Background & Aims

The cingulate cortex (CC) has been reported to be involved in processing pain of esophageal origin. However, little is known about molecular changes and cortical activation that arise from early-life, esophageal acid reflux. Excitatory neurotransmission via activation of the N-methyl-D-aspartate (NMDA) receptor and its interaction with post-synaptic density protein-95 (PSD-95) at the synapse appears to mediate neuronal development and plasticity. We investigated the effect of early-life esophageal acid exposure on NMDA receptor subunits and PSD-95 expression in the developing CC.

Methods

We assessed NMDA receptor subunits and PSD-95 protein expression in rostral CC (rCC) tissues of rats exposed to esophageal acid or saline (control), either during post-natal days 7–14 (P7–P14) and/or acutely, at adult stage (P60), using immunoblot and immunoprecipitation analyses.

Results

Compared with controls, acid exposure from P7 to P14 significantly increased expression of NR1, NR2A, and PSD-95, measured 6 weeks after exposure. However, acute exposure at P60 caused a transient increase in expression of NMDA receptor subunits. These molecular changes were more robust in animals exposed to acid neonatally and rechallenged, acutely, at P60. Esophageal acid exposure induced calcium calmodulin kinase II-mediated phosphorylation of the subunit NR2B at Ser1303.

Conclusions

Esophageal acid exposure during early stages of life has long-term effects, because of phosphorylation of the NMDA receptor and overexpression in the rCC. This molecular alteration in the rCC might mediate sensitization of patients with acid-induced esophageal disorders.

Background

The objective of this study was to determine if neonatal cystitis alters colonic sensitivity later in life and to investigate the role of peripheral mechanisms.

Methods

Neonatal rats received intravesical zymosan, normal saline, or anesthesia only for three consecutive days (postnatal days 14th–16th). The estrous cycle phase was determined prior to recording the visceromotor response (VMR) to colorectal distension (CRD) in adult rats. Eosinophils and mast cells were examined from colon and bladder tissue. CRD or urinary bladder distension (UBD)-sensitive pelvic nerve afferents (PNAs) were identified and their responses to distension were examined. The relative expression of N-methyl-D-aspartic acid (NMDA) NR1 subunit in the L6-S1 spinal cord was examined using Western blot.

Results

The VMR to CRD (≥10mmHg) in the neonatal zymosan group was significantly higher than control in both the diestrus, estrus phase and in all phases combined. There was no difference in the total number of eosinophils, mast cells or number of degranulated mast cells between groups. The spontaneous firing of UBD, but not CRD-sensitive PNAs from the zymosan rats was significantly higher than the control. However, the mechanosensitive properties of PNAs to CRD or UBD were no different between groups (p > 0.05). The expression of spinal NR1 subunit was significantly higher in zymosan-treated rats compared to saline treated rats (p <0.05).

Conclusion

Neonatal cystitis results in colonic hypersensitivity in adult rats without changing tissue histology or the mechanosensitive properties of CRD-sensitive PNAs. Neonatal cystitis does results in overexpression of spinal NR1 subunit in adult rats.

The transient receptor potential vanilloid 1 receptor (TRPV1) is an important nociceptor involved in neurogenic inflammation. We aimed to examine the role of TRPV1 in experimental colitis and in the development of visceral hypersensitivity to mechanical and chemical stimulation. Male Sprague-Dawley rats received a single dose of trinitrobenzenesulfonic acid (TNBS) in the distal colon. In the pre-emptive group, rats received the TRPV1 receptor antagonist JYL1421 (10μmol/kg, iv) or vehicle 15 minutes prior to TNBS followed by daily doses for 7days. In the post-inflammation group, rats received JYL1421 daily for 7 days starting on day 7 following TNBS. The visceromotor response (VMR) to colorectal distension (CRD), intraluminal capsaicin, capsaicin vehicle (pH6.7) or acidic saline (pH5.0) was assessed in all groups and compared to controls and naïve rats. Colon inflammation was evaluated with H&E staining and myeloperoxidase (MPO) activity. TRPV1 immunoreactivity was assessed in the thoraco-lumbar (TL) and lumbo-sacral (LS) DRGs neurons. In the pre-emptive vehicle group, TNBS resulted in a significant increase in the VMR to CRD, intraluminal capsaicin and acidic saline compared the JYL1421 treated group (p<0.05). Absence of microscopic colitis and significantly reduced MPO activity was also evident compared to vehicle treated rats (p<0.05). TRPV1 immunoreactivity in the TL (69.1± 4.6%) and LS (66.4± 4.2%) DRG in vehicle treated rats was increased following TNBS but significantly lower in the pre-emptive JYL treated group (28.6±3.9 and 32.3±2.3 respectively, p<0.05). JYL1421 in the post-inflammation group improved microscopic colitis and significantly decreased the VMR to CRD compared to vehicle (p<0.05, ≥30mmHg) but had no effect on the VMR to chemical stimulation. TRPV1 immunoreactivity in the TL and LS DRG was no different from vehicle or naïve controls. These results suggest an important role for TRPV1 channel in the development of inflammation and subsequent mechanical and chemical visceral hyperalgesia.

The ubiquitous fungus Aspergillus fumigatus causes allergic rhinitis, asthma, sinusitis and allergic bronchopulmonary aspergillosis. A number of major allergens from A. fumigatus are purified, but their structure-function role in the pathogenesis of disease is not known. Such information is essential for devising alternative therapy of fungal allergic diseases. In the present study, N-terminal and C-terminal deletion mutants ofAsp f 3 were constructed and their immunopathological responses studied in a mice model of allergy. Three mutants viz,Asp f 3 (aa 33–168), (aa 1–142), and (aa 23–142) were made by deleting certain amino acids from epitopic regions of full lengthAsp f 3, a major allergen of A. furnigatus. TheAsp f 3 and three mutated proteins were expressed in pET vector. The C-terminal deletion mutantAsp f 3 (aa 1–142) induced elevated IFN-γ but low levels of IL-4 by spleen cells. This mutant also showed significant downregulation of peripheral blood eosinophils and lung inflammation in immunized mice. The N-terminal deletion mutantAsp f 3 (aa 33–168) also exhibited an immuno-suppressive effect in terms of IgE production and induction of Th2 cytokine. The results indicate thatrAsp f 3 and its deletion mutants induced distinct immune-inflammatory responses in mice on challenge with these proteins. The non-IgE binding deletion mutants ofAsp f 3 (aa 1–142 and aa 33–168) could deviate Th2 immune response with a concomitant reduction in airway inflammation and infiltration of inflammatory cells.

Allergic aspergillosis is a Th2 T-lymphocyte-mediated pulmonary complication in patients with atopic asthma and cystic fibrosis. Therefore, any therapeutic strategy that selectively inhibits Th2 T-cell activation may be useful in downregulating allergic lung inflammation in asthma. In the present study, we developed a CpG oligodeoxynucleotide (ODN)-based immune intervention of allergic inflammation in a mouse model of allergic aspergillosis. Four different groups of mice were used in a short-term immunization protocol. Three experimental groups of animals (groups 1 to 3) were sensitized with Aspergillus fumigatus antigens. Animals in group 1 were immunized with A. fumigatus antigen alone, while those in group 2 were treated with CpG-ODN 1 day before the first antigen immunization, and the animals in group 3 received the first CpG-ODN administration between the antigen treatments. The animals in group 4 served as controls and were given phosphate-buffered saline. Allergen-specific serum immunoglobulins and total immunoglobulin E in different groups of animals were measured by enzyme-linked immunosorbent assay, while airway remodeling and cytokine production were studied by immunohistochemistry. The results demonstrated that CpG-ODN administration either before (group 2) or between (group 3) antigen treatments resulted in reduced total immunoglobulin E levels and peripheral blood eosinophil numbers compared to A. fumigatus allergen-sensitized group 1 animals. Similarly, treatment with CpG-ODN also downregulated inflammatory cell infiltration, goblet cell hyperplasia, and basement membrane thickening compared to A. fumigatus-sensitized mice. The distinct reduction in peripheral blood eosinophilia and airway remodeling in CpG-ODN-treated mice emphasized its usefulness as an immunomodulating agent for allergic fungal diseases.

Among the several allergens cloned and expressed from Aspergillus fumigatus, Asp f 4 is a major one associated with allergic bronchopulmonary aspergillosis (ABPA). The structure-function relationship of allergens is important in understanding the immunopathogenesis, diagnosis, and treatment of allergic diseases. These include the epitopes, conformational or linear, deletion of the N or C terminus or both N and C termini, and glycosylation or nonglycosylation, all of which affect immune responses. Similarly, the role of cysteine residues present in allergens may yield useful information regarding the conformational structure of allergens and the immunoglobulin E (IgE) epitope interaction. Such information may help in developing new strategies towards immunotherapy. In order to define the role of cysteine in the interaction of the antibody with Asp f 4, we have constructed mutants by selectively deleting cysteine residues from the C-terminal region of the Asp f 4. Immunological evaluation of these engineered recombinant constructs was conducted by using sera from patients with ABPA, Aspergillus skin test-positive asthmatics, and healthy controls. The results demonstrate strong IgE binding with Asp f 4 and two truncated mutants, Asp f 41-234 (amino acids [aa] 1 to 234) and Asp f 41-241 (aa 1 to 241), while another mutant, Asp f 41-196 (aa 1 to 196), showed reactivity with fewer patients. The result suggests that deletion of cysteines and the alteration of IgE epitopes at the C-terminal end resulted in conformational changes, which may have a potential role in the immunomodulation of the disease.

Fungi are non-chlorophyllus microorganisms, which constitutes the main source of outdoor and indoor allergens. The antigens present in the spores and fragments of hyphae induce allergic responses in sensitized patients. The frequently recognized fungi associated with asthma include Alternaria, Cladosporium, Aspergillus, and Penicillium. With the advent of molecular biology techniques a number of fungal genes encoding relevant allergens have been cloned and the expressed allergens purified and characterized. In this review, we have presented the recent developments, where recombinant allergens have been used in the precise diagnosis of fungal allergy. We have also discussed the role played by these allergens and the T- and B-cell epitopes in the immune mechanism in fungal allergy.

Asp f 2 is a major Aspergillus fumigatus allergen involved in allergic bronchopulmonary aspergillosis. Knowledge of the B-cell epitopes may contribute to the understanding of immunoregulation and immunodiagnosis. To elucidate the immunoglobulin E (IgE) binding epitopes in the linear sequence of Asp f 2, we synthesized decamer peptides spanning the whole molecule of Asp f 2 on derivatized cellulose membranes and evaluated IgE binding in ABPA patient and control sera. Peptides three to five amino acids long were synthesized based on amino acid sequences within the IgE binding regions and evaluated for the specificity of epitope antibody interactions. Nine IgE binding regions were recognized in this protein of 268 amino acid residues. Of the nine epitopes, seven (ATQRRQI, RKYFG, HWR, YTTRR, DHFAD, ALEAYA, and THEGGQ) are present in the hydrophilic regions of Asp f 2. Immunologic evaluation of the three recombinant fragments, Asp f 2A encompassing the N-terminal epitope region, Asp f 2B without N- and C-terminal regions of the protein, and Asp f 2C representing C-terminal epitopes, revealed that either the N- or C-terminal region of the protein is essential for the correct folding and conformation for IgE antibody binding.

The 37-kDa recombinant protein Asp f 2, encoding an allergen of Aspergillus fumigatus, was expressed in a prokaryotic expression system and immunologically evaluated for its functional and structural properties. The open reading frame for a 310-amino-acid-long protein was shown to encode a signal peptide of 31 amino acids. A native 37-kDa culture filtrate protein and a 55-kDa mycelial glycoprotein (gp55) exhibited complete N-terminal sequence homology to Asp f 2. A GenBank search for homologous proteins revealed 60 and 44% sequence homologies to the cytosolic protein ASPND1 from Aspergillus nidulans and fibrinogen binding protein from Candida albicans, respectively. The glycosylation sites and cysteine molecules are conserved in all the three proteins. The extracellular matrix protein laminin showed a dose-dependent interaction with Asp f 2. This protein, expressed as a major cell-associated protein within 24 h of in vitro fungal culture, comprises 20 to 40% of total fungal protein. Furthermore, both native and recombinant Asp f 2 exhibited specific immunoglobulin (IgE) binding with allergic bronchopulmonary aspergillosis (ABPA) and cystic fibrosis-ABPA patients, whereas A. fumigatus-sensitized allergic asthma and normal control subjects failed to show IgE binding with Asp f 2. These results indicate that Asp f 2 is a major allergen of A. fumigatus exhibiting IgE antibody binding with sera from patients with ABPA. The antigen should be explored further for its potential role in the differential diagnosis of A. fumigatus-associated allergic diseases.