BACKGROUND

The condition of concomitant upper lobe emphysema and lower lobe fibrosis as identified by computer tomography is known as combined pulmonary fibrosis and emphysema (CPFE). CPFE has distinct clinical characteristics compared with emphysema alone (EA) and idiopathic pulmonary fibrosis (IPF) without emphysema. However, the pulmonary inflammation characteristics of CPFE are not well known, and the differences between CPFE and the other two diseases with regards to pulmonary inflammation need to be explored. The pulmonary inflammatory characteristics were investigated in CPFE patients and compared with EA and IPF.

METHODS

Fraction exhaled nitric oxide (Fe,NO) and differential cell counts, the concentrations of monokine induced by interferon gamma (MIG/CXCL9), interferon-inducible protein 10 (IP-10/CXCL10), and interferon-inducible T cell alpha chemoattractant (I-TAC/CXCL11) were measured in induced sputum obtained from subjects with CPFE (n = 22), EA (n = 22), IPF (n = 14), and healthy volunteers (HV, n = 12). In addition, immunohistochemistry was used to quantify the expression of nitric oxide synthases in alveolar macrophages in 23 lung tissues from patients and control subjects.

RESULTS

The CPFE group had higher alveolar NO than subjects in the EA and HV groups (P = 0.009, P = 0.001, respectively) but not than the IPF group (P>> 0.05). Numbers of sputum eosinophils were significantly elevated in CPFE and IPF groups compared with the HV group (P = 0.001, P = 0.008). In contrast, eosinophil counts in EA group did not differ from those in the HV group. Compared with the EA and HV groups, the CPFE group had a lower concentration of I-TAC/CXCL11 in sputum supernatants (P = 0.003, P = 0.004). Immunoreactivity for inducible nitric oxide synthase (iNOS) was higher in the CPFE group than in the EA group (P = 0.018, P = 0.006, respectively).

CONCLUSIONS

The pulmonary inflammation of CPFE group is more similar to IPF group, while the distal airway inflammation is more significant in CPFE and IPF groups than in EA group. Lung eosinophil cell infiltration and high NOS expression in alveolar macrophage might participate in this pathogenesis.

Inbred stains of mice display differential susceptibility to infection with the common foodborne pathogen Listeria monocytogenes (Lm). Previously, Listr1 and Listr2, two genetic loci that control differential sensitivity to Lm infection between BALB/cByJ and C57BL/6ByJ mice, were identified. To analyze the role of Listr1 in innate immune responses, we employed congenic mice (C.B6By-Listr1/Rag2 (-/-) ) bearing the C57BL/6ByJ-derived Listr1 locus on a BALB/c-Rag2 (-/-) background. Consistent with the results of a previous genetic analysis, the congenic mice showed increased susceptibility to Lm infection. The bacterial burden in the liver between the congenic and control lines was significantly different (P < 0.05) from 24 h postinfection with Lm. Analysis of genes within the Listr1 locus identified a frameshift mutation in the Cxcl11 gene of the C57BL/6 strain that prevents production of the mature chemokine CXCL11. No differences in inflammatory cell infiltration or cells expressing CXCR3 and CXCR7 which are the receptors of CXCL11 occurred because of CXCL11 deficiency in the congenic mice spleens. However, these mice lacked a distinct population of CD14(+) positive resident mononuclear cells that express intermediate levels of CXCR3 and CXCR7 in the liver. There were fewer microabscesses in the liver of CXCL11-deficient mice during the early stage of infection, which is consistent with their decreased ability to resist Lm. Our results, when taken together, show that the Listr1 locus plays an important role in early control of Lm infection in the mouse liver and that Cxcl11 is a candidate gene for disease severity within this locus.

BACKGROUND

It is well established that macrophage infiltration is involved in concanavalin A (conA)-induced liver injury. However, the role of macrophages in conA-induced renal injury remains unknown. The aims of this study were to investigate macrophage infiltration in conA-induced renal injury and determine whether paeoniflorin (PF) could inhibit macrophage infiltration into the kidney.

METHODS

BALB/C mice were pre-treated with or without PF 2 h (h) before conA injection. At 8 h after con A injection, all the mice were sacrificed; The liver and kidney histology were studied. The renal CD68 expression was detected by immunohistochemical and real-time PCR analysis. The level of expression of C-X-C chemokine receptor type 3 (CXCR3) was analyzed by western blot, immunohistochemical and real-time PCR. The pathophysiological involvement of CXCR3 in macrophage infiltration were investigated using dual-colour immunofluorescence microscopy.

RESULTS

PF administration significantly reduced the elevated serum levels of alanine transaminase (ALT), blood urea nitrogen (BUN), creatinine (Cr) and the severity of liver and renal damage compared with that in the conA-vehicle group. PF administration inhibited the increase in renal IL1β mRNA expression and concentration. Furthermore, immunohistochemical analysis showed that macrophages secreted CXCR3 in the kidneys of the conA-vehicle mice. Immunofluorescence microscopy demonstrated CXCR3 bound tightly to C-X-C motif ligand 11 (CXCL11) in the kidneys of the conA-vehicle mice and showed that PF treatment could suppress CXCR3/CXCL11 over-activation.

CONCLUSIONS

Macrophage infiltration was a notable pathological change in the kidneys of conA-treated mice. PF administration attenuated conA-induced renal damage, at least in part, by inhibiting the over-activated CXCR3/CXCL11 signal axis.

Retinoic acid-inducible gene-I (RIG-I) is a member of the DExH box family proteins, which have diverse roles in the regulation of gene expression and cellular functions. RIG-I is one of the factors regulated by interferon (IFN)-gamma and regarded as an intracellular signaling molecule for IFN-gamma. IFN-gamma is a major cytokine and also suggested to be involved in embryonal implantation and pregnancy. It is demonstrated that IFN-gamma stimulates endometrial epithelial cells to produce CXCL11, which is implicated in implantation. The aim of the present study was to investigate the effect of IFN-gamma on RIG-I expression in HeLa cells, a cell line derived from human uterine carcinoma. We found that RIG-I mRNA and protein were expressed in HeLa cells stimulated with IFN-gamma. The effect of IFN-gamma was observed in concentration- and time-dependent manners. The RNA interference against RIG-I resulted in the suppression of the IFN-gamma-induced CXCL11 expression. Immunohistochemical studies revealed the RIG-I expression in the normal human endometrium, suggesting a possible role of RIG-I in human reproductive organs.

In order to understand human inflammatory diseases and to develop and assess new therapeutic strategies targeting leukocyte recruitment to tissue, it is necessary to study human lymphocyte interactions with endothelium. It is often not practical to carry out assays on fresh human samples and therefore cells may be cryopreserved and batched for later study. Furthermore, many forms of adoptive cell therapy use cryopreserved cells that are required to migrate to tissue after infusion in vivo. The consequences of cryopreservation on the adhesion and migration of leukocytes is not known leading us to study the effects of cryopreservation on lymphocyte phenotype, migration, and adhesion. Cryopreservation and subsequent thawing did not alter the proportion of retrieved T cell subsets. Overall levels of expression of β1 or β2 integrins were unaffected but marked changes were observed in other relevant receptors. Expression of CD69, a transmembrane protein that plays a critical role in lymphocyte egress from tissues and the chemokine receptor CXCR4, increased on thawed populations and levels of CD62L and CXCR3 were reduced on thawed cells but restored if cells were allowed to recover after thawing. These changes were associated with modulation of the ability of lymphocytes to migrate across cytokine-stimulated monolayers of endothelium toward recombinant CXCL11 and CXCL12. Thus cryopreservation and thawing of lymphocytes induces changes in their adhesive phenotype and modulates their ability to migrate across endothelial monolayers. These findings have implications for in vitro experimentation and for cell therapy in which cryopreserved cells are expected to migrate when reinfused into patients.

The current study was undertaken to explore the correlation of adjuvanticity and local inflammatory response elicited in the murine vagina and the draining lymph nodes following local administration of two candidate vaginal adjuvants, Toll like receptor (TLR) 9 agonist CpG ODN, and a non-TLR targeting molecule alpha-galactosylceramide (alpha-GalCer). Using real-time PCR array analysis, we could show that a group of 13 common cytokine genes are activated in the vagina within 24h after vaginal administration of these adjuvants, including Ccl2, Ccl7, Ccl12, Ccl19, Ccl20, Ccl22, Cxcl1, Cxcl5, Il10 and the Th1-inducing molecules Ifng, Cxcl9, Cxcl10 and Cxcl11. A high degree of inflammation in and damage to the epithelium was exclusively observed in the vagina of the CpG ODN treated mice, which was reversed within 48h. These results indicate that there is a group of common genes that correlate with the adjuvanticity of CpG ODN and alpha-GalCer in the vagina, and that alpha-GalCer induces less of local inflammatory reactions in the murine vagina compared to CpG ODN.

Keratinocytes of the oral mucosa and epidermis play key roles in host defense. In addition to functioning as a physical barrier, they also produce cytokines to elicit inflammation in response to infection or injury. We recently established that receptor-interacting protein kinase 4 (RIPK4) and interferon regulatory factor 6 (IRF6) function as a cell-intrinsic signaling axis to regulate keratinocyte differentiation. In this study, we have demonstrated a functional relationship between RIPK4 and IRF6 in the control of proinflammatory cytokine expression in keratinocytes. The overexpression of RIPK4 by oral keratinocytes induced the strong expression of CCL5 and CXCL11. In contrast, the expression of other cytokines (e.g. IL8 and TNF) was largely unaffected, thus demonstrating specificity in the induction of proinflammatory cytokine expression by RIPK4. CCL5 and CXCL11 expression were also induced in response to the activation of the PKC pathway, and gene silencing experiments indicated that their inducible expression was dependent on RIPK4 and IRF6. Moreover, gene reporter assays suggested that RIPK4 induces CCL5 and CXCL11 expression by stimulating the transactivation of their promoters by IRF6. Accordingly, our findings suggest that the RIPK4-IRF6 signaling axis plays a multifaceted role in barrier epithelial homeostasis through its regulation of both keratinocyte inflammation and differentiation.

Tumor progression in the colon moves from aberrant crypt foci to adenomatous polyps to invasive carcinomas. The composition of the tumor-infiltrating leukocyte population affects the ability of the immune system to fight the tumor. T cell infiltration into colorectal adenocarcinomas, particularly T helper 1 (Th1) type T cells as well as increased regulatory T cell (Treg) frequencies, is correlated with improved prognosis. However, whether Th1 cells and Tregs are already present at the adenoma stage is not known. In this study, the APC(Min/+) mouse model of intestinal adenomatous polyposis was used to investigate tumor-associated lymphocyte subsets and the mechanisms of their accumulation into gastrointestinal adenomas. Compared to unaffected tissue, adenomas accumulated CD4(+)FoxP3(+) putative Treg in parallel with lower frequencies of conventional T cells and B cells. The accumulation of Treg was also observed in human adenomatous polyps. Despite high Treg numbers, the function of conventional T cells present in the APC(Min/+) adenomas was not different from those in the unaffected tissue. Adenomas displayed an altered chemokine balance, with higher CCL17 and lower CXCL11 and CCL25 expression than in the unaffected tissue. In parallel, CXCR3(+) Tregs were largely absent from adenomas. The data indicate that already in early stages of tumor development, the balance of lymphocyte-recruiting chemokines is altered possibly contributing to the observed shift toward higher frequencies of Treg.

OBJECTIVE

To examine the expression of the three interferon-gamma-inducible CXCR3-binding chemokines, CXCL10/IP-10 (interferon-gamma-inducible protein of 10 KDa), CXCL9/Mig (monokine induced by interferon-gamma), and CXCL11/I-TAC (interferon-inducible T-cell alpha chemoattractant) in the conjunctiva of patients with vernal keratoconjunctivitis (VKC). These chemokines exhibit potent T-lymphocyte chemoattractant activity.

METHODS

Immunohistochemical study.

METHODS

Conjunctival biopsy specimens from 16 patients with active VKC and nine control subjects were studied by immunohistochemical techniques using monoclonal antibodies directed against IP-10, Mig, and I-TAC. The phenotype of inflammatory cells expressing chemokines was examined by double immunohistochemistry.

RESULTS

In the normal conjunctiva, very weak Mig immunoreactivity was observed on basal epithelial cells and on vascular endothelial cells in the upper substantia propria. There was no immunoreactivity for the other chemokines. In all VKC specimens, strong immunoreactivity for Mig was expressed by epithelial cells, vascular endothelial cells, and inflammatory mononuclear cells. Inflammatory mononuclear cells expressing IP-10 and I-TAC were noted in 10 and nine specimens, respectively. The numbers of Mig(+) inflammatory cells were significantly higher than the numbers of IP-10(+) and I-TAC(+) inflammatory cells (P <.001). Inflammatory cells expressing Mig were CD4(+) T-helper/inducer cells (71.6 +/- 3.2%), CD8(+) T-cytotoxic/suppressor cells (19.5 +/- 1.5%), and CD68(+) monocytes/macrophages (5.3 +/- 5%). All inflammatory cells expressing IP-10 and I-TAC were CD68(+) monocytes/macrophages.

CONCLUSIONS

The CXC chemokine Mig is selectively and highly expressed in VKC suggesting a pathogenic role of the chemokine receptor CXCR3 and the ligand Mig in the recruitment of activated T lymphocytes.

Significant health benefits have been demonstrated for certain probiotic strains through intervention studies; however, there is a shortage of experimental evidence relative to the mechanisms of action. Here, noninvasive experimental procedure based on a colon organ culture system has been used that, in contrast to most experimental in vitro models reported, can preserve natural immunohistochemical features of the human mucosa. This system has been used to test whether commensal lactobacilli (Lactobacillus paracasei BL23, Lactobacillus plantarum 299v and L. plantarum 299v (A(-))) were able to hinder inflammation-like signals induced by phorbol 12-myristate 13-acetate (PMA)/ionomycin (IO). Whole genome microarrays have been applied to analyze expression differences, from which mRNA markers could be inferred to monitor the effect of putative probiotic strains under such conditions. Regarding the gene expression, PMA/IO treatment induced not only interleukin (IL)-2 and interferon gamma (IFN-γ), as expected, but also other relevant genes related to immune response and inflammation, such as IL-17A, chemokine (C-X-C motif) ligand (CXCL) 9 and CXCL11. The ex vivo culturing did not modify the pattern of expression of those genes or others related to inflammation. Interestingly, this study demonstrated that lactobacilli downregulated those genes and triggered a global change of the transcriptional profile that indicated a clear homeostasis restoring effect and a decrease in signals produced by activated T cells.

OBJECTIVE

To find previously unknown properties of ML3000, a competitive inhibitor of the cyclooxygenase and the lipoxygenase (LO) pathway.

METHODS

Gene expression of ML3000 treated and untreated rheumatoid arthritis synovial fibroblasts were measured with Affymetrix gene arrays. Downregulation of chemokine (C-X-C motif) ligands CXCL9, CXCL10 and CXCL11 was verified with Real-time polymerase chain reaction, CXCL10 protein levels were determined with ELISA. Rheumatoid arthritis synovial fibroblasts were treated with the cyclooxygenase inhibitor naproxen, the 5-LO inhibitor BWA4C and the 5-lipoxygenase-activating protein (FLAP) inhibitor MK886, and consecutive changes in CXCL10 protein levels measured. 5-LO expression was determined by polymerase chain reaction and Western blot.

RESULTS

In synovial fibroblasts and monocyte-derived macrophages ML3000 inhibited the tumour necrosis factor induced expression of CXCL9, CXCL10 and CXCL11, which are all ligands of the chemokine receptor CXCR3. No effect was observed in monocytes. Whereas inhibition of the cyclooxygenase pathway or the FLAP protein showed no effect, blockade of 5-LO significantly downregulated CXCL10 protein levels. 5-LO mRNA was detected in monocytes and in monocyte-derived macrophages. All tested cell types expressed 5-LO protein.

CONCLUSIONS

ML3000 effectively downregulates CXCR3 ligands. This study confirms that a thorough analysis of the impact of a drug on its target cells cannot only reveal unexpected properties of a substance, but also helps to understand the underlying molecular mechanisms. Accordingly, our data provide the basis for further clinical studies testing the application of ML3000 in diseases such as rheumatoid arthritis or multiple sclerosis.

Chemokines constitute a protein family that exhibit a variety of biological activities involved in normal and pathological physiological processes. CCL11 (eotaxin), CCL19 (MIP-3beta), CCL22 (MDC), CXCL11 (I-TAC) and CXCL12 (SDF-1alpha) chemokines, modified with the Alexa Fluor 647 fluorescent dye at specific positions along their sequence, were produced by a chemical route and their biological activities were characterized. In a migration assay, fluorescent chemokines were as biologically active as the unmodified forms. All labeled chemokines specifically stained cell lines transfected with the appropriate human chemokine receptors. The specificity of binding was further established by showing that the unlabeled ligands efficiently competed with the labeled chemokines for binding to their respective receptor. A low molecular weight antagonist of CXCR4 prevented binding of labeled CXCL12 to CXCR4 comparably to a neutralizing anti-CXCR4 antibody. Finally, labeled CCL19 was used for the staining of primary cells, illustrating that this reagent can be used for studying CCR7 expression on different cell types. Together, these results demonstrate that fluorescent synthetic chemokines constitute promising ligands for the development of chemokine receptor-binding assays on intact cells, for applications such as cell-based, high throughput screening, and studies of chemokine receptor expression by primary cells.

BACKGROUND

Respiratory viral infections are the most common trigger of acute exacerbations in patients with allergic asthma. The anti-viral response of airway epithelial cells (AEC) may be impaired in asthmatics, while cytokines produced by AEC may drive the inflammatory response. We investigated whether AEC cultured in the presence of Th2 cytokines associated with an allergic environment exhibited altered responses to double-stranded RNA, a virus-like stimulus.

METHODS

We undertook preliminary studies using the MLE-12 cell line derived from mouse distal respiratory epithelial cells, then confirmed and extended our findings using low-passage human AEC. Cells were cultured in the absence or presence of the Th2 cytokines IL-4 and IL-13 for 48 hours, then stimulated with poly I:C for 4 hours. Expression of relevant anti-viral response and cytokine genes was assessed by quantitative real-time PCR. Secretion of cytokine proteins was assessed by immunoassay.

RESULTS

Following stimulation with poly I:C, MLE-12 cells pre-treated with Th2 cytokines exhibited significantly higher levels of expression of mRNA for the cytokine genes Cxcl10 and Cxcl11, as well as a trend towards increased expression of Cxcl9 and Il6. Expression of anti-viral response genes was mostly unchanged, although Stat1, Ifit1 and Ifitm3 were significantly increased in Th2 cytokine pre-treated cells. Human AEC pre-treated with IL-4 and IL-13, then stimulated with poly I:C, similarly exhibited significantly higher expression of IL8, CXCL9, CXCL10, CXCL11 and CCL5 genes. In parallel, there was significantly increased secretion of CXCL8 and CCL5, as well as a trend towards increased secretion of CXCL10 and IL-6. Again, expression of anti-viral response genes was not decreased. Rather, there was significantly enhanced expression of mRNA for type III interferons, RNA helicases and other interferon-stimulated genes.

CONCLUSIONS

The Th2 cytokine environment appears to promote increased production of pro-inflammatory chemokines by AEC in response to double-stranded RNA, which could help explain the exaggerated inflammatory response to respiratory viral infection in allergic asthmatics. However, any impairment of anti-viral host defences in asthmatics appears unlikely to be a consequence of Th2 cytokine-induced downregulation of the expression of viral response genes by AEC.

Background. Pediatric Kawasaki disease (KD) and human immunodeficiency virus (HIV)+ adult Kawasaki-like syndrome (KLS) are dramatic vasculitides with similar physical findings. Both syndromes include unusual arterial histopathology with immunoglobulin (Ig)A+ plasma cells, and both impressively respond to pooled Ig therapy. Their distinctive presentations, histopathology, and therapeutic response suggest a common etiology. Because blood is in immediate contact with inflamed arteries, we investigated whether KD and KLS share an inflammatory signature in serum. Methods. A custom multiplex enzyme-linked immunosorbent assay (ELISA) defined the serum cytokine milieu in 2 adults with KLS during acute and convalescent phases, with asymptomatic HIV+ subjects not taking antiretroviral therapy serving as controls. We then prospectively collected serum and plasma samples from children hospitalized with KD, unrelated febrile illnesses, and noninfectious conditions, analyzing them with a custom multiplex ELISA based on the KLS data. Results. Patients with KLS and KD subjects shared an inflammatory signature including acute-phase reactants reflecting tumor necrosis factor (TNF)-α biologic activity (soluble TNF receptor I/II) and endothelial/smooth muscle chemokines Ccl1 (Th2), Ccl2 (vascular inflammation), and Cxcl11 (plasma cell recruitment). Ccl1 was specifically elevated in KD versus febrile controls, suggesting a unique relationship between Ccl1 and KD/KLS pathogenesis. Conclusions. This study defines a KD/KLS inflammatory signature mirroring a dysfunctional response likely to a common etiologic agent. The KD/KLS inflammatory signature based on elevated acute-phase reactants and specific endothelial/smooth muscle chemokines was able to identify KD subjects versus febrile controls, and it may serve as a practicable diagnostic test for KD.

Target specific short single-stranded DNA (ssDNA) molecules, called aptamers, are auspicious ligands for numerous in vivo applications. However, aptamers are synthetic molecules, which might be recognized by the immune cells in vivo and induce an activation of the innate immune system. Thus, immune activation potential of synthetic ssDNA oligonucleotides (ODNs) was determined using a well established closed-loop circulation model. Fresh human blood was incubated at 37°C for 2 or 4 hours with ssDNA ODNs (SB_ODN) or CpG ODN as positive control. Transcriptional changes were determined by microarray analyses. Blood samples containing SB_ODN demonstrated after 4 hours a significant regulation of 295 transcripts. Amongst others, CCL8, CXCL10, CCL7 and CXCL11 were highest regulated genes. Gene Ontology terms and KEGG pathway analyses exhibited that the differentially expressed genes belong to the transcripts that are regulated during an immune and inflammatory response, and were overrepresented in TLR signaling pathway. This study shows for the first time the potential of aptamers to activate immune system after systemic application into the human blood. Thus, we highly recommend performing of these preclinical tests with potential aptamer-based therapeutics.

Ribavirin has proven to be a key component of hepatitis C therapies both involving IFNs and new direct-acting antivirals. The hepatitis C virus-mediated interference with intrahepatic immunity by cleavage of mitochondrial antiviral signaling protein (MAVS) and T cell protein tyrosine phosphatase (TCPTP) suggests an avenue for compounds that may counteract these effects. We therefore studied the effects of ribavirin, with or without inhibition of the nonstructural (NS)3/4A protease, on intrahepatic immunity. The intrahepatic immunity of wild-type and NS3/4A-transgenic mice was determined by Western blot, ELISA, flow cytometry, and survival analysis. Various MAVS or TCPTP constructs were injected hydrodynamically to study their relevance. Ribavirin pretreatment was performed in mice expressing a functional or inhibited NS3/4A protease to analyze its effect on NS3/4A-mediated changes. Intrahepatic NS3/4A expression made mice resistant to TNF-α-induced liver damage and caused an alteration of the intrahepatic cytokine (IFN-γ and IL-10) and chemokine (CCL3, CCL17, CCL22, CXCL9, and CXCL11) profiles toward an anti-inflammatory state. Consistent with this, the number of intrahepatic Th1 cells and IFN-γ(+) T cells in NS3/4A-transgenic mice decreased, whereas the amount of Th2 cells increased. These effects could be reversed by injection of uncleavable TCPTP but not uncleavable MAVS and were absent in a mouse expressing a nonfunctional NS3/4A protease. Importantly, the NS3/4A-mediated effects were reversed by ribavirin treatment. Thus, cleavage of TCPTP by NS3/4A induces a shift of the intrahepatic immune response toward a nonantiviral Th2-dominated immunity. These effects are reversed by ribavirin, supporting that ribavirin complements the effects of direct-acting antivirals as an immunomodulatory compound.

The chemokines (CXCL9, CXCL10 and CXCL11) and associated CXCR3 receptor are expressed during the inflammatory process from multiple sclerosis, atherosclerosis or organ transplantation resulting in the recruitment of lymphocytes leading to tissue damage. It is hypothesized that blocking of the ligand/CXCR3 receptor interaction has potential to provide opportunity for development of agents that would block tissue rejection. In this paper, four classes of natural product inhibitors (IC50 ranging 0.1-41 microM) have been described that block the CXCR3 receptor interaction of IP-10 ligand. These include a cyclic thiopeptide (duramycin), polyketide glycosides (roselipins), steroidal glycosides (hypoglausin A and dioscin) and a novel alkyl pyridinium alkaloid that were isolated by bioassay-guided fractionation of the organic extracts derived from actinomycete, fungal, plant and marine sources and discovered using 125I IP-10/CXCR3 binding assay. Duramycin was the most potent with an IC50 of 0.1 microM. Roselipins 2A, 2B and 1A showed IC50 values of 14.6, 23.5, and 41 microM, respectively. Diosgenin glycosides dioscin, hypoglaucin A and kallstroemin D exhibited IC50 values of 2.1, 0.47 and 3 microM, respectively. A novel cyclic 3-alkyl pyridinium salt isolated from a sponge displayed a binding IC50 of 0.67 microM.

Butyrate (NaBu), a product of intestinal microbial metabolism, has been proposed as an anti-inflammatory agent for treating inflammatory bowel diseases. However, the molecular mechanisms implicated in the modulation of intestinal epithelial cell inflammatory response to NaBu remain unknown. Here, microarray analysis performed on nontransformed human crypt intestinal epithelial cells (HIEC) shows that NaBu regulated specifically the short-term IL-1beta -dependent induction of different inflammatory genes. While NaBu significantly increased the IL-1beta -induction of genes like SAA2, C3, and IL-1alpha , other inflammatory genes like CXCL5, CXCL11, and IL-1beta were decreased. Induction of various genes such as CXCL8, CCL20, and IL-6 was unaffected by NaBu. We show that, compared to genes that are upregulated or downregulated by NaBu, genes that are unaffected by NaBu were induced more rapidly after IL-1beta treatment and contained a higher concentration of transcription factor binding sites in their promoter region. In addition, transient treatment with IL-1beta was sufficient for subsequent induction of NaBu-upregulated and NaBu-unaffected classes of genes, while a continuous presence of IL-1beta was required for NaBu-downregulated gene expression. In conclusion, our results suggest that fundamental differences predispose inflammatory genes to specific regulation by NaBu in intestinal epithelial cells, thereby allowing precise control of inflammation.

BACKGROUND

The mechanism by which viruses cause exacerbations of chronic airway disease and the capacity of patients with cystic fibrosis (CF) to respond to viral infection are not precisely known.

OBJECTIVE

To determine the antiviral response to infection in patients with CF.

METHODS

Sputum was collected from patients with CF with respiratory exacerbation. Viruses were detected in multiplex polymerase chain reaction (PCR)-based assays. Gene expression of 84 antiviral response genes was measured, using a focused quantitative PCR gene array.

RESULTS

We examined 36 samples from 23 patients with respiratory exacerbation. Fourteen samples tested virus-positive and 22 virus-negative. When we compared exacerbations associated with rhinovirus (RV, n = 9) and influenza (n = 5) with virus-negative specimens, we found distinct patterns of antiviral gene expression. RV was associated with greater than twofold induction of five genes, including those encoding the monocyte-attracting chemokines CXCL10, CXCL11, and CXCL9. Influenza was associated with overexpression of 20 genes, including those encoding the cytokines tumor necrosis factor and IL-12; the kinases MEK, TBK-1, and STAT-1; the apoptosis proteins caspase-8 and caspase-10; the influenza double-stranded RNA receptor RIG-I and its downstream effector MAVS; and pyrin, an IFN-stimulated protein involved in influenza resistance.

CONCLUSIONS

We conclude that virus-induced exacerbations of CF are associated with immune responses tailored to specific infections. Influenza induced a more potent response consisting of inflammation, whereas RV infection had a pronounced effect on chemokine expression. As far as we are aware, this study is the first to compare specific responses to different viruses in live patients with chronic airway disease.

BACKGROUND

To utilize the large volume of gene expression information generated from different microarray experiments, several meta-analysis techniques have been developed. Despite these efforts, there remain significant challenges to effectively increasing the statistical power and decreasing the Type I error rate while pooling the heterogeneous datasets from public resources. The objective of this study is to develop a novel meta-analysis approach, Consistent Differential Expression Pattern (CDEP), to identify genes with common differential expression patterns across different datasets.

RESULTS

We combined False Discovery Rate (FDR) estimation and the non-parametric RankProd approach to estimate the Type I error rate in each microarray dataset of the meta-analysis. These Type I error rates from all datasets were then used to identify genes with common differential expression patterns. Our simulation study showed that CDEP achieved higher statistical power and maintained low Type I error rate when compared with two recently proposed meta-analysis approaches. We applied CDEP to analyze microarray data from different laboratories that compared transcription profiles between metastatic and primary cancer of different types. Many genes identified as differentially expressed consistently across different cancer types are in pathways related to metastatic behavior, such as ECM-receptor interaction, focal adhesion, and blood vessel development. We also identified novel genes such as AMIGO2, Gem, and CXCL11 that have not been shown to associate with, but may play roles in, metastasis.

CONCLUSIONS

CDEP is a flexible approach that borrows information from each dataset in a meta-analysis in order to identify genes being differentially expressed consistently. We have shown that CDEP can gain higher statistical power than other existing approaches under a variety of settings considered in the simulation study, suggesting its robustness and insensitivity to data variation commonly associated with microarray experiments.

BACKGROUND

CDEP is implemented in R and freely available at: http://genomebioinfo.musc.edu/CDEP/.

BACKGROUND

zhengw@musc.edu.

The JAK/STAT signaling pathway is suggested to play an important role in the pathogenesis of psoriasis, and recently JAK/STAT inhibitors have shown promising results in psoriasis treatment. The present study aimed to characterize the role of STAT2 in psoriasis. We demonstrated an increased expression of STAT2 and an increased level of phosphorylated/activated STAT2 in lesional compared with nonlesional psoriatic skin. Gene silencing of STAT2 by siRNA in human keratinocytes revealed that upon IFNα stimulation CXCL11 and CCL5 were the only two cytokines, among 102 analyzed, found to be regulated through a STAT2-dependent mechanism. Moreover, the regulation of CXCL11 and CCL5 depended on IRF9, but not on STAT1 and STAT6. The CXCL11 and CCL5 expression was increased in lesional compared with nonlesional psoriatic skin, and analysis demonstrated positive correlation between the expression of CXCL11 and IFNγ and between the expression of CCL5 and IFNγ in lesional psoriatic skin. In contrast, no correlation between the expression of CXCL11 and IL-17A and the expression of CCL5 and IL-17A in lesional psoriatic skin was found. Our data suggest that STAT2 plays a role in the psoriasis pathogenesis by regulating the expression of CXCL11 and CCL5, and thereby attracting IFNγ-producing immune cells to the skin.

The chemokine, CXCL11, is highly expressed in different solid tumors and controls tumor growth, metastasis, and lymphocyte infiltration. Although of potential clinical interest, it is presently unknown whether these tumor-promoting activities involve the CXCL11 receptors, CXCR3 and/or CXCR7. This issue is further intrigued by the fact that CXCR3 exists in the two functionally divergent splice variants, CXCR3A and CXCR3B, which exert pro- and anti-tumorigenic influences, respectively. To unravel the role of the various CXCL11 receptors in tumor progression, we have now defined their role in CXCL11-induced chemotaxis of the tumor cell lines, A549, C33-A, DLD-1, MDA-MB-231, and PC-3. CXCL11-induced cell migration was either sensitive to the CXCR3 antagonist, ÀMG487 (DLD-1), the CXCR7 antagonist, CCX771 (C33-A, PC-3), or both (A549, MDA-231). Moreover, in C33-A and PC-3 cells, but not in the other tumor cells, pharmacological activation and inhibition of CXCR3B prevented and potentiated CXCL11-induced cell migration, respectively. Both immunocytochemistry and Western blot analysis finally revealed that the observed cell type specific organization of the CXCL11 system is not the result of differences in expression levels or subcellular location of CXCL11 receptors. Our findings imply that the therapeutic use of CXCR3 antagonists in cancer patients requires exact knowledge of the organization of the CXCR3 system in the respective tumor.

BACKGROUND

Our purpose was to obtain genome-wide expression data for the rabbit species on the responses of peripheral blood mononuclear cells (PBMCs) after in vitro stimulation by lipopolysaccharide (LPS) or phorbol myristate acetate (PMA) and ionomycin. This transcriptome profiling was carried out using microarrays enriched with immunity-related genes, and annotated with the most recent data available for the rabbit genome.

RESULTS

The LPS affected 15 to 20 times fewer genes than PMA-Ionomycin after both 4 hours (T4) and 24 hours (T24), of in vitro stimulation, in comparison with mock-stimulated PBMCs. LPS induced an inflammatory response as shown by a significant up-regulation of IL12A and CXCL11 at T4, followed by an increased transcription of IL6, IL1B, IL1A, IL36, IL37, TNF, and CCL4 at T24. Surprisingly, we could not find an up-regulation of IL8 either at T4 or at T24, and detected a down-regulation of DEFB1 and BPI at T24. A concerted up-regulation of SAA1, S100A12 and F3 was found upon stimulation by LPS. PMA-Ionomycin induced a very early expression of Th1, Th2, Treg, and Th17 responses by PBMCs at T4. The Th1 response increased at T24 as shown by the increase of the transcription of IFNG and by contrast to other cytokines which significantly decreased from T4 to T24 (IL2, IL4, IL10, IL13, IL17A, CD69) by comparison to mock-stimulation. The granulocyte-macrophage colony-stimulating factor (CSF2) was by far the most over-expressed gene at both T4 and T24 by comparison to mock-stimulated cells, confirming a major impact of PMA-Ionomycin on cell growth and proliferation. A significant down-regulation of IL16 was observed at T4 and T24, in agreement with a role of IL16 in PBMC apoptosis.

CONCLUSIONS

We report new data on the responses of PBMCs to LPS and PMA-Ionomycin in the rabbit species, thus enlarging the set of mammalian species for which such reports exist. The availability of the rabbit genome assembly together with high throughput genomic tools should pave the way for more intense genomic studies for this species, which is known to be a very relevant biomedical model in immunology and physiology.