Immunological characterization of the American cockroach allergen Per a 9 expressed in baculovirus-infected insect cells.

American cockroach (CR) allergy has been recognized as important IgE-mediated type I hypersensitivity. Per a 9 is an arginine kinase, reacting with IgE in sera of all CR allergic Thai patients. Per a 9 gene was cloned and expressed in eukaryotic systems (baculovirus-infected insect cells). The expressed Per a 9 was purified by Nickel column. The antigenicities were analyzed by ELISA, immunoblot analysis and basophile activation test. The results show that 13 out of 16 (81.3%) sera from American CR patients reacted to Per a 9, confirming that Per a 9 is a major allergen of CR. The IgE reactivity of Per a 9 in the sera from American CR patients was increased 8.3-fold in comparison with the sera from healthy controls. Per a 9 at 1.0 µg/ml induced an approximately up to 5.6-fold increase in CD63 and CCR3 double positive cells when incubating with passively sensitized basophils from by sera from American CR patients.

[Expression of LRG-1 in clinical specimens and Tca8113 cell line of tongue carcinoma].

OBJECTIVE: To investigate the expression of LRG-1 in clinical specimens and Tca8113 cell line of tongue carcinoma and analyze the relationship between LRG-1 expression and the clinicopathological parameters. METHODS: LRG-1 expression was detected in 40 tongue squamous cell carcinoma (TSCC) tissues and paired normal adjacent tissues, 20 atypical hyperplasia tissues of the tongue, and 20 tissues of tongue cancer in situ using immunohistochemical method. The expression of LRG-1 in Tca8113 cell line was detected using flow cytometry. The expression of LRG-1 was also detected in human TSCC tissues and Tca8113 cells with Western blotting. The effect of LRG-1 on the proliferation of HUVECs was determined using MTT assay, and its effect on angiogenesis was evaluated with Matrigel tube formation assays. RESULTS: Human TSCC tissues had a significantly higher rate of positive expression for LRG-1 (85%, 34/40) than the adjacent tissues (10%, 4/40), invasive tongue cancer (30%, 6/20), and tongue cancer in situ (50%, 10/20) (P<0.05). LRG-1 expression was correlated with the degree of tumor differentiation, clinical stage and lymph node metastasis of the tumor (P<0.05) but not with the patients' age or gender. In the in vitro experiment, LRG-1 promoted HUVEC proliferation and angiogenesis. CONCLUSION: Abnormal LRG-1 expression is present in the human TSCC tissue and Tca8113 cells. LRG-1 can promote HUVEC proliferation and angiogenesis in vitro, suggesting its possible role in promoting tumor angiogenesis.

Mast cells and basophils are essential for allergies: mechanisms of allergic inflammation and a proposed procedure for diagnosis.

The current definition of allergy is a group of IgE-mediated diseases. However, a large portion of patients with clinical manifestations of allergies do not exhibit elevated serum levels of IgE (sIgEs). In this article, three key factors, ie soluble allergens, sIgEs and mast cells or basophils, representing the causative factors, messengers and primary effector cells in allergic inflammation, respectively, were discussed. Based on current knowledge on allergic diseases, we propose that allergic diseases are a group of diseases mediated through activated mast cells and/or basophils in sensitive individuals, and allergic diseases include four subgroups: (1) IgE dependent; (2) other immunoglobulin dependent; (3) non-immunoglobulin mediated; (4) mixture of the first three subgroups. According to our proposed definition, pseudo-allergic-reactions, in which mast cell or basophil activation is not mediated via IgE, or to a lesser extent via IgG or IgM, should be non-IgE-mediated allergic diseases. Specific allergen challenge tests (SACTs) are gold standard tests for diagnosing allergies in vivo, but risky. The identification of surface membrane activation markers of mast cells and basophils (CD203c, CCR3, CD63, etc) has led to development of the basophil activation test (BAT), an in vitro specific allergen challenge test (SACT). Based on currently available laboratory allergy tests, we here propose a laboratory examination procedure for allergy.

IFN-λ inhibits HIV-1 integration and post-transcriptional events in vitro, but there is only limited in vivo repression of viral production.

The lambda interferons (IL-28a, 28b, and IL-29) inhibit the replication of many viruses, but their role in the inhibition of HIV-1 infection remains unclear. During this study, we monitored IL-29 production in HIV-1 infected individuals and analyzed the in vitro and in vivo inhibition of HIV-1 production. Prior treatment with IL-28a or IL-29 induced an antiviral state in cultured primary T-cells, which suppressed HIV-1 integration and post-transcriptional events. The antiviral factors MxA, OAS, and PKR were up-regulated. In HIV-1 infected patients, IL-29 level was increased along with the depletion of CD4⁺ T-cells in peripheral blood, while the elevated IL-29 did not show a significantly negative correlation with viral load. Further analysis of HIV-1 infected individuals showed that IL-29 was positively correlated with IFN-ß and anti-inflammatory cytokine IL-10, and was negatively correlated with IFN-γ, which might suggest that IFN-λ participates in modulating antiviral immune responses during HIV-1 infection in vivo. Together, although IFN-λ impeded HIV-1 infection of T-cells in vitro, IFN-λ showed only limited in vivo repression of viral production. The modulation of IFN-λ on inflammatory factors might be worthy for further concentrating on for better understanding the host immune response during HIV-1 infection.

Eosinophil-derived interferon-lambda contributes to initiation of allergen-related inflammation in the intestine.

BACKGROUND AND AIMS: Epithelial barrier dysfunction plays a critical role in the initiation of a number of immune diseases; the causative factors are not fully understood. The present study aimed to elucidate the mechanism by which the eosinophil-derived interferon (IFN)-lambda induced the gut epithelial barrier dysfunction. METHODS: The duodenal biopsies were obtained from patients with or without food allergies. The eosinophils and IFNλ expression were observed by immune staining. Intestinal epithelial cell line, T84 cells, and a mouse model were employed to observe the effect of IFNλ on the epithelial barrier function and the initiation of skewed T helper (Th)2 polarization in the mouse intestine. RESULTS: IFNλ expression was observed in over 80% human eosinophils of the subjects with or without food allergies. Exposure to microbial products, lipopolysaccharide or peptidoglycan, could induce eosinophils to release IFNλ. Exposure to IFNλ could induce intestinal epithelial barrier dysfunction via inducing the epithelial cell apoptosis. Concurrent exposure to microbial products and food antigens could induce aberrant antigen specific Th2 polarization and Th2 pattern inflammation in the intestine. CONCLUSIONS: Eosinophils express IFNλ that can induce intestinal epithelial barrier dysfunction and promotes the initiation of the aberrant Th2 polarization in the intestine.

[Signal transduction pathway for TNF-induced IL-6 release from mast cells].

AIM: To investigate the effect of TNF on release of IL-6, IL-10 and histamine from mast cells and to explore its potential signal transduction pathway. METHODS: After stimulation of various concentrations of TNF, the P815 cells were analyzed by cellular activation of signal ELISA(CASE) to detect phosphorylation of ERK, p38 and STAT3, and the supernatants were collected and analyzed by ELISA to quantify release of IL-6, IL-10 and histamine from the cells. RESULTS: Compared with the untreated control, increased IL-6 release was detected in TNF-stimulated P815 cells (P<0.05). Pretreatment of PD98059 or U0126 inhibited TNF-induced IL-6 release and ERK phosphorylation in P815 cells (P<0.05), whereas pretreatment of SB203580 or AG490 hardly affect IL-6 release, with little effect on phosphorylation of p38 and STAT3 respectively. CONCLUSION: TNF induced IL-6 release from P815 cells may involve activation of ERK signalling pathway.

[Induction of monocyte-derived dendritic cell differentiation by asthmatic serum in a transendothelial trafficking model].

OBJECTIVE: To explore the effect of asthmatic and healthy serum on differentiation and function of monocyte-derived dendritic cells (MDDC) in a transendothelial trafficking model. METHODS: The sera and peripheral blood mononuclear cells (PBMC) were separated from 12 asthmatic patients and 12 healthy volunteers, and monocytes were selected from PBMC using magnetic beads. The trypsin-digested human umbilical vein endothelial cells (HUVEC) at passage 2 from 5 healthy lying-in women were used to construct the transendothelial trafficking model under asthmatic or healthy serum, wherein MDDC were identified by silver nitrate staining and scanning electron microscopy. Nuclear factor κB (NF-κB) activity was determined by electrophoretic mobility shift assay. Flow cytometry, ELISA and mixed leukocyte reaction were relevantly utilized to detect the phenotype, cytokine and T cell proliferation. RESULTS: (1) Monocytes traversed through HUVEC monolayer after 2 h, and reverse-transmigrated to develop into DC 48 h later. (2) The healthy serum stimulated monocytes into immature MDDC with lower CD(14) [(20 ± 5)%] (F = 49.01, P < 0.05), and higher HLA-DR, CD(80), CD(86) and CD(83) [(43 ± 4)%, (17.9 ± 3.5)%, (43 ± 11)% and (6.7 ± 1.8)%, respectively] (F = 10.35 - 40.17, all P < 0.05) than monocytes did before transmigration at 0 h [CD(14) (81 ± 6)%, HLA-DR (24 ± 5)%, CD(80) (2.8 ± 2.0)%, CD(86) (14 ± 4)% and CD(83) (0.9 ± 0.8)%, respectively]. (3) The asthmatic serum stimulated monocytes into mature MDDC, characteristic of dendrites, with similar HLA-DR and CD(86) [(55 ± 6)% and (59 ± 12)%] (F = 15.29 and 35.97, all P > 0.05), higher CD(80) and CD(83) [(49.7 ± 10.2)% and (30.2 ± 6.8)%] (F = 4.01 and 20.68, all P < 0.05), accompanied by increased levels of NF-κB activity, IL-12 p70 and T cell proliferation [(100 ± 11)%, (568 ± 43) ng/L and (2033 ± 198) cpm, respectively] (F = 49.23 - 350.84, all P < 0.05) relative to the healthy serum-stimulated immature MDDC [(12 ± 3)%, (220 ± 35) ng/L and (952 ± 64) cpm, respectively]. CONCLUSION: The asthmatic serum induces mature MDDC in association with NF-κB overactivation in the transendothelial trafficking model, which provides a promising experimental platform for both investigation of immunological mechanisms in asthma and screening of novel anti-asthma drugs in vitro.

Interferon-λ mediates oral tolerance and inhibits antigen-specific, T-helper 2 cell-mediated inflammation in mouse intestine.

BACKGROUND & AIMS: Oral tolerance is an important component of gastrointestinal homeostasis, but mechanisms of its development are not fully understood. Loss of oral tolerance occurs during food allergen-related inflammation in the gastrointestinal tract. Interferon (IFN)-λ regulates immunity, but its role in oral tolerance is not clear. We investigated the role and the mechanism of IFN-λ in the development of oral tolerance and its effect on antigen-induced, T-helper (Th)-2 cell-mediated inflammation in the intestine. METHODS: Expression of IFN-λ and its receptor were analyzed by immunohistochemical, flow cytometric, or immunoblot analyses. Tolerogenic dendritic cells (DCs) and regulatory T cells were examined in vitro and in vivo. A mouse model of antigen-induced, Th2 cell-mediated intestinal inflammation was used to examine the role of IFN-λ and T cells in oral tolerance in the intestine. RESULTS: CD3+ cells expressed the IFN-λ receptor, which was up-regulated following antigen-specific or nonspecific activation. Interaction between IFN-λ and its receptor induced apoptosis of T cells and their subsequent phagocytosis by DCs. This led to the generation of tolerogenic DCs and T regulatory cells in vitro and in vivo. Passive transfer of IFN-λ-primed CD3+ cells inhibited Th2 cell-mediated inflammation in the intestine. CONCLUSIONS: IFN-λ is involved in development and maintenance of oral tolerance in the intestines of mice; it might be used to suppress antigen-specific Th2 cell-mediated inflammation in patients.

Unusual life-threatening Rosai-Dorfman disease of the trachea: role of NF-κB.

Rosai-Dorfman disease (RDD) is a rare non-neoplastic histioproliferative disorder characterised by painless lymphadenopathy, low fever, high erythrocyte sedimentation rate, leucocytosis and hypergammaglobulinaemia. Overactivity of nuclear factor κB (NF-κB) is linked with inflammatory, cancerous and autoimmune diseases. The first case is described of an unusual life-threatening RDD of the trachea with no lymphadenopathy at risk of suffocation in a 39-year-old Chinese woman. A diagnosis of RDD was made following CT scans, thoracotomy and histological examination. Gel shift assay revealed an essential role for NF-κB overactivity in RDD. The patient remains well with no evidence of progression without treatment. Histological confirmation should be sought in all cases as the clinical manifestation of RDD is similar to asthma or lung carcinoma.

Induction of inflammatory cell accumulation by TM-N49 and promutoxin, two novel phospholipase A(2).

Local inflammation is a prominent characteristic of snakebite wound. Snake venom phospholipase A(2)s (PLA(2)s) are one of the main components which contribute to accumulation of inflammatory cells. We have isolated TM-N49 and promutoxin from Protobothrops mucrosquamatus venom and investigated their ability in induction of cell accumulation by using an in vivo mouse model. The results showed that both TM-N49 and promutoxin are potent stimuli for induction of neutrophil, lymphocyte, macrophage and eosinophil accumulation in the mouse peritoneum. The TM-N49- and promutoxin-induced inflammatory cell accumulation was inhibited by pretreatment of animals with cyproheptadine, terfenadine and Ginkgolide B, indicating that histamine and PAF is likely to contribute to the cells accumulation. Pre-injection of antibodies against adhesion molecules ICAM-1, CD18, CD11a and L-selectin showed that ICAM-1 is a key adhesion molecule of TM-N49- and promutoxin-induced lymphocyte, macrophage and eosinophil accumulation; CD18 and CD11a plays an important role in the migration of neutrophils, eosinophils and macrophages; and L-selectin is involved in the neutrophil and eosinophil accumulation. In conclusion, induction of inflammatory cell accumulation by TM-N49 and promutoxin confirms that group II PLA(2)s is pivotal stimulus for cell infiltration, through which they participate in the formation of snakebite inflammation.

Induction of microvascular leakage and histamine release by promutoxin, an Arg49 phospholipase A2.

It has been recognized that phospholipase A(2) (PLA(2)) is a crucial factor of snake venom induced inflammation. Recently, promutoxin, a novel member of minor subgroup of snake venom PLA(2) (R49 PLA(2)) has been characterized in our laboratory, but its roles in induction of inflammation remain uninvestigated. Using highly purified promutoxin, we found this enzymatically inactive PLA(2) provoked a dose-dependent increase in microvascular leakage in the skin of rats. Pretreatment of rats with compound 48/80 diminished promutoxin-induced skin reaction and reduced mast cell numbers in rats. Cyproheptadine, terfenadine, Ginkgolide B and heparin inhibited promutoxin elicited microvascular leakage when they were co-injected with the stimulus to rat skin. Moreover, promutoxin was found to induce histamine release from human colon, lung and tonsil mast cells, and both metabolic inhibitors and pertussis toxin were capable of inhibiting promutoxin elicited histamine release. Provocation of microvascular leakage and mast cell activation by promutoxin suggests further that snake venom induced inflammation is related to mast cell activation and certain anti-inflammatory drugs could be therapeutic effective in treating snake wound.

Targeted NF-kappaB inhibition of asthmatic serum-mediated human monocyte-derived dendritic cell differentiation in a transendothelial trafficking model.

Transendothelial trafficking model mimics in vivo differentiation of monocytes into dendritic cells (DC). The serum from patients with systemic lupus erythematosus promotes the differentiation of monocytes into mature DC. We have shown that selective inhibition of NF-kappaB by adenoviral gene transfer of a novel mutated IkappaBalpha (AdIkappaBalphaM) in DC contributes to T cell tolerance. Here we demonstrated for the first time that asthmatic serum facilitated human monocyte-derived DC (MDDC) maturation associated with increased NF-kappaB activation in this model. Furthermore, selective blockade of NF-kappaB by AdIkappaBalphaM in MDDC led to increased apoptosis, and decreased levels of CD80, CD83, CD86, and IL-12 p70 but not IL-10 in asthmatic serum-stimulated MDDC, accompanied by reduced proliferation of T cells. These results suggest that AdIkappaBalphaM-transferred MDDC are at a more immature stage which is beneficial to augment the immune tolerance in asthma.

Purification, characterization and biological activities of the L-amino acid oxidase from Bungarus fasciatus snake venom.

L-amino acid oxidases (LAAOs) are widely distributed in snake venoms, which contribute to the toxicity of venoms. However, LAAO from Bungarus fasciatus (B. fasciatus) snake venom has not been isolated previously. In the present study, LAAO from B. fasciatus snake venom was purified by SP-Sepharose HP anion exchange chromatography followed by Heparin-Sepharose FF affinity chromatography procedure and the purified enzyme was named BF-LAAO. BF-LAAO presented an estimated molecular weight of 55kDa in SDS-PAGE and an apparent molecular weight of 70kDa in size-exclusion chromatography suggesting that BF-LAAO is a monomeric protein. Kinetics studies showed that BF-LAAO was very active against L-Tyr, L-Asp, L-Phe, L-Glu, L-Trp, L-His, L-Gln, L-Ile, L-Met, L-Leu and moderately active against L-Lys, L-Arg, L-Ala and L-Asn. BF-LAAO exhibited a cytotoxic effect on A549 cells and caused up to 41.2% apoptosis of A549 cells following 12h incubation period. In the mouse peritoneum, BF-LAAO provoked a marked increase in the number of neutrophils, lymphocytes and macrophages following injection. It also induced rabbit platelet aggregation in a dose-dependent manner. At 3h following injection, BF-LAAO elicited severe inflammation in the gastrocnemius muscles of mice, but failed to induce significant organ damage. In conclusion, the cytotoxic and proinflammatory activities of BF-LAAO could be the main cause of the local inflammation, which helps us to understand the pathogenesis of snakebite.

Induction of mast cell accumulation, histamine release and skin edema by N49 phospholipase A2.

BACKGROUND: It has been recognized that phospholipase A2 (PLA2) is a crucial component of snake venom, which contributes greatly to snake venom induced inflammation in man. However, the mechanisms through which N49 PLA2 provoke inflammation remain unclear. Recently, a N49 PLA2, TM-N49 from Protobothrops mucrosquamatus crude venom was characterized in our laboratory. Since the purification procedure developed is able to supply us with relatively large quantity of highly purified TM-N49, we investigated the ability of TM-N49 in induction of inflammation. RESULTS: The results showed that TM-N49 provoked a dose dependent increase in microvascular leakage in the skin of rats. The potency of TM-N49 in induction of skin edema appeared similar potency of bradykinin and histamine. Pretreatment of rats with compound 48/80 diminished TM-N49 induced skin reaction and reduced mast cell numbers in rats. Ginkgolide B and cyproheptadine, but not terfenadine and quinacrine, inhibited TM-N49 elicited microvascular leakage when they were co-injected with the stimulus to rat skin. Moreover, TM-N49 was found to induce histamine release from human colon, lung and tonsil mast cells, and both metabolic inhibitors and pertussis toxin were capable of inhibiting TM-N49 elicited histamine release. TM-N49 induced mast cell accumulation in the peritoneum of mice, which was inhibited by co-injection of ginkgolide B, cyproheptadine and terfenadine. Intravenous injection of monoclonal antibodies against CD18, ICAM-1 and CD11a also blocked TM-N49 induced mast cell accumulation. CONCLUSION: TM-N49 is a potent stimulus for skin edema, mast cell activation and accumulation.

Expression of integrin alphavbeta6 in the intestinal epithelial cells of patients with inflammatory bowel disease.

BACKGROUND AND AIMS: The prevalence of inflammatory bowel disease (IBD) is about 0.05% in industrialized countries. The pathogenesis of IBD remains to be further understood. The present study aims to elucidate the expression of integrin αvß6 in the intestinal mucosa of patients with IBD. MATERIALS AND METHODS: Colonic biopsy was obtained from a group of IBD patients. The expression of αvß6 in the intestinal mucosa was detected by Western blotting. Human colonic epithelial cell line T84 cells were stimulated by microbial antigen flagellin. The expression of αvß6 in T84 cells was evaluated by quantitative RT-PCR and Western blotting. RESULTS: The levels of αvß6 in the intestinal mucosa were much lower than it in normal control subjects. The serum levels of myeloperoxidase (MPO) were higher in IBD patients that were negatively correlated with the levels of αvß6 in the intestinal mucosa. The expression of αvß6 was detectable in T84 cells at naοve status that could be upregulated by exposure to microbial antigen flagellin. Pretreatment with MPO dramatically suppressed the expression of αvß6 in T84 cells. CONCLUSIONS: We conclude that the expression of αvß6 was suppressed in IBD intestinal mucosa, which could be resulted from the high levels of MPO.

Glucuronoxylomannan promotes the generation of antigen-specific T regulatory cell that suppresses the antigen-specific Th2 response upon activation.

T regulatory cells (Treg) have the capability to suppress the skewed immune response, but the generation of antigen (Ag)-specific Treg for therapeutic purpose is a challenge; the mechanism of Ag-specific Treg activation remains obscure. Here, we report that glucuronoxylomannan (GXM) is capable of promoting the development of human tolerogenic dendritic cells (DC). GXM-pulsed DCs increased the expression of forkhead box P3 (Foxp3) in naïve human CD4(+)CD25(-) T cells via activating Fc gamma receptor IIb and activator protein-1 and promoting the expression of transforming growth factor beta in dendritic cells. Furthermore, the conjugated complex of house dust mite Ag, Dermatophagoides pteronyssinus (Der p) 1, and GXM-pulsed DCs to drive the naïve human CD4(+)CD25(-) T cells to develop into the Der p 1-specific Tregs, which efficiently suppressed the Ag-specific Th2 responses. We conclude that GXM-conjugated specific Ag have the capacity to up-regulate the tolerogenic property of DCs and promote the generation of Ag-specific Tregs; the latter can be activated upon the re-exposure to specific Ag and suppress the skewed Ag-specific T helper (Th)2 responses.

Disruption of T-cell immunoglobulin and mucin domain molecule (TIM)-1/TIM4 interaction as a therapeutic strategy in a dendritic cell-induced peanut allergy model.

BACKGROUND: Recent reports indicate that dendritic cell (DC)-derived T-cell immunoglobulin and mucin domain molecule (TIM)-4 plays an important role in the initiation of T(H)2 polarization. This study aims to elucidate the mechanisms of peanut allergy mediated by microbial products and DCs and the relationship between peanut allergy and TIM4. METHODS: Mouse bone marrow-derived DCs (BMDCs) were generated and exposed to cholera toxin (CT) or/and peanut extract (PE) for 24 hours and then adoptively transferred to naive mice. After re-exposure to specific antigen PE, the mice were killed; intestinal allergic status was determined. RESULTS: Increased expression of TIM4 and costimulatory molecules was detected in BMDCs after concurrent exposure to CT and PE. Adoptively transferred CT/PE-conditioned BMDCs resulted in the increases in serum PE-specific IgE and skewed T(H)2 polarization in the intestine. Oral challenge with specific antigen PE induced mast cell activation in the intestine. Treating with Toll-like receptor 4 small interfering RNA abolished increased expression of TIM4 and costimulatory molecules by BMDCs. Pretreatment with anti-TIM1 or anti-TIM4 antibody abolished PE-specific T(H)2 polarization and allergy in the intestine. CONCLUSION: Concurrent exposure to microbial product CT and food antigen PE increases TIM4 expression in DCs and promotes DC maturation, which plays an important role in the initiation of PE-specific T(H)2 polarization and allergy in the intestine. Modulation of TIM4 production in DCs represents a novel therapeutic approach for the treatment of peanut allergy.

Induction of inflammatory cytokine release from human umbilical vein endothelial cells by agonists of proteinase-activated receptor-2.

1. Human endothelial cells express proteinase-activated receptor-2 (PAR-2), inflammatory cytokines and trypsin (EC 3.4.21.4). However, little is known about the mechanism through which trypsin induces cytokine release from endothelial cells. 2. In the present study, we investigated the effect of trypsin on cytokine release from primary cultures of human umbilical vein endothelial cells (HUVEC) using an antibody based protein microarray and ELISA. 3. The results showed that 1 microg/mL trypsin induced release of 32 different inflammatory factors, whereas 100 micromol/L Ser-Leu-Ile-Gly-Lys-Val-NH2 (SLIGKV-NH2) only stimulated secretion of 16 inflammatory factors from HUVEC, as assessed by an antibody based protein microarray. Because the release of interleukin (IL)-1a, IL-8, IL-10 and IL-12 was markedly increased following PAR-2 activation, their release was investigated further using ELISA. Increases in release of up to approximately 4.8-, 4.3-, 4.1- and 1.8-fold were observed for IL-1a, IL-10, IL-12 and IL-8, respectively, when HUVEC were challenged with trypsin for 16 h. Agonist peptides of PAR-2, namely SLIGKV-NH2 and trans-cinnamoyl-Leu-Ile-Gly-Arg-Leu-Orn-NH2 (tc-LIGRLO-NH2), also provoked significant release of IL-8. Trypsin-induced cytokine release was inhibited by its inhibitors soybean trypsin inhibitor, alpha1-antitrypsin and the inhibitor peptide of PAR-2 Phe-Ser-Leu-Leu-Arg-Tyr-NH2 (FSLLRY-NH2). 4. These data indicate the action of trypsin on HUVEC is most likely through activation of PAR-2, suggesting that PAR-2-related mechanisms are involved in the inflammatory process in humans.

Evaluation of FORS-D analysis: a comparison with the statistically significant stem-loop potential.

The stem-loop potential of a nucleic acid segment (expressed as a FONS value), decomposes into base composition-dependent and base order-dependent components. The latter, expressed as a FORS-D value, is derived by subtracting the value of the base composition-dependent component (FORS-M) from the FONS value. FORS-D analysis is the use of FORS-D values to estimate the potential of local base order to contribute to a stem-loop structure, and it has been used to investigate the relationship between stem-loop structure and other selective pressures on genomes. In the present study, we evaluated the reliability of FORS-D analysis by comparing it with statistically significant stem-loop potential, another robust method developed by Le and Maizel for examining stem-loop structure. We found that FORS-M values calculated using 10 randomized sequences are as reliable as those calculated using 100 randomized sequences. The resulting FORS-D values have a similar trend and distribution as statistically significant stem-loop potential, implying that FORS-D analysis is as reliable as the latter in measuring the distribution of base order-dependent stem-loop potential. Since the calculation of the FORS-M values is time consuming, the integrated program Bodslp developed by us will become a convenient tool for large-scale FORS-D analysis. The results also suggest that for some purposes the online program SigStb developed by Le and Maizel may be used as an alternative tool for FORS-D analysis.