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BACKGROUND: Ulcerative colitis (UC) is a chronic, idiopathic inflammatory condition affecting the colonic epithelium, with the inflammasome, T cells, complement activation, and microbiome dysbiosis contributing to pathogenesis. AIMS: We applied a previously established method of microarray molecular analysis to a set of 71 UC biopsies (from 61 patients), to elucidate the molecular changes associated with active UC, specifically comparing UC to T-cell mediated rejection (TCMR), as a prototype of a sterile, T-cell mediated disease. METHODS: 71 for-cause UC colonic biopsies were collected at the U of A Hospital in Edmonton, AB and Cedars-Sinai Hospital in LA, California. These biopsies were processed using Affymetrix GeneChip microarrays and the data analyzed in R programming language. Gene expression data was displayed using volcano plots (showing the fold change and association between the genes and endoscopic Mayo score) and heatmaps (showing expression of the top 30 genes in a cell panel). We then analyzed overexpression of the top genes using the DAVID tool(1), and compared the results to similar results for top genes overexpressed in TCMR. RESULTS: The volcano plot (Figure 1) showed strong associations between the endoscopic Mayo score and decay accelerating factor (CD55), and moderate associations with calprotectin genes (S100A8/S100A9), with lesser associations for effector T cell transcripts (i.e. CTLA4, interferon gamma (IFNG), and chemokine ligand (CXCL13)), many IFNG inducible transcripts, inflammasome transcripts (CASP1), and toll-like receptors (TLR5). NLRP3 (incriminated in mouse UC models) did not pass the IQR filter and was not significant. Expression of the top genes in a cell panel showed primary expression in monocytes, macrophages, and dendritic cells, with some expression in epithelial and endothelial cells, while minimal expression was found in CD4/CD8 T cells, or in NK cells. Pathway analysis showed major differences between pathway terms. CONCLUSIONS: Our findings show that while transcripts associated with cognate T cell inflammatory processes (TCMR) are expressed in UC, there are substantial differences between the pathogenesis of a pure T cell-mediated disease and UC. In our cohort, the top genes associated with the endoscopic Mayo score in UC were not those associated with effector T cells: T cell-associated transcripts were only moderately associated with the Mayo score, and pathway analysis showed significant differences between TCMR and UC, which is much more an inflammatory environment with strong associations to CD55. This suggests that cognate T cell recognition is present in UC but supplemented by a second source of inflammation. REFERENCES: 1. Huang et al. Systematic and integrative analysis of large gene lists using DAVID bioinformatics resources. Nature Protocols (2009). FUNDING AGENCIES: None

trans-Urocanic acid (UCA) acts as a chromophore for UV radiation in the epidermis and isomerizes to cis-UCA which then initiates some of the changes leading to UV-induced immunosuppression. The mechanism of the immunomodulation by cis-UCA is unknown at present, but one possibility is that the interaction between cis-UCA and keratinocytes causes the release of immunosuppressive cytokines locally. To test this hypothesis, PAM-212 cells, a murine keratinocyte cell line, were incubated with 0.10–100 μg/mL trans- and cis-UCA for 6 or 24 h, respectively. The expression of interleukin (IL)-10, transforming growth factor (TGF)-β and tumor necrosis factor (TNF)-α messenger RNA (mRNA) was then measured by reverse transcription-polymerase chain reaction in comparison with the mRNA for the house-keeping gene, β-actin. No change or significant induction of any of the cytokine messages occurred. However, the expression of IL-10 messenger RNA (mRNA) was induced 24 h after UVB irradiation (300 J/m2) and that of TNF-α mRNA occurred 6 h after treatment with phorbol myristate acetate. The expression of IL-10 protein was also examined by immunostaining in both PAM-212 cells and B16-F10 murine melanoma cells between 3 and 48 h after incubation with 10 and 100 μg/mL cis- and trans-UCA. No alteration was seen with either isomer at either concentration. In contrast, UVB irradiation of both cell lines resulted in a marked increase in intracellular IL-10 protein at 24 and 48 h. Therefore the upregulation of the immunosuppressive cytokines, IL-10, TNF-α and TGF-β, in keratinocytes is unlikely to be the mechanism by which cis-UCA induces immunosuppression in mice.

The binding of unfractionated heparin to endothelium is thought to be responsible for the rapid and saturable phase of unfractionated heparin clearance. Thrombin can induce endothelial cells to express and/or secrete a number of heparin binding proteins that have the potential to increase the binding of unfractionated heparin and to a lesser extent the binding of low molecular weight heparin. To explore this possibility, we examined the binding of unfractionated heparin and low molecular weight heparin to thrombin-activated endothelial cells. Cultured human umbilical vein endothelial cells were used to determine the binding of 125 I-labeled unfractionated heparin and low molecular weight heparin to untreated and to thrombin-activated cells. After thrombin treatment, we obtained a time-dependent increase in the binding of radiolabeled unfractionated heparin. In contrast, there was much less binding of low molecular weight heparin, and a time-dependent increase was not apparent. After 30, 45, and 60 minutes of thrombin treatment, the binding of unfractionated heparin was significantly higher than that of low molecular weight heparin. The increase in binding of unfractionated heparin to thrombin-activated cells also was demonstrated using fluorescently labeled unfractionated heparin followed by fluorescence microscopy. The average fluorescence intensity of thrombin-treated cells increased by 44% when compared with resting cells. The present results indicate that thrombin can increase the binding of unfractionated heparin to human umbilical vein endothelial cells. Thus, an activated endothelium may contribute to the variability of the anticoagulant response to unfractionated heparin. In contrast, the binding of low molecular weight heparin is much less affected, which may account for its better bioavailability and longer half-life.

SummaryPreviously, we demonstrated in a rat model of heparin-induced osteoporosis that low molecular weight heparin (LMWH) produces less bone loss than unfractionated heparin, and that only heparin increases osteoclast number and activity. In contrast, both heparin and LMWH were found to decrease osteoblast function to a similar extent, possibly because at the doses tested both agents produced maximal inhibition. To examine the relative effects of heparin and LMWH on osteoblast function more closely we used an in vitro bone nodule assay, together with measurements of alkaline phosphatase (ALP) activity. Both agents inhibited bone nodule formation and ALP activity in a concentration-dependent manner, but 6 to 8-fold higher concentrations of LMWH were required to achieve equivalent effects. The effect of heparin on osteoblast function was both chain-length and negative charge-dependent because the ability of defined heparin fragments to inhibit nodule formation correlated with their molecular weight (r = 0.98), and N-desulfated heparin was less inhibitory than heparin. In contrast, the effect of heparin on osteoblast function was pentasaccharide-independent because heparin with low affinity for antithrombin had similar activity to heparin with high antithrombin activity. These findings help to explain mounting clinical evidence that the risk of osteoporosis is lower with LMWH than with heparin.

Abstract We have previously demonstrated that the nonspecific binding of unfractionated heparin (UFH) to plasma proteins has a marked modulating effect on its anticoagulant activity. Since some heparin-binding proteins are also acute-phase-reactant proteins, we explored the possibility that the induction of the acute-phase response can increase the plasma concentrations of heparin-binding proteins. The recovery of a fixed amount of UFH or low-molecular-weight heparin (LMWH) added in vitro to rat plasma samples obtained at various time intervals after the administration of intravenous endotoxin or subcutaneous turpentine was compared with that of saline-treated control animals. The anti–factor Xa activity was measured in the plasma samples before and after the addition of a chemically modified low-affinity heparin (LAH) to displace the proportion of the added heparin that is reversibly bound to plasma proteins. Our results show that at 6 hours post–endotoxin and at 24 hours post–turpentine treatment, virtually no anti–factor Xa activity could be measured in the plasma samples, while the expected levels were obtained for control plasma. After the addition of LAH to displace protein-bound UFH, essentially the same anti–factor Xa levels were measured in the plasma from all three treatment groups. These results indicate that induction of the acute-phase reaction can dramatically increase the levels of heparin-binding proteins in rat plasma. In addition, we compared the anti–factor Xa recovery of UFH with that of an LMWH from the plasma of endotoxin- and saline-treated rats and demonstrated that LMWH binds less to plasma proteins than UFH, even in plasma in which the levels of heparin-binding proteins are markedly elevated. The recovery of a fixed amount of UFH added in vitro to human plasma from septic patients was also reduced, but not to the same extent as seen in rat plasma. Removal of candidate heparin-binding and acute-phase proteins by immunodepletion indicated that vitronectin plays an important role in the nonspecific binding of UFH in patient plasma.

Cyclosporine A (CsA) has been shown to be an effective therapeutic agent for a wide variety of cutaneous diseases yet its exact mechanism of action is still unclear, although one well-defined effect of CsA is the inhibition of T-cell-derived cytokine expression. We recently demonstrated in vitro that CsA inhibits cell proliferation and suppresses cytokine gene expression in keratinocytes. In this study, we report the in vivo effects of CsA on skin cytokine gene expression as determined by reverse-transcriptase polymerase chain reaction. C57BL6 mice (female, 8-10 weeks old) were subcutaneously injected with CsA in olive oil (0, 5 and 10 mg/kg) every other day for 3 weeks. Treatment with 5 mg/kg CsA inhibited both interleu-kin (IL)-1Α and tumor necrosis factor Α gene expression by about 70 and 90%, respectively, relative to vehicle control levels. However, IL-6 gene expression did not significantly change. Injection of 10 mg/kg CsA inhibited expression of all three genes by 80-90% relative to control levels. These data show that CsA can inhibit constitutive cytokine gene expression in mouse skin.

Ultraviolet radiation can induce the transcription and release of cytokines from keratinocytes (KC's). These cytokines have the potential to modulate local and systemic immunologic responses. In this paper we report that northern blotting showed that human KC and KC lines expressed a 1.2-1.4 kb transcript for the chemokine and melanoma growth-stimulatory protein, GRO-alpha and that ultraviolet B radiation (UVB) could upregulate the expression of GRO-alpha mRNA and protein in the KC line A431. The GRO-alpha gene response to UVB was maximal at 48h post-irradiation with 70 J/m2. Reverse transcription-polymerase chain reaction (RT-PCR) revealed a 4.5-fold increase in GRO-alpha mRNA over basal levels (p < 0.001). GRO-alpha protein was measured in the culture media by enzyme-linked immunosorbent assay (ELISA). Media from unirradiated cultures contained 1166 +/- 83 pg/ml GRO-alpha protein. After UVB, a time-dependent increase in GRO-alpha protein was seen in the culture media from 6-48h. At 48h post-irradiation the GRO-alpha protein content was 27583 +/- 678 pg/ml, or 23 times the basal level. This protein release could be inhibited by 70% when the cells were pre-incubated with 10 micrograms/ml interleukin-1 receptor antagonist (IL-1RA). We also show that another potent leukocyte chemoattractant, Interleukin-8 (IL-8), was induced in A431 cells by UVB. This induction of IL-8 mRNA began as early as 3h post-irradiation, when it reached twice basal levels (p < 0.05) and reached 4.5-fold basal levels at 48h post-irradiation (p < 0.005).(ABSTRACT TRUNCATED AT 250 WORDS)