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.

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.

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.

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.

[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.

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.

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.

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.

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.

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.

Staphylococcal enterotoxin B increases TIM4 expression in human dendritic cells that drives naïve CD4 T cells to differentiate into Th2 cells.

Aberrant T helper (Th)2 polarization plays a critical role in the pathogenesis of allergic disorders; the etiology remains unclear. Dendritic cells (DCs) express T cell immunoglobulin mucin domain (TIM)4 that ligates TIM1 on CD4 T cells to drive them to become Th2 cells, but the pathogenic source of TIM4 is unknown. Here we report that a significant increase in TIM4 expression in human DCs was observed in response to Staphylococcal enterotoxin B (SEB) stimulation via Toll-like receptor (TLR)2 and nucleotide-binding oligomerization domain (NOD)1 pathway. Coculture SEB-conditioned DCs with naïve CD4 T cells induced Th2 responses that could be abolished using TLR2 or NOD1 or TIM4 or TIM1 with counterpart antibodies or RNA interference. The results demonstrate that Staphylococcus aureus derived SEB promotes the TIM4 production in human DCs. The interaction between TIM4 and TIM1 drives naïve CD4 T cells to develop to Th2 cells.

N49 phospholipase A2, a unique subgroup of snake venom group II phospholipase A2.

A novel phospholipase A2 (PLA2) with Asn at its site 49 was purified from the snake venom of Protobothrops mucrosquamatus by using SP-Sephadex C25, Superdex 75, Heparin-Sepharose (FF) and HPLC reverse-phage C18 chromatography and designated as TM-N49. It showed a molecular mass of 13.875 kDa on MALDI-TOF. TM-N49 does not possess enzymatic, hemolytic and hemorrhagic activities. It fails to induce platelet aggregation by itself, and does not inhibit the platelet aggregation induced by ADP. However, it exhibits potent myotoxic activity causing inflammatory cell infiltration, severe myoedema, myonecrosis and myolysis in the gastrocnemius muscles of BALB/c mice. Phylogenetic analysis found that that TM-N49 combined with two phospholipase A2s from Trimeresurus stejnegeri, TsR6 and CTs-R6 cluster into one group. Structural and functional analysis indicated that these phospholipase A2s are distinct from the other subgroups (D49 PLA2, S49 PLA2 and K49 PLA2) and represent a unique subgroup of snake venom group II PLA2, named N49 PLA2 subgroup.

Purification, characterization and potent lung lesion activity of an L-amino acid oxidase from Agkistrodon blomhoffii ussurensis snake venom.

L-amino acid oxidases (LAOs) are one of the major components of snake venoms, which possess numerous biological functions. However, little is known of the influence of LAOs on organ lesions. In the present study, a unique LAO from Agkistrodon blomhoffii ussurensis snake venom named ABU-LAO was purified by Heparin-Sepharose FF chromatography followed by an ion-exchange chromatography procedure. The purified ABU-LAO appears a dimer with a molecular mass of approximately 108.8kDa. Kinetics studies showed that ABU-LAO is very active towards its substrates L-Asn, L-Phe, L-Tyr, L-Leu, L-Ile and L-Trp. The most striking observation in the present study is that ABU-LAO causes severe pneumorrhagia, pulmonary interstitial edema, fusion of pulmonary alveoli, cardiac interstitial edema and bleeding when being intravenously injected into BALB/c mice. ABU-LAO also induces liver cell necrosis and release of cytokines including IL-6, IL-12 and IL-2 from highly purified human peripheral blood monocytes and T cells, respectively. In conclusion, ABU-LAO potently induces lesions in lungs and livers. The ability of ABU-LAO will contribute to the understanding of the pathogenesis of snakebite wound.

Modulation of expression and function of Toll-like receptor 3 in A549 and H292 cells by histamine.

It was reported recently that histamine induced Toll-like receptor (TLR)2 and TLR4 expression in endothelial cells and enhanced their sensitivity to Gram-positive and Gram-negative bacteria; and that TLRs were expressed in airway epithelial cells and that several inflammatory mediators modulated their expression. However, little is known of potential influence of histamine on TLRs in pulmonary epithelial cells. In the present study, effects of histamine on expression of TLRs in both human A549 and NCI-H292 cell lines were examined by using real-time quantitative RT-PCR analysis, flow cytometry and immunofluorescent staining. The results revealed that both cell types constitutively expressed mRNAs for TLR1-TLR10. Histamine up-regulated the expression of TLR3 mRNA by 12.3- and 11.6-fold, respectively in both cell types. The time course showed that histamine induced TLR3 mRNA expression was initiated at 30 min, nearly reached peak levels after 2 h and was sustained at least until 12 h. Histamine also induced TLR3 protein expression in A549 and NCI-H292 cells. Histamine and poly (I:C), a specific TLR3 ligand stimulated interleukin (IL)-8 secretion from both cell types. Moreover, histamine enhanced poly (I:C)-induced IL-8 secretion and phosphorylation of NF-kappaB in the two cell types, and histamine H1 receptor antagonists inhibited the action of histamine. In conclusion, histamine selectively up-regulated expression of TLR3, and stimulated IL-8 secretion from the cells. Histamine also enhanced poly (I:C) induced IL-8 secretion and phosphorylation of NF-kappaB. These observations suggest that histamine might play an important role in enhancing the innate immune responses of airway to viral infection.

Potent histamine-releasing activity of atrahagin, a novel snake venom metalloproteinase.

Poisonous snakebite wound is a popular disease worldwide. However, the pathogenesis remains unclear. In the present study, a novel metalloproteinase atrahagin in Chinese cobra (Naja atra) snake venom was purified, using heparin-sepharose followed by Superdex 75 gel filtration chromatography. Apart from its alpha-fibrinogenase activity, atrahagin potently activated human colon, lung and tonsil mast cells with the net histamine release being 25.9+/-4.4, 17.0+/-1.9, 13.2+/-3.6%, respectively. Time course studies revealed that the peak histamine release induced by atrahagin occurred at 12, 12 and 8 min following incubation of the enzyme with colon, lung and tonsil mast cells, respectively. The response of mast cells to atrahagin was abolished by preincubation of the cells with metabolic inhibitors or pertussis toxin, and by removal of Ca2+ and Mg2+ from the challenge buffer. In conclusion, activation of human mast cells by atrahagin indicated that the enzyme might contribute to the pathogenesis of snakebite wound.

Purification, characterization and cytokine release function of a novel Arg-49 phospholipase A(2) from the venom of Protobothrops mucrosquamatus.

Group IIA phospholipase A(2) (PLA(2)) are major components in Viperidae/Crotalidae venom. In the present study, a novel PLA(2) named promutoxin with Arg at the site 49 has been purified from the venom of Protobothrops mucrosquamatus by chromatography. It consists of 122 amino acid residues with a molecular mass of 13,656 Da assessed by MALDI-TOF. It has the structural features of snake venom group IIA PLA(2)s, but has no PLA(2) enzymatic activity. Promutoxin shows higher amino acid sequence identity to the K49 PLA(2)s (72-95%) than to D49 PLA(2)s (52-58%). Promutoxin exhibits potent myotoxicity in the animal model with as little as 1 microg of promutoxin causing myonecrosis and myoedema in the gastrocnemius muscle of mice. Promutoxin is also able to stimulate the release of IL-12, TNFalpha, IL-6 and IL-1beta from human monocytes, and induce IL-2, TNFalpha and IL-6 release from T cells, indicating that this snake venom group IIA PLA(2) is actively involved in the inflammatory process in man caused by snake venom poisoning.

Adaptive evolution of the spike gene of SARS coronavirus: changes in positively selected sites in different epidemic groups.

BACKGROUND: It is believed that animal-to-human transmission of severe acute respiratory syndrome (SARS) coronavirus (CoV) is the cause of the SARS outbreak worldwide. The spike (S) protein is one of the best characterized proteins of SARS-CoV, which plays a key role in SARS-CoV overcoming species barrier and accomplishing interspecies transmission from animals to humans, suggesting that it may be the major target of selective pressure. However, the process of adaptive evolution of S protein and the exact positively selected sites associated with this process remain unknown. RESULTS: By investigating the adaptive evolution of S protein, we identified twelve amino acid sites (75, 239, 244, 311, 479, 609, 613, 743, 765, 778, 1148, and 1163) in the S protein under positive selective pressure. Based on phylogenetic tree and epidemiological investigation, SARS outbreak was divided into three epidemic groups: 02-04 interspecies, 03-early-mid, and 03-late epidemic groups in the present study. Positive selection was detected in the first two groups, which represent the course of SARS-CoV interspecies transmission and of viral adaptation to human host, respectively. In contrast, purifying selection was detected in 03-late group. These indicate that S protein experiences variable positive selective pressures before reaching stabilization. A total of 25 sites in 02-04 interspecies epidemic group and 16 sites in 03-early-mid epidemic group were identified under positive selection. The identified sites were different between these two groups except for site 239, which suggests that positively selected sites are changeable between groups. Moreover, it was showed that a larger proportion (24%) of positively selected sites was located in receptor-binding domain (RBD) than in heptad repeat (HR)1-HR2 region in 02-04 interspecies epidemic group (p = 0.0208), and a greater percentage (25%) of these sites occurred in HR1-HR2 region than in RBD in 03-early-mid epidemic group (p = 0.0721). These suggest that functionally different domains of S protein may not experience same positive selection in each epidemic group. In addition, three specific replacements (F360S, T487S and L665S) were only found between 03-human SARS-CoVs and strains from 02-04 interspecies epidemic group, which reveals that selective sweep may also force the evolution of S genes before the jump of SARS-CoVs into human hosts. Since certain residues at these positively selected sites are associated with receptor recognition and/or membrane fusion, they are likely to be the crucial residues for animal-to-human transmission of SARS-CoVs, and subsequent adaptation to human hosts. CONCLUSION: The variation of positive selective pressures and positively selected sites are likely to contribute to the adaptive evolution of S protein from animals to humans.

Rhinosinusitis derived Staphylococcal enterotoxin B plays a possible role in pathogenesis of food allergy.

BACKGROUND: Staphylococcal enterotoxin B (SEB) is a potent immunomodulator and implicated with pathogenesis of inflammatory diseases mediated by Th1 or Th2 dominant immune responses. The objective of this study is to determine a possible association between rhinosinusitis derived SEB and pathogenesis of food allergy (FA). METHODS: The study included chronic rhinosinusitis (CRS) patients with FA (N = 46) or without FA (N = 33). Controls included FA patients without CRS (N = 26) and healthy volunteers (N = 25). In CRS patients, we assessed the parameters associated with FA including prick skin test (PST) reactivity to food allergens, serum levels of allergen-specific IgE and cytokines (IL-4, IL-13, IFN-I3), and the number/reactivity of food-allergen specific Th1/Th2 cells in the peripheral blood before and 2 months after sinus surgery. Changes of these parameters were evaluated in comparison with changes in SEB concentration in the sinus lavage and stool samples and also in vitro reactivity to SEB. In CRS patients with FA, we also assessed changes in reactivity to oral challenge of offending food before and after sinus surgery. RESULTS: Two months following sinus surgery, we observed statistically significant reduction in PST and oral challenge reactivity in CRS patients with FA in parallel to decrease in serum levels of Th2 cytokines (IL-4 and IL-13) and allergen specific IgE. Improvement of reactivity to food allergens was positively associated with decline in SEB concentrations in the sinus lavage and stool samples. In vitro study results also indicated a role of SEB in aggravation of Th2 skewed responses to food allergens. Such changes were not observed in CRS-non FA patients or control FA patients. CONCLUSION: The rhinosinusitis derived SEB plays a certain role in the pathogenesis of FA by augmenting and/or maintaining polarized Th2 responses. Removal of SEB-producing pathogens from the rhinosinuses may be beneficial for attenuating the FA symptoms in patients with CRS-FA.