Despite differences between dendritic cells and Langerhans cells in the mechanism of papillomavirus-like particle antigen uptake, both cells cross-prime T cells

As human papillomavirus-like particles (HPV-VLP) represent a promising vaccine delivery vehicle, delineation of the interaction of VLP with professional APC should improve vaccine development. Differences in the capacity of VLP to signal dendritic cells (DC) and Langerhans cells (LC) have been demonstrated, and evidence has been presented for both clathrin-coated pits and proteoglycans (PG) in the uptake pathway of VLP into epithelial cells. Therefore, we compared HPV-VLP uptake mechanisms in human monocyte-derived DC and LC, and their ability to cross-present HPV VLP-associated antigen in the MHC class I pathway. DC and LC each took up virus-like particles (VLP). DC uptake of and signalling by VLP was inhibited by amiloride or cytochalasin D (CCD), but not by filipin treatment, and was blocked by several sulfated and non-sulfated polysaccharides and anti-CD16. In contrast, LC uptake was inhibited only by filipin, and VLP in LC were associated with caveolin, langerin, and CD1a. These data suggest fundamentally different routes of VLP uptake by DC and LC. Despite these differences, VLP taken up by DC and LC were each able to prime naive CD8(+) T cells and induce cytolytic effector T cells in vitro. (C) 2004 Elsevier Inc. All rights reserved.

Regulation of adult olfactory neurogenesis by insulin-like growth factor-I

Insulin-like growth factor-I (IGF-I) has multiple effects within the developing nervous system but its role in neurogenesis in the adult nervous system is less clear. The adult olfactory mucosa is a site of continuing neurogenesis that expresses IGF-I, its receptor and its binding proteins. The aim of the present study was to investigate the roles of IGF-I in regulating proliferation and differentiation in the olfactory mucosa. The action of IGF-I was assayed in serum-free culture combined with bromodeoxyuridine-labelling of proliferating cells and immunochemistry for specific cell types. IGF-I and its receptor were expressed by globose basal cells (the neuronal precursor) and by olfactory neurons. IGF-I reduced the numbers of proliferating neuronal precursors, induced their differentiation into neurons and promoted morphological differentiation of neurons. The evidence suggests that IGF-I is an autocrine and/or paracrine signal that induces neuronal precursors to differentiate into olfactory sensory neurons. These effects appear to be similar to the cellular effects of IGF-I in the developing nervous system.

Role of growth hormone receptor signaling in osteogenesis from murine bone marrow progenitor cells

Growth hormone (GH) regulates many of the factors responsible for controlling the development of bone marrow progenitor cells (BMPCs). The aim of this study was to elucidate the role of GH in osteogenic differentiation of BMPCs using GH receptor null mice (GHRKO). BMPCs from GHRKO and their wild-type (WT) littermates were quantified by flow cytometry and their osteogenic differentiation in vitro was determined by cell morphology, real-time RT-PCR, and biochemical analyses. We found that freshly harvested GHRKO marrow contains 3% CD34 (hernatopoietic lineage), 43.5% CD45 (monocyte/macrophage lineage), and 2.5% CD106 positive (CFU-F/BMPC) cells compared to 11.2%, 45%, and 3.4% positive cells for (WT) marrow cells, respectively. When cultured for 14 days under conditions suitable for CFU-F expansion, GHRKO marrow cells lost CD34 positivity, and were markedly reduced for CD45, but 3- to 4-fold higher for CD106. While WT marrow cells also lost CD34 expression, they maintained CD45 and increased CD106 levels by 16-fold. When BMPCs from GHRKO mice were cultured under osteogenic conditions, they failed to elongate, in contrast to WT cells. Furthermore, GHRKO cultures expressed less alkaline phosphatase, contained less mineralized calcium, and displayed lower osteocalcin expression than WT cells. However, GHRKO cells displayed similar or higher expression of cbfa-1, collagen 1, and osteopontin mRNA compared to WT. In conclusion, we show that GH has an effect on the proportions of hematopoietic and mesenchymal progenitor cells in the bone marrow, and that GH is essential for both the induction and later progression of osteogenesis. (c) 2005 Elsevier Inc. All rights reserved.

Basal-like breast carcinomas: clinical outcome and response to chemotherapy

Background: Grade-III invasive ductal carcinomas of no special type (IDCs-NST) constitute a heterogeneous group of tumours with different clinical behaviour and response to chemotherapy. As many as 25% of all grade-III IDCs-NST are known to harbour a basal-like phenotype, as defined by gene expression profiling or immunohistochemistry for basal cytokeratins. Patients with basal-like breast carcinomas (BLBC) are reported to have a shorter disease-free and overall survival. Material and methods: A retrospective analysis of 49 patients with BLBC (as defined by basal cytokeratin expression) and 49 controls matched for age, nodal status and grade was carried out. Histological features, immunohistochemical findings for oestrogen receptor (ER), progesterone receptor (PgR) and HER2, and clinical outcome and survival after adjuvant chemotherapy were compared between the two groups. Results: It was more likely for patients with BLBCs to be found negative for ER (p < 0.0001), PgR (p < 0.0001) and HER2 (p < 0.01) than controls. Patients with BLBCs were found to have a significantly higher recurrence rate (p < 0.05) and were associated with significantly shorter disease-free and overall survival (both p, 0.05). In the group of patients who received anthracycline-based adjuvant chemotherapy (BLBC group, n = 47; controls, n = 49), both disease-free and overall survival were found to be significantly shorter in the BLBC group (p < 0.05). Conclusions: BLBCs are a distinct clinical and pathological entity, characterised by high nuclear grade, lack of hormone receptors and HER2 expression and a more aggressive clinical course. Standard adjuvant chemotherapy seems to be less effective in these tumours and new therapeutic approaches are indicated.

Suppression and overexpression of adenosylhomocysteine hydrolase-like protein 1 (AHCYL1) influences zebrafish embryo development A - Possible role for AHCYL1 in inositol phospholipid signaling

Adenosylhomocysteine hydrolase-like protein 1 (AHCYL1) is a novel intracellular protein with similar to 50% protein identity to adenosyl homocysteine hydrolase (AHCY), an important enzyme for metabolizing S-adenosyl-L-homocysteine, the by-product of S-adenosyl-L-homomethionine-dependent methylation. AHCYL1 binds to the inositol 1,4,5-trisphosphate receptor, suggesting that AHCYL1 is involved in intracellular calcium release. We identified two zebrafish AHCYL1 orthologs(zAHCYL1A and -B) by bioinformatics and reverse transcription-PCR. Unlike the ubiquitously present AHCY genes, AHCYL1 genes were only detected in segmented animals, and AHCYL1 proteins were highly conserved among species. Phylogenic analysis suggested that the AHCYL1 gene diverged early from AHCY and evolved independently. Quantitative reverse transcription-PCR showed that zAHCYL1A and -B mRNA expression was regulated differently from the other AHCY-like protein zAHCYL2 and zAHCY during zebrafish embryogenesis. Injection of morpholino antisense oligonucleotides against zAHCYL1A and -B into zebrafish embryos inhibited zAHCYL1A and -B mRNA translation specifically and induced ventralized morphologies. Conversely, human and zebrafish AHCYL1A mRNA injection into zebrafish embryos induced dorsalized morphologies that were similar to those obtained by depleting intracellular calcium with thapsigargin. Human AHCY mRNA injection showed little effect on the embryos. These data suggest that AHCYL1 has a different function from AHCY and plays an important role in embryogenesis by modulating inositol 1,4,5-trisphosphate receptor function for the intracellular calcium release.

Expression of human DEC-205 (CD205) multilectin receptor on leukocytes

DEC-205 (CD205) belongs to the macrophage mannose receptor family of C-type lectin endocytic receptors and behaves as an antigen uptake/processing receptor for dendritic cells (DC). To investigate DEC-205 tissue distribution in human leukocytes, we generated a series of anti-human DEC-205 monoclonal antibodies (MMRI-5, 6 and 7), which recognized epitopes within the C-type lectin-like domains 1 and 2, and the MMRI-7 immunoprecipitated a single similar to 200 kDa band, identified as DEC-205 by mass spectrometry. MMRI-7 and another DEC-205 mAb (MG38), which recognized the epitope within the DEC-205 cysteine-rich and fibronectin type II domain, were used to examine DEC-205 expression by human leukocytes. Unlike mouse DEC-205, which is reported to have predominant expression on DC, human DEC-205 was detected by flow cytometry at relatively high levels on myeloid blood DC and monocytes, at moderate levels on B lymphocytes and at low levels on NK cells, plasmacytoid blood DC and T lymphocytes. MMRI-7 F(ab')(2) also labeled monocytes, B lymphocytes and NK cells similarly excluding reactivity due to non-specific binding of the mAb to Fc gamma R. Tonsil mononuclear cells showed a similar distribution of DEC-205 staining on the leukocytes. DEC-205-specific semiquantitative immunoprecipitation/western blot and quantitative reverse transcriptase-PCR analysis established that these leukocyte populations expressed DEC-205 protein and the cognate mRNA. Thus, human DEC-205 is expressed on more leukocyte populations than that were previously assumed based on mouse DEC-205 tissue localization studies. The broader DEC-205 tissue expression in man is relevant to clinical DC targeting strategies and DEC-205 functional studies.

Papillomavirus virus-like particles activate the PI3-kinase pathway via alpha-6 beta-4 integrin upon binding

We have previously shown that human papillomavirus virus-like particles (VLPs) are able to activate the Ras/MAP kinase pathway. Ras can also elicit an anti-apoptotic signal via PI3-kinase so we investigated this further. Here we show that binding of VLPs from HPV types 6b, 18, 3 1, 35 and BPV1 results in activation of PI3-kinase. Activation was achieved by either L1 or L1/L2 VLPs and was dependent on both VLP-cell interaction and correct conformation of the virus particle. VLP-induced PI3-kinase activity resulted in efficient downstream signaling to Akt and consequent phosphorylation of FKHR and GSK3 beta. We also present evidence that PV signaling is activated via the alpha 6 beta 4 integrin. These data suggest that papillomaviruses use a common receptor that is able to signal through to Ras. Combined activation of the Ras/MAP kinase and PI3-kinase pathways may be beneficial for the virus by increasing cell numbers and producing an environment more conducive to infection. (c) 2006 Elsevier Inc. All rights reserved

Protease-activated receptor-2 peptides activate neurokinin-1 receptors in the mouse isolated trachea

Protective roles for protease-activated receptor-2 (PAR2) in the airways including activation of epithelial chloride (Cl-) secretion are based on the use of presumably PAR(2)-selective peptide agonists. To determine whether PAR(2) peptide-activated Cl- secretion from mouse tracheal epithelium is dependent on PAR(2), changes in ion conductance across the epithelium [short-circuit current (I-SC)] to PAR(2) peptides were measured in Ussing chambers under voltage clamp. In addition, epithelium and endothelium-dependent relaxations to these peptides were measured in two established PAR(2) bioassays, isolated ring segments of mouse trachea and rat thoracic aorta, respectively. Apical application of the PAR(2) peptide SLIGRL caused increases in I-SC, which were inhibited by three structurally different neurokinin receptor-1 (NK1R) antagonists and inhibitors of Cl- channels but not by capsaicin, the calcitonin gene-related peptide (CGRP) receptor antagonist CGRP(8-37), or the nonselective cyclooxygenase inhibitor indomethacin. Only high concentrations of trypsin caused an increase in I-SC but did not affect the responses to SLIGRL. Relaxations to SLIGRL in the trachea and aorta were unaffected by the NK1R antagonist nolpitantium (SR 140333) but were abolished by trypsin desensitization. The rank order of potency for a range of peptides in the trachea I-SC assay was 2-furoyl-LIGRL > SLCGRL > SLIGRL > SLIGRT > LSIGRL compared with 2-furoyl-LIGRL > SLIGRL > SLIGRT > SLCGRL (LSIGRL inactive) in the aorta relaxation assay. In the mouse trachea, PAR(2) peptides activate both epithelial NK1R coupled to Cl- secretion and PAR(2) coupled to prostaglandin E-2-mediated smooth muscle relaxation. Such a potential lack of specificity of these commonly used peptides needs to be considered when roles for PAR(2) in airway function in health and disease are determined.

Solution structure and novel insights into the determinants of the receptor specificity of human relaxin-3

Relaxin- 3 is the most recently discovered member of the relaxin family of peptide hormones. In contrast to relaxin- 1 and - 2, whose main functions are associated with pregnancy, relaxin- 3 is involved in neuropeptide signaling in the brain. Here, we report the solution structure of human relaxin- 3, the first structure of a relaxin family member to be solved by NMR methods. Overall, relaxin- 3 adopts an insulin- like fold, but the structure differs crucially from the crystal structure of human relaxin- 2 near the B- chain terminus. In particular, the B- chain C terminus folds back, allowing Trp(B27) to interact with the hydrophobic-core. This interaction partly blocks the conserved RXXXRXXI motif identified as a determinant for the interaction with the relaxin receptor LGR7 and may account for the lower affinity of relaxin- 3 relative to relaxin for this receptor. This structural feature is likely important for the activation of its endogenous receptor, GPCR135.

Characterization of receptors for calcitonin gene-related peptide and adrenomedullin on the guinea-pig vas deferens

1. The receptors which mediate the effects of calcitonin gene-related peptide (CGRP), amylin and adrenomedullin on the guinea-pig vas deferens have been investigated. 2. All three peptides cause concentration dependant inhibitions of the electrically stimulated twitch response (pD 2s for CGRP, amylin and adrenomedullin of 7.90 ± 0.11, 7.70 ± 0.19 and 7.25 ± 0.10 respectively). 3. CGRP 8-37 (1 μM) and AC187 (10 μM) showed little antagonist activity against adrenomedullin. 4. Adrenomedullin 22-52 by itself inhibited the electrically stimulated contractions of the vas deferens and also antagonized the responses to CGRP, amylin and adrenomedullin. 5. [ 125I]-adrenomedullin labelled a single population of binding sites in vas deferens membranes with a pIC 50 of 8.91 and a capacity of 643 fmol mg -1. Its selectivity profile was adrenomedullin > AC187 > CGRP = amylin. It was clearly distinct from a site labelled by [ 125I]-CGRP (pIC 50 = 8.73, capacity = 114 fmol mg -1, selectivity CGRP > amylin = AC187 > adrenomedullin). [ 125I]-amylin bound to two sites with a total capacity of 882 fmol mg -1. 6. Although CGRP has been shown to act at a CGRP 2 receptor on the vas deferens with low sensitivity to CGRP 8-37, this antagonist displaced [ 125I]-CGRP with high affinity from vas deferens membranes. This affinity was unaltered by increasing the temperature from 4°C to 25°C, suggesting the anomalous behaviour of CGRP 8-37 is not due to temperature differences between binding and functional assays.

Structural determinants for binding to CGRP receptors expressed by human SK-N-MC and Col 29 cells:Studies with chimeric and other peptides

1. Structure-activity relationships for the binding of human α-calcitonin gene-related peptide 8-37 (hαCGRP8-37) have been investigated at the CGRP receptors expressed by human SK-N-MC (neuroblastoma) and Col 29 (colonic epithelia) cells by radioligand binding assays and functional assays (hαCGRP stimulation of adenylate cyclase). 2. On SK-N-MC cells the potency order was hαCGRP8-37 > hαCGRP19-37 = AC187 > rat amylin8-37 > hα[Tyr0]-CGRP28-37 (apparent pKBS of 7.49 ± 0.25, 5.89 ± 0.20, 6.18 ± 0.19, 5.85 ± 0.19 and 5.25 ± 0.07). The SK-N-MC receptor appeared CGRP1-like. 3. On Col 29 cells, only hαCGRP8-37 of the above compounds was able to antagonize the actions of hαCGRP (apparent pKB = 6.48 ± 0.28). Its receptor appeared CGRP2-like. 4. hα[Ala11,18]-CGRP8-37, where the amphipathic nature of the N-terminal α-helix has been reduced, bound to SK-N-MC cells a 100 fold less strongly than hαCGRP8-37. 5. On SK-N-MC cells, hαCGRP(8-18, 28-37) (M433) and mastoparan-hαCGRP28-37 (M432) had apparent pKBS of 6.64 ± 0.16 and 6.42 ± 0.26, suggesting that residues 19-27 play a minor role in binding. The physico-chemical properties of residues 8-18 may be more important than any specific side-chain interactions. 6. M433 was almost as potent as hαCGRP8-37 on Col 29 cells (apparent pKB = 6.17 ± 0.20). Other antagonists were inactive.

The selectivity and structural determinants of peptide antagonists at the CGRP receptor of rat, L6 myocytes

1. Potency orders were determined for a series of agonists and antagonists on the calcitonin gene-related peptide (CGRP) receptor of rat L6 myocytes. The agents tested were all shown to have been active against CGRP, amylin or adrenomedullin receptors. 2. AC187 had a PIC50 Of 6.8 ± 0.10, making it 14 fold less potent as an antagonist than CGRP8-37 (PIC50, 7.95 ± 0.14). Amyline8-37 was equipotent to AC187 (PIC50, 6.6 ± 0.16) and CGRP19-37 was a fold less potent than either (pIC50 6.1 ± 0.24). 3. [Ala11]-CGRP8-37 was 6 fold less potent than CGRP8-37, (pIC50 7.13 ± 0.14), whereas [Ala18] CGRP8-37 was approximately equipotent to CGRP8-37 (pIC50, 7.52 ± 0.15). However, [Ala11,Ala18]- CGRP8-37 was over 300 fold less potent than CGRP8-37 (pIC50, 5.30 ± 0.04). 4. [Tyr0]-CGRP28-37, amylin19-37 and adrenomedullin22-52 were inactive as antagonists at concentrations of up to 1 μM. 5. Biotinyl-human α-CGRP was 150 fold less potent than human α-CGRP itself (EC50 values of 48 ± 17 nM and 0.31 ± 0.13 nM, respectively). At 1 μM, [Cys(acetomethoxy)(2'7)]-CGRP was inactive as an agonist. 6. These results confirm a role for Arg11 in maintaining the high affinity binding of CGRP8-37. Arg18 is of less direct significance for high affinity binding, but it may be important in maintaining the amphipathic nature of CGRP and its analogues.

Multiple receptors for calcitonin gene-related peptide and amylin on guinea-pig ileum and vas deferens

1. The responses of the electrically stimulated guinea-pig ileum and vas deferens to human and rat calcitonin gene-related peptide (CGRP) and amylin were investigated. 2. The inhibition of contraction of the ileum produced by human alpha CGRP was antagonized by human alpha CGRP8-37 (apparent pA2 estimated at 7.15 +/- 0.23) > human alpha CGRP19-37 (apparent pA2 estimated as 6.67 +/- 0.33) > [Tyr0]-human alpha CGRP28-37. The amylin antagonist, AC187, was three fold less potent than CGRP8-37 in antagonizing human alpha CGRP. 3. Both human beta- and rat alpha CGRP inhibited contractions of the ileum, but this was less sensitive to inhibition by CGRP8-37 than the effect of human alpha CGRP. However, CGRP19-37 was twenty times more effective in inhibiting the response to rat alpha CGRP (apparent pA2 estimated as 8.0 +/- 0.1) compared to human alpha CGRP. 4. Rat amylin inhibited contractions in about 10% of ileal preparations; this effect was not antagonized by any CGRP fragment. Human amylin had no action on this preparation. 5. Both human and rat alpha CGRP inhibited electrically stimulated contractions of the vas deferens, which were not antagonized by 3 microM CGRP8-37 or 10 microM AC187. 6. Rat amylin inhibited the stimulated contractions of the vas deferens (EC50 = 77 +/- 9 nM); human amylin was less potent (EC50 = 213 +/- 22 nM). The response to rat amylin was antagonized by 10 microM CGRP8-37 (EC50 = 242 +/- 25 nM) and 10 microM AC187 (EC50 = 610 +/- 22 nM). 7. It is concluded that human alpha CGRP relaxes the guinea-pig ileum via CGRP1-like receptors, but that human beta CGRP and rat alpha CGRP may use additional receptors. These are distinct CGRP2-like and amylin receptors on guinea-pig vas deferens.

Calcitonin gene-related peptide:multiple actions, multiple receptors

Calcitonin gene-related peptide (CGRP) shows diversity both in its effects and its receptors. It is likely to have roles as a neurotransmitter, neuromodulator, local hormone and trophic factor. Its effects include rapid changes in neuronal activity, relaxation of many type of smooth muscle, actions on metabolism and changes in gene expression. Receptor heterogenecity has been revealed from experiments comparing agonist potency ratios and antagonists affinities. the evidence from these approaches is reviewed in this article and a speculative receptor classification scheme is proposed. Some of the likely future directions for CGRP research are discussed. © 1993.