Inhibition Effect of Eucommia ulmoides Leaf Extract on Interleukin 8 Production by A549 Cells.

Asthma is characterized by chronic inflammation of the airway mucosa. As Eucommia ulmoides Oliv. leaf extract (ELE) has been known to have anti-inflammatory properties, herein, we investigated the effect of ELE on interleukin (IL-) 8 production in A549 cells, a human airway epithelial cell line. The addition of ELE 1 h before tumor necrosis factor-alpha (TNFα) stimulation inhibited IL-8 production by A549 cells in a concentration-dependent manner. The addition of geniposidic acid, the main component of ELE, also inhibited IL-8 production. To further investigate the mechanism by which ELE inhibits IL-8 production, the effect of ELE or geniposidic acid on TNFα-stimulated p38 phosphorylation was examined by Western blotting. After 30 min of TNFα stimulation, p38 phosphorylation was inhibited by the addition of ELE or geniposidic acid, suggesting that ELE inhibited IL-8 production in TNFα-stimulated A549 cells by suppressing one of the signal transducers of p38 phosphorylation. These results indicate that ELE can be used as an effective measure against asthma, particularly neutrophilic asthma.

Toll-like receptor 3 ligand specifically induced bronchial epithelial cell death in caspase dependent manner and functionally upregulated Fas expression.

BACKGROUND: Viral infections are the most common cause of asthma exacerbation. Virally infected epithelial cells undergo apoptosis. Although in healthy conditions, apoptosis may have a host-defensive role in limiting virus spread, this process may have a detrimental effect on damaged epithelium in asthma. Toll-like receptors (TLRs) are the receptors for various pathogens, and viruses possess several components that can activate TLR3, TLR4, and TLR7/8. However, as it has not been determined as to which component is responsible for virus-induced epithelial cell apoptosis, we comprehensively analyzed the effects of all TLR ligands on apoptosis. METHODS: BEAS-2B cells or primary cultured human bronchial epithelial cells (PBECs) were stimulated by TLR 2, 3, 4, 5, 7/8, and 9 ligands and cell death was analyzed by flow cytometry. Chemokine generations induced by these ligands were also analyzed. RESULTS: The TLR3 ligand polyinosinic-polycytidylic acid (poly I:C) specifically induced chemokine generation and apoptosis, while other TLR ligands including those for TLR5, 7/8, and 9 had no effect. The response to poly I:C had two phases, which included rapid secretion of chemokines and subsequent apoptosis in a later phase. Poly I:C induced apoptosis in a caspase-dependent manner and functionally upregulated the expression of Fas. CONCLUSIONS: Previous findings indicating that viruses induced caspase-dependent death and upregulated Fas expression were reproduced by poly I:C, suggesting the central role of dsRNA/TLR3 in virus-induced apoptosis. Since these processes may have detrimental effects on pre-existing epithelial damage, the dsRNA/TLR3 pathway may be potential novel treatment target for virus-induced exacerbation of asthma.

Apoptosis occurs during early development of the bursa of Fabricius in chicken embryos.

The bursa of Fabricius (BF) is a unique primary lymphoid organ, and among vertebrates is unique to birds. Despite its importance to the immune systems of various avian species, little is known of the molecular mechanisms underlying early BF development. In the present study, we demonstrated that apoptosis occurs during early development of the bursa of Fabricius in chicken embryos. Initial histological analyses of BF morphogenesis in chicken embryos led to the hypothesis that formation of the bursal lumen correlates with fusion of vacuoles, which appear in the cloacal epithelial bud. Using terminal deoxynucleotidyl transferase deoxyuridine triphosphate (dUTP) nick-end labeling (TUNEL) analysis and immunostaining with an anti-cleaved (activated) caspase-3 antibody, we detected multiple apoptotic cells around these vacuoles. In further experiments, treatments with a caspase inhibitor caused abnormal bursal lumen in vivo. The present data indicate that apoptosis may play important roles in BF morphogenesis in chickens.

Thymic stromal lymphopoietin gene promoter polymorphisms are associated with susceptibility to bronchial asthma.

Thymic stromal lymphopoietin (TSLP) triggers dendritic cell--mediated T helper (Th) 2 inflammatory responses. A single-nucleotide polymorphism (SNP), rs3806933, in the promoter region of the TSLP gene creates a binding site for the transcription factor activating protein (AP)-1. The variant enhances AP-1 binding to the regulatory element, and increases the promoter--reporter activity of TSLP in response to polyinosinic-polycytidylic acid (poly[I:C]) stimulation in normal human bronchial epithelium (NHBE). We investigated whether polymorphisms including the SNP rs3806933 could affect the susceptibility to and clinical phenotypes of bronchial asthma. We selected three representative (i.e., Tag) SNPs and conducted association studies of the TSLP gene, using two independent populations (639 patients with childhood atopic asthma and 838 control subjects, and 641 patients with adult asthma and 376 control subjects, respectively). We further examined the effects of corticosteroids and a long-acting ß(2)-agonist (salmeterol) on the expression levels of the TSLP gene in response to poly(I:C) in NHBE. We found that the promoter polymorphisms rs3806933 and rs2289276 were significantly associated with disease susceptibility in both childhood atopic and adult asthma. The functional SNP rs3806933 was associated with asthma (meta-analysis, P = 0.000056; odds ratio, 1.29; 95% confidence interval, 1.14-1.47). A genotype of rs2289278 was correlated with pulmonary function. Moreover, the induction of TSLP mRNA and protein expression induced by poly(I:C) in NHBE was synergistically impaired by a corticosteroid and salmeterol. TSLP variants are significantly associated with bronchial asthma and pulmonary function. Thus, TSLP may serve as a therapeutic target molecule for combination therapy.

Cimetidine enhances antigen-specific IgE and Th2 cytokine production.

BACKGROUND: Treatment with anti-ulcer drugs has been shown to enhance IgE production against food antigens. However, little is known about the immunological effects of cimetidine, a histamine receptor type 2 (H2R) antagonist that is widely used as an anti-ulcer drug, in allergy. Therefore, the present study investigated the role of cimetidine in Th2 immune responses in mice. METHODS: BALB/c mice were immunized intraperitoneally with ovalbumin (OVA) with and without cimetidine. The levels of cytokines in supernatants of spleen cells cultured in the presence of OVA for 4 days and the levels of total and OVA-specific IgG(1), IgG(2a) and/or IgE in sera from these mice were determined by ELISA. RESULTS: Administration of cimetidine to OVA-sensitized BALB/c mice promoted Th2 cytokine secretion by OVA-stimulated spleen cells in vitro and increased serum levels of OVA-specific IgE, IgG(1) and IgG(2a). CONCLUSIONS: These results indicate that cimetidine can enhance Th2 responses, suggesting that cimetidine may contribute to IgE production in allergies.

Corneal and conjunctival fibroblasts are major sources of eosinophil-recruiting chemokines.

BACKGROUND: Differential expression of chemokine genes were investigated in various types of ocular surface cells. METHODS: Primary cultures of human corneal epithelial cells (n = 3), corneal fibroblasts (n = 2), conjunctival epithelial cells (n = 2) and conjunctival fibroblasts (n = 2) were established and incubated with or without interleukin (IL)-4 (30ng/ml) and tumor necrosis factor (TNF)-alpha(30ng/ml) for 24 hours. Gene transcription levels of 33 chemokines and production of 4 chemokines were analyzed. RESULTS: After stimulation, chemokine expression increased for 18 of 33 coded chemokine gene transcripts. In stimulated conjunctival and corneal cells, CC chemokine genes increased in fibroblasts (expression of 6 out of 8 genes), while CXC chemokine genes increased in both epithelial cells (expression of 4 out of 9 genes in conjunctival epithelial cells and 7 out of 9 genes in corneal epithelial cells) and in fibroblasts (expression of 8 out of 9 genes in conjunctival and corneal fibroblasts). Except for MCP-1, gene transcription levels for most CC chemokines were inducible and, except for IP-10 and I-TAC, most CXC chemokines were constitutively expressed. Corneal epithelial cell and fibroblast production patterns for eotaxin-1, MCP-1 and IP-10 were comparable to the mRNA expression pattern. CONCLUSIONS: Corneal and conjunctival fibroblasts exhibited marked increases in the expression of chemokines upon stimulation with TNF-alpha and IL-4, suggesting that fibroblasts may be one of the primary sources of chemokines in allergic conjunctival diseases. Therefore, regulation of chemokine production from these cells may be an effective strategy for treating such diseases.

Profiling gene expression ratios of paired cancerous and normal tissue predicts relapse of esophageal squamous cell carcinoma.

Esophageal squamous cell carcinoma has heterogeneous clinical outcomes that cannot be predicted well using any existing clinical or molecular prognostic factors. Gene expression profiling may enable more precise prediction of the clinical outcome of these patients. We developed a new approach using gene expression ratios of paired cancerous and normal tissue specimens from the same patient to reduce the effects of variation among individuals. Using oligonucleotide microarrays, we analyzed total RNA expression levels corresponding to 12,600 transcript sequences in 24 paired cancerous and normal tissue operative specimens from 12 patients with esophageal squamous cell carcinoma. Hierarchical clustering analysis using gene expression ratios (cancer:normal) divided the 12 patients into two groups; all 7 patients in the first cluster survived without relapse (median follow-up, 483 days), whereas all 5 patients in the second cluster relapsed (median relapse-free survival time, 280 days; log-rank test, P = 0.006). In contrast, clustering either with cancerous tissue alone or with normal tissue alone did not show significant differences in the outcomes. The expressions of a variety of genes related with cell cycle, gene-repair, apoptosis and chemoradiotherpay resistance were up-regulated in the poor prognostic cluster. These results suggest that ratios of paired gene expression profiles may more efficiently predict relapse-free survival of esophageal squamous cell carcinoma than existing prognostic factors or than gene expression profiling with cancerous tissue alone.

The implications of the upregulation of ICAM-1/VCAM-1 expression of corneal fibroblasts on the pathogenesis of allergic keratopathy.

OBJECTIVE: The present study investigated the expression of ICAM-1 and VCAM-1 on fibroblasts with interleukin (IL)-4 and/or tumor necrosis factor (TNF)-alpha stimulation and assessed the effect of eosinophil adhesion on fibroblast viability. METHODS: Primary cultured human corneal fibroblasts were incubated with IL-4, TNF-alpha, or their combination for 24 hours. Expression of ICAM-1 and VCAM-1 was examined by real-time quantitative PCR and flow cytometric analysis. Purified eosinophils were cocultured with activated fibroblasts, and the number of eosinophils adhered to fibroblasts and the number of damaged fibroblasts were counted using microscopy. In a separate trial, conjunctival and corneal impression cytology was performed on patients with atopic keratoconjunctivitis and corneal ulcers (eight eyes) to assess the status of the ocular surface epithelium and the presence of inflammatory cell infiltrates. RESULTS: Real-time quantitative PCR and flow cytometric analysis revealed that both mRNA and protein of VCAM-1 and ICAM-1 were upregulated by IL-4 and TNF-alpha. IL-5-primed eosinophils adhered to the corneal fibroblasts treated with IL-4 and TNF-alpha, and the fibroblasts were damaged by eosinophil adherence. Anti-ICAM-1 antibody and anti-VCAM-1 antibody inhibited the eosinophil adherence to fibroblasts and the fibroblast damage. Impression cytology revealed extensive infiltration of neutrophil and eosinophils among isolated ocular surface epithelial cells with advanced squamous metaplasia. CONCLUSIONS: Corneal fibroblasts expressed ICAM-1 and VCAM-1 when activated with IL-4 and TNF-alpha. Eosinophils can adhere to the activated fibroblasts and can induce subsequent fibroblast damage through these adhesion molecules. Eosinophil adhesion to fibroblasts may possibly contribute to the pathogenesis of severe persistent allergic corneal ulcers.

Novel genetic polymorphisms in the NR3C1 (glucocorticoid receptor) gene in a Japanese population.

Glucocorticoid receptor, encoded by NR3C1, is a transcriptional regulator of many drug metabolizing enzymes and anti-inflammatory molecules. In order to identify genetic variations of the NR3C1 gene, genomic DNA from 265 Japanese individuals was sequenced. Fifty genetic polymorphisms were identified, including 32 novel ones [3 were in coding exons, 17 in the introns, 4 in the 5'-untranslated region (UTR), and 8 in the 5'-flanking region]. The novel nonsynonymous variation was 420G>T (Lys140Asn), and the allele frequency was 0.004. We did not detect any nonsynonymous polymorphism reported previously in other races, including a relatively frequent SNP Asn363Ser found in Caucasians and African-Americans. Thus, ethnic differences between Japanese and other races are suggested to exist in NR3C1.

Single nucleotide polymorphisms and haplotype frequencies of CYP3A5 in a Japanese population.

In order to identify single nucleotide polymorphisms (SNPs) and haplotype frequencies of CYP3A5 in a Japanese population, we sequenced the proximal promoter region, all exons, and the surrounding intronic regions using genomic DNA from 187 Japanese subjects. Thirteen SNPs, including seven novel ones: 13108T>C, 16025A>G, 16903A>G, 16993C>G, 27448C>A, 29782A>G, and 31551T>C (A of the translational start codon of GenBank Accession # NG_000004.2 is numbered 1 according to the CYP Allele Nomenclature), were identified. The most common SNP was 6986A>G (key SNP for CYP3A5*3), with a 0.759 frequency. Two novel SNPs, 29782A>G (I456V) and 31551T>C (I488T), as well as 12952T>C (*5 marker) were found, but these alterations were always associated with the *3A marker SNPs, 6986A>G and 31611C>T. Using these 13 SNPs, haplotype analysis was performed and five novel *1 haplotypes (subtypes) (*1e to *1i) and six novel *3 haplotypes (subtypes) (*3d to *3i) were identified. Our findings suggest that CYP3A5*3 is the major defective allele and that other functional exonic SNPs are rare in the Japanese.

Haplotypes of CYP3A4 and their close linkage with CYP3A5 haplotypes in a Japanese population.

In order to identify single nucleotide polymorphisms (SNPs) and haplotype frequencies of CYP3A4 in a Japanese population, the distal enhancer and proximal promoter regions, all exons, and the surrounding introns were sequenced from genomic DNA of 416 Japanese subjects. We found 24 SNPs, including 17 novel ones: two in the distal enhancer, four in the proximal promoter, one in the 5'-untranslated region (UTR), seven in the introns, and three in the 3'-UTR. The most common SNP was c.1026+12G>A (IVS10+12G>A), with a 0.249 frequency. Four non-synonymous SNPs, c.554C>G (p.T185S, CYP3A4(*)16), c.830_831insA (p.E277fsX8, (*)6), c.878T>C (p.L293P, (*)18), and c.1088 C>T (p.T363M, (*)11) were found with frequencies of 0.014, 0.001, 0.028, and 0.002, respectively. No SNP was found in the known nuclear transcriptional factor-binding sites in the enhancer and promoter regions. Using these 24 SNPs, 16 haplotypes were unambiguously identified, and nine haplotypes were inferred by aid of an expectation-maximization-based program. In addition, using data from 186 subjects enabled a close linkage to be found between CYP3A4 and CYP3A5 SNPs, especially among the SNPs at c.1026+12 in CYP3A4 and c.219-237 (IVS3-237, a key SNP site for CYP3A5(*)3), c.865+77 (IVS9+77) and c.1523 in CYP3A5. This result suggested that CYP3A4 and CYP3A5 are within the same gene block. Haplotype analysis between CYP3A4 and CYP3A5 revealed several major haplotype combinations in the CYP3A4-CYP3A5 block. Our findings provide fundamental and useful information for genotyping CYP3A4 (and CYP3A5) in the Japanese, and probably Asian populations.

TNF-alpha and IL-4 regulate expression of fractalkine (CX3CL1) as a membrane-anchored proadhesive protein and soluble chemotactic peptide on human fibroblasts.

The CX(3)C chemokine, fractalkine (FKN, CX(3)CL1), has multiple functions and exists as two distinct forms, a membrane-anchored protein and a soluble chemotactic peptide that cleaves from the cell surface FKN. In this study, we first demonstrated the expression of FKN in tumor necrosis factor (TNF)-alpha- and interleukin (IL)-4-stimulated human fibroblasts. The induction of FKN was observed for both forms. We also demonstrated monocyte chemotactic activity in the culture supernatant from the fibroblasts stimulated with these cytokines. These results suggest that TNF-alpha- and IL-4-stimulated fibroblasts may play an important role in accumulation of monocytes at inflammatory sites.

CC-chemokine receptor 3: a possible target in treatment of allergy-related corneal ulcer.

PURPOSE: To determine the suppressive effects of antibodies (Abs) against CC-chemokine receptor (CCR)-1 and CCR-3 on eosinophil chemotaxis induced by culture supernatant from corneal keratocytes and by tears from severely allergic patients with corneal ulcer. METHODS: Primary cultures of human corneal keratocytes were incubated with interleukin (IL)-4 (33.3 ng/mL) and tumor necrosis factor (TNF)-alpha (33.3 ng/mL) for 48 hours. In tear samples collected from five severely allergic patients and three nonallergic control subjects, eosinophils were immunostained for CCR. Next, eosinophils purified from peripheral blood were preincubated with or without anti-CCR-1 and anti-CCR-3 Abs before a Boyden chamber assay was conducted. Recombinant human (rh) eotaxin, rh-regulated on activation normal T-cell expressed and secreted (rh-RANTES), culture supernatant from human corneal keratocytes, and tear samples were used as chemoattractants. RESULTS: Eosinophils in tears from allergic patients expressed CCR-1 and -3 on their surfaces. Anti-CCR-1 and -3 Abs each inhibited eosinophil chemotaxis induced by rh-RANTES. Anti-CCR-3 Ab (but not anti-CCR-1 Ab) also inhibited eosinophil chemotaxis induced by rh-eotaxin. Anti-CCR-1 and -3 Abs, respectively, inhibited up to 75.2% and 94.6% of eosinophil chemotaxis induced by culture supernatant, as well as 27.8% and 74.5% of chemotaxis induced by tear samples. CONCLUSIONS: Anti-CCR-1 and -3 Abs inhibited eosinophil chemotaxis induced by culture supernatant from corneal keratocytes and tear samples from severely allergic patients. Anti-CCR-3 Ab was more effective than anti-CCR-1 Ab. Inhibition of CCR-3 on eosinophils may be a treatment for corneal ulcer in patients with ocular allergy.

Twelve novel single nucleotide polymorphisms in the CES2 gene encoding human carboxylesterase 2 (hCE-2).

Twelve novel single nucleotide polymorphisms (SNPs) were found in the CES2 gene from 153 Japanese individuals, who were administered irinotecan or steroidal drugs. The detected SNPs were as follows:1) SNP, MPJ6_CS2001; GENE NAME, CES2; ACCESSION NUMBER, NT_010498.13; LENGTH, 25 bases; 5'-CTGGAACAACTCG/CCTCCCCTCGGAA-3'. 2) SNP, MPJ6_CS2002; GENE NAME, CES2; ACCESSION NUMBER, NT_010498.13; LENGTH, 25 bases; 5'-AACCACCACCGCT/CGATCCTAGCAGG-3'. 3) SNP, MPJ6_CS2003; GENE NAME, CES2; ACCESSION NUMBER, NT_010498.13; LENGTH, 25 bases; 5'-AAATGTTTGTCAA/GGTGGATAAATGA-3'. 4) SNP, MPJ6_CS2004; GENE NAME, CES2; ACCESSION NUMBER, NT_010498.13; LENGTH, 25 bases; 5'-CCTCCTATCGATC/GCCCCAGCGCGCT-3'. 5) SNP, MPJ6_CS2005; GENE NAME, CES2; ACCESSION NUMBER, NT_010498.13; LENGTH, 25 bases; 5'-GCCAGTCCCATCC/TGGACCACACACA-3'. 6) SNP, MPJ6_CS2006; GENE NAME, CES2; ACCESSION NUMBER, NT_010498.13; LENGTH, 25 bases; 5'-GCTGGGCAACCCG/AGGCTGAGCGGGG-3'. 7) SNP, MPJ6_CS2007; GENE NAME, CES2; ACCESSION NUMBER, NT_010498.13; LENGTH, 25 bases; 5'-CAAGCACGCAACC/TGGCAACTGGGGC-3'. 8) SNP, MPJ6_CS2008; GENE NAME, CES2; ACCESSION NUMBER, NT_010498.13; LENGTH, 25 bases; 5'-CATGGAGAGTGGC/TGTGGCCCTCCTG-3'. 9) SNP, MPJ6_CS2009; GENE NAME, CES2; ACCESSION NUMBER, NT_010498.13; LENGTH, 25 bases; 5'-CCTGTTCTTGGCC/TAGGGCCTTGGGC-3'. 10) SNP, MPJ6_CS2010; GENE NAME, CES2; ACCESSION NUMBER, NT_010498.13; LENGTH, 25 bases; 5'-CCCATCCCCAGCT/AACAGACTCTCTC-3'. 11) SNP, MPJ6_CS2011; GENE NAME, CES2; ACCESSION NUMBER, NT_010498.13; LENGTH, 25 bases; 5'-TCCACCTGGGGTA/GGATGTTGCCTCC-3'. 12) SNP, MPJ6_CS2013; GENE NAME, CES2; ACCESSION NUMBER, NT_010498.13; LENGTH, 25 bases; 5'-GGACTGGGGACCG/AAGGTCTCGGGGG-3'The frequencies were 0.029 for MPJ6_CS2004 and CS2013, 0.026 for MPJ6_CS2009, 0.013 for MPJ6_CS2001, 0.007 for MPJ6_CS2003, and 0.003 for the other 7 SNPs. Among these SNPs, MPJ6_CS2005 (100C>T) resulted in an amino acid alteration (R34W), and MPJ6_CS2007 (579C>T) and MPJ6_CS2008 (765C>T) were synonymous (T193T and G255G, respectively). Furthermore, MPJ6_CS2011 (IVS8-2A>G) resulted in variation at ther 3' splice acceptor site.

Eleven novel single nucleotide polymorphisms in the NR1I2 (PXR) gene, four of which induce non-synonymous amino acid alterations.

Eleven novel single nucleotide polymorphisms (SNPs) were found in the NR1I2 (PXR/SXR) gene from 205 Japanese subjects. The detected SNPs were as follows: 1) SNP, MPJ6_1I2001; GENE NAME, NR1I2; ACCESSION NUMBER, AF364606; LENGTH, 25 bases; 5'-TTTCTACCTCTAC/TTATTGAAAGGGC-3'. 2) SNP, MPJ6_1I2004; GENE NAME, NR1I2; ACCESSION NUMBER, AF364606; LENGTH, 25 bases; 5'-AGGCCCAAATGTG/AAGTGATGCATAG-3'. 3) SNP, MPJ6_1I2007; GENE NAME, NR1I2; ACCESSION NUMBER, AF364606; LENGTH, 25 bases; 5'-TGCCAGGCCTGCC/TGCCTGCGCAAGT-3'. 4) SNP, MPJ6_1I2008; GENE NAME, NR1I2; ACCESSION NUMBER, AF364606; LENGTH, 25 bases; 5'-GAGTGAGCAGTGG/CGCGCGCGGGCGG-3'. 5) SNP, MPJ6_1I2010; GENE NAME, NR1I2; ACCESSION NUMBER, AF364606; LENGTH, 25 bases; 5'-CAGAGGAGCAGCG/AGATGATGATCAG-3'. 6) SNP, MPJ6_1I2011; GENE NAME, NR1I2; ACCESSION NUMBER, AF364606; LENGTH, 25 bases; 5'-CTGGAAGTGGCCA/GGGAGGTTCAAAG-3'. 7) SNP, MPJ6_1I2013; GENE NAME, NR1I2; ACCESSION NUMBER, AF364606; LENGTH, 25 bases; 5'-TCTTCCTCTCGCC/TCCCAACTTCTGG-3'. 8) SNP, MPJ6_1I2017; GENE NAME, NR1I2; ACCESSION NUMBER, AF364606; LENGTH, 25 bases; 5'-ATTGAATGCAATC/TGGCCCCAGCCTG-3'. 9) SNP, MPJ6_1I2018; GENE NAME, NR1I2; ACCESSION NUMBER, AF364606; LENGTH, 25 bases; 5'-GGTGAGCACAGCA/GGGGGGTGAGGAC-3'. 10) SNP, MPJ6_1I2019; GENE NAME, NR1I2; ACCESSION NUMBER, AF364606; LENGTH, 25 bases; 5'-GAGCTCCGCAGCA/GTCAATGCTCAGC-3'. 11) SNP, MPJ6_1I2021; GENE NAME, NR1I2; ACCESSION NUMBER, AF364606; LENGTH, 25 bases; 5'-GGTGACACCTCCG/AAGAGGCAGCCAG-3'. The frequencies were 0.0293 for MPJ6_1I2021, 0.0073 for MPJ6_1I2011, and 0.0024 for the other 9 SNPs. All SNPs were found as heterozygous. Among these SNPs, MPJ6_1I2007, MPJ6_1I2010, MPJ6_1I2017 and MPJ6_1I2019 induce non-synonymous amino acid alterations (R98C, R148Q, R381W and I403V, respectively, in PAR1).

Semimature stage: a checkpoint in a dendritic cell maturation program that allows for functional reversion after signal-regulatory protein-alpha ligation and maturation signals.

CD47 on live cells actively engages signal-regulatory protein-alpha (SIRP-alpha) on phagocytes and delivers a negative signal that prevents their elimination. We evaluated the biological consequences of SIRP-alpha ligation on the dendritic cell (DC) response to maturation signals and the potential interplay with the IL-10/IL-10R inhibitory pathway. At first, CD47/SIRP-alpha allowed the generation of mature migratory DCs not producing IL-12, IFN-gamma-inducible protein-10, and CCL19. Rather, they secreted neutrophils attracting chemokine CXCL5 and IL-1beta, reflecting a partial block in functional DC maturation. Afterward, semimature DCs functionally regressed in an IL-10-independent fashion toward cells that retrieved the cardinal features of immature DCs: re-expression of CCR5, loss of DC-lysosome-associated membrane protein, high endocytosis, and impaired allostimulatory functions. The global gene expression profile of IL-10 and SIRP-alpha-ligated DC demonstrated two distinct molecular pathways. IL-10R and SIRP-alpha expression were reciprocally down-regulated by CD47 and IL-10, respectively. These results emphasize that the SIRP-alpha pathway might be part of the molecular machinery used by the DC to dampen or resolve an inflammatory response in an IL-10-independent manner.

Thrombospondin/CD47 interaction: a pathway to generate regulatory T cells from human CD4+ CD25- T cells in response to inflammation.

Thymus-derived CD4+ CD25+ T regulatory cells (Tregs) are essential for the maintenance of self-tolerance. What critical factors and conditions are required for the extra-thymic development of Tregs remains an important question. In this study, we show that the anti-inflammatory extracellular matrix protein, thrombospondin-1, promoted the generation of human peripheral regulatory T cells through the ligation of one of its receptor, CD47. CD47 stimulation by mAb or a thrombospondin-1 peptide induced naive or memory CD4+ CD25- T cells to become suppressive. The latter expressed increased amounts of CTLA-4, OX40, GITR, and Foxp3 and inhibited autologous Th0, Th1, and Th2 cells. Their regulatory activity was contact dependent, TGF-beta independent, and partially circumvented by IL-2. This previously unknown mechanism to induce human peripheral Tregs in response to inflammation may participate to the limitation of collateral damage induced by exacerbated responses to self or foreign Ags and thus be relevant for therapeutic intervention in autoimmune diseases and transplantation.

Hierarchical organization of photoresponsive hydrogen-bonded rosettes.

Hydrogen-bonded disk-shaped aggregates (rosettes) composed of azobenzene-appended melamine and barbiturate or cyanurate are investigated in view of their hierarchical organization and photoresponsive behavior by (1)H NMR and UV/vis spectroscopies, dynamic light scattering, and gelation behavior in aliphatic solvents and liquid crystalline behavior in bulk state. In the bulk state the rosette possessing a sterically bulky tridodecyloxyphenyl substituent in the barbiturate component stacks in an offset arrangement to form a rectangular columnar mesophase, whereas in aliphatic solvents it does not hierarchically organize into higher-order columnar aggregates. This drawback is improved by exchanging the barbiturate component into a more sterically nondemanding N-dodecylcyanurate component. The resulting new rosette stacks in a face-to-face arrangement to form a hexagonal columnar mesophase in the bulk state and hierarchically organizes into elongated fibrous aggregates in cyclohexane, which eventually leads to the formation of organogel. Dynamic light scattering and UV-vis experiments upon UV-irradiation of the columnar aggregates in cyclohexane revealed that the dissociation and the reformation of columnar aggregates can be controlled by the trans-cis isomerization of the azobenzene moiety. Molecular modeling indicates that the rosette possessing cis-azobenzene side chains loses its planarity. Using this photoinduced morphological change of the rosette, photoresponsive organogel is created by the use of a disk-shaped supramolecule the first time.

A target selection of somatic hypermutations is regulated similarly between T and B cells upon activation-induced cytidine deaminase expression.

Activation-induced cytidine deaminase (AID) is essential for somatic hypermutations (SHM) and class switch recombination. Overexpression of AID in non-B cells can induce SHM in artificial constructs inserted in various loci in the genome. AID overexpression was thus proposed to introduce mutations in a wide variety of genes with little specificity. We previously showed that AID transgenic mice developed T cell lymphomas in which the variable region beta genes of the T cell receptor and c-myc were mutated as frequently as SHM in activated B cells. To understand the target specificity of SHM in AID-expressing T lymphomas, we sequenced six oncogenes (c-myc, pim1, p53, atm, tgfbr-2, and k-ras) and two genes (cd4 and cd5) that are actively transcribed in T lymphomas. SHM was found only in c-myc, pim1, cd4, and cd5, which share the E47 binding motif in the enhancer/promoter. The rest that are not mutated in B cells were not mutated in AID-induced T lymphomas either, although they are transcribed in T and B cells. Comparison of several features of SHM, including selection of targets and mutation distribution, suggests that the regulatory mechanism of SHM is similar between T and B cells. SHM base specificities in the CD4 and CD5 genes were biased to AT, indicating that the preference of target bases of the mutations generated by overexpression of AID is not always GC bases but variable between target genes.