Cross­reactivity between group-5 and -21 mite allergens from Dermatophagoides farinae, Tyrophagus putrescentiae and Blomia tropicalis.

Group-5 and group-21 allergens, produced by house dust mites and storage mites are 36.6-55.8% identical in their sequences and are recognized by at least 50% of immunoglobulin (Ig)E from the sera of individuals allergic to dust mites. In the present study, recombinant group-5 and ­21 allergens from three mite species, Dermatophagoides farinae (rDer f 5 and 21), Tyrophagus putrescentiae (rTyr p 5 and 21), and Blomia tropicalis (rBlo t 5 and 21), were purified from Escherichia coli, and the IgE reactivities and cross­reactivities of these allergen variants were assessed. The IgE binding frequencies of rDer f 5, rDer f 21, rTyr p 5, rTyr p 21, rBlo t and rBlo t 21 proteins were 64.95, 65.98, 30.41, 41.24, 30.93 and 21.65%, respectively. The IgE reactivity of rDer f 5 correlated highly with that of rDer f 21 (r=0.733). rTyr p 5 exhibited the highest level of correlation with rTyr p 21 (r=0.950), while the correlation of rBlo t 5 with rBlo t 21 was the lowest observed (r=0.104). The binding of IgE to rDer f 5 and rDer f 21 was not inhibited by any allergens but themselves. While rDer f 5 inhibited only 60.3% of IgE binding to rBlo t 5, rDer f 21 exhibited a high inhibitory effect against rTyr p 5 (93.01%), rTyr p 21 (92.12%), rBlo t 5 (86.97%) and rBlo t 21 (70.30%), implying cross­reactivity among mite species. The results of the present study demonstrated that the majority of the IgE reactivity to group-5 and -21 storage mite allergens is due to cross­reaction. It is therefore imperative to develop an accurate, component­resolved diagnosis for dust mite allergies.

House dust mite allergen Der f 1 induces IL-8 in human basophilic cells via ROS-ERK and p38 signal pathways.

Der f 1, a major house dust mite allergen and member of the papain-like cysteine protease family, can provoke immune responses with its proteolytic activity. To understand the role of Der f 1 in inflammatory immune responses, we studied the mechanism of the regulation of interleukin (IL)-8 expressions in human basophilic cell KU812 by proteolytically active recombinant Der f 1. Not only production of IL-8 mRNA was induced but also the DNA binding activity of activator protein-1 (AP-1) and phosphorylation of NF-κB p65 were increased in Der f 1-treated KU812. Furthermore, Der f 1 induction of IL-8 expression was sensitive to pharmacological inhibition of ERK and p38 mitogen activated protein kinase (MAPK) pathways. Der f 1 also activated ERK and p38 MAPK phosphorylation and rapidly induced reactive oxygen species (ROS) production. The antioxidant N-acetyl-cysteine (NAC) inhibited phosphorylation of ERK, but not p38, suggesting that secretion of IL-8 in KU812 cells treated with Der f 1 is dependent on ROS, ERK MAPK and p38 MAPK. We describe the mechanism of Der f 1-induced IL-8 secretion from human basophilic cells, which are thought to be important for allergic inflammation independent of IgE antibodies. These findings improve our understanding of the inflammatory immune response in human basophils to protease allergens.

Identification of novel allergenic components from German cockroach fecal extract by a proteomic approach.

BACKGROUND: Cockroaches produce potent allergens, and cockroach feces are known to be especially rich in allergens. In this study, we analyze the allergenic components from cockroach feces and evaluate allergenicity of recombinant α-amylase identified from fecal extract. METHODS: IgE-reactive proteins from German cockroach fecal extract were analyzed by proteomic analysis and immunoblotting. Recombinant α-amylase was produced and its allergenicity was evaluated by ELISA. RESULTS: Analysis of German cockroach fecal extracts identified 12 IgE-reactive components. Most of these allergens were found to be digestive enzymes such as α-amylase, trypsin, chymotrypsin, metalloprotease, and midgut carboxypeptidase A, but the identity of 3 IgE-reactive proteins is still unknown. Glycinin-like proteins, which were likely derived from the cockroach diet, were also identified. German cockroach α-amylase shares the highest identity with pig α-amylase (55.8%), followed by mite group 4 allergens (Blo t 4, 50.4%; Der p 4, 49.8%; Eur m 4, 47.4%). In this study, recombinant α-amylase from German cockroach was expressed, and its allergenicity was examined by ELISA. Specific IgE against recombinant amylase was detected in 41.4% (12/29) of serum samples from German cockroach-sensitized subjects. Recombinant α-amylase was able to inhibit 55% of specific IgE to German cockroach whole-body extract. CONCLUSIONS: Amylase was found to be an important novel allergen in cockroach feces. It is hoped that recombinant α-amylase will be useful for further studies and clinical applications.

IgE binding epitopes of Bla g 6 from German cockroach.

Bla g 6, a German cockroach allergen, shows homology to muscle protein troponin C. It contains four calcium-binding domains at amino acid (aa) residues 20-30, 56-67, 96-107, and 132-143, and its immunoglobulin E (IgE) reactivity is dependent upon calcium ion level. However, the IgE binding epitopes of Bla g 6 have not been investigated. This study aimed to analyze the IgE binding epitopes from the five peptide fragments of Bla g 6. The full-length of three Bla g 6 isoallergens (Bla g 6.0101, Bla g 6.0201, and Bla g 6.0301) and five peptide fragments (P1: aa 1-111, P2: aa 1-95, P3: aa 33-111, P4: aa 80-151, and P5: aa 33-151) of Bla g 6.0101 were generated by polymerase chain reaction (PCR) and expressed in Escherichia coli. Enzyme-linked immunosorbent assay (ELISA) was performed on 24 patients' sera that adjusted the final concentration 10 mM of CaCl(2) to determine the IgE activities of Bla g 6. Eight sera (33.3%), 9 sera (37.5%), and 11 sera (45.8%) showed IgE reactivity to Bla g 6.0101, Bla g 6.0201, and Bla g 6.0301, respectively. Among the sera from the positive IgE reactivity, three patients' sera were selected and the IgE reactivity was measured by ELISA with the five peptide fragments of Bla g 6. Based on IgE responses, one patient's serum exhibited the strongest IgE reactivity. We assumed that the aa between 96-151 residues, including the calcium binding domains III and IV, would be important for IgE binding. These results may provide information that will yield safe diagnostic methods and immunotherapeutics.

IgE-binding epitope analysis of Bla g 5, the German cockroach allergen.

Cockroach infestations have been linked with allergic diseases such as asthma in humans. Bla g 5, sigma class glutathione S-transferase (GST), is the major cockroach allergen which has the highest IgE response value of all cockroach allergens. Although several cockroach allergens have been identified and cloned, information regarding their B ell and T cell IgE-binding epitopes is limited. In order to analyze the IgE binding epitopes of Bla g 5, full-length and five peptide fragments (A, 1-100 amino acid residue; B, 91-200; Ba, 1-125; Bb, 1-150; Bc, 1-175) were expressed. Twelve (37.5%) of 32 sera from cockroach-sensitized subjects showed positive IgE reactivity to the recombinant Bla g 5 (rBla g 5). Six strong positive sera were selected for the epitope study. Recombinant proteins not containing 176-200 amino acid residues were unable to react to sera from cockroach sensitized individuals, suggesting that this region contains the IgE-binding epitope. Despite strong IgE reactivity to rBla g 5, the pooled serum from 5 cockroach-sensitized patients did not show IgE reactivity to all synthetic peptides consisting of 15 residues covering 161-200 amino acids. These results suggest the possibility that Bla g 5 may have a conformational epitope in the C-terminal region. GST is the important target for the development of vaccines and drugs against allergic diseases because of high cross-reactivity among insect species. This study will aid recombinant allergen research for immunotherapy of cockroach allergens and other insect allergens.

Allergenicity of recombinant troponin C from Tyrophagus putrescentiae.

BACKGROUND: The storage mite, Tyrophagus putrescentiae, produces potent allergens, many of which have not been characterized. This study was undertaken to characterize the allergenicity of troponin C from T. putrescentiae. METHODS: A cDNA encoding 17.7 kDa troponin C, with homology to cockroach allergen Bla g 6, was identified from T. putrescentiae-expressed sequence tags. Recombinant troponin C was expressed and IgE responses to the recombinant protein were assessed in the presence and absence of 10 mM CaCl(2). Cross-reactivity between T. putrescentiae troponin C and Bla g 6 was tested using an inhibition ELISA. RESULTS: Recombinant T. putrescentiae troponin C shares 62.7-85.5% homology with troponin C from various arthropods. Sera from 5 of 47 subjects in our study group (10.6%) showed IgE binding to the recombinant protein. Interestingly, addition of 10 mM CaCl(2) increased the intensity of IgE binding approximately 2-fold. In an immune-inhibition ELISA with these sera, T. putrescetiae troponin C and Bla g 6 did not cross-react significantly. CONCLUSIONS: Troponin C is a new mite allergen with calcium-dependent IgE reactivity.

IgE-binding reactivity of peptide fragments of Bla g 1.02, a major German cockroach allergen.

Cockroaches cause allergic diseases and are closely linked with the development of asthma. Bla g 1 is one of the major allergen proteins produced by German cockroaches. It consists of tandem repeats of approximately 100 amino acids. The aim of the present study was to identify linear IgE-binding epitopes of Bla g 1.02. RT-PCR was used to clone a cDNA sequence encoding Bla g 1.02 (EF202179) which shared 98.6-99.8% identity with a previously reported Bla g 1.02 (AF072220). To investigate IgE binding regions, five separate but overlapping Bla g 1.02 peptide fragments (A: aa 1-111, B: aa 102-215, C: aa 206-299, D: aa 289-403, E: aa 394-491) were amplified and cloned. The full-length and five peptide fragments were overexpressed in Pichia pastoris and E. coli, respectively, and their IgE binding reactivities were measured by ELISA using 37 serum samples isolated from cockroach-sensitized patients. The sera of 24 patients (64.9%) recognized the full-length Bla g 1.02 recombinant protein. Among 19 selected serum samples, 11 sera (57.9%) reacted to peptide fragment A, 5 sera (31.3%) to B, 4 sera (21.1%) to C, 9 sera (47.4%) to D, and 10 sera (52.6%) to peptide fragment E. IgE-binding epitopes are found to be distributed to each tandem repeat of Bla g 1. The combination of peptide fragments A, D, and E may able to detect all Bla g 1-sensitized subjects. We suggest that these peptide fragments may be useful in allergy diagnosis and the design of novel immunotherapeutics.

UV band Splitting of chromogenic azo-coupled calix[4]crown upon cation complexation.

Calixcrown-6 compounds carrying a pair of phenylazo moieties on the upper rim and two OH groups, one OH group, and two OR groups on the lower rim have been prepared in both the cone and partial cone conformations. UV/vis spectral measurements showed a red shift upon the addition of Ca2+ to the calixcrown carrying two OH groups and a blue shift for the calixcrown carrying two OR groups. For the compounds with two OR groups on the lower rim and a fixed partial cone conformation, a blue shift caused by electrostatic interaction between the oxygen atoms of OR and the metal ion as well as a red shift caused by the pi-metal complexation between the rotated calix benzene and the metal ion were observed.