Molecular evolution of CXCR1, a G protein-coupled receptor involved in signal transduction of neutrophils

Human neutrophils are a type of white blood cell, which forms an early line of defense against bacterial infections. Neutrophils are highly responsive to the chemokine, interleukin-8 (IL-8) due to the abundant distribution of CXCR1, one of the IL-8 receptors on the neutrophil cell surface. As a member of the GPCR family, CXCR1 plays a crucial role in the IL-8 signal transduction pathway in neutrophils. We sequenced the complete coding region of the CXCR1 gene in worldwide human populations and five representative nonhuman primate species. Our results indicate accelerated protein evolution in the human lineage, which was likely caused by Darwinian positive selection. The sliding window analysis and the codon-based neutrality test identified signatures of positive selection at the N-terminal ligand/receptor recognition domain of human CXCR1.

Molecular characterization and expression profiles in response to bacterial infection of Chinese soft-shelled turtle interleukin-8 (IL-8), the first reptilian chemokine gene

In this study, an IL-8 homologue has been cloned and identified from a reptile, Chinese soft-shelled turtle for the first time. The full-length cDNA of turtle IL-8 was 1188 bp and contained a 312 bp open reading frame (ORF) coding for a protein of 104 amino acids. The chemokine CXC domain, which contained Glu-Leu-Arg (ELR) motif and four cysteine residues, was well conserved in turtle IL-8. The 4924 bp genomic DNA of turtle IL-8 contained four exons and three introns. Phylogenetic analysis showed that the amino acid sequence of turtle IL-8 clustered together with birds. RT-PCR analysis showed that turtle IL-8 mRNA was constitutively expressed liver, spleen, kidney, heart, blood and intestine tissues of control turtles. Real-time quantitative PCR analysis further indicated that the turtle IL-8 mRNA expression was apparent in various tissues at 8 h and up-regulated significantly during 8 h-7 d after Aeromonas hydrophila infection. The present studies will help us to understand the evolution of IL-8 molecule and the inflammatory response mechanism in reptiles. (C) 2009 Elsevier Ltd. All rights reserved.

Molecular cloning and expression of interleukin 1beta (IL-1 beta) from red seabream (Pagrus major)

The interleukin 1beta (IL-1beta) cDNA was cloned from the red seabream (Pagrus major) by homology cloning strategy. A cDNA fragment was amplified by PCR using two degenerated primers, which were designed according to the conserved regions of other known IL-1beta sequences, and elongated by 3' ends and 5' ends RACE PCR to get the full length coding sequence of red seabream IL-1beta (RS IL-1beta). The sequence contained 1252 nucleotides that included a 5' untranslated region (UTR) of 84 bp, a 3' UTR of 410 bp and an open reading frame (ORF) of 759 nucleotides which could be translated into a putative peptide of 253 amino acids with molecular weight of 28.6 kD and putative isoelectric point pI of 5.29. The deduced peptide contained two potential N-glycosylation sites and an identifiable IL1 family signature, but lacked the signal peptide and the clear ICE cut site, which were common in other nonmammalian IL-1beta genes. The RS IL-1beta had the highest homology with piscine IL-1beta according to phylogenetic tree analysis. The transcript expression was detected in blood, brain, gill, heart, head kidney, kidney, liver, muscle and spleen in the pathogen challenged and healthy red seabream by RTPCR. Results showed that the RS IL-1beta mRNA was constitutively expressed in most of the tissues both in stimulated and un-stimulated fish, and the expression could be enhanced by pathogen challenging.

The role of the IL-1 type 1 receptor in the life, death and differentiation of adult hippocampal neural precursor cells

Neurogenesis occurs in two distinct regions of the adult brain; the subgranular zone (SGZ) of the dentate gyrus (DG) in the hippocampus, and the subventricular zone (SVZ) lining the lateral ventricles. It is now well-known that adult hippocampal neurogenesis can be modulated by a number of intrinsic and extrinsic factors e.g. local signalling molecules, exercise, environmental enrichment and learning. Moreover, levels of adult hippocampal neurogenesis decrease with age, at least in rodents, and alterations in hippocampal neurogenesis have been reported in animal models and human studies of neuropsychiatric and neurodegenerative conditions. Neuroinflammation is a common pathological feature of these conditions and is also a potent modulator of adult hippocampal neurogenesis. Recently, the orphan nuclear receptor TLX has been identified as an important regulator of adult hippocampal neurogenesis as its expression is necessary to maintain the neural precursor cell (NPC) pool in the adult DG. Likewise, exposure of animals to voluntary exercise has been consistently demonstrated to promote adult hippocampal neurogenesis. Lentivirus (LV)- mediated gene transfer is a useful tool to elucidate gene function and to explore potential therapeutic candidates across an array of conditions as it facilitates sustained gene expression in both dividing and post-mitotic cell populations. Both intrinsic and extrinsic factors are important regulators of adult hippocampal neurogenesis. Examining how these factors are affected by an inflammatory stimulus, and the subsequent effects on adult hippocampal neurogenesis provides important information for the development of novel treatment strategies for neuropsychiatric and neurodegenerative conditions in which adult hippocampal neurogenesis is impaired. The aims of the series of experiments presented in this thesis were to examine the effect of the pro-inflammatory cytokine interleukin-1β (IL-1β) on adult hippocampal NPCs both in vitro and in vivo. In vitro, we have shown that IL-1β reduces proliferation of adult hippocampal NPCs in a dose and time-dependent manner. In addition, we have demonstrated that TLX expression is reduced by IL-1β. Blockade of IL-1β signalling prevented both the IL-1β-induced reduction in cell proliferation and TLX expression. In vivo, we examined the effect of short term and long term exposure to LV-IL-1β in sedentary mice and in mice exposed to voluntary running. We demonstrated that impaired hippocampal neurogenesis is only evident after long term exposure to IL-1β. In mice exposed to voluntary running, hippocampal neurogenesis is significantly increased following short-term but not long-term exposure to running. Moreover, short-term running effectively prevents any IL-1β-induced effects on hippocampal neurogenesis; however, no such effects are seen following long-term exposure to running.

The role of presenilin 1 and presenilin 2 in IL-1β-, LPS- and TNFα- mediated signalling events

The importance of γ-secretase protease activities in development, neurogenesis and the immune system are highlighted by the diversity of its substrates and phenotypic characterization of Presenilin (PS)-deficient transgenic animals. Since the discovery of Amyloid precursor protein (APP) and it’s cleavage by γ-secretase complexes, over 90 other type I membrane proteins have been identified as γ-secretase substrates. We have identified interleukin-1 (IL-1) receptor type I (IL-1R1), toll-like receptor 4 (TLR4) and tumour necrosis factor-α (TNFα) receptor-1 (TNFR1) as novel substrates for - secretase cleavage, which play an important role in innate immunity. In this study, using PS-deficient cells and PS-knockout animal models we examined the role of PS proteins, PS1 and PS2, in IL-1R1-, TLR4- and TNFR1- mediated inflammatory responses. Data presented show that in response to IL- 1β, lipopolysaccharide (LPS) or TNFα, immortalised fibroblasts from PS2- deficient animals have diminished production of specific cytokines and chemokine, with differential reduction in nuclear factor-κB (NF-κB) and (mitogen activated protein kinase) MAPK activities. In contrast, no defect in the response to IL-1β, LPS or TNFα was observed in PS1-deficient immortalised fibroblasts. These observations were confirmed using bone marrow-derived macrophages from PS2-null mice, which also display impaired responsiveness to IL-1β- and LPS, with decreased production of inflammatory cytokines. Furthermore, in whole animal in vivo responses, we show that PS2-deficient animals display ligand (IL-1β, LPS and TNFα)-dependent alterations in the production of specific pro-inflammatory cytokines or chemokines. Importantly, this reduced responsiveness to IL-1β, LPS or TNFα is independent of γ- secretase protease activity and γ-secretase cleavage of TNFR1, IL-1R1 or TLR4. These observations suggest a novel γ-secretase-independent role of PS2 in the regulation of innate immune responsiveness and challenge current concepts regarding the regulation of IL-1β-, LPS- and TNFα-mediated immune signalling.

Anti-CTLA-4 treatment induces IL-10-producing ICOS+ regulatory T cells displaying IDO-dependent anti-inflammatory properties in a mouse model of colitis.

BACKGROUND AND AIMS: Cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) has been shown to act as a negative regulator of T cell function and has been implicated in the regulation of T helper 1 (Th1)/Th2 development and the function of regulatory T cells. Tests were carried out to determine whether anti-CTLA-4 treatment would alter the polarisation of naive T cells in vivo. METHODS: Mice were treated with anti-CTLA-4 monoclonal antibody (mAb) (UC10-4F10) at the time of immunisation or colonic instillation of trinitrobenzene sulfonic acid (TNBS). The cytokines produced by lymph node cells after in vitro antigenic stimulation and the role of indoleamine 2,3 dioxygenase (IDO) and of interleukin-10 (IL-10) were tested, and the survival of mice was monitored. RESULTS: Injection of anti-CTLA-4 mAb in mice during priming induced the development of adaptive CD4(+) regulatory T cells which expressed high levels of ICOS (inducible co-stimulator), secreted IL-4 and IL-10. This treatment inhibited Th1 memory responses in vivo and repressed experimental intestinal inflammation. The anti-CTLA-4-induced amelioration of disease correlated with IDO expression and infiltration of ICOS(high) Foxp3(+) T cells in the intestine, suggesting that anti-CTLA-4 acted indirectly through the development of regulatory T cells producing IL-10 and inducing IDO. CONCLUSIONS: These observations emphasise the synergy between IL-10 and IDO as anti-inflammatory agents and highlight anti-CTLA-4 treatment as a potential novel immunotherapeutic approach for inducing adaptive regulatory T cells.

Role of mast cells in inflammatory bowel disease and inflammation-associated colorectal neoplasia in IL-10-deficient mice.

BACKGROUND: Inflammatory bowel disease (IBD) is hypothesized to result from stimulation of immune responses against resident intestinal bacteria within a genetically susceptible host. Mast cells may play a critical role in IBD pathogenesis, since they are typically located just beneath the intestinal mucosal barrier and can be activated by bacterial antigens. METHODOLOGY/PRINCIPAL FINDINGS: This study investigated effects of mast cells on inflammation and associated neoplasia in IBD-susceptible interleukin (IL)-10-deficient mice with and without mast cells. IL-10-deficient mast cells produced more pro-inflammatory cytokines in vitro both constitutively and when triggered, compared with wild type mast cells. However despite this enhanced in vitro response, mast cell-sufficient Il10(-/-) mice actually had decreased cecal expression of tumor necrosis factor (TNF) and interferon (IFN)-gamma mRNA, suggesting that mast cells regulate inflammation in vivo. Mast cell deficiency predisposed Il10(-/-) mice to the development of spontaneous colitis and resulted in increased intestinal permeability in vivo that preceded the development of colon inflammation. However, mast cell deficiency did not affect the severity of IBD triggered by non-steroidal anti-inflammatory agents (NSAID) exposure or helicobacter infection that also affect intestinal permeability. CONCLUSIONS/SIGNIFICANCE: Mast cells thus appear to have a primarily protective role within the colonic microenvironment by enhancing the efficacy of the mucosal barrier. In addition, although mast cells were previously implicated in progression of sporadic colon cancers, mast cells did not affect the incidence or severity of colonic neoplasia in this inflammation-associated model.

Report on Archaeological Investigations Conducted at the St. Mary's Site (18AP45), 107 Duke of Gloucester Street, Annapolis, Maryland, 1987-1990

In celebration of the 250th anniversary of the birth of Charles Carroll of Carrollton, Archaeology in Annapolis was invited to excavate the Carroll House and garden on 107 Duke of Gloucester Street in Annapolis, Maryland. The site, named the St. Mary's Site (18AP45) for the Catholic church on the property, is currently owned by the Redemptorists, a Roman Catholic congregation of priests and brothers who have occupied the site since 1852. Prior to the Redemptorists' tenure, the site was owned by the Carroll family from 1701-1852 and is perhaps best known as the home of Charles Carroll of Carrollton (1737-1832), signer of the Declaration of Independence. Excavations at the site were conducted during four consecutive summer seasons from 1987-1990. The investigation focused on three research questions. The first line of inquiry were questions surrounding the dating, architectural configuration, and artifact deposits of the "frame house," a structure adjoining the west wall of the brick Carroll House via a "passage" and later a three story addition. The frame house was partially demolished in the mid-nineteenth century but the construction was thought to pre-date the brick portion of the house. The second research question was spurred by documentary research which indicated that the property might have been the location of Proctor's Tavern, a late 17th-century tavern which served as the meeting place of the Maryland Provincial Assembly. Archaeological testing hoped to determine its location and, if found, investigate Annapolis' early Euro-American occupation. The third research question focused on the landscape of the site as it was shaped by its occupants over the past three hundred years. The research questions included investigating the stratigraphy, geometry, and architectural and planting features of Charles Carroll of Carrollton's terraced garden built during the 1770s, and investigating the changes to the landscape made by the Redemptorists in the nineteenth and twentieth centuries. While no structural evidence associated with Proctor’s Tavern was uncovered during limited excavations along Spa Creek, the historic shore of Spa Creek was identified, buried beneath deep fill deposits laid down during construction of the Carroll Garden. Features and deposits associated with this period likely remain intact in a waterlogged environment along the southeastern sea wall at the St. Mary’s Site. Evidence of extensive earth moving by Carroll is present in the garden and was identified during excavation and coring. This strongly suggests that the garden landscape visible at the St. Mary’s Site is the intact Carroll Garden, which survives beneath contemporary and late nineteenth century strata. The extant surviving garden should be considered highly sensitive to ground-disturbing activities, and is also highly significant considering demonstrable associations with the Carroll family. Other garden-related features were also discovered, including planting holes, and a brick pavilion or parapet located along Spa Creek to the south of the site. The Duke of Gloucester Street wall was shown to be associated with the Carroll occupation of the site. Finally, intensive archaeological research was directed at the vicinity of a frame house constructed and occupied by the Carrolls to the east of the existing brick house, which was replaced by the Redemptorists in the nineteenth century with a greenhouse. These superimposed buildings were documented in detail and remain highly significant features at the St. Mary’s Site.

When the library is located in prime real estate: a case study on the loss of space from the Duke University Medical Center Library and Archives.

The Duke University Medical Center Library and Archives is located in the heart of the Duke Medicine campus, surrounded by Duke Hospital, ambulatory clinics, and numerous research facilities. Its location is considered prime real estate, given its adjacency to patient care, research, and educational activities. In 2005, the Duke University Library Space Planning Committee had recommended creating a learning center in the library that would support a variety of educational activities. However, the health system needed to convert the library's top floor into office space to make way for expansion of the hospital and cancer center. The library had only five months to plan the storage and consolidation of its journal and book collections, while working with the facilities design office and architect on the replacement of key user spaces on the top floor. Library staff worked together to develop plans for storing, weeding, and consolidating the collections and provided input into renovation plans for users spaces on its mezzanine level. The library lost 15,238 square feet (29%) of its net assignable square footage and a total of 16,897 (30%) gross square feet. This included 50% of the total space allotted to collections and over 15% of user spaces. The top-floor space now houses offices for Duke Medicine oncology faculty and staff. By storing a large portion of its collection off-site, the library was able to remove more stacks on the remaining stack level and convert them to user spaces, a long-term goal for the library. Additional space on the mezzanine level had to be converted to replace lost study and conference room spaces. While this project did not match the recommended space plans for the library, it underscored the need for the library to think creatively about the future of its facility and to work toward a more cohesive master plan.

The East Asian Collection at Duke University: Dynamics of Change.

Traces the history of Duke's East Asian Studies program and associated library collections from the beginning of the twentieth century to the present. Describes the strengths of the Japanese, Chinese and Korean collections, materials in special collections and cooperation with the University of North Carolina.

Association of Common Genetic Polymorphisms with Melanoma Patient IL-12p40 Blood Levels, Risk, and Outcomes.

Recent investigation has identified association of IL-12p40 blood levels with melanoma recurrence and patient survival. No studies have investigated associations of single-nucleotide polymorphisms (SNPs) with melanoma patient IL-12p40 blood levels or their potential contributions to melanoma susceptibility or patient outcome. In the current study, 818,237 SNPs were available for 1,804 melanoma cases and 1,026 controls. IL-12p40 blood levels were assessed among 573 cases (discovery), 249 cases (case validation), and 299 controls (control validation). SNPs were evaluated for association with log[IL-12p40] levels in the discovery data set and replicated in two validation data sets, and significant SNPs were assessed for association with melanoma susceptibility and patient outcomes. The most significant SNP associated with log[IL-12p40] was in the IL-12B gene region (rs6897260, combined P=9.26 × 10(-38)); this single variant explained 13.1% of variability in log[IL-12p40]. The most significant SNP in EBF1 was rs6895454 (combined P=2.24 × 10(-9)). A marker in IL12B was associated with melanoma susceptibility (rs3213119, multivariate P=0.0499; OR=1.50, 95% CI 1.00-2.24), whereas a marker in EBF1 was associated with melanoma-specific survival in advanced-stage patients (rs10515789, multivariate P=0.02; HR=1.93, 95% CI 1.11-3.35). Both EBF1 and IL12B strongly regulate IL-12p40 blood levels, and IL-12p40 polymorphisms may contribute to melanoma susceptibility and influence patient outcome.

Molecular dissociation of FHA-induced IL-10 and IL-12 secretion by human dendritic cells.

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Role of the IL-10/IL-12 balance in the primary cellular immune response of infants to pertussis vaccines

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Effects of different molecular forms of FHA on IL-10 and IL-12 secretions by human dendritic cells.

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