Identification and characterization of rostral ventromedial medulla neurons synaptically connected to the urinary bladder afferents in female rats with or without neonatal cystitis.

The neurons in the rostral ventromedial medulla (RVM) play a major role in pain modulation. We have previously shown that early-life noxious bladder stimuli in rats resulted in an overall spinal GABAergic disinhibition and a long-lasting bladder/colon sensitization when tested in adulthood. However, the neuromolecular alterations within RVM neurons in the pathophysiology of early life bladder inflammation have not been elucidated. In this study, we have identified and characterized RVM neurons that are synaptically linked to the bladder and colon and examined the effect of neonatal bladder inflammation on molecular expressions of these neurons. A transient bladder inflammation was induced by intravesicular instillation of protamine sulfate and zymosan during postnatal days 14 through 16 (P14-16) followed by pseudorabies virus PRV-152 and PRV-614 injections into the bladder and colon, respectively, on postnatal day P60. Tissues were examined 96 h postinoculation for serotonergic, GABAergic, and enkephalinergic expressions using in situ hybridization and/or immunohistochemistry techniques. The results revealed that > 50% of RVM neurons that are synaptically connected to the bladder (i.e., PRV-152+) were GABAergic, 40% enkephalinergic, and about 14% expressing serotonergic marker tryptophan hydroxylase 2 (TpH2). Neonatal cystitis resulted in a significant increase in converging neurons in RVM receiving dual synaptic inputs from the bladder and colon. In addition, neonatal cystitis significantly downregulated vesicular GABA transporter (VGAT) with a concomitant increase in TpH2 expression in bladder-linked RVM neurons, suggesting an alteration in supraspinal signaling. These alterations of synaptic connectivity and GABAergic/serotonergic expressions in RVM neurons may contribute to bladder pain modulation and cross-organ visceral sensitivity.

Peripheral antinociceptive effects of a bifunctional µ and δ opioid receptor ligand in rat model of inflammatory bladder pain.

There is a need to develop a novel analgesic for pain associated with interstitial cystitis/painful bladder syndrome (IC/PBS). The use of the conventional µ-opioid receptor agonists to manage IC/PBS pain is controversial due to adverse CNS effects. These effects are attenuated in benzylideneoxymorphone (BOM), a low-efficacy µ-opioid receptor agonist/δ-opioid receptor antagonist that attenuates thermal pain and is devoid of reinforcing effects. We hypothesize that BOM will inhibit bladder pain by attenuating responses of urinary bladder distension (UBD)-sensitive afferent fibers. Therefore, the effect of BOM was tested on responses of UBD-sensitive afferent fibers in L6 dorsal root from inflamed and non-inflamed bladder of rats. Immunohistochemical (IHC) examination reveals that following the induction of inflammation there were significant high expressions of µ, δ, and µ-δ heteromer receptors in DRG. BOM dose-dependently (1-10 mg/kg, i.v) attenuated mechanotransduction properties of these afferent fibers from inflamed but not from non-inflamed rats. In behavioral model of bladder pain, BOM significantly attenuated visceromotor responses (VMRs) to UBD only in inflamed group of rats when injected either systemically (10 mg/kg, i.v.) or locally into the bladder (0.1 ml of 10 mg/ml). Furthermore, oxymorphone (OXM), a high-efficacy µ-opioid receptor agonist, attenuated responses of mechanosensitive bladder afferent fibers and VMRs to UBD. Naloxone (10 mg/kg, i.v.) significantly reversed the inhibitory effects of BOM and OXM on responses of bladder afferent fibers and VMRs suggesting µ-opioid receptor-related analgesic effects of these compounds. The results reveal that a low-efficacy, bifunctional opioid-based compound can produce analgesia by attenuating mechanotransduction functions of afferent fibers innervating the urinary bladder.

MicroRNA-mediated downregulation of potassium-chloride-cotransporter and vesicular γ-aminobutyric acid transporter expression in spinal cord contributes to neonatal cystitis-induced visceral pain in rats.

Loss of GABAergic inhibition in pain pathways has been considered to be a key component in the development of chronic pain. In the present study, we intended to examine whether miR-92b-mediated posttranscriptional dysregulation of spinal potassium chloride cotransporter (KCC2) and vesicular γ-aminobutyric acid transporter (VGAT) plays a major role in the development and maintenance of long-term visceral hyperalgesia in neonatal zymosan-treated rats. Neonatal cystitis was induced by transurethral zymosan administration from postnatal (P) days 14 to 16 (protocol 1). Two other zymosan protocols were also used: adult rechallenge on P57 to 59 following neonatal P14 to 16 exposures (protocol 2), and adult zymosan exposures on P57 to 59 (protocol 3). Both neonatal and adult bladder inflammation protocols demonstrated an increase in spinal miR-92b-3p expression and subsequent decrease in KCC2 and VGAT expression in spinal dorsal horn neurons. In situ hybridization demonstrated a significant upregulation of miR-92b-3p in the spinal dorsal horn neurons of neonatal cystitis rats compared with saline-treated controls. In dual in situ hybridization and immunohistochemistry studies, we further demonstrated coexpression of miR-92b-3p with targets KCC2 and VGAT in spinal dorsal horn neurons, emphasizing a possible regulatory role both at pre- and post-synaptic levels. Intrathecal administration of lentiviral pLSyn-miR-92b-3p sponge (miR-92b-3p inhibitor) upregulated KCC2 and VGAT expression in spinal dorsal horn neurons. In behavioral studies, intrathecal administration of lentiviral miR-92b-3p sponge attenuated an increase in visceromotor responses and referred viscerosomatic hypersensitivity following the induction of cystitis. These findings indicate that miR-92b-3p-mediated posttranscriptional regulation of spinal GABAergic system plays an important role in sensory pathophysiology of zymosan-induced cystitis.

Neonatal bladder inflammation induces long-term visceral pain and altered responses of spinal neurons in adult rats.

Painful events early in life have been shown to increase the incidence of interstitial cystitis/painful bladder syndrome in adulthood. However, the intrinsic mechanism is not well studied. We previously reported that neonatal bladder inflammation causes chronic visceral hypersensitivity along with molecular disruption of spinal GABAergic system in rats. The present study investigates whether these molecular changes affect the integrative function and responses of bladder-sensitive primary afferent and spinal neurons. Neonatal bladder inflammation was induced by intravesicular injection of zymosan during postnatal (P) days 14-16. In adulthood (P60), the viscero-motor response (VMR) to visceral stimuli was significantly inhibited by intrathecal (i.t) HZ166 (GABAAα-2 agonist) only in neonatally saline-treated, but not in neonatally zymosan-treated rats. HZ166 significantly inhibited the responses of bladder-responsive lumbosacral (LS) spinal neurons to urinary bladder distension (UBD) and slow infusion (SI) in neonatally saline-treated rats. Similar results were also observed in naïve adult rats where HZ166 produced significant inhibition of bladder-responsive spinal neurons. However, HZ166 did not inhibit responses of UBD-responsive spinal neurons from neonatally zymosan-treated rats. The drug did not attenuate the responses of UBD-sensitive pelvic nerve afferent (PNA) fibers to UBD and SI in either group of rats tested. Immunohistochemical studies showed a significantly lower level of GABAAα-2 receptor expression in the LS spinal cord of neonatally zymosan-treated rats compared to saline-treated rats. These findings indicate that neonatal bladder inflammation leads to functional and molecular alteration of spinal GABAAα-2 receptor subtypes, which may result in chronic visceral hyperalgesia in adulthood.

Role of MicroRNA in Visceral Pain.

The long-lasting nociceptive transmission under various visceral pain conditions involves transcriptional and/or translational alter-ation in neurotransmitter and receptor expression as well as modification of neuronal function, morphology and synaptic connections. Although it is largely unknown how such changes in posttranscriptional expression induce visceral pain, recent evi-dence strongly suggests an important role for microRNAs (miRNAs, small non-coding RNAs) in the cellular plasticity underlying chronic visceral pain. MicroRNAs are small noncoding RNA endogenously produced in our body and act as a major regulator of gene expression by either through cleavage or translational repression of the target gene. This regulation is essential for the normal physiological function but when disturbed can result in pathological conditions. Usually one miRNA has multiple targets and target mRNAs are regulated in a combinatorial fashion by multiple miRNAs. In recent years, many studies have been per-formed to delineate the posttranscriptional regulatory role of miRNAs in different tissues under various nociceptive stimuli. In this review, we intend to discuss the recent development in miRNA research with special emphases on miRNAs and their tar-gets responsible for long term sensitization in chronic pain conditions. In addition, we review miRNAs expression and function data for different animal pain models and also the recent progress in research on miRNA-based therapeutic targets for the treatment of chronic pain.

NMDA receptor mediates chronic visceral pain induced by neonatal noxious somatic stimulation.

NMDA receptors (NMDAR) are important in the development and maintenance of central sensitization. Our objective was to investigate the role of spinal neurons and NMDAR in the maintenance of chronic visceral pain. Neonatal rats were injected with acidic saline adjusted to pH 4.0 in the gastrocnemius muscle every other day for 12 days. In adult rats, NR1 and NR2B subunits were examined in the lumbo-sacral (LS) spinal cord. A baseline, visceromotor response (VMR) to graded colorectal distension (CRD) was recorded before and after administration of the NMDA antagonist, CGS-19755. Extracellular recordings were performed from CRD-sensitive LS spinal neurons and pelvic nerve afferents (PNA) before and after CGS-19755. Rats that received pH 4.0 saline injections demonstrated a significant increase in the expression NR2B subunits and VMR response to CRD>20 mmHg. CGS-19755 (i.v. or i.t.) had no effect in naïve rats, but significantly decreased the response to CRD in pH 4.0 saline injected rats. CGS-19755 had no effect on the spontaneous firing of SL-A, but decreased that of SL-S. Similarly, CGS-19755 attenuates the responses of SL-S neurons to CRD, but had no effect on SL-A neurons or on the response characteristics of PNA fibers. Neonatal noxious somatic stimulation results in chronic visceral hyperalgesia and sensitizes a specific subpopulation of CRD-sensitive spinal neurons. The sensitization of these SL-S spinal neurons is attenuated by the NMDAR antagonist. The results of this study suggest that spinal NMDARs play an important role in the development of hyperalgesia early in life.

Analgesic effect of minocycline in rat model of inflammation-induced visceral pain.

The present study investigates the analgesic effect of minocycline, a semi-synthetic tetracycline antibiotic, in a rat model of inflammation-induced visceral pain. Inflammation was induced in male rats by intracolonic administration of tri-nitrobenzenesulphonic acid (TNBS). Visceral hyperalgesia was assessed by comparing the viscero-motor response (VMR) to graded colorectal distension (CRD) prior and post 7 days after TNBS treatment. Electrophysiology recordings from CRD-sensitive pelvic nerve afferents (PNA) and lumbo-sacral (LS) spinal neurons were performed in naïve and inflamed rats. Colonic inflammation produced visceral hyperalgesia characterized by increase in the VMRs to CRD accompanied with simultaneous activation of microglia in the spinal cord and satellite glial cells (SGCs) in the dorsal root ganglions (DRGs). Selectively inhibiting the glial activation following inflammation by araC (Arabinofuranosyl Cytidine) prevented the development of visceral hyperalgesia. Intrathecal minocycline significantly attenuated the VMR to CRD in inflamed rats, whereas systemic minocycline produced a delayed effect. In electrophysiology experiments, minocycline significantly attenuated the mechanotransduction of CRD-sensitive PNAs and the responses of CRD-sensitive LS spinal neurons in TNBS-treated rats. While the spinal effect of minocycline was observed within 5min of administration, systemic injection of the drug produced a delayed effect (60min) in inflamed rats. Interestingly, minocycline did not exhibit analgesic effect in naïve, non-inflamed rats. The results demonstrate that intrathecal injection of minocycline can effectively attenuate inflammation-induced visceral hyperalgesia. Minocycline might as well act on neuronal targets in the spinal cord of inflamed rats, in addition to the widely reported glial inhibitory action to produce analgesia.

MicroRNA-mediated GABA Aα-1 receptor subunit down-regulation in adult spinal cord following neonatal cystitis-induced chronic visceral pain in rats.

The nociceptive transmission under pathological chronic pain conditions involves transcriptional and/or translational alteration in spinal neurotransmitters, receptor expressions, and modification of neuronal functions. Studies indicate the involvement of microRNA (miRNA) - mediated transcriptional deregulation in the pathophysiology of acute and chronic pain. In the present study, we tested the hypothesis that long-term cross-organ colonic hypersensitivity in neonatal zymosan-induced cystitis is due to miRNA-mediated posttranscriptional suppression of the developing spinal GABAergic system. Cystitis was produced by intravesicular injection of zymosan (1% in saline) into the bladder during postnatal (P) days P14 through P16 and spinal dorsal horns (L6-S1) were collected either on P60 (unchallenged groups) or on P30 after a zymosan re-challenge on P29 (re-challenged groups). miRNA arrays and real-time reverse transcription-polymerase chain reaction (RT-PCR) revealed significant, but differential, up-regulation of mature miR-181a in the L6-S1 spinal dorsal horns from zymosan-treated rats compared with saline-treated controls in both the unchallenged and re-challenged groups. The target gene analysis demonstrated multiple complementary binding sites in miR-181a for GABA(A) receptor subunit GABA(Aα-1) gene with a miRSVR score of -1.83. An increase in miR-181a concomitantly resulted in significant down-regulation of GABA(Aα-1) receptor subunit gene and protein expression in adult spinal cords from rats with neonatal cystitis. Intrathecal administration of the GABA(A) receptor agonist muscimol failed to attenuate the viscero-motor response (VMR) to colon distension in rats with neonatal cystitis, whereas in adult zymosan-treated rats the drug produced significant decrease in VMR. These results support an integral role for miRNA-mediated transcriptional deregulation of the GABAergic system in neonatal cystitis-induced chronic pelvic pain.

Neuronal plasticity in the cingulate cortex of rats following esophageal acid exposure in early life.

BACKGROUND & AIMS: The cingulate cortex 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 postsynaptic density protein 95 (PSD-95) at the synapse appear 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 cingulate cortex. METHODS: We assessed NMDA receptor subunits and PSD-95 protein expression in rostral cingulate cortex (rCC) tissues of rats exposed to esophageal acid or saline (control), either during postnatal day (P) 7 to 14 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 as a result 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.

Altered expression of P2X3 in vagal and spinal afferents following esophagitis in rats.

Purinergic P2X(3) receptors are predominantly expressed in small diameter primary afferent neurons and activation of these receptors by adenosine triphosphate is reported to play an important role in nociceptive signaling. The objective of this study was to investigate the expression of P2X(3) receptors in spinal and vagal sensory neurons and esophageal tissues following esophagitis in rats. Two groups of rats were used including 7 days fundus-ligated (7D-ligated) esophagitis and sham-operated controls. Esophagitis was produced by ligating the fundus and partial obstruction of pylorus that initiated reflux of gastric contents. The sham-operated rats underwent midline incision without surgical manipulation of the stomach. Expressions of P2X(3) receptors in thoracic dorsal root ganglia (DRGs), nodose ganglia (NGs), and esophageal tissues were evaluated by RT-PCR, western blot and immunohistochemistry. Esophageal neurons were identified by retrograde transport of Fast Blue from the esophagus. There were no significant differences in P2X(3) mRNA expressions in DRGs (T1-T3) and NGs between 7D-ligated and sham-operated rats. However, there was an upregulation of P2X(3) mRNA in DRGs (T6-T12) and in the esophageal muscle. At protein level, P2X(3) exhibited significant upregulation both in DRGs and in NGs of rats having chronic esophagitis. Immunohistochemical analysis exhibited a significant increase in P2X(3) and TRPV1 co-expression in DRGs and NGs in 7D-ligated rats compared to sham-operated rats. The present findings suggest that chronic esophagitis results in upregulation of P2X(3) and its co-localization with TRPV1 receptor in vagal and spinal afferents. Changes in P2X(3) expression in vagal and spinal sensory neurons may contribute to esophageal hypersensitivity following acid reflux-induced esophagitis.

Intramucosal distribution of WNT signaling components in human esophagus.

BACKGROUND: The molecular mechanisms governing the biology and pathobiology of esophageal squamous mucosa in health and disease are not completely understood. Earlier genome-wide expression study of normal-looking esophageal squamous mucosa has shown differential expression of the Wingless-type MMTV integration site family (Wnt) modulators Dickkopf (Dkk) homologs among healthy individuals and patients with reflux esophagitis and Barrett metaplasia suggesting that the Wnt pathway may be involved in esophageal mucosal biology. STUDY: Seven full-thickness human donor esophagi were cryosectioned for immunohistochemical analysis, and lamina propria (LP), basal (BC), intermediate (IC), and superficial (SC) cells were also dissected by laser-capture microdissection for real-time polymerase chain reaction. RESULTS: Wnt1, 2b, and 3a were expressed primarily in BC, Wnt3, and 5b in LP, and Wnt5a in IC. Frizzled 1, low-density lipoprotein receptor-related protein 6, secreted frizzled-related protein 1, T-cell-specific transcription factor 3, and dishevelled 3 were expressed highest in LP decreasing precipitously medially toward SC. Dkk1 predominantly expressed in SC was more than 100-folds greater than other layers (P<0.001). Dkk4 was expressed primarily in SC but Dkk3 was opposite with greatest expression in LP. Immunohistochemical analysis showed Wnt1 and 3a in BC, Wnt5a in IC and SC, Dkk1 predominantly in SC, Dkk4 in SC and IC, and Dkk3 and SFRP1 in LP and BC

Alterations in N-methyl-D-aspartate receptor subunits in primary sensory neurons following acid-induced esophagitis in cats.

The excitatory amino acid glutamate plays an important role in the development of neuronal sensitization and the ionotropic N-methyl-d-aspartate receptor (NMDAR) is one of the major receptors involved. The objective of this study was to use a cat model of gastroesophageal reflux disease (GERD) to investigate the expression of the NR1 and NR2A subunits of NMDAR in the vagal and spinal afferent fibers innervating the esophagus. Two groups of cats (Acid-7D and PBS-7D) received 0.1 N HCl (pH 1.2) or 0.1 M PBS (pH 7.4) infusion in the esophagus (1 ml/min for 30 min/day for 7 days), respectively. NR1 splice variants (both NH(2) and COOH terminals) and NR2A in the thoracic dorsal root ganglia (DRGs), nodose ganglia (NGs), and esophagus were evaluated by RT-PCR, Western blot, and immunohistochemistry. Acid produced marked inflammation and a significant increase in eosinophil peroxidase and myeloperoxidase contents compared with PBS-infused esophagus. The NR1-4 splice variant gene exhibited a significant upregulation in DRGs and esophagus after acid infusion. In DRGs, NGs, and esophagus, acid infusion resulted in significant upregulation of NR1 and downregulation of NR2A subunit gene expression. A significant increase in NR1 polypeptide expression was observed in DRGs and NGs from Acid-7D compared with control. In conclusion, long-term acid infusion in the cat esophagus resulted in ulcerative esophagitis and differential expressions of NR1 and NR2A subunits. It is possible that these changes may in part contribute to esophageal hypersensitivity observed in reflux esophagitis.

Immune response to n-terminal and c-terminal deletion mutants of Aspergillus fumigatus major allergen ASP F 3.

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.

Modulation of airway inflammation by immunostimulatory CpG oligodeoxynucleotides in a murine model of allergic aspergillosis.

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.

Role of C-terminal cysteine residues of Aspergillus fumigatus allergen Asp f 4 in immunoglobulin E binding.

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 4(1-234) (amino acids [aa] 1 to 234) and Asp f 4(1-241) (aa 1 to 241), while another mutant, Asp f 4(1-196) (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.

Molecular biology of Aspergillus allergens.

Allergic Bronchopulmonary Aspergillosis (ABPA) is a severe respiratory disease caused by the fungus Aspergillus fumigatus (Af). It occurs as secondary complication mostly in patients with atopy and cystic fibrosis. The standardized and well-characterized allergens are essential for the immunodiagnosis of ABPA as well as for understanding the pathophysiology of the disease. Molecular cloning was resorted to obtain purified Af allergens for such studies. Currently, twenty-two recombinant Af allergens have been identified and characterized and several of these can be used as standardized allergens in in vitro and in vivo diagnosis of ABPA. The knowledge of primary, secondary, and tertiary structures of these allergens may facilitate the identification of immunodominant T and B cell epitopes and may be used to unravel the structure function relationship of these allergens. Such findings may open up novel avenues in the immune modulatory therapy and other effective intervention of the disease.

Aspergillus antigens associated with allergic bronchopulmonary aspergillosis.

Among the molds causing allergy, Aspergillus fumigatus (Af) constituted a major species present both indoors and outdoors. The antigens both secreted and bound to hyphae and conidia have been isolated for immunodiagnosis of allergic aspergillosis. The crude extracts have been used to demonstrate IgE antibody in vitro and for skin testing. The crude extracts contain many antigenic and non-antigenic components and toxins and have demonstrated inconsistent reactivities. In addition, similarity with other allergens also may complicate specific diagnosis. A number of methods including conventional purification and fractionation methods have been used to obtain relevant antigens. In recent years, monoclonal antibody dependent affinity purification and molecular biology methods have obtained considerable progress in the allergen purification and in developing specific and reliable immunoassays. However, international standards are still lacking and hence, comparison of results among laboratories are not possible yet.

C-terminal cysteine residues determine the IgE binding of Aspergillus fumigatus allergen Asp f 2.

The knowledge of the structure function relationship of the allergen is essential to design allergenic variants with reduced IgE binding capacity but intact T cell reactivity. Asp f 2 is a major allergen from the fungus Aspergillus fumigatus and >90% of A. fumigatus-sensitized individuals displayed IgE binding to Asp f 2. In the present study, we evaluated the involvement of C-terminal cysteine residues in IgE binding conformation of Asp f 2. The deletion mutants were constructed by adding three C-terminal cysteines of the native Asp f 2 one at a time to the non-IgE binding Asp f 2 (68-203). The point mutants of Asp f 2 (68-268) with C204A and C257A substitutions were constructed to study the role of C-terminal cysteines in IgE binding. Immunological evaluation of reduced and alkylated Asp f 2 and its mutants were conducted to determine the contribution of free sulfhydryl groups as well as the disulfide bonds in allergen Ab interaction. Four-fold increase in IgE Ab binding of Asp f 2 (68-267) compared with Asp f 2 (68-266) and complete loss in IgE binding of C204A mutant of Asp f 2 (68-268) indicate the involvement of C(204) and C(267) in IgE binding conformation of Asp f 2. A significant reduction in IgE binding of wild and mutated Asp f 2 after reduction and alkylation emphasizes the importance of cysteine disulfide bonds in epitope Ab interaction. The hypoallergenic variants may be explored further to develop safe immunotherapeutic strategy for allergic disorders.

IgE binding conformational epitopes of Asp f 3, a major allergen of Aspergillus fumigatus.

sp f 3 has been identified as one of the major allergens of Aspergillus fumigatus associated with the sensitization and immune responses in allergic bronchopulmonary aspergillosis (ABPA). In order to understand the structure/function relationship of Asp f 3, we studied synthetic peptides and constructed mutants deleted of specific IgE binding regions. The mutated allergens were obtained by expressing the genes and studied by ELISA for their reactivity with IgE from patients with ABPA. Seven linear IgE binding regions spanning the whole Asp f 3 molecule were demonstrated. The results demonstrated strong binding of IgE from ABPA patients with Asp f 3 and one mutant, Asp f 3(1-150), but not with other mutant constructs. The results identified 12 amino acids at the N-terminal end and 8 amino acids (143-150) at the C-terminal end as significant in the conformational constraints for IgE binding. The Fourier transfer spectra showed comparable beta-sheet structure of Asp f 3(1-150) and Asp f 3, indicating the role of secondary structure in IgE binding. The primary and secondary structures may help understanding of the functional role the allergens play in the disease and may have implications in immunodiagnosis and probably immunotherapy.