Myung-hee Yi

Allergenicity of sigma and delta class glutathione S-transferases from the German cockroach.

Background: Cockroach glutathione S-transferases (GSTs) are known to elicit strong IgE responses. This study was undertaken to compare the IgE reactivity of German cockroach GSTs, Bla g 5 (σ class) and δ class GST (BgGSTD1). Methods: Full-length Bla g 5 and BgGSTD1 were cloned, and their recombinant proteins were expressed and purified. Their IgE reactivities and cross-reactivities were examined by ELISA using sera from cockroach-sensitized subjects. Results: A predominant variant of Bla g 5 cDNA has amino acid substitutions at positions 10 (C to F) and 42 (N to K). BgGSTD1 has substitutions at positions 27 (E to N) and 207 (K to R). Sera from cockroach-sensitized patients showed 20.5% IgE reactivity to Bla g 5 and 17.9% IgE reactivity to BgGSTD1. However, inhibition studies using 1 serum sample with the highest IgE reactivity showed limited cross-reactivity. Conclusions: IgE-binding frequency to the cockroach GSTs was low, but the titer of IgE reactivity was strong in some sera. The inclusion of different classes of GSTs could be helpful for the delicate diagnosis and immunotherapy of cockroach allergy.

Sequence Polymorphisms of Major German Cockroach Allergens Bla g 1, Bla g 2, Bla g 4, and Bla g 5

Background: The allergenicity of allergens could be influenced by amino acid substitutions in B- or T-cell epitope regions. The German cockroach is known to produce potent allergens inducing strong IgE-mediated allergic reactions. This study was performed to investigate sequence variations in major allergens of the German cockroach. Methods: Reverse transcriptase PCR was used to amplify the cDNA sequences encoding major allergens of the German cockroach (Bla g 1, Bla g 2, Bla g 4, and Bla g 5). Results: The deduced amino acid sequences revealed 38 Bla g 1 variants with 1–7 amino acid substitutions (98.6–99.8% identity), 28 Bla g 2 variants with 1–3 substitutions (99.1–99.7%), 27 Bla g 4 variants with 0–32 substitutions (82.4–100%), and 8 Bla g 5 variants with 1–2 substitutions (99.0–99.5%), respectively. Bla g 1 and Bla g 2 showed sporadic amino acid substitutions despite the divergence in their sequences. Bla g 4 exhibited frequent variations, with clusters of substitutions in residues 29–38, 52–80, and 132–155. Sequence variations in Bla g 4 imply the presence of multiple isoforms and isoallergens, which may in turn have various effects on the IgE-binding capacity and T-cell responsiveness. Only 8 variants were found in Bla g 5, with infrequent amino acid changes of one or two residues. Conclusions: Analyses of T-cell and IgE-binding epitope regions would clarify the effect of sequence polymorphisms on allergenicity, which in turn will aid in the design of allergen formulations for diagnosis and immunotherapy for cockroach allergies.

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 CaCl2. 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 CaCl2 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.

Sequence diversity of the Bla g 4 cockroach allergen, homologous to lipocalins, from Blattella germanica.

Background: The cockroach allergen Bla g 4, a putative lipocalin, is known to exhibit frequent sequence variations. However, the previously reported cDNA sequences are truncated at the N terminus. This study was undertaken to investigate the mechanisms by which these sequence variations are generated. Methods: Rapid amplification of cDNA ends PCR and RT-PCR were performed to obtain the full sequence of the Bla g 4 cDNA, and PCR was also used to clone the Bla g 4 genomic DNA. In addition, Bla g 4 protein variants were analyzed by two-dimensional gel electrophoresis. Results: Nine additional amino acid residues at the N terminus of Bla g 4 were identified, and 2 genes encoding Bla g 4, both of which consisted of 5 exons, were cloned. Examination of 34 clones of Bla g 4 cDNA obtained by RT-PCR revealed 14 variants. In particular, Bla g 4 sequences showed frequent clusters of variations in residues 38–45, 61–82 and 144–163. Differences in cDNA sequences may imply that RNA sequences are edited after transcription. More than 10 spots were identified between pH 5 and 7 upon two-dimensional gel electrophoresis, indicating that multiple variants of Bla g 4 are produced at the protein level. Conclusions: Genetic polymorphisms among individual cockroaches, the existence of multiple genes and sequence variations caused by RNA editing produce sequence diversity of Bla g 4, which may influence its allergenicity. The sequence information obtained in this study will be helpful for the standardization of the cockroach allergen and thereby aid in the development of diagnostics and immunotherapeutics.

16S rRNA profiling of the Dermatophagoides farinae core microbiome: Enterococcus and Bartonella

Department of Environmental Medical Biology, Institute of Tropical Medicine, and Arthropods of Medical Importance Resource Bank, Yonsei University College of Medicine, Seoul, Korea Brain Korea 21 PLUS Project for Medical Science, Yonsei University College of Medicine, Seoul, Korea Department of Laboratory Medicine and Research Institute of Bacterial Resistance, Yonsei University College of Medicine, Seoul, Korea

Intestinal fluke Metagonimus yokogawai infection increases probiotic Lactobacillus in mouse cecum

Helminth infection can alleviate immune-mediated disorders such as allergies and autoimmune diseases, by altering the gut microbiome. However, changes in gut microbiome due to intestinal trematodes remain unelucidated. Here, we evaluated the changes in the gut microbiome of ICR mice infected with Metagonimus yokogawai, a hypo-virulent intestinal trematode. Four weeks after infection, mouse cecal content was analyzed by 16S rRNA amplicon analysis. Although there was no apparent difference in species richness and diversity, the microbiome composition was different in the infected and control groups. Furthermore, several Lactobacillus species with known immunomodulatory role in immune-mediated diseases were increased in the infected group.