Serum proteomic profiling at diagnosis predicts clinical course, and need for intensification of treatment in inflammatory bowel disease.

BACKGROUND: Success in personalized medicine in complex disease is critically dependent on biomarker discovery. We profiled serum proteins using a novel proximity extension assay [PEA] to identify diagnostic and prognostic biomarkers in inflammatory bowel disease [IBD]. METHODS: We conducted a prospective case-control study in an inception cohort of 552 patients [328 IBD, 224 non-IBD], profiling proteins recruited across six centres. Treatment escalation was characterized by the need for biological agents or surgery after initial disease remission. Nested leave-one-out cross-validation was used to examine the performance of diagnostic and prognostic proteins. RESULTS: A total of 66 serum proteins differentiated IBD from symptomatic non-IBD controls, including matrix metallopeptidase-12 [MMP-12; Holm-adjusted p = 4.1 × 10-23] and oncostatin-M [OSM; p = 3.7 × 10-16]. Nine of these proteins are associated with cis-germline variation [59 independent single nucleotide polymorphisms]. Fifteen proteins, all members of tumour necrosis factor-independent pathways including interleukin-1 (IL-1) and OSM, predicted escalation, over a median follow-up of 518 [interquartile range 224-756] days. Nested cross-validation of the entire data set allowed characterization of five-protein models [96% comprising five core proteins ITGAV, EpCAM, IL18, SLAMF7 and IL8], which define a high-risk subgroup in IBD [hazard ratio 3.90, confidence interval: 2.43-6.26], or allowed distinct two- and three-protein models for ulcerative colitis and Crohn's disease respectively. CONCLUSION: We have characterized a simple oligo-protein panel that has the potential to identify IBD from symptomatic controls and to predict future disease course. Further prospective work is required to validate our findings.

Anti-inflammatory potential of melanocortin receptor-directed drugs.

Melanocortin receptor-based drug discovery is particularly active in the field of neuroendocrine systems and is mostly related to food intake and novel obesity therapies. The immunomodulatory and anti-inflammatory effects of nonpeptidic, low molecular weight compounds activating the melanocortin-1 receptor (MC1R) provide a new principle for treating various types of inflammation, such as dermal, joint, and gastrointestinal, probably by virtue of the effects acting through modulation of proinflammatory and anti-inflammatory cytokines. Several reports demonstrate that alpha-MSH, for example, has anti-inflammatory effects in different models. The aim of our study was to design, synthesize, and characterize compounds that bind to and activate the MC1R in vitro. The binding affinities are submicromolar to this receptor, and activation of the receptor (cAMP assay) varies from full agonists to partial agonists as well as antagonists. In vivo, the compounds exert prominent anti-inflammatory effects, with efficacy in the same range as that of dexamethasone, for example. The potential advantages of MC1R-based anti-inflammatory effects versus glucocorticosteroids, for example, are that the latter, albeit exerting prominent anti-inflammatory effects, also have many side effects that most likely will not characterize an MC1R-based anti-inflammatory drug.

Latanoprost-induced increase of tyrosinase transcription in iridial melanocytes.

PURPOSE: Latanoprost, the active principle of Xalatan eye drops, has been shown to cause increased iridial pigmentation as a side-effect in some patients. The purpose of the present study was to investigate whether latanoprost affects tyrosinase, the rate limiting enzyme in melanogenesis, at the gene transcription level in the iridial melanocytes. METHODS: Four cynomolgus monkeys were treated unilaterally with 3 or 11 microg latanoprost once daily for 10 days. The contralateral eye received the vehicle only. Tyrosinase mRNA was visualized by in situ hybridization using radio-labelled riboprobes. The transcription of tyrosinase was also studied in vitro using cultivated human iridial melanocytes. Tyrosinase RNA was quantified by Northern blotting. RESULTS: In the monkeys transcription of tyrosinase was found to be increased in iridial melanocytes of the treated eyes compared to the control eyes. Increased transcription of tyrosinase was in addition found in the iridial pigment epithelium and in melanocytes of the anterior choroid. Latanoprost was also found to increase the transcription of tyrosinase in melanocytes isolated from at least one human eye. CONCLUSIONS: Although the tyrosinase enzyme has to undergo complex post-translational modification to become biologically active, which we have not studied, it appears that latanoprost treatment may increase the transcription of the tyrosinase gene in some individuals, consistent with increased melanogenesis in the iridial melanocytes leading to darker eye colour.

Effect of latanoprost on the extracellular matrix of the ciliary muscle. A study on cultured cells and tissue sections.

Prostaglandin F2alpha and its analogue latanoprost, both prostanoid FP receptor agonists, reduce the intraocular pressure mainly by enhancing uveoscleral outflow. Changes in the extracellular matrix of the ciliary muscle may be involved in the increased outflow. The effect of latanoprost and prostaglandin F2alpha on the extracellular matrix of the ciliary muscle was investigated. Cell cultures of human ciliary muscle were treated with latanoprost acid or prostaglandin F2alpha for 1-2 days and were immunostained against various extracellular matrix components and metalloproteinases. Proteinases were also analysed by zymography and by measuring plasmin generating ability. For comparison, matrix components were immunolocalized on tissue sections from monkey eyes, treated topically once daily with latanoprost for 10 days. In response to both prostaglandins collagens I, III, and IV, fibronectin, laminin and hyaluronan were reduced, while metalloproteinase -2 and -3 were increased. Zymography demonstrated the presence of functionally active metalloproteinase -2. Both prostaglandins enhanced the generation of plasmin, an activator of metalloproteinases. In the anterior part of the ciliary muscle in latanoprost-treated eyes immunostained collagen VI was decreased in 5 out of 5 monkeys and collagen IV was decreased in 4 of the 5 monkeys. These results suggest a role for latanoprost in the remodeling of extracellular matrix in the ciliary muscle. A latanoprost-induced change in the extracellular matrix might augment the flow of aqueous humour through the ciliary muscle bundles of the uveoscleral pathway.

The molecular biology and ocular distribution of prostanoid receptors.

Enormous progress has been made in the characterization of prostanoid receptors during the past five years. Molecular biological studies have enabled structural identification of all the human prostanoid receptors that had been proposed according to pharmacological criteria. The pharmacological classification proposed different receptor subtypes for prostaglandins D2, E2, F2 alpha, I2 and thromboxane A2 which were termed DP, EP, FP, IP and TP, respectively. Further subdivision for only the EP receptor has been reported and EP1, EP2, EP3, and EP4 subtypes have been unequivocally identified. The molecular structure of all prostanoid receptors is typical of that for G protein-coupled receptors and consists of seven alpha-helical transmembrane domains, three extracellular loops and an amino terminus, and three intracellular loops and a carboxyl terminus. Interestingly, mRNA alternative splice variants of the carboxyl termini have been found to determine G protein interactions for the EP3 receptor. Application of molecular biological techniques is beginning to make an impact in ocular research, where precise localization of receptors is difficult by more traditional methods because of the diminutive size of most ocular tissues. In situ hybridization and immunohistochemical studies using antibodies against the cloned human FP receptor have already suggested an unexpectedly wide distribution in the monkey eye. Transgenic studies involving FP receptor knock-out animals may provide future insight into the role of this receptor in glaucoma. However, since prostaglandins are extraordinarily effective in reducing intraocular pressure, it follows that traditional physiological and pharmacological studies retain a key role in glaucoma research. Studies in perfused human anterior segment organ culture have revealed that although prostaglandin F2 alpha does not facilitate trabecular aqueous humor outflow, prostaglandin E1 does increase trabecular outflow. Thus, different prostanoids may lower intraocular pressure by distinctly different mechanisms of action. Recent studies also indicate that prostanoids may exert a beneficial effect on retinal blood perfusion and may even act as neuroprotective agents.

Localization of the prostaglandin F2 alpha receptor in rat tissues.

The localization of the prostaglandin F2alpha (FP) receptor was examined in rat tissues by immunohistochemistry and in situ hybridization. Immunohistochemistry on paraffin sections was performed with a rabbit polyclonal antiserum raised against a synthetic peptide derived from the rat FP receptor sequence. In situ hybridization on cryosections was done with 35S-labelled rat FP receptor antisense and sense riboprobes. The most intense FP receptor-like immunoreactivity was observed in granulosa luteal cells, muscle and epithelial cells, e.g. cardiac, skeletal and smooth muscle, and hepatocytes. Weaker immunoreactivity was found in connective tissue fibroblasts. In the eye, intense immunostaining was associated with the corneal and conjunctival epithelium and moderate staining with the ciliary body, retina, iris and connective tissues. In situ hybridization generally confirmed the results. The riboprobe hybridized weakly with the heart, skeletal muscle, uterus, liver, lung and corpus luteum. Thus, the prostaglandin FP receptor was found to be widely distributed in rat tissues.

Localization of the prostaglandin F2 alpha receptor messenger RNA and protein in the cynomolgus monkey eye.

PURPOSE: To determine the distribution of the prostaglandin F2 alpha (FP) receptor within the monkey eye. METHODS: The expression and localization of the FP receptor was studied by in situ hybridization and immunohistochemistry. Cryosections of the eye were hybridized with a 35S-labeled FP receptor riboprobe, and paraffin sections were immunostained with polyclonal antibodies against an FP receptor peptide. Reverse transcriptase-polymerase chain reaction (RT-PCR) was performed on cultured cell populations from the eye. RESULTS: Results of the three methods largely correlated with each other. The highest expression of FP receptor mRNA and protein was found in the corneal, conjunctival, and iridial epithelium, the ciliary muscle, and ciliary processes. Iridial and choroidal melanocytes, the retina, and the optic nerve expressed lower levels of both FP receptor message and protein. Using immunohistochemistry, the FP receptor protein was found in connective tissue fibroblasts, the corneal endothelium, and the vasculature; however, FP receptor expression was not detected using in situ hybridization. With RT-PCR, cultured retinal pigment epithelial and ciliary muscle cells from the cynomolgus monkey eye were found to express the FP receptor. CONCLUSIONS: The FP receptor was found to be distributed widely in the ocular tissues, suggesting an array of autocrine and paracrine functions of PGF2 alpha in the eye.

Immortalization of cat iris sphincter smooth muscle cells by SV40 virus: growth, morphological, biochemical and pharmacological characteristics.

The purpose of this study was to establish immortalized cell cultures of cat iris sphincter smooth muscle cells for a model investigating ocular receptors and their signal transduction pathways. Cultured cat iris sphincter muscle cells were immortalized by viral transformation with SV40 virus and the morphological and immunocytochemical properties of the normal and immortalized cells were investigated. The transformed cell clone, SV-CISM-2, was further characterized biochemically and pharmacologically. The normal muscle cells showed characteristics of smooth muscle cells, as judged by their growth and the presence of smooth muscle alpha-actin and desmin. After seven passages the normal cells ceased to proliferate. In contrast, the immortalized cells retained their proliferative ability for more than 220 population doublings over 55 passages. The transformation phenotype in these cells was confirmed by their expression of the large T-antigen, the incorporation of viral DNA into cellular DNA, growth in agarose and in low-serum medium, and complete loss of contact inhibition. The immortalized cells expressed smooth muscle alpha-actin, desmin and MLC protein. Biochemical and pharmacological studies on the SV-CISM cells revealed the presence of several functional receptors including muscarinic cholinergic, beta-adrenergic, peptidergic (substance P and endothelin). Platelet-activating factor, and prostaglandin (PG). Muscarinic stimulation of these cells resulted in: (a) a dose-dependent increase in the release of arachidonic acid (AA) and (PGs) and enhancement in the production of inositol trisphosphate (IP3); and (b) a substantial increase in MLC phosphorylation (118%), an indicator of smooth muscle contractility. The stimulatory effects of carbachol on these responses were completely blocked by atropine, a muscarinic receptor antagonist. This study constitutes the first successful immortalization of iris sphincter smooth muscle cells. The SV-CISM-2 cells can serve as an important model system for investigations on the biochemical and pharmacological properties of receptors and their signal transduction pathways in smooth muscle. The advantage of these cells over normal iris sphincter cells is that they can be propagated over many generations without alterations in their morphological, biochemical and physiological characteristics.

Inflammatory response in the rabbit eye after intraocular implantation with poly(methyl methacrylate) and heparin surface modified intraocular lenses.

Early inflammatory responses in rabbit eyes after anterior chamber implantation with poly(methyl methacrylate) (PMMA) and heparin surface modified intraocular lenses (IOLs) and after cataract extraction and implantation with PMMA and heparin surface modified intraocular lenses (IOLs) were investigated between 1 and 30 days postoperatively. The number of white blood cells in the aqueous humor, the distribution of white blood cell subsets, and the interleukin-1 (IL-1b) levels were studied. At one day postoperatively, there was a significantly smaller number of white blood cells in eyes with heparin surface modified IOLs than in eyes with PMMA IOLs after both anterior chamber implantation and cataract surgery and capsular bag implantation. At one day postoperatively, 87% of the cell population in eyes with PMMA IOLs were neutrophilic granulocytes. Two days later the neutrophilic granulocytes had decreased to 13% and at seven days they were gone. At 14 days the frequency was 7% and at 30 days it was 27%. The fraction of monocytes/macrophages in the cell population was 9% (day 1), 79% (day 3), 94% (day 7), 81% (day 14), and 72% (day 30). The fraction of lymphocytes was low throughout the test period (1% to 10%). The percentages were almost the same in eyes with heparin surface modified IOLs and there was no significant difference in the distribution of white blood cell subsets between the two lens materials. The IL-1 was not detected in any of the samples (day 3, day 7, and day 30). Inactivated macrophages and/or levels under detection limits could be the reason.(ABSTRACT TRUNCATED AT 250 WORDS)