John Donnie A. Ramos

cDNA cloning and expression of Blo t 11, the Blomia tropicalis allergen homologous to paramyosin

Blomia tropicalis is an important mite species in many parts of the world and the most predominant mite species in tropical countries. The prevalence of sensitization to this species has probably been underestimated because commercial extracts are largely unavailable. Identification and characterization of B. tropicalis allergens is an important step toward understanding the role of this species in allergic sensitization and could provide appropriate reagents for diagnostic and therapeutic procedures. This paper describes the isolation, sequence analysis, expression and allergenicity of a cDNA gene coding for a B. tropicalis allergen with homology to paramyosin, a high-molecular-weight allergen previously identified in Dermatophagoides farinae. The full-length Blo t 11 cDNA gene was isolated by cDNA library screening, 5′-rapid amplification of cDNA ends and long-distance PCR. Sequence analysis was performed with a combination of CLUSTAL W, CGC and BLAST program packages. The cDNA gene was expressed as a GST fusion protein in Escherichia coli and purified by affinity chromatography using the glutathione Sepharose column. Allergenicity of the rBlo t 11 was tested by human IgE dot blot immunoassay. Blo t 11 is a 3,111-bp cDNA gene with a 2,625-bp open reading frame coding for an 875-amino acid protein, exhibiting significant homology with different invertebrate paramyosins. The human IgE dot blot immunoassay showed that the rBlo t 11 reacted positively to 52% (33/63) of sera from asthmatic patients. Blo t 11 is the homolog of Der f 11 exhibiting potentially important allergenic activity.

Lack of human IgE cross-reactivity between mite allergens Blo t 1 and Der p 1

Background:  The group 1 mite allergens are the most significant indoor allergens and they belong to the papain‐like cysteine protease family. To date there is only one published report on the isolation and characterization of group 1 allergens from Blomia tropicalis mites. The aims of the study are to determine the cross‐reactivity between group 1 allergens and to evaluate their clinical importance in allergic patients.

Comparative allergenicity studies of native and recombinant Blomia tropicalis Paramyosin (Blo t 11)

Background: The complementary DNA (cDNA) encoding for Blo t 11, a 102 kD allergen from Blomia tropicalis (Bt) was isolated, expressed and characterized previously. This study aimed to isolate the native Blo t 11 allergen and compare its allergenicity with the recombinant forms.

Mite Amylase from Blomia tropicalis (Blo t 4): Differential Allergenicity Linked to Geographical Regions

Background:Blomia tropicalis is an important domestic dust mite in the tropics and subtropics. This study describes cDNA cloning of the group 4 allergen of B. tropicalis, and the evaluation of the sensitization of this allergen in atopic populations from 2 geographic regions. Methods: cDNA cloning was carried out using the Smart RACE cDNA amplification kit. The full-length Blo t 4 cDNA was isolated by cDNA library screening, 5′ and 3′ rapid amplification of cDNA ends and long-distance PCR. Sequence analysis was performed with a combination of the Clustal W, CGC and Blast program packages. The cDNA was expressed in Pichia pastoris yeast. The skin prick test was used to evaluate the sensitization profile of recombinant Blo t 4, crude dust mite allergen extracts and major B. tropicalis recombinant allergen Blo t 5. Results: The cloned Blo t 4 had a molecular weight of 56 kDa and had 68% amino acid homology with group 4 allergens of Dermatophagoides pteronyssinus and 65% with those of Euroglyphus maynei. A sensitization profile to the expressed recombinant Blo t 4 allergen (28%) showed an unusually higher frequency than to the major allergen Blo t 5 (22%) in allergic subjects from Chengdu, PR China. In comparison, the subjects from Singapore showed very low sensitization to Blo t 4 (4%) compared with Blo t 5 (84%). Conclusions: Group 4 allergens of B. tropicalis may be an important dust mite allergen in certain distinct populations.

Peptide mapping of immunoglobulin E and immunoglobulin G immunodominant epitopes of an allergenic Blomia tropicalis paramyosin, Blo t 11

Background The identification of immunodominant peptides containing the IgE and IgG epitopes on allergen molecules is an important step in understanding the interaction of the allergen with the immune system and, thus, essential for the development of effective immunotherapeutic and diagnostic reagents. The present study aimed to map the IgE and IgG immunodominant peptides of Blomia tropicalis (Bt) allergen Blo t 11, a high molecular weight allergen homologous to paramyosin, exhibiting important allergenic activity.

Production of Monoclonal Antibody by DNA Immunization with Electroporation

DNA immunization with in vivo electroporation is an efficient alternative protocol for the production of monoclonal antibodies (mAb). Generation of mAb by DNA immunization is a novel approach to circumvent the following technical hurdles associated with problematic antigens: low abundance and protein instability and use of recombinant proteins that lack posttranslational modifications. This chapter describes the use of a DNA-based immunization protocol for the production of mAb against a house dust mite allergen, designated as Blo t 11, which is a paramyosin homologue found in Blomia tropicalis mites. The Blo t 11 cDNA fused at the N terminus to the sequence of a signal peptide was cloned into the pCI mammalian expression vector. The DNA construct was injected intramuscularly with in vivo electroporation into mice, and the specific antibody production in mice was analyzed by enzyme-linked immunosorbent assay (ELISA). Hybridomas were generated by fusing mouse splenocytes with myeloma cells using the ClonaCell-HY Hybridoma Cloning Kit. Six hybridoma clones secreting Blo t 11 mAb were successfully generated, and these mAb are useful reagents for immunoaffinity purification and immunoassays.

Production of monoclonal antibodies for immunoaffinity purification and quantitation of Blo t 1 allergen in mite and dust extracts

Background Blo t 1 is a cysteine protease‐like allergen from Blomia tropicalis. Recombinant Blo t 1 binds up to 90% of IgE from allergic patients and shows limited cross‐reactivity to Der p 1. The generation of monoclonal antibodies (mAbs) against Blo t 1 is important for the detection, isolation and characterization of the native form of the allergen.

DNA immunization for the production of monoclonal antibodies to Blo t 11, a paramyosin homolog from Blomia tropicalis

Background:  Blo t 11 is a high molecular weight allergen from Blomia tropicalis with significant immunoglobulin (Ig)E binding frequency. Native and recombinant Blo t 11 are susceptible to degradation and the isolation and expression of the allergen is problematic thus obtaining sufficient amounts of purified Blo t 11 for antibody production is limiting. DNA‐based immunization is an attractive alternative strategy that bypasses antigen purification for antibody production.

Overall Survival of Filipino Patients with Squamous Cell Carcinoma of the Head and Neck: A Single-Institution Experience

This paper is the first to present the incidence and overall survival of patients with squamous cell carcinoma of the head and neck (SCCHN) from the extreme northern part of the Philippines. We retrospectively retrieved the records of patients with histologically-confirmed squamous cell carcinoma of the oral cavity, oropharynx, hypopharynx and larynx at the Mariano Marcos Memorial Hospital and Medical Center, Ilocos Norte, Philippines, from 2003 to 2012 and analysed prognostic factors associated with survival. Of the 150 cases, only 80 (53.3%) were still living when the study was terminated. Median age at initial diagnosis was 61.5 years and the male to female ratio was 7:3. The majority of the cases had tumours in the oral cavity (50.7%), followed by the larynx (36.7%). Sex (log rank=1.94, p value/α=0.16), tumor site (log rank=0.02, p value/α=0.90), tumor grade (log rank=1.74, p value/α=0.42), and node stage (log rank=0.07, p value/α=0.80) were not shown to be associated with the survival of our cases. Only 45 (30.0%) had no regional lymph node involvement (N0) at presentation and 12 (8.0%) had already developed distant metastases. Among the 150 patients, 71 (47.3%) were not able to receive treatment of any kind. Oddly, treatment (log rank=1.65, p value/α=0.20) was also shown to be not associated with survival. The survival rate of those who underwent surgery, radiotherapy, or both was not statistically different from those who did not receive any treatment. Only the tumor stage (log rank=4.51, p value/α=0.03) was associated with patient survival. The overall mean survival was 49.3 months, with survival rate diminishing from 88.3% during the 1st year to 1.80% by end of the study. This relatively low survival rate of our cases only reflects their poor access to quality diagnostic and treatment facilities.

Association of house dust mite-specific IgE with asthma control, medications and household pets

Background Evidence is conflicting regarding the effectiveness of creating a low-allergen environment or reducing allergen exposure to control asthma exacerbations. Objective This study determined the association of house dust mite (HDM)-specific IgE levels with asthma symptom control, selected medications, family history of allergic disease, and exposure to second-hand smoke and household pets. Methods Serum samples from 102 doctor-diagnosed allergic asthma patients and 100 non-atopic controls were subjected to enzyme-linked immunosorbent assay using the HDM species Dermatophagoides pteronyssinus (Dp), Dermatophagoides farinae (Df), and Blomia tropicalis (Bt) allergens. Point-biserial correlation coefficient, Pearson R correlation, and logistic regression analyses were used to determine association of HDM-specific IgE levels with the abovementioned variables. Results Of the 102 cases, 38.24%, 47.06%, and 33.33% were sensitized to Bt, Df, and Dp, respectively. Sensitized patients showed greater probability [Bt (OR = 1.21), Df (OR = 1.14), and Dp (OR = 1.35)] to manifest symptoms than those who were not. Obtained p-values [Bt (p = 0.73), Df (p = 0.83), and Dp (p = 0.59)], however, proved that HDM-specific IgE levels had no significant contribution in predicting or explaining occurrence of asthma symptoms. Bt- and Df-specific IgEs showed moderately weak but significant relationship with bambuterol HCl and expectorant, respectively. Patients currently on said medications registered higher HDM-specific IgE levels than those who were not. No significant correlation between IgE levels and family history of allergic disease or with exposure to second-hand smoke was seen. Dp-specific IgE levels of patients exposed to household pets were significantly lower compared to those without exposure. Conclusion This study proves that sensitization to Bt, Df, and Dp allergens is not significantly associated with asthma symptoms and control. Although cases were shown to be sensitized to HDMs, their current medications were at least effective in controlling their asthma symptoms.