred to other bacterial species and thus provides a conceptual advance in the field of molecular MRI.

Keywords: Bacterial cell labeling, Bacteria tracking, Infectious diseases, Mouse models of infection, Cellular and
molecular MRI

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Gene regulation by the act of long non-coding RNA transcription

Aleksandra E Kornienko, Philipp M Guenzl, Denise P Barlow and Florian M Pauler

Abstract
Long non-protein-coding RNAs (lncRNAs) are proposed to be the largest transcript class in the mouse and human transcriptomes. Two important questions are whether all lncRNAs are functional and how they could exert a function. Several lncRNAs have been shown to function through their product, but this is not the only possible mode of action. In this review we focus on a role for the process of lncRNA transcription, independent of the lncRNA product, in regulating protein-coding-gene activity in cis. We discuss examples where lncRNA transcription leads to gene silencing or activation, and describe strategies to determine if the lncRNA product or its transcription causes the regulatory effect.

Keywords: Gene expression regulation, Histone modifications, lincRNA, lncRNA, Silencing,Transcriptional interference

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Demecology in the Cambrian: synchronized molting in arthropods from the Burgess Shale

Joachim T Haug, Jean-Bernard Caron and Carolin Haug

Abstract
Background: The Burgess Shale is well known for its preservation of a diverse soft-bodied biota dating from the Cambrian period (Series 3, Stage 5). While previous paleoecological studies have focused on particular species (autecology) or entire paleocommunities (synecology), studies on the ecology of populations (demecology) of Burgess Shale organisms have remained mainly anecdotal.

Results: Here, we present evidence for mass molting events in two unrelated arthropods from the Burgess Shale Walcott Quarry, Canadaspis perfecta and a megacheiran referred to as Alalcomenaeus sp.

Conclusions: These findings suggest that the triggers for such supposed synchronized molting appeared early on during the Cambrian radiation, and synchronized molting in the Cambrian may have had similar functions in the past as it does today. In addition, the finding of numerous juvenile Alalcomenaeus sp. molts associated with the putative alga Dictyophycus suggests a possible nursery habitat. In this nursery habitat a population of this animal might have found a more protected environment in which to spend critical developmental phases, as do many modern species today.

Keywords: Burgess Shale, Cambrian bioradiation, ‘Cambrian explosion’, Demecology, Molting, Nursery habitats

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Mitochondrial metabolism of sexual and asexual blood stages of the malaria parasite Plasmodium falciparum

James I MacRae, Matthew WA Dixon, Megan K Dearnley, Hwa H Chua, Jennifer M Chambers,Shannon Kenny, Iveta Bottova, Leann Tilley and Malcolm J McConville

Abstract
Background: The carbon metabolism of the blood stages of Plasmodium falciparum, comprising rapidly dividing asexual stages and non-dividing gametocytes, is thought to be highly streamlined, with glycolysis providing most of the cellular ATP. However, these parasitic stages express all the enzymes needed for a canonical mitochondrial tricarboxylic acid (TCA) cycle, and it was recently proposed that they may catabolize glutamine via an atypical branched TCA cycle. Whether these stages catabolize glucose in the TCA cycle and what is the functional significance of mitochondrial metabolism remains unresolved.

Results: We reassessed the central carbon metabolism of P. falciparum asexual and sexual blood stages, by metabolically labeling each stage with 13C-glucose and 13C-glutamine, and analyzing isotopic enrichment in key pathways using mass spectrometry. In contrast to previous findings, we found that carbon skeletons derived from both glucose and glutamine are catabolized in a canonical oxidative TCA cycle in both the asexual and sexual blood stages. Flux of glucose carbon skeletons into the TCA cycle is low in the asexual blood stages, with glutamine providing most of the carbon skeletons, but increases dramatically in the gametocyte stages. Increased glucose catabolism in the gametocyte TCA cycle was associated with increased glucose uptake, suggesting that the
energy requirements of this stage are high. Significantly, whereas chemical inhibition of the TCA cycle had little effect on the growth or viability of asexual stages, inhibition of the gametocyte TCA cycle led to arrested development and death.

Conclusions: Our metabolomics approach has allowed us to revise current models of P. falciparum carbon metabolism. In particular, we found that both asexual and sexual blood stages utilize a conventional TCA cycle to catabolize glucose and glutamine. Gametocyte differentiation is associated with a programmed remodeling of
central carbon metabolism that may be required for parasite survival either before or after uptake by the mosquito vector. The increased sensitivity of gametocyte stages to TCA-cycle inhibitors provides a potential target for transmission-blocking drugs.

Keywords: Malaria, Central carbon metabolism, TCA cycle, Metabolomics, Gametocyte

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DNA damage strength modulates a bimodal switch of p53 dynamics for cell-fate control

Xi Chen, Jia Chen, Siting Gan, Huaji Guan, Yuan Zhou, Qi Ouyang and Jue Shi

Abstract
Background: The p53 pathway is differentially activated in response to distinct DNA damage, leading to alternative phenotypic outcomes in mammalian cells. Recent evidence suggests that p53 expression dynamics play an important role in the differential regulation of cell fate, but questions remain as to how p53 dynamics and the subsequent cellular response are modulated by variable DNA damage.

Results: We identified a novel, bimodal switch of p53 dynamics modulated by DNA-damage strength that is crucial for cell-fate control. After low DNA damage, p53 underwent periodic pulsing and cells entered cell-cycle arrest. After high DNA damage, p53 underwent a strong monotonic increase and cells activated apoptosis. We found that the damage dose-dependent bimodal switch was due to differential Mdm2 upregulation, which controlled the alternative cell fates mainly by modulating the induction level and pro-apoptotic activities of p53.

Conclusions: Our findings not only uncover a new mode of regulation for p53 dynamics and cell fate, but also suggest that p53 oscillation may function as a suppressor, maintaining a low level of p53 induction and pro-apoptotic activities so as to render cell-cycle arrest that allows damage repair.

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Mitochondrial network morphology: building an integrative, geometrical view

Susanne M Rafelski

Abstract
The morphology of mitochondrial networks is complex and highly varied, yet vital to cell function.The first step toward an integrative understanding of how mitochondrial morphology is generated and regulated is to define the interdependent geometrical features and their dynamics that together generate
the morphology of a mitochondrial network within a cell. Distinct aspects of the size, shape, position, and dynamics of mitochondrial networks are described
and examples of how these features depend on one another discussed.

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Macromolecular juggling by ubiquitylation enzymes

Sonja Lorenz, Aaron J Cantor, Michael Rape and John Kuriyan

Abstract
The posttranslational modification of target  proteins with ubiquitin and ubiquitin-like proteins  is accomplished by the sequential action of E1, E2, and E3 enzymes. Members of the E1 and E3 enzyme  families can undergo particularly large conformational  changes during their catalytic cycles, involving the  remodeling of domain interfaces. This enables the  efficient, directed and regulated handover of ubiquitin  from one carrier to the next one. We review some of
these conformational transformations, as revealed by crystallographic studies.

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Baculum morphology predicts reproductive success of male house mice under sexual selection

Paula Stockley, Steven A Ramm, Amy L Sherborne, Michael D F Thom, Steve Paterson and Jane L Hurst

Abstract
Background: Diversity in penile morphology is characterised by extraordinary variation in the size and shape of the baculum (penis bone) found in many mammals. Although functionally enigmatic, diversity in baculum form is hypothesised to result from sexual selection. According to this hypothesis, the baculum should influence the outcome of reproductive competition among males within promiscuous mating systems. However, a test of this key prediction is currently lacking.

Results: Here we show that baculum size explains significant variation in the reproductive success of male house mice under competitive conditions. After controlling for body size and other reproductive traits, the width (but not length) of the house mouse baculum predicts both the mean number of offspring sired per litter and total number of offspring sired.

Conclusions: By providing the first evidence linking baculum morphology to male reproductive success, our results support the hypothesis that evolutionary diversity in baculum form is driven by sexual selection.

Keywords: Baculum, Cryptic female choice, Genital evolution, Os penis, Penile morphology, Postcopulatory sexual
selection, Sperm competition

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Intact cluster and chordate-like expression of ParaHox genes in a sea star

Rossella Annunziata, Pedro Martinez and Maria Ina Arnone

Abstract
Background: The ParaHox genes are thought to be major players in patterning the gut of several bilaterian taxa.Though this is a fundamental role that these transcription factors play, their activities are not limited to the endoderm and extend to both ectodermal and mesodermal tissues. Three genes compose the ParaHox group: Gsx, Xlox and Cdx. In some taxa (mostly chordates but to some degree also in protostomes) the three genes are arranged into a genomic cluster, in a similar fashion to what has been shown for the better-known Hox genes. Sea urchins possess the full complement of ParaHox genes but they are all dispersed throughout the genome, an arrangement that, perhaps, represented the primitive condition for all echinoderms. In order to understand the evolutionary history of this group of genes we cloned and characterized all ParaHox genes, studied their expression patterns and identified their genomic loci in a member of an earlier branching group of echinoderms, the asteroid Patiria miniata.

Results: We identified the three ParaHox orthologs in the genome of P. miniata. While one of them, PmGsx is provided as maternal message, with no zygotic activation afterwards, the other two, PmLox and PmCdx are expressed during embryogenesis, within restricted domains of both endoderm and ectoderm. Screening of a Patiria bacterial artificial chromosome (BAC) library led to the identification of a clone containing the three genes. The transcriptional directions of PmGsx and PmLox are opposed to that of the PmCdx gene within the cluster.

Conclusions: The identification of P. miniata ParaHox genes has revealed the fact that these genes are clustered in the genome, in contrast to what has been reported for echinoids. Since the presence of an intact cluster, or at least a partial cluster, has been reported in chordates and polychaetes respectively, it becomes clear that within echinoderms, sea urchins have modified the original bilaterian arrangement. Moreover, the sea star ParaHox domains of expression show chordate-like features not found in the sea urchin, confirming that the dynamics of gene expression for the respective genes and their putative regulatory interactions have clearly changed over evolutionary time within the echinoid lineage.

Keywords: Patiria miniata, Asteroid, ParaHox, Cluster, Gsx, Xlox, Cdx, Colinearity, Gut

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Time is of the essence for ParaHox homeobox gene clustering

Myles Garstang and David EK Ferrier

Abstract
ParaHox genes, and their evolutionary sisters the Hox genes, are integral to patterning the anterior-posterior axis of most animals. Like the Hox genes, ParaHox genes can be clustered and exhibit the phenomenon of colinearity - gene order within the cluster matching gene activation. Two new instances of ParaHox
clustering provide the first examples of intact clusters outside chordates, with gene expression lending weight to the argument that temporal colinearity is the key to understanding clustering.

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The cilium in lights: new views of an ancient organelle

Iain A Drummond

Abstract
Nearly all cells have cilia, microtubule based projections from the apical cell surface, and nearly all cells use them in essential and unique ways. How
mammalian cilia function in vivo as mechanosensors, signal transducers, or in fluid propulsion has been difficult to study due to lack of tools for live imaging
of cilia. A new transgenic mouse that enables tissuespecific GFP labeling of cilia creates new opportunities for using live imaging to understand cilium function
and to better characterize the many genetic models of ciliopathies.

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A unique swim bladder-inner ear connection in a teleost fish revealed by a combined high-resolution microtomographic and three-dimensional histological study

Tanja Schulz-Mirbach, Martin Heß, Brian D Metscher and Friedrich Ladich

Abstract
Background: In most modern bony fishes (teleosts) hearing improvement is often correlated with a close morphological relationship between the swim bladder or other gas-filled cavities and the saccule or more rarely with the utricle. A connection of an accessory hearing structure to the third end organ, the lagena, has not yet been reported. A recent study in the Asian cichlid Etroplus maculatus provided the first evidence that a swim bladder may come close to the lagena. Our study was designed to uncover the swim bladder-inner ear relationship in this species. We used a new approach by applying a combination of two high-resolution techniques, namely microtomographic (microCT) imaging and histological serial semithin sectioning, providing the basis for subsequent three-dimensional reconstructions. Prior to the morphological study, we additionally measured auditory evoked potentials at four frequencies (0.5, 1, 2, 3 kHz) to test the hearing abilities of the fish.

Results: E. maculatus revealed a complex swim bladder-inner ear connection in which a bipartite swim bladder extension contacts the upper as well as the lower parts of each inner ear, a condition not observed in any other teleost species studied so far. The gas-filled part of the extension is connected to the lagena via a thin bony lamella and is firmly attached to this bony lamella with connective material. The second part of the extension, a pad-like structure, approaches the posterior and horizontal semicircular canals and a recessus located posterior to the utricle.

Conclusions: Our study is the first detailed report of a link between the swim bladder and the lagena in a teleost species. We suggest that the lagena has an auditory function in this species because the most intimate contact exists between the swim bladder and this end organ. The specialized attachment of the saccule to the cranial bone and the close proximity of the swim bladder extension to the recessus located posterior to the utricle indicate that the saccule and the utricle also receive parallel inputs from the swim bladder extension. We further showed that a combination of non-destructive microCT imaging with histological analyses on the same specimen provides a powerful tool to decipher and interpret fine structures and to compensate for methodological artifacts.

Keywords: Cichlidae, Otolithic end organs, Accessory auditory structures, High-resolution microCT,Histological serial sectioning, Lagena

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What has driven the evolution of multiple cone classes in visual systems: object contrast enhancement or light flicker elimination?

Shai Sabbah1* and Craig W Hawryshyn

Abstract
Background: Two competing theories have been advanced to explain the evolution of multiple cone classes in vertebrate eyes. These two theories have important, but different, implications for our understanding of the design and tuning of vertebrate visual systems. The ‘contrast theory’ proposes that multiple cone classes evolved in shallow-water fish to maximize the visual contrast of objects against diverse backgrounds. The competing ‘flicker theory’ states that multiple cone classes evolved to eliminate the light flicker inherent in shallow-water environments through antagonistic neural interactions, thereby enhancing object detection. However, the selective pressures that have driven the evolution of multiple cone classes remain largely obscure.

Results: We show that two critical assumptions of the flicker theory are violated. We found that the amplitude and temporal frequency of flicker vary over the visible spectrum, precluding its cancellation by simple antagonistic interactions between the output signals of cones. Moreover, we found that the temporal frequency of flicker matches the frequency where sensitivity is maximal in a wide range of fish taxa, suggesting that the flicker may actually enhance
the detection of objects. Finally, using modeling of the chromatic contrast between fish pattern and background under flickering illumination, we found that the spectral sensitivity of cones in a cichlid focal species is optimally tuned to maximize the visual contrast between fish pattern and background, instead of to produce a flicker-free visual signal.

Conclusions: The violation of its two critical assumptions substantially undermines support for the flicker theory as originally formulated. While this alone does not support the contrast theory, comparison of the contrast and flicker theories revealed that the visual system of our focal species was tuned as predicted by the contrast theory rather than by the flicker theory (or by some combination of the two). Thus, these findings challenge key assumptions of the flicker theory,leaving the contrast theory as the most parsimonious and tenable account of the evolution of multiple cone classes.

Keywords: Contrast hypothesis, Cone photoreceptors, Critical fusion frequency, Temporal contrast sensitivity,Opponent mechanisms, Color vision, Retina, Fish

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Ascl1b and Neurod1, instead of Neurog3, control pancreatic endocrine cell fate in zebrafish

Lydie C Flasse, Justine L Pirson, David G Stern, Virginie Von Berg, Isabelle Manfroid, Bernard Peers and Marianne L Voz

Abstract
Background: NEUROG3 is a key regulator of pancreatic endocrine cell differentiation in mouse, essential for the generation of all mature hormone producing cells. It is repressed by Notch signaling that prevents pancreatic cell differentiation by maintaining precursors in an undifferentiated state.

Results: We show that, in zebrafish, neurog3 is not expressed in the pancreas and null neurog3 mutant embryos do not display any apparent endocrine defects. The control of endocrine cell fate is instead fulfilled by two basic helixloop-helix factors, Ascl1b and Neurod1, that are both repressed by Notch signaling. ascl1b is transiently expressed in the mid-trunk endoderm just after gastrulation and is required for the generation of the first pancreatic endocrine precursor cells.Neurod1 is expressed afterwards in the pancreatic anlagen and pursues the endocrine cell differentiation program initiated by Ascl1b. Their complementary role in endocrine differentiation of the dorsal bud is demonstrated by the loss of all hormone-secreting cells following their simultaneous inactivation. This defect is due to a blockage of the initiation of endocrine cell differentiation.

Conclusions: This study demonstrates that NEUROG3 is not the unique pancreatic endocrine cell fate determinant in vertebrates. A general survey of endocrine cell fate determinants in the whole digestive system among vertebrates indicates that they all belong to the ARP/ASCL family but not necessarily to the Neurog3 subfamily. The identity of the ARP/ASCL factor involved depends not only on the organ but also on the species. One could, therefore, consider differentiating stem cells into insulin-producing cells without the involvement of NEUROG3 but via another ARP/ASCL factor.

Keywords: Pancreas, Endocrine, Zebrafish, Ascl1b, Neurod1, Neurog3

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Plasmodium falciparum encodes a conserved active inhibitor-2 for Protein Phosphatase type 1: perspectives for novel anti-plasmodial therapy

Aline Fréville, Katia Cailliau-Maggio, Christine Pierrot, Géraldine Tellier, Hadidjatou Kalamou, Sophia Lafitte,Alain Martoriati, Raymond J Pierce  , Jean-François Bodart and Jamal Khalife

Abstract
Background: It is clear that the coordinated and reciprocal actions of kinases and phosphatases are fundamental in the regulation of development and growth of the malaria parasite. Protein Phosphatase type 1 is a key enzyme playing diverse and essential roles in cell survival. Its dephosphorylation activity/specificity is governed by the interaction of its catalytic subunit (PP1c) with regulatory proteins. Among these, inhibitor-2 (I2) is one of the most evolutionarily ancient PP1 regulators. In vivo studies in various organisms revealed a defect in chromosome segregation and cell cycle progression when the function of I2 is blocked.

Results: In this report, we present evidence that Plasmodium falciparum, the causative agent of the most deadly form of malaria, expresses a structural homolog of mammalian I2, named PfI2. Biochemical, in vitro and in vivo studies revealed that PfI2 binds PP1 and inhibits its activity. We further showed that the motifs 12KTISW16 and 102HYNE105 are critical for PfI2 inhibitory activity. Functional studies using the Xenopus oocyte model revealed that PfI2 is able to overcome the G2/M cell cycle checkpoint by inducing germinal vesicle breakdown. Genetic manipulations in P. falciparum suggest an essential role of PfI2 as no viable mutants with a disrupted PfI2 gene were detectable.Additionally, peptides derived from PfI2 and competing with RVxF binding sites in PP1 exhibit anti-plasmodial activity
against blood stage parasitesin vitro.

Conclusions: Taken together, our data suggest that the PfI2 protein could play a role in the regulation of the P. falciparum cell cycle through its PfPP1 phosphatase regulatory activity. Structure-activity studies of this regulator led to the identification of peptides with anti-plasmodial activity against blood stage parasitesin vitro suggesting that PP1cregulator interactions could be a novel means to control malaria.

Keywords: PP1, Plasmodium, RVXF motifs, Inhibitor-2, G2/M cell cycle

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Juvenile hormone regulation of Drosophila aging

Rochele Yamamoto, Hua Bai, Adam G Dolezal, Gro Amdam and Marc Tatar

Abstract
Background: Juvenile hormone (JH) has been demonstrated to control adult lifespan in a number of non-model insects where surgical removal of the corpora allata eliminates the hormone’s source. In contrast, little is known about how juvenile hormone affects adult Drosophila melanogaster. Previous work suggests that insulin signaling may modulate Drosophila aging in part through its impact on juvenile hormone titer, but no data yet address whether reduction of juvenile hormone is sufficient to control Drosophila life span. Here we adapt a genetic approach to knock out the corpora allata in adult Drosophila melanogaster and characterize adult life history phenotypes produced by reduction of juvenile hormone. With this system we test potential explanations for how juvenile hormone modulates aging.

Results: A tissue specific driver inducing an inhibitor of a protein phosphatase was used to ablate the corpora allata while permitting normal development of adult flies. Corpora allata knockout adults had greatly reduced fecundity, inhibited oogenesis, impaired adult fat body development and extended lifespan. Treating these adults with the juvenile hormone analog methoprene restored all traits toward wildtype. Knockout females remained relatively long-lived even when crossed into a genotype that blocked all egg production. Dietary restriction further extended the lifespan of knockout females. In an analysis of expression profiles of knockout females in fertile and sterile backgrounds, about 100 genes changed in response to loss of juvenile hormone independent of
reproductive state.

Conclusions: Reduced juvenile hormone alone is sufficient to extend the lifespan of Drosophila melanogaster. Reduced juvenile hormone limits reproduction by inhibiting the production of yolked eggs, and this may arise because juvenile hormone is required for the post-eclosion development of the vitellogenin-producing adult fat body. Our data do not support a mechanism for juvenile hormone control of longevity simply based on reducing the physiological costs of egg production. Nor does the longevity benefit appear to function through mechanisms by which dietary restriction extends longevity. We identify transcripts that change in response to juvenile hormone independent of reproductive state and suggest these represent somatically expressed genes that could modulate how juvenile hormone controls persistence and longevity.

Keywords: Juvenile hormone, Drosophila, Lifespan, Fecundity, Fat body, Gene expression

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