Safety evaluation of human umbilical cord-mesenchymal stem cells in type 2 diabetes mellitus treatment: A phase 2 clinical trial.

BACKGROUND: Progressive pancreatic ß cell dysfunction is a fundamental aspect of the pathology underlying type 2 diabetes mellitus (T2DM). Recently, mesenchymal stem cell (MSC) transplantation has emerged as a new therapeutic method due to its ability to promote the regeneration of pancreatic ß cells. However, current studies have focused on its efficacy, and there are few clinical studies on its safety. AIM: To evaluate the safety of human umbilical cord (hUC)-MSC infusion in T2DM treatment. METHODS: An open-label and randomized phase 2 clinical trial was designed to evaluate the safety of hUC-MSC transplantation in T2DM in a Class A hospital. Ten patients in the placebo group received acellular saline intravenously once per week for 3 wk. Twenty-four patients in the hUC-MSC group received hUC-MSCs (1 × 106 cells/kg) intravenously once per week for 3 wk. Diabetic clinical symptoms and signs, laboratory findings, and imaging findings were evaluated weekly for the 1st mo and then at weeks 12 and 24 post-treatment. RESULTS: No serious adverse events were observed during the 24-wk follow-up. Four patients (16.7%) in the hUC-MSC group experienced transient fever, which occurred within 24 h after the second or third infusion; this did not occur in any patients in the placebo group. One patient from the hUC-MSC group experienced hypoglycemic attacks within 1 mo after transplantation. Significantly lower lymphocyte levels (weeks 2 and 3) and thrombin coagulation time (week 2) were observed in the hUC-MSC group compared to those in the placebo group (all P < 0.05). Significantly higher platelet levels (week 3), immunoglobulin levels (weeks 1, 2, 3, and 4), fibrinogen levels (weeks 2 and 3), D-dimer levels (weeks 1, 2, 3, 4, 12, and 24), and neutrophil-to-lymphocyte ratios (weeks 2 and 3) were observed in the hUC-MSC group compared to those in the placebo group (all P < 0.05). There were no significant differences between the two groups for tumor markers (alpha-fetoprotein, carcinoembryonic antigen, and carbohydrate antigen 199) or blood fat. No liver damage or other side effects were observed on chest X-ray. CONCLUSION: Our study suggested that hUC-MSC transplantation has good tolerance and high safety in the treatment of T2DM. It can improve human immunity and inhibit lymphocytes. Coagulation function should be monitored vigilantly for abnormalities.

Glomus mosseae improved the adaptability of alfalfa (Medicago sativa L.) to the coexistence of cadmium-polluted soils and elevated air temperature.

The coexistence of heavy metal-polluted soils and global warming poses serious threats to plants. Many studies indicate that arbuscular mycorrhizal fungi (AMF) can enhance the resistance of plants to adverse environments such as heavy metals and high temperature. However, few studies are carried out to explore the regulation of AMF on the adaptability of plants to the coexistence of heavy metals and elevated temperature (ET). Here, we investigated the regulation of Glomus mosseae on the adaptability of alfalfa (Medicago sativa L.) to the coexistence of cadmium (Cd)-polluted soils and ET. G. mosseae significantly enhanced total chlorophyll and carbon (C) content in the shoots by 15.6% and 3.0%, respectively, and Cd, nitrogen (N), and phosphorus (P) uptake by the roots by 63.3%, 28.9%, and 85.2%, respectively, under Cd + ET. G. mosseae significantly increased ascorbate peroxidase activity, peroxidase (POD) gene expression, and soluble proteins content in the shoots by 13.4%, 130.3%, and 33.8%, respectively, and significantly decreased ascorbic acid (AsA), phytochelatins (PCs), and malondialdehyde (MDA) contents by 7.4%, 23.2%, and 6.5%, respectively, under ET + Cd. Additionally, G. mosseae colonization led to significant increases in POD (13.0%) and catalase (46.5%) activities, Cu/Zn-superoxide dismutase gene expression (33.5%), and MDA (6.6%), glutathione (22.2%), AsA (10.3%), cysteine (101.0%), PCs (13.8%), soluble sugars (17.5%), and proteins (43.4%) contents in the roots and carotenoids (23.2%) under ET + Cd. Cadmium, C, N, G. mosseae colonization rate, and chlorophyll significantly influenced shoots defenses and Cd, C, N, P, G. mosseae colonization rate, and sulfur significantly affected root defenses. In conclusion, G. mosseae obviously improved the defense capacity of alfalfa under ET + Cd. The results could improve our understanding of the regulation of AMF on the adaptability of plants to the coexistence of heavy metals and global warming and phytoremediation of heavy metal-polluted sites under global warming scenarios.

[Consecutive 4-year Elevated Atmospheric CO2 on Shaped Microbial Communities in the Rhizosphere Soil of Robinia pseudoacacia L. Seedlings Grown in Pb-contaminated Soils].

Elevated atmospheric CO2 could affect the speciation of heavy metals in rhizosphere soils by changing root exudates, thereby influencing soil microecosystem in the rhizosphere. Therefore, understanding the function of heavy metals in soils on rhizospheric ecology under elevated atmospheric CO2 scenarios is highly important. Here, we investigated the combined effects of a four-year period of elevated air CO2 concentrations[(700±27) µmol·L-1] and Pb-contamination (15.6 mg·kg-1 and 515.6 mg·kg-1) on the soil rhizopheric microbial community of Robinia pseudoacacia L. seedlings. Significant (P<0.05) effects of CO2, Pb, and their interaction on bacterial richness and fungal diversity were observed. Relative to Pb exposure alone, elevated CO2 significantly increased pH, total C, total N, and water-soluble organic carbon, and the C/N ratio under Pb exposure (P<0.05) and significantly decreased total and soluble Pb content (P<0.05). The richness and diversity of bacteria increased (P<0.05), fungal richness decreased (P<0.05), and microbial diversity increased (P<0.05) under the combined treatments relative to Pb contamination alone. The changes in the relative abundance of the top two dominant bacterial and fungal genera were not significant; however, differences in the relative abundances of other groups, such as Anaerolineaceae, Solirubrobacterales, Eurotiomycetes, Aspergillus, and Trichocomaceae, were significant between the different treatments. According to a redundancy analysis, total C and soluble Pb had a significant influence (P<0.05) on the dominant bacterial genera, and total C affected (P<0.05) the dominant genera in the fungal community. These results suggest that the responses of soil environmental factors to the combination of elevated atmospheric CO2 and Pb could shape soil microbial community structure in the rhizosphere of R. pseudoacacia seedlings.

Oreocharis flavovirens, a new species of Gesneriaceae from Southern Gansu Province, China.

Oreocharis flavovirens is a new species of Gesneriaceae from Gansu, China and is described and illustrated here. It is morphologically similar to O. glandulosa, O. humilis and O. farreri, but those congeners of this new taxon can be distinguished by several salient characters. A description of O. flavovirens, together with illustrations and photos, are presented.

Brain injury in combination with tacrolimus promotes the regeneration of injured peripheral nerves.

Both brain injury and tacrolimus have been reported to promote the regeneration of injured peripheral nerves. In this study, before transection of rat sciatic nerve, moderate brain contusion was (or was not) induced. After sciatic nerve injury, tacrolimus, an immunosuppressant, was (or was not) intraperitoneally administered. At 4, 8 and 12 weeks after surgery, Masson's trichrome, hematoxylin-eosin, and toluidine blue staining results revealed that brain injury or tacrolimus alone or their combination alleviated gastrocnemius muscle atrophy and sciatic nerve fiber impairment on the experimental side, simultaneously improved sciatic nerve function, and increased gastrocnemius muscle wet weight on the experimental side. At 8 and 12 weeks after surgery, brain injury induction and/or tacrolimus treatment increased action potential amplitude in the sciatic nerve trunk. Horseradish peroxidase retrograde tracing revealed that the number of horseradish peroxidase-positive neurons in the anterior horn of the spinal cord was greatly increased. Brain injury in combination with tacrolimus exhibited better effects on repair of injured peripheral nerves than brain injury or tacrolimus alone. This result suggests that brain injury in combination with tacrolimus promotes repair of peripheral nerve injury.

Puerarin Prevents LPS-Induced Osteoclast Formation and Bone Loss via Inhibition of Akt Activation.

Osteolysis induced by chronic Gram-negative bacterial infection underlies many bone diseases such as osteomyelitis, septic arthritis, and periodontitis. Drugs that inhibit lipopolysaccharide (LPS)-induced osteolysis are critically needed for the prevention of bone destruction in infective bone diseases. In this study, we assessed the effect of puerarin, a natural isoflavone isolated from Pueraria lobata OHWI root, on LPS-induced osteoclastogenesis and bone loss. Our in vitro study showed that puerarin significantly inhibited LPS-induced osteoclast differentiation from osteoclast precursor RAW264.7 cells. The inhibition occurred through suppressing the production of osteoclast activating factor tumor necrosis factor (TNF)-α, interleukin (IL)-1ß and prostaglandin E2 (PGE2), which led to down-regulating mRNA expression of osteoclastogenic genes including tartrate-resistant acid phosphatase (TRAP), cathepsin K and matrix metalloprotein 9 (MMP-9). Furthermore, LPS triggered activation of Akt in osteoclast precursor RAW264.7 cells, which was inhibited by puerarin treatment. In vivo, puerarin attenuated LPS-induced bone loss in a murine calvarial osteolysis model. Collectively, puerarin prevents LPS-induced osteoclast formation, function and bone loss, where the inhibition of Akt activation plays an important role. These findings provide evidences that puerarin might be beneficial as a promising candidate drug for the prevention and treatment of bacteria-induced bone destruction disease, and give new insights for understanding its possible mechanism.

Hypoglycemic effect of D-chiro-inositol in type 2 diabetes mellitus rats through the PI3K/Akt signaling pathway.

In this investigation, a model of type 2 diabetes mellitus (T2DM) was used on Sprague-Dawley (SD) rats to clarify more details of the mechanism in the therapy of T2DM. D-chiro-inositol (DCI) was administrated to the diabetic rats as two doses [30, 60 mg/(kg·body weight·day)]. The biochemical indices revealed that DCI had a positive effect on hypoglycemic activity and promoted the glycogen synthesis. The rats in DCI high-dosage group had a blood glucose reduction rate of 21.5% after 5 weeks of treatment, and had insulin content in serum about 15.3 ± 2.37 mIU/L which was significantly decreased than diabetes control group. Real-time polymerase chain reaction (RT-PCR) results revealed that DCI gave a positive regulation on glycogen synthase (GS) and protein glucose transporter-4 (Glut4). Western blotting suggested that DCI could up-regulated the expression of the phosphatidylinositol-3-kinase (PI3K) p85, PI3Kp110, GS as well as the phosphorylation of protein kinase B (Akt) both in the liver and the skeletal muscle. The results also revealed that DCI enhanced the Glut4 expression on skeletal muscle. Above all, DCI played a positive role in regulating insulin-mediated glucose uptake through the PI3K/Akt signaling pathway in T2DM rats.

The longitudinal epineural incision and complete nerve transection method for modeling sciatic nerve injury.

Injury severity, operative technique and nerve regeneration are important factors to consider when constructing a model of peripheral nerve injury. Here, we present a novel peripheral nerve injury model and compare it with the complete sciatic nerve transection method. In the experimental group, under a microscope, a 3-mm longitudinal incision was made in the epineurium of the sciatic nerve to reveal the nerve fibers, which were then transected. The small, longitudinal incision in the epineurium was then sutured closed, requiring no stump anastomosis. In the control group, the sciatic nerve was completely transected, and the epineurium was repaired by anastomosis. At 2 and 4 weeks after surgery, Wallerian degeneration was observed in both groups. In the experimental group, at 8 and 12 weeks after surgery, distinct medullary nerve fibers and axons were observed in the injured sciatic nerve. Regular, dense myelin sheaths were visible, as well as some scarring. By 12 weeks, the myelin sheaths were normal and intact, and a tight lamellar structure was observed. Functionally, limb movement and nerve conduction recovered in the injured region between 4 and 12 weeks. The present results demonstrate that longitudinal epineural incision with nerve transection can stably replicate a model of Sunderland grade IV peripheral nerve injury. Compared with the complete sciatic nerve transection model, our method reduced the difficulties of micromanipulation and surgery time, and resulted in good stump restoration, nerve regeneration, and functional recovery.

Crystal structure of Der f 7, a dust mite allergen from Dermatophagoides farinae.

BACKGROUND: Der f 7 is the group 7 allergen from the dust mite Dermatophagoides farinae, homologous to the major allergen Der p 7 from D. pteronyssinus. Monoclonal antibody that bind to residues Leu48 and Phe50 was found to inhibit IgE binding to residue Asp159, which is important for the cross-reactivity between Der f 7 and Der p 7. METHODOLOGY/PRINCIPAL FINDINGS: Here, we report the crystal structure of Der f 7 that shows an elongated and curved molecule consisting of two anti-parallel ß-sheets--one 4-stranded and the other 5-stranded--that wrap around a long C-terminal helix. The overall fold of Der f 7 is similar to Der p 7 but key difference was found in the ß1-ß2 loop region. In Der f 7, Leu48 and Phe50 are in close proximity to Asp159, explaining why monoclonal antibody binding to Leu48 and Phe50 can inhibit IgE binding to Asp159. Both Der f 7 and Der p 7 bind weakly to polymyxin B via a similar binding site that is formed by the N-terminal helix, the 4-stranded ß-sheet and the C-terminal helix. The thermal stability of Der f 7 is significantly lower than that of Der p 7, and the stabilities of both allergens are highly depend on pH. CONCLUSION/SIGNIFICANCE: Der f 7 is homologous to Der p 7 in terms of the amino acid sequence and overall 3D structure but with significant differences in the region proximal to the IgE epitope and in thermal stability. The crystal structure of Der f 7 provides a basis for studying the function and allergenicity of this group of allergens.

NMR structure and IgE epitopes of Blo t 21, a major dust mite allergen from Blomia tropicalis.

Blo t 21 is a paralogue of the group 5 allergen, Blo t 5, a major allergen from the dust mite Blomia tropicalis. Blo t 21 has moderate sequence identity (40.7%) to Blo t 5 and low to moderate cross-reactivity to Blo t 5. In B. tropicalis, the most prevalent and allergenic allergens are in the order of Blo t 21, Blo t 5, and Blo t 7. Here, we determined the NMR solution structure of Blo t 21, which represents the first structure of the group 21 dust mite allergen. The structure of Blo t 21 closely resembles the structures of Blo t 5 and Der p 5, comprising three anti-parallel α-helices arranged in a helical bundle. Using site-directed mutagenesis and specific IgE binding ELISA, Blo t 21 was found to contain both conserved and unique charged IgE epitope residues at the L2 loop region and on helix α3. Cross-inhibition assays confirmed that Blo t 21 has a low to moderate cross-reactivity with Blo t 5 and Der p 5 and represents a novel group of major allergen in B. tropicalis. In addition to group 5 allergens, Blo t 21 has also a low to moderate cross-reactivity with group 21 allergens from Dermatophagoides mites, confirming that B. tropicalis is a major and distinct source of dust mite allergens.

Identification and functional characterization of a putative 17ß-hydroxysteroid dehydrogenase 12 in abalone (Haliotis diversicolor supertexta).

The 17ß-hydroxysteroid dehydrogenases (17ß-HSDs) are key enzymes in the downstream process of steroid hormone biosynthesis. To date, relatively little is known about the role of 17ß-HSDs in marine gastropods. In the present study, a putative cDNA sequence encoding type 12 17ß-HSD (17ß-HSD-12) was identified in abalone (Haliotis diversicolor supertexta). The full-length cDNA was 1,978 bp, including an open reading frame (ORF) of 963 bp that encoded a protein of 321 amino acids. Comparative structural analysis revealed that abalone 17ß-HSD-12 shared 39.8-42.8% amino acid identity with other 17ß-HSD-12 homologues and that the functional domains were well conserved. Phylogenetic analysis revealed that abalone 17ß-HSD-12 belonged to the short-chain dehydrogenases/reductases (SDRs) family. Functional analysis following transient transfection of the ORF in human embryonic kidney-293 (HEK-293) cells indicated that abalone 17ß-HSD-12 had the ability to convert estrone (E1) into estradiol (E2). Expression analysis in vivo demonstrated that abalone 17ß-HSD-12 was differentially expressed during the three reproductive stages (pre-spawning, spawning, and post-spawning). These results indicate that abalone 17ß-HSD-12 is an SDR family member with a key role in steroidogenesis during the reproductive period.

Mite component-specific IgE repertoire and phenotypes of allergic disease in childhood: the tropical perspective.

Sensitization to perennial aeroallergens correlates with the risk of persistent asthma (AS) in children. In tropical Singapore, multiple codominant species of mites abound in the indoor environment, and preferential species-specific sensitization has been associated with different phenotypes of allergic disease. We investigated the pattern of mite component-specific IgE (mcsIgE) in children with different phenotypes of clinical allergic disease in an environment with multiple mite species exposure. A prospective evaluation of newly diagnosed patients with clinical diagnosis of allergic rhinitis (AR), atopic dermatitis (AD), or AS and sensitization to one or more aeroallergens were performed. Sera were tested for specific IgE against an extensive panel of Dermatophagoides pteronyssinus and Blomia tropicalis allergens. A total of 253 children were included, mean age 7.3 yr, 79% fulfilled criteria for AR, 46% AS, 71% AD, and 31% for all three. Sensitization to one or both mites was observed in 91% of children, 89% were sensitized to D. pteronyssinus, and 70% to B. tropicalis. The most common mite allergens recognized by these atopic children were Der p 1 (64%), Der p 2 (71%), Blo t 5 (45%), Blo t 7 (44%), and Blo t 21 (56%). Specific IgE responses to an increased number of distinct mite allergens correlated with the complexity of the allergic phenotype. In multivariate analysis, an increased risk for the multi-systemic phenotype (AR + AS + AD) was associated with sensitization to an increased repertoire of mite components (three or more) (OR 4.3, 95% CI 2.1-8.8, p = 0.001) and a positive parental history of AS (OR 2.4, 95% CI 1.2-2.9, p = 0.013). A highly pleiomorphic IgE response to the prevalent indoor mites is associated with the presence of a multi-systemic allergic phenotype in childhood in a tropical environment.

[The study of effects and mechanism of U50, 488H on electrical coupling during ischemia in the perfused isolated rat heart].

OBJECTIVE: To determine the effect of activation of lambda-opioid receptor with U50, 488H, a selective kappa-opioid receptor agonist, on the changes in electrical coupling during prolonged ischemia and to explore the possible mechanism. METHODS: The isolated rat heart was perfused in a Langendorff apparatus. The effect of U50, 488H on electrical coupling parameters including onset of uncoupling, plateau time, slope and fold increase in r(t) was observed in isolated perfused rat heart subjected to global no-flow ischemia. The effect of U50, 488H on connexin 43 (Cx43) expression of ventricular muscle during ischemia was determined by immunohistochemistry. RESULTS: In the prolonged ischemia model, U50, 488H concentration dependently delayed the onset of uncoupling, increased time to plateau, and decreased the maximal rate of uncoupling during ischemia. The effect of U50, 488H on electrical uncoupling parameters during ischemia was abolished by a selective kappa-opioid receptor antagonist nor-BNI or a PKC inhibitor chelerythrine. The amount of Cx43 immunoreactive signal in ventricular muscle was greatly reduced after ischemia. U50, 488H markedly increased Cx43 expression during ischemia and its effect was also attenuated by nor-BNI or chelerythrine. CONCLUSION: These results demonstrated that U50, 488H delayed the onset of uncoupling and plateau time, decreased the maximal rate of uncoupling and increased Cx43 expression of ventricular muscle during ischemia, and these effects of U50, 488H were mediated by kappa-opioid receptor, in which activation of PKC was involved. The effect of U50, 488H on electrical coupling during ischemia was probably correlated with preservation of Cx43 in cardiac muscle.

Innate immune parameters and haemolymph protein expression profile to evaluate the immunotoxicity of tributyltin on abalone (Haliotis diversicolor supertexta).

The immunotoxicity of tributyltin (TBT) on marine gastropods has been comparatively little studied although risks to wildlife associated with this compound are well known. In this study, a 30-day trial was conducted to evaluate the immunotoxic effects on abalone (Haliotis diversicolor supertexta) by exposing a range of doses of TBT (0, 2, 10, and 50 ng/L). Innate immune parameters, including phagocytic ability (PA), lysozyme activity, phenoloxidase (PO) level and superoxide dismutase (SOD) activity were monitored at intervals of 5, 15 and 30 days. Haemolymph protein expression profile was also examined at the end of the experiment. The results showed that PA value, lysozyme activity and PO level significantly decreased compared with the controls (P < 0.05), which indicated that TBT exposure markedly suppressed non-specific immune competence. Exposure to TBT also caused variation in protein expression patterns of haemolymph. Among the protein spots of differential expressions, seven proteins from the haemolymph of TBT-treated abalone were successfully identified by MALDI-TOF-MS analysis. Three protein spots increased and were identified as carrier-like peptide, peroxidase 21 precursor and creatine phosphokinase. These proteins are believed to up-regulate in expression as a response to detoxification and antioxidative stress mechanisms. The other four protein spots that down-regulated in TBT-treated groups were identified as aromatase-like protein, protein kinase C, ceruloplasmin and microtubule-actin crosslinking factor 1, and these proteins play an important role in endocrine regulation and immune defense. Taken together, the results demonstrate that TBT impair abalone immunological ability and is a potential immune disruptor.

A proteomics based approach to assessing the toxicity of bisphenol A and diallyl phthalate to the abalone (Haliotis diversicolor supertexta).

The contamination of marine ecosystems by endocrine disrupting compounds (EDCs) is of great concern. Protein expression profile maybe a good method to help us understand the molecular mechanisms of EDCs-toxicity to aquatic organisms. In this study, the abalone (Haliotis diversicolor supertexta), was selected as the target organism. Toxicological effects of two reference endocrine disruptors: diallyl phthalate (DAP, 50microgL(-1)) and bisphenol-A (BPA, 100microgL(-1)) were investigated after a three months static-renewal exposure on abalones using proteomics to analyze their hepatopancreas tissues. Some enzyme activity parameters of hepatopancreas extracts were also performed, including Na(+)-K(+)-ATPase, Ca(2+)-Mg(2+)-ATPase, peroxidase (POD) and malondialdehyde (MDA) production. After analyzing the proteomics profile of hepatopancreas by 2D gel electrophoresis, we found that 24 spots significantly increased or decreased at protein expression level (2-fold difference) in the 2D-maps from the treatment groups. Eighteen out of 24 protein spots were successfully identified by matrix-assisted laser desorption/ionization tandem time-of-flight mass spectrometry (MALDI-TOF-MS). These proteins can be roughly categorized into diverse functional classes such as detoxification, oxidative stress, hormone regulating, cellular metabolism and innate immunity. In addition, the enzymatic results indicated that DAP/BPA exposure affected the oxidative stress status and the cellular homeostasis, which partly corroborated the proteomics' results. Taken together, these data demonstrate that proteomics is a powerful tool to provide valuable insights into possible mechanisms of toxicity of EDCs contaminants in aquatic species. Additionally, the results highlight the potential of abalone as a valuable candidate for investigating EDCs impacts on marine ecosystems.

Preparation and PEGylation of exendin-4 peptide secreted from yeast Pichia pastoris.

Exendin-4, a peptide analogue of glucagon-like peptide (GLP-1), has been developed for treatment of type 2 diabetes. Herein, the secretive exendin-4 peptide, expressed by methanol induction in Pichia pastoris, was purified to near homogeneity by Ni-NTA agarose chromatography. 103.6 mg of protein was obtained from 1 L of the supernatant and its purity was 96.1%. Subsequently, the PEGylated exendin-4 was prepared. The bioactivity of exendin-4 was determined by examining the glucose-lowering and insulin-releasing ability in plasma. Then, a safety evaluation was performed by histological examination of the main organs (liver, kidney and pancreas). PEGylated exendin-4 displayed glucose-lowering and insulin-stimulating action in vivo without obvious damage to the above organs. The results suggest that the P. pastoris expression could be used to produce large quantities of exendin-4, and PEGylation is a useful tool to maintain and enhance bioactivity of the peptide.

Nuclear magnetic resonance structure and IgE epitopes of Blo t 5, a major dust mite allergen.

A high incidence of sensitization to Blomia tropicalis, the predominant house dust mite species in tropical regions, is strongly associated with allergic diseases in Singapore, Malaysia, and Brazil. IgE binding to the group 5 allergen, Blo t 5, is found to be the most prevalent among all B. tropicalis allergens. The NMR structure of Blo t 5 determined represents a novel helical bundle structure consisting of three antiparallel alpha-helices. Based on the structure and sequence alignment with other known group 5 dust mite allergens, surface-exposed charged residues have been identified for site-directed mutagenesis and IgE binding assays. Four charged residues, Glu76, Asp81, Glu86, and Glu91 at around the turn region connecting helices alpha2 and alpha3 have been identified to be involved in the IgE binding. Using overlapping peptides, we have confirmed that these charged residues are located on a major putative linear IgE epitope of Blo t 5 from residues 76-91 comprising the sequence ELKRTDLNILERFNYE. Triple and quadruple mutants have been generated and found to exhibit significantly lower IgE binding and reduced responses in skin prick tests. The mutants induced similar PBMC proliferation as the wild-type protein but with reduced Th2:Th1 cytokines ratio. Mass screening on a quadruple mutant showed a 40% reduction in IgE binding in 35 of 42 sera of atopic individuals. Findings in this study further stressed the importance of surface-charged residues on IgE binding and have implications in the cross-reactivity and use of Blo t 5 mutants as a hypoallergen for immunotherapy.

Identification and characterization of a novel allergen from Blomia tropicalis: Blo t 21.

BACKGROUND: Allergenic components from Blomia tropicalis are important triggers of allergies in the tropics. OBJECTIVE: We sought to identify and characterize a novel allergen, Blo t 21, from B tropicalis. METHODS: Blo t 21 was initially identified from an expressed sequence tag database generated from a B tropicalis cDNA library. Allergenicity of this antigen was examined by means of skin prick testing, ELISA, and IgE immuno-dot blotting. We evaluated whether Blo t 21 and Blo t 5 were cross-reactive by using IgE inhibition ELISAs. RESULTS: Blo t 21, a 129-amino-acid protein sharing 39% identity with Blo t 5, is a product of a single-copy gene. It has an alpha-helical secondary structure and localizes to midgut and hindgut contents of B tropicalis, as well as fecal particles. Positive responses to Blo t 21 were shown in 93% (40/43) by means of ELISA and 95% (41/43) by means of skin prick testing when assayed in 43 adult patients with ongoing persistent allergic rhinitis. However, sera of 494 consecutive individuals attending outpatient allergy clinics over 1(1/2) years showed 57.9% (286/494) had positive responses to Blo t 21. Although the majority (>75%) of sensitized individuals were cosensitized to both Blo t 5 and Blo t 21, these 2 allergens had a low-to-moderate degree of cross-reactivity. CONCLUSION: Blo t 21 is a major allergen in B tropicalis that is not highly cross-reactive to Blo t 5, despite sharing some sequence and structural identity. CLINICAL IMPLICATIONS: Blo t 21, representing a new group of allergens, is an important B tropicalis allergen.