Functional Interleukin-17 Receptor A is Expressed in Central Nervous System Glia and Upregulated in Experimental Autoimmune Encephalomyelitis

Background: Interleukin-17A (IL-17A) is the founding member of a novel family of inflammatory cytokines that plays a critical role in the pathogenesis of many autoimmune diseases, including multiple sclerosis (MS) and its animal model, experimental autoimmune encephalomyelitis (EAE). IL-17A signals through its receptor, IL-17RA, which is expressed in many peripheral tissues; however, expression of IL-17RA in the central nervous system (CNS) and its role in CNS inflammation are not well understood. Methods: EAE was induced in C57Bl/6 mice by immunization with myelin oligodendroglial glycoprotein. IL-17RA expression in the CNS was compared between control and EAE mice using RT-PCR, in situ hybridization, and immunohistochemistry. Cell-type specific expression was examined in isolated astrocytic and microglial cell cultures. Cytokine and chemokine production was measured in IL-17A treated cultures to evaluate the functional status of IL-17RA. Results: Here we report increased IL-17RA expression in the CNS of mice with EAE, and constitutive expression of functional IL-17RA in mouse CNS tissue. Specifically, astrocytes and microglia express IL-17RA in vitro, and IL-17A treatment induces biological responses in these cells, including significant upregulation of MCP-1, MCP-5, MIP-2 and KC chemokine secretion. Exogenous IL-17A does not significantly alter the expression of IL-17RA in glial cells, suggesting that upregulation of chemokines by glial cells is due to IL-17A signaling through constitutively expressed IL-17RA. Conclusion: IL-17RA expression is significantly increased in the CNS of mice with EAE compared to healthy mice, suggesting that IL-17RA signaling in glial cells can play an important role in autoimmune inflammation of the CNS and may be a potential pathway to target for therapeutic interventions. © 2009 Sarma et al; licensee BioMed Central Ltd.

Role of behaviour in biological invasions and species distributions; lessons from interactions between the invasive Gammarus pulex and the native G-duebeni (Crustacea : Amphipoda)

In attempting to understand the distributions of both introduced species and the native species on which they impact, there is a growing trend to integrate studies of behaviour with more traditional life history/ecological approaches. The question of what mechanisms drive the displacement of the freshwater amphipod Gammarus duebeni by the often introduced G pulex is presented as a case study Patterns of displacement are well documented throughout Europe, but the speed and direction of displacement between these species can be varied. From early studies proposing interspecific competition as causal in these patterns, I review research progress to date. I show there has been no evidence for interspecific competition operating, other than the field patterns themselves, a somewhat tautological argument. Rather, the increased recognition of behavioural attributes with respect to the cannibalistic and predatory nature of these species gave rise to a series of studies unravelling the processes driving field patterns. Both species engage in 'intraguild predation' (IGP), with moulting females particularly vulnerable to predation by congeneric males. G pulex is more able both to engage in and avoid this interaction with G duebeni. However, several factors mediate the strength and asymmetry of this IGP, some biotic (e.g. parasitism) and others abiotic (e.g. water chemistry). Further, a number of alternative hypotheses that may account for the displacement (hybridization; parasite transmission) have been tested and rejected. While interspecific competition has been modelled mathematically and found to be a weak interaction relative to IGP, mechanisms of competition between these Gammarus species remain largely untested empirically. Since IGP may be finely balanced in some circumstances, I conclude that the challenge to detect interspecific competition remains and we require assessment of its role, if any, in the interaction between these species. Appreciation of behavioural attributes and their mediation should allow us to more fully understand, and perhaps predict, species introductions and resultant distributions.

The 67-kd laminin receptor is preferentially expressed by proliferating retinal vessels in a murine model of ischemic retinopathy.

Endothelial cell association with vascular basement membranes is complex and plays a critical role in regulation of cell adhesion and proliferation. The interaction between the membrane-associated 67-kd receptor (67LR) and the basement membrane protein laminin has been studied in several cell systems where it was shown to be crucial for adhesion and attachment during angiogenesis. As angiogenesis in the pathological setting of proliferative retinopathy is a major cause of blindness in the Western world we examined the expression of 67LR in a murine model of hyperoxia-induced retinopathy that exhibits retinal neovascularization. Mice exposed to hyperoxia for 5 days starting at postnatal day 7 (P7) and returned to room air (at P12) showed closure of the central retinal vasculature. In response to the ensuing retinal ischemia, there was consistent preretinal neovascularization starting around P17, which persisted until P21, after which the new vessels regressed. Immunohistochemistry was performed on these retinas using an antibody specific for 67LR. At P12, immunoreactivity for 67LR was absent in the retina, but by P17 it was observed in preretinal proliferating vessels and also within the adjacent intraretinal vasculature. Intraretinal 67LR immunoreactivity diminished beyond P17 until by P21 immunoreactivity was almost completely absent, although it persisted in the preretinal vasculature. Control P17 mice (not exposed to hyperoxia) failed to demonstrate any 67LR immunoreactivity in their retinas. Parallel in situ hybridization studies demonstrated 67LR gene expression in the retinal ganglion cells of control and hyperoxia-exposed mice. In addition, the neovascular intra- and preretinal vessels of hyperoxia-treated P17 and P21 mice labeled strongly for 67LR mRNA. This study has characterized 67LR immunolocalization and gene expression in a murine model of ischemic retinopathy. Results suggest that, although the 67LR gene is expressed at high levels in the retinal ganglion cells, the mature receptor protein is preferentially localized to the proliferating retinal vasculature and is almost completely absent from quiescent vessels. The differential expression of 67LR between proliferating and quiescent retinal vessels suggests that this laminin receptor is an important and novel target for future chemotherapeutic intervention during proliferative vasculopathies.

Wave mixing of hybrid Bogoliubov modes in a Bose-Einstein condensate

Mode-mixing of coherent excitations of a trapped Bose-Einstein condensate is modeled using the Bogoliubov approximation. Calculations are presented for second-harmonic generation between the two lowest-lying even-parity m=0 modes in an oblate spheroidal trap. Hybridization of the modes of the breather (l=0) and surface (l=4) states leads to the formation of a Bogoliubov dark state near phase-matching resonance so that a single mode is coherently populated. Efficient harmonic generation requires a strong coupling rate, sharply-defined and well-separated frequency spectrum, and good phase matching. We find that in all three respects the quantal results are significantly different from hydrodynamic predictions. Typically the second-harmonic conversion rate is half that given by an equivalent hydrodynamic estimate.

Desmoplastic Small Round Cell Tumor of Major Salivary Glands: Report of 1 Case and a Review of the Literature

Desmoplastic small round cell tumor is a rare malignant neoplasm mostly occurring in the vicinity of or within the peritoneal cavity, and is uncommon in the head and neck region. Tumor location within a major salivary gland is exceptional. We report a case of a 41-year-old Chinese man with a history of diabetes mellitus and end-stage renal failure on peritoneal dialysis with a desmoplastic small round cell tumor occurring in the left submandibular gland. Fine-needle aspiration cytology showed variably cohesive clusters of small cells with hyperchromatic nuclei and fine granular chromatin. On histology the neoplasm displayed classic features of a desmoplastic small round cell tumor with angulated nests of small round blue cells in a fibromyxoid/desmoplastic stroma. Neoplastic cells were immunoreactive for cytokeratins (AE1/3), desmin (paranuclear dot-like), WT-1 (nuclear), epithelial membrane antigen, and CD56. EWS gene translocation and EWS-WT1 gene fusion were detected by fluorescence in situ hybridization and reverse transcriptase polymerase chain reaction, respectively. The case presented is the sixth case of and the oldest reported patient with a desmoplastic small round cell tumor occurring in a major salivary gland to date. Desmoplastic small round cell tumor should be considered in the differential diagnosis of a salivary gland neoplasm with a basaloid or small cell pattern on fine-needle aspiration cytology.

Progressive loss of epidermal growth factor receptor in a subpopulation of breast cancers: implications in target-directed therapeutics

Understanding the molecular etiology and heterogeneity of disease has a direct effect on cancer therapeutics. To identify novel molecular changes associated with breast cancer progression, we conducted phosphoproteomics of the MCF10AT model comprising isogenic, ErbB2- and ErbB3-positive, xenograft-derived cell lines that mimic different stages of breast cancer. Using in vitro animal model and clinical breast samples, our study revealed a marked reduction of epidermal growth factor receptor (EGFR) expression with breast cancer progression. Such diminution of EGFR expression was associated with increased resistance to Gefitinib/Iressa in vitro. Fluorescence in situ hybridization showed that loss of EGFR gene copy number was one of the key mechanisms behind the low/null expression of EGFR in clinical breast tumors. Statistical analysis on the immunohistochemistry data of EGFR expression from 93 matched normal and breast tumor samples showed that (a) diminished EGFR expression could. be detected as early as in the preneoplastic lesion (ductal carcinoma in situ) and this culminated in invasive carcinomas; (b) EGFR expression levels could distinguish between normal tissue versus carcinoma in situ and invasive carcinoma with high statistical significance (P

Reliability of tissue microarrays in detecting protein expression and gene amplification in breast cancer

Tissue microarrays allow high throughput molecular profiling of diagnostic or predictive markers in cancer specimens and rapid validation of novel potential candidates identified from genomic and proteomic analyses in a large number of tumor samples. To validate the use of tissue microarray technology for all the main biomarkers routinely used to decide breast cancer prognostication and postsurgical adjuvant therapy, we constructed a tissue microarray from 97 breast tumors, with a single 0.6 mm core per specimen. Inummostaining; of tissue microarray sections and conventional full sections of each tumor were performed using well-characterized prognostic markers (estrogen receptor ER, progesterone receptor PR and c-erbB2). The full section versus tissue microarray concordance for these stains was 97% for ER, 98% for PR, and 97% for c-erbB2, respectively, with a strong statistical association (kappa value more than 0.90). Fluorescence in situ hybridization analysis for HER-2/neu gene amplification from the single-core tissue microarray was technically successful in about 90% (87/97) of the cases, with a concordance of 95% compared with parallel analyses with the full sections. The correlation with other pathological parameters was not significantly different between full-section and array-based results. It is concluded that the constructed tissue microarray with a single core per specimen ensures full biological representativeness to identify the associations between biomarkers and clinicopathological parameters, with no significant associated sampling bias.

Evaluation of HER-2/neu oncogene status in breast tumors on tissue microarrays

The amplification and/or overexpression of the HER-2/neu oncogene and its encoded receptor protein are increasingly used for prognostication and prediction of therapeutic response to Herceptin in breast cancer. However, large-scale examination of archival tumor blocks by immunohistochemistry (IHC) or fluorescence in situ hybridization (FISH) is prohibitively laborious and technically challenging. The tissue microarray (TMA) technique enables hundreds of tumors to be studied simultaneously in a single experiment. To evaluate the HER-2/neu status of a selection of the breast tumors in our tumor bank, we constructed a TMA from 97 breast tumors, with a single 0.6-mm core per specimen. HER-2/neu gene amplification by FISH was found in 20 of the 87 interpretable cases (23%): in 14 of 14 IHC 3+ cases (100%), 5 of 8 IHC 2+ cases (62.5%) and 1 of 65 IHC 0/1+ cases (1.5%). Three of the 67 cases with no evidence of HER-2/neu gene amplification by FISH were moderately positive (2+) by IHC. A close relationship was observed between these 2 assays as applied to the TMA (95.4% concordance: 95% CI, - 2.2% to 6.8%; P

FMRFamide-like peptides in root knot nematodes and their potential role in nematode physiology

FMRFamide-like peptides (FLPs) are a diverse group of neuropeptides that are expressed abundantly in nematodes. They exert potent physiological effects on locomotory, feeding and reproductive musculature and also act as neuromodulators. However, little is known about the specific expression patterns and functions of individual peptides. The current study employed rapid amplification of cDNA ends-polymerase chain reaction (RACE-PCR) to characterize flp genes from infective juveniles of the root knot nematodes, Meloidogyne incognita and Meloidogyne minor. The peptides identified from these transcripts are sequelogs of FLPs from the free-living nematode, Caenorhabditis elegans; the genes have therefore been designated as Mi-flp-1, Mi-flp-7, Mi-flp-12, Mm-flp-12 and Mi-flp-14. Mi-flp-1 encodes five FLPs with the common C-terminal moiety, NFLRFamide. Mi-flp-7 encodes two copies of APLDRSALVRFamide and APLDRAAMVRFamide and one copy of APFDRSSMVRFamide. Mi-flp-12 and Mm-flp-12 encode the novel peptide KNNKFEFIRFamide (a longer version of RNKFEFIRFamide found in C. elegans). Mi-flp-14 encodes a single copy of KHEYLRFamide (commonly known as AF2 and regarded as the most abundant nematode FLP), and a single copy of the novel peptide KHEFVRFamide. These FLPs share a high degree of conservation between Meloidogyne species and nematodes from other clades, including those of humans and animals, perhaps suggesting a common neurophysiological role which may be exploited by novel drugs. FLP immunoreactivity was observed for the first time in Meloidogyne, in the circumpharyngeal nerve ring, pharyngeal nerves and ventral nerve cord. Additionally, in situ hybridization revealed Mi-flp-12 expression in an RIR-like neuron and Mi-flp-14 expression in SMB-like neurons, respectively. These localizations imply physiological roles for FLP-12 and FLP-14 peptides, including locomotion and sensory perception.

A MOLECULAR AND MORPHOLOGICAL ANALYSIS OF THE GYMNOGONGRUS-DEVONIENSIS (RHODOPHYTA) COMPLEX IN THE NORTH-ATLANTIC

The Gymnogongrus devoniensis (Greville) Schotter complex in the North Atlantic Ocean was elucidated by comparative molecular, morphological, and culture studies. Restriction fragment length patterns and hybridization data on organellar DNA revealed two distinct taxa in samples from Europe and eastern Canada. Nucleotide sequences for the intergenic spacer between the large and small subunit genes of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco), and the adjoining regions of both genes, differed by 12.5-13.4% between the two taxa. One of the taxa, which included material from the type locality of G. devoniensis at Torbay, Devon, England, was taken to represent authentic G. devoniensis. Within this taxon, samples from Ireland, England, northern France, northern Spain, and southern Portugal showed great morphological variation, particularly in habit, but their Rubisco spacer sequences were identical or differed by only a single nucleotide. Constant morphological features included the development, from a single auxiliary cell, of the spherical cystocarp with a thick mucilage sheath that appears to be typical of Gymnogongrus species with internal cystocarps. Two life-history types were found. Northern isolates underwent a direct-type life history, recycling apomictic females by carpospores, whereas the Portuguese isolate followed a heteromorphic life history in which carpospores gave rise to a crustose tetrasporophyte.

Expression of C5a receptor in mouse brain: Role in signal transduction and neurodegeneration

In this study we explored the potential role of the complement derived anaphylatoxin C5a and the expression of its receptor in mouse brain. Using in situ hybridization, we found that C5a receptor messenger RNA is expressed in mouse brain. In response to intraventricular kainic acid injection, there was marked increase in the C5a receptor messenger RNA expression, particularly in hippocampal formation and cerebral cortex. C5a ligand-binding autoradiography confirmed the functional expression and elevation of the C5a receptor post-lesioning. The expression of Cia receptor messenger RNA in brain was confirmed by northern blot hybridization of total RNA from neuronal and glial cells in vitiro. Based on these findings we explored the role of C5a in mechanisms of signal transduction in brain cells. Treatment of primary cultures of mouse astrocytes with human recombinant C5a resulted in the activation of mitogen-activated extracellular signal-regulated protein kinase. This response appeared to be mediated by the C5a receptor since astrocyte cultures derived from C5a receptor knockout mice were not responsive to the treatment. Understanding the regulation of C5a receptor in brain and mechanisms by which pro-inflammatory C5a modulates specific signal transduction pathways in brain cells is crucial to studies of inflammatory mechanisms in neurodegeneration. (C) 1998 IBRO. Published by Elsevier Science Ltd.

Identification of a general secretory pathway in a human isolate of Burkholderia vietnamiensis (formerly B. cepacia complex genomovar V) that is required for the secretion of hemolysin and phospholipase C activities

Members of the Burkholderia cepacia complex can secrete proteases, lipases, and hemolysins. We report in this study the identification of a general secretory pathway present in a B. vietnamiensis (formerly genomovar V) clinical isolate, which is required for the efficient secretion of phospholipase C and hemolysin activities. Southern blot hybridization experiments revealed that this general secretion pathway is highly conserved among the different genomovars of the B. cepacia complex and is homologous to a similar system described in B. pseudomallei. We also show that this pathway appears not to be necessary for intracellular survival of B. vietnamiensis within Acanthamoeba polyphaga.

Diphenylamine increases cloacin DF13 sensitivity in avian septicemic strains of Escherichia coli

Thirteen avian septicemic isolates of Escherichia coli were examined for the presence of the aerobactin iron transport system. All of the strains possessed a functional aerobactin system and hybridization experiments showed that the aerobactin genes were located on ColV-type plasmids in all cases. The expression of the aerobactin receptor IutA was also studied by determining the bacterial susceptibility to the bacteriocin cloacin DF13. Twelve of the 13 isolates were cloacin-resistant but became sensitive to this bacteriocin upon treatment with diphenylamine which caused a reduction in the amount of O-side chain lipopolysaccharide.

Relatedness of O-specific lipopolysaccharide side chain genes from strains of Shigella boydii type 12 belonging to two clonal groups and from Escherichia coli O7:K1

The O-specific lipopolysaccharide side chains of Escherichia coli O7 and Shigella boydii type 12 possess similar but not identical chemical structures. We investigated the genetic relatedness between the O-specific side chain genes in members of these two species. Examination of outer membrane protein and lipopolysaccharide (LPS) banding patterns demonstrated that five strains which had been identified as S. boydii type 12 fell into two clonal groups, SB1 and SB2. Hybridizations with O7-specific radiolabeled probes derived from the chromosomal DNA of an E. coli O7 strain detected identical fragments among the three SB1 strains of S. boydii type 12 and the two E. coli O7 reference isolates. The two other S. boydii type 12 strains, which belonged to the SB2 clone, did not show homologies with the O7 probe under high-stringency conditions of hybridization. The homology between the O7 and type 12 LPS gene regions from the SB1 strains was further confirmed by the construction of O-specific side chain-deficient mutations in these strains by homologous recombination of a suicide plasmid containing O7-specific DNA sequences. Immunoblot experiments with O7 antiserum gave a weak cross-reaction with LPS purified from the SB2 strains but a very strong cross-reaction with the LPS from SB1 isolates. Antiserum raised to one of the SB2 strains cross-reacted only with S. boydii type 12 LPS from the SB1 clone but failed to react with O7 LPS.

Molecular cloning and expression in Escherichia coli K-12 of chromosomal genes determining the O7 lipopolysaccharide antigen of a human invasive strain of E. coli O7:K1

We have cloned and studied the expression in Escherichia coli K-12 of chromosomal rfb genes determining the biosynthesis of the O7 lipopolysaccharide (LPS) antigen from E. coli K1 strain VW187. Two E. coli K-12 strains carrying recombinant cosmids gave positive coagglutination reactions with protein A-rich staphylococcal particles bearing an O7-specific rabbit polyclonal antiserum. Silver-stained polyacrylamide gels of total membranes extracted with hot phenol showed O side chain material which had O7 specificity as determined by immunoblotting experiments. However, the amount of O7 LPS expressed in E. coli K-12 was considerably lower than that produced by the wild-type strain VW187. Deletion and transposition experiments identified a region of about 17 kilobase pairs which is essential for the expression of O7 LPS. The existence of homologies between the O7 LPS genes and other E. coli O side chain genes was investigated by Southern blot hybridization experiments. An O7-specific probe fragment of 15 kilobase pairs did not hybridize to genomic DNA digests of E. coli strains belonging to several different O types, demonstrating that the O7 LPS genes are unique.