[Validation of the digital integration technology for evaluating the nasolabial morphology variation after the cross-arch fixed restoration of maxillary implant-supported prostheses].

OBJECTIVE: To explore the applicability of integration between three-dimensional (3D) facial and dental data to evaluate the nasolabial morphology variation before and after the cross-arch fixed restoration of the maxillary implant-supported prostheses. METHODS: Twelve patients (4 women and 8 men), mean age (54.82±5.50) years (from 45 to 62 years) referred to the Department of Oral Implan-tology, Peking University School and Hospital of Stomatology, were selected and diagnosed with edentulous maxilla. For all the patients, 4 to 6 implants were inserted into the maxilla. Six months later, the final cross-arch fixed prostheses were delivered. The 3D facial images were collected before and after the final restoration. The 3D data of prostheses were also captured. All the 3D data were registered and measured in the same coordinate system. Then the displacement of all the landmarks [cheilion left (CHL), cheilion right (CHR), crista philtri left (CPHL), crista philtri right (CPHR), labrale supe-rius (LS), subnasale (SN), stomion (STO), upper incisor (UI), upper flange border of the prostheses (F-point, F)], and the variation of the distances between these landmarks (SN-LS, CPHR-CPHL, CHR-CHL, LS-STO) were analyzed and compared. RESULTS: The consistency test among three measurements of the length of F-SN indicated that the integration method of the dental prostheses and soft tissue had the good repetitiveness, ICC=0.983 (95%CI: 0.957-0.995). After wearing the final cross-arch maxillary implant-supported prostheses, all the landmarks on the soft tissue moved forward. The nasal base area changed minimally, and the shift of SN in the sagittal direction was only (0.61±0.44) mm. But the sagittal shift of LS was (3.12±1.38) mm. In the vertical direction, SN, LS, CPHL, and CPHR moved upward. But STO, CHL, and CHR moved downward a little. Except for the slight decrease of the length of philtrum (SN-LS), the length of CHL-CHR, CPHL-CPHR, and the height of upper lip were increased together (P < 0.01). In the direction of Z axis, the strong correlations were found not only between the movements of SN and F (r=0.904 3) but also between the movements of LS and UI (r=0.958 4). CONCLUSION: The integration method of 3D facial and dental data showed good repetitiveness. And the strong correlations between the landmarks of prostheses and nasolabial soft tissue in the sagittal direction were found by this new method.

[Digital workflow coupling conic retention for the immediate restoration of adjacent posterior implants].

OBJECTIVE: To evaluate the digital workflow coupling conic retention for the immediate restoration of adjacent posterior implants. METHODS: The patients with adjacent teeth missing in the posterior jaw seeking for implant restoration in the Department of Implantology, Peking University School and Hospital of Stomatology from March, 2017 to February, 2018 were recruited. After implant placement and commercial conic retention coping delivery, the patient had an intraoral scan for digital impression, and the computer-assisted design/computer-assisted manufacturing (CAD/CAM) technology was used for the fabrication of the immediate splinted prosthesis, which was made of polymethyl methacrylate (PMMA) and loaded immediately after delivery. Six months later, all the temporary prostheses were replaced by the permanent ones made of monolithic zirconia with CAD/CAM technology as well. The parallel periapical films were taken for the temporary and permanent prostheses post-delivery. The clinical effect of this workflow was evaluated by indices including the survival rates of implants and prostheses, the change of marginal bone level, and the implant-related and prosthesis-related complications; before the final restoration, the Visual Analogue Score (VAS) was used to evaluate the satisfaction of the patients. RESULTS: Ten patients (4 males and 6 females, 55.5 years old for average) were recruited. Totally 34 implants were placed; 14 prostheses were fabricated, temporary and permanent, respectively. After an observation period from 4 to 14 months, the survival rate for implants and prostheses were both 100%; the marginal bone level of the implants were (1.06±0.97) mm and (0.96±0.82) mm, immediate post-operation and 6 months later, respectively. The difference was not statistically significant (P>0.05). Neither implant- nor prostheses- related complications were observed. And the VAS of the patients' satisfaction was 87.2. CONCLUSION: For the adjacent posterior implants, the immediate prostheses manufactured by digital workflow, coupling conic retention, were clinically feasible and patient-satisfactory.

[Complications of implant-supported full-arch immediate prosthesis: a retrospective analysis of 114 cases].

Objective: To evaluate the feasibility of long-term function of implant-supported full-arch immediate prosthesis via assessing the complications and risk factors. Methods: This historical cohort study included patients treated with implant-supported full-arch restoration under immediate loading protocol between April, 2008 to June, 2016 and wearing the immediate prosthesis for more than 6 months. Medical charts were reviewed for patients' general information, implant information, prosthetic information and details of prosthetic complications. COX proportional hazards ratio model was adopted to analyze the potential risk factors for prosthesis fracture. Results: A total of 114 patients with a mean age of (56.7±10.2) years old and 144 prostheses were included. The median wearing time of immediate prosthesis was 17.6 months. Sixty-two (54%) patients experienced prosthetic complication, 30 of them suffered more than once. Artificial teeth fractures were more common in anterior region while resin base fractured more often in the posterior region. The possibility of immediate prosthesis fracture in the first year was high but declined over the following years. COX regression analysis showed that fibre-reinforcement (HR=0.486, P=0.017) and rigid opposing dentition (HR=2.272, P=0.016) were significantly related to the prosthesis fracture. Conclusions: Long-term function of implant-supported full-arch immediate prosthesis renders a high prosthetic complication prevalence, featuring the prosthesis fracture as the most common complication and the first year of highest fracture probability. Fibre-reinforced acrylic immediate prosthesis may function well in cases with a removable denture restored opposing jaw.

Asp159 is a critical core amino acid of an IgE-binding and cross-reactive epitope of a dust mite allergen Der f 7.

Der f 7 and Der p 7 are important house dust mite allergens with known structure and suggested biological function recently. However, their IgE-binding determinants remain unknown. The purpose of this study is to identify the IgE-reactive epitopes of Der f 7 and the determinants of IgE-mediated cross-reactivity between Der f 7 and Der p 7. IgE-reactive determinants were identified by immunodot blot inhibition using synthetic overlapping peptides, allergen mutants, and a Der f 7 structural model. Our results showed that synthetic peptides with sequence (156)SILDP(160) on Der f 7 bind IgE in two of the 30 asthmatic serum samples tested. Recombinant Der f 7 I157A, L158A, or D159A mutants have reduced IgE-binding activity. Inhibition experiments confirmed Asp159 as a critical core residue for IgE-binding. Among Der p 7, Der f 7 and Der f 7 mutants with single substitution between residues 156 and 160, only the D159A mutant cannot inhibit significantly IgE-binding against Der p 7. Therefore, Asp159 contributes to IgE-mediated cross-reactivity between Der f 7 and Der p 7. The structural model constructed for Der f 7 suggests that the IgE-binding epitope forms a loop-like structure on the surface of the molecule. In conclusion, Asp 159 is a critical core residue of an IgE-binding and IgE-mediated cross-reactive epitope (156)SILDP(160) of Der f 7. Results obtained from this study provide more information on molecular and structural features related to allergenicity, underlying basis of IgE cross-reactivity between allergens, and in designing safer immunotherapy.

Transaldolases are novel and immunoglobulin E cross-reacting fungal allergens.

BACKGROUND: Mould-induced atopic respiratory diseases are a worldwide problem. Characterization of fungal allergens is of major clinical importance. OBJECTIVE: We identified a novel transaldolase family allergen of Cladosporium and Penicillium species. METHODS: Fungal allergens were identified by immunoblotting, peptide mass mapping and partial sequencing, cDNA cloning and IgE epitope mapping. RESULTS: A 36.5 kDa IgE-binding component in a partially purified C. cladosporioides preparation was identified. Mass spectrometric analyses suggest that this novel IgE-reacting allergen is a transaldolase. A corresponding full-length 1246 bp cDNA encoding a polypeptide of 325 residues was isolated. The newly identified transaldolase allergen has been designated as Cla c 14.0101. The cDNA encoding the Pencillium chrysogenum transaldolase was isolated by RT-PCR according to the cDNA sequence encoding a P. chrysogenum Wisconsin 54-1255 hypothetical protein. The purified rCla c 14.0101 protein reacted with IgE antibodies in 10 (38%) of 26 Cladosporium cladosporioides-sensitized asthmatic patients. Nine of the 10 rCla c 14.0101-positive sera have IgE binding against the recombinant Penicillium transaldolase (rPen ch 35.0101). Among the eight fungal transaldolase-positive sera tested, three showed IgE binding against the recombinant human transaldolase. To determine cross-reactivity between the Cladosporium and Penicillium fungi, IgE cross-reactivity was detected between these two fungal transaldolase allergens by inhibition assays. Both the N- and the C-terminal fragments of Cla c 14.0101 were recognized by IgE antibodies. The C-terminal IgE-reacting determinant was narrowed down to a region encompassing Thr257 to Ser278 of Cla c 14.0101. It was mapped onto a loop-like structure of a 3D model constructed for Cla c 14.0101. CONCLUSION AND CLINICAL RELEVANCE: We identified transaldolase as a novel and IgE cross-reactive allergen family of C. cladosporioides and P. chrysogenum. In addition, an IgE-reacting fragment (Thr257 to Ser278) was pinpointed to a loop-like structure on Cla c 14.0101. Results obtained provide important information in clinical mould allergy.

Pen ch 13 allergen induces secretion of mediators and degradation of occludin protein of human lung epithelial cells.

BACKGROUND: Alkaline serine proteases from six prevalent airborne Penicillium and Aspergillus species have been identified as a group of major allergens (group 13). After entering human airways, the allergens are in initial contacts with respiratory epithelial cells. The purpose of this study is to investigate interactions between the Pen ch 13 allergen from P. chrysogenum and human lung epithelial cells. METHODS: A549 cells, 16HBE14o- cells and primary cultures of human bronchial epithelial cells (HBEpC) were exposed to purified Pen ch 13 and mediators released into culture supernatants were assayed with enzyme-linked immunosorbent assay (ELISA) kits. Cleavage of occludin in 16HBE14o- cells was analysed by immunofluorescent staining of whole cells and immunoblot analysis of whole cell extracts. Fragments generated by incubating Pen ch 13 and a synthetic peptide carrying the occludin sequence were analysed by mass spectrometry. RESULTS: Pen ch 13 induced productions of prostaglandin-E2 (PGE2), interleukin (IL)-8 and transforming growth factor (TGF)-beta1 by A549 cells, 16HBE14o- cells and primary cultures of HBEpC. The protease activity of Pen ch 13 is needed for the induction of PGE2 IL-8, TGF-beta1 and cyclo-oxygenase (COX)-2 expression. A tight junction protein occludin of 16HBE14o- cells can be cleaved by Pen ch 13 at Gln202 and Gln211 which are within the second extracellular domain of the protein. CONCLUSION: Pen ch 13 may contribute to asthma by damaging the barrier formed by the airway epithelium and stimulating the release of mediators that orchestrate local immune responses and inflammatory process from HBEpC.

Molecular cloning and characterization of full-length cDNAs encoding a novel high-molecular-weight Dermatophagoides pteronyssinus mite allergen, Der p 11.

BACKGROUND: Dermatophagoides pteronyssinus (Dp) and D. farinae (Df) mites are the most important source of indoor aeroallergens. Most Dp mite allergens identified to date have relatively low molecular weights (MWs). Identification of high-MW mite allergens is a crucial step in characterizing the complete spectrum of mite allergens and to provide appropriate tools for diagnostic and therapeutic application. METHODS: The full-length Der p 11 cDNA clone was isolated using cDNA library immunoscreening, the 5'-3' rapid amplification of cDNA ends (RACE) system and polymerase chain reactions (PCR). The whole cDNA insert and its PCR-derived DNA fragments (p1 to p4) were generated and expressed in the Escherichia coli expression system. The allergenicity of the recombinant protein and its peptide fragments was examined by IgE immunodot assays. The IgE-binding reactivity of rDer p 11 was analyzed in the serum of 50 asthmatic children with positive reactivity to Dp mite extract. Its recombinant peptide fragments were also examined by immunodot assays in 30 mite-allergic children. RESULTS: Der p 11 cDNA consists of a 2625-bp open reading frame encoding a 103-kDa protein with 875 amino acids. It exhibits significant homology with the paramyosin of other invertebrates. The protein sequence alignment of this newly identified Dp mite allergen (denominated as Der p 11) revealed over 89% identity with Der f 11 and Blo t 1. Among 50 Dp-sensitive asthmatic children, rDer p 11 showed positive IgE-binding reactivity to 39 patients (78%). Using immunodot assays, multiple human IgE-binding activities were demonstrated in all four fragments of Der p 11. Using immunoblot assays, the dominant IgG-binding epitope for monoclonal antibody (mAb642) was located in fragment p3 only. In immunoblot assays, cross-inhibition between rDer p 11 and rDer f 11 was up to 73-80% at concentrations of 100 microg/ml. CONCLUSIONS: This study confirms that the newly identified recombinant Der p 11 is a novel and important high-MW Dp mite allergen for asthmatic children. Our data also indicates that human IgE-binding major epitopes are scattered over the entire molecule of Der p 11.

cDNA cloning and immunologic characterization of a novel EF-1beta allergen from Penicillium citrinum.

BACKGROUND: We have identified previously that Penicillium citrinum is the most prevalent Penicillium species in the Taipei area. It is important to delineate the whole spectrum of allergenic components of this prevalent airborne fungus. The purpose of this study was to identify novel P. citrinum allergens through molecular cloning of allergen genes using a cDNA library of P. citrinum and sera from patients with bronchial asthma. METHODS: A lambda-Uni-ZAP XR-based cDNA library of P. citrinum was screened with sera from asthmatic patients. An IgE-binding cDNA clone was isolated and heterologously expressed in Escherichia coli. The frequency of IgE-binding to the expressed protein and the IgE reactivity to allergen subunits were analyzed by immunoblotting. RESULTS: An IgE-reactive cDNA clone (clone B) was isolated by plaque immunoassay. The cDNA insert is 876-bp long and encodes a 228-amino acid polypeptide with a calculated molecular mass of 25 035 Da. Protein database search with the deduced clone B sequence revealed that 121 (53%) and 82 (36%) of the 228 amino acids were identical to those of the elongation factor 1-beta (EF-1beta) proteins from the yeast Saccharomyces cerevisiae and the parasite Echinococcus granulosus, respectively. His-tagged recombinant clone B proteins were constructed and expressed in E. coli. Seven (8%) of the 92 serum samples from patients with bronchial asthma showed IgE-binding to the recombinant clone B protein. Among these seven positive sera, five demonstrated IgE-binding to the C-terminal fragment (aa 119-228) while the other two sera showed IgE reactivity to the N-terminal fragment (aa 1-118) of this newly identified EF-1betaPenicillium allergen. CONCLUSIONS: A novel P. citrinum allergen (Pen c 24) was identified and characterized in the present study. Results obtained provide more information about allergens of prevalent airborne fungi and a basis to understand more about the IgE responses in human atopic disorders and in parasitic infections.

Molecular and structural analysis of immunoglobulin E-binding epitopes of Pen ch 13, an alkaline serine protease major allergen from Penicillium chrysogenum.

BACKGROUND: Through proteomic and genomic approaches we have previously identified and characterized an alkaline serine protease that is a major allergen (88% frequency of IgE binding) of Penicillium chrysogenum (Pen ch 13). OBJECTIVE: The aim of the present study is to identify the linear IgE-binding epitopes of Pen ch 13. METHODS: IgE-binding regions were identified by dot-blot immunoassay using 11 phage-displayed peptide fragments spanning the whole molecule of Pen ch 13. The minimal epitope requirements for IgE binding were further defined with overlapping peptides synthesized on derivatized cellulose membranes using SPOTs technology. The critical residues on the immunodominant epitopes were mapped through site-directed mutagenesis. The locations of the IgE epitopes identified were correlated with a three-dimensional structure of Pen ch 13. RESULTS: IgE antibodies in 35 serum samples reacted with at least one of the 11 peptide fragments of Pen ch 13. Peptide f-2n (residues 31-61) showed a high-intensity and the highest frequency (77%) of IgE binding. The frequencies of IgE binding to peptide f-4 (residues 93-133), f-1 (residues 1-37) and f-7 (residues 168-206) were 51%, 34% and 31%, respectively. SPOTs assay narrowed down the region of IgE binding of f-2n to residues 48-55 (GHADFGGR). Three, two and one epitope(s) that are four to nine amino acids in length, within f-4, f-1 and f-7, respectively, were found. Site-directed mutagenesis of Pen ch 13 revealed that substitution of His49 and/or Phe52 on Pen ch 13 with methionine resulted in proteins with drastic loss of IgE binding in seven sera tested. Proteins with amino acid replacements at residues 15-18 (RISS), or at residues 112 (I) and 116 (D) have lower IgE-binding reactivity in one of the two patient's sera tested. Substituting residues 117 (W), 119 (V) and 120 (K) also block most of the IgE binding in one of the two patient's sera tested. In addition, replacing residues 203 (V) and 204 (D) along with a deletion at residue 206 (Y) diminished the IgE binding in two serum samples tested. A model was constructed based on the structure of P. cyclopium subtilisin protease that has >90% (256 out of 283 amino acids) sequence identity with Pen ch 13. The major epitope (GHADFGGR) on Pen ch 13 formed a loop-like structure and was located at the surface of the allergen. CONCLUSIONS: Several linear IgE-reactive epitopes and their critical core amino acid residues were identified for the Pen ch 13 allergen. The major linear IgE-binding epitope, 48GHADFGGR55, formed a loop-like structure at the surface of the allergen. Substitution of His49 and/or Phe52 with methionine significantly reduced IgE-binding to Pen ch 13. Mapping of these results on a 3D model of the allergen provides valuable information about the molecular basis of allergenicity for Pen ch 13 and for designing specific immunotherapeutics.

Characterization of a novel Candida albicans 29 kDa IgE-binding protein--purification, cDNA isolation and heterologous expression of Cand a 3.

BACKGROUND: Candida albicans has been implicated in human allergic disorders. However, many of its immunoglobulin E (IgE)-reacting components have not yet been identified. The purpose of the present study is to characterize a novel 29 kDa IgE-binding protein from C. albicans. METHODS: The 29 kDa protein was partially purified and its tryptic digests subjected to mass spectrometric analysis. The cDNA encoding this protein was isolated and heterologously expressed in Escherichia coli. Monoclonal antibodies (MoAbs) were raised against the 29 kDa protein purified from C. abicans extracts. RESULTS: We isolated a 29 kDa IgE-reacting component from C. albicans. The protein was digested on-gel with trypsin and the masses of the resulting fragments were determined in a MALDI-TOF mass spectrometer. The data were searched against protein sequences deduced from the C. albicans genome. An open reading frame that possibly encodes the 29 kDa IgE-reacting component was identified. The cDNA corresponding to the open reading frame was isolated. It encodes a 236 residues protein that has 62% sequence identity to that of a hypothetical protein (YDR533c) from Saccharomyces cerevisiae. Conserved domain search suggests that the encoded protein belongs to the ThiJ/PfpI family. The cDNA isolated was inserted into a pQE-30 vector for protein expression in Escherichia coli. The recombinant protein can react with IgE antibodies in sera from asthmatic patients and two MoAbs that were generated against the purified native 29 kDa protein from C. albicans. CONCLUSIONS: We identified and cloned a novel 29 kDa IgE-reacting component (Cand a 3) from C. albicans. The recombinant proteins produced from this clone and the MoAbs prepared may be useful in the standardization of diagnostic extracts. They are also instrumental in elucidating the role of C. albicans in clinical allergy.

Molecular and immunological characterization of Pen ch 18, the vacuolar serine protease major allergen of Penicillium chrysogenum.

BACKGROUND: We have suggested previously that the 32 and 34 kDa major allergens of Penicillium chrysogenum (also known as P. notatum) are the vacuolar (Pen ch 18) and the alkaline (Pen ch 13) serine proteases, respectively, of P. chrysogenum. The purpose of this study is to characterize the 32 kDa allergen of P. chrysogenum and its immunoglobulin E (IgE)cross-reactivity with Pen ch 13 allergen. METHODS: The full-length cDNA of Pen ch 18 was isolated by reverse transcriptase-polymerase chain reaction and the 5'-rapid amplification cDNA end reaction. Recombinant Pen ch 18 was expressed as his-tagged proteins in Escherichia coli. Its reactivity with IgE and monoclonal antibodies against fungal serine protease allergens was analyzed by immunoblotting. The IgE cross-reactivity between Pen ch 18 and Pen ch 13 was analyzed by immunoblot inhibition. Overlapping recombinant fragments and synthetic peptides were used to map the B cell epitopes on Pen ch 18. RESULTS: In this study, we isolated a 1857 bp cDNA fragment containing an open reading frame of 494 amino acids that encodes the preproenzyme of Pen ch 18. Similar to other vacuolar serine proteases, this precursor appears to undergo N- and possibly C-terminal cleavage upon maturation. The his-tagged recombinant Pen ch 18 containing the putative sequence of the mature protein reacted with IgE antibodies in serum samples from asthmatic patients. In addition, IgE-binding to the 32 kDa major allergen of P. chrysogenum was inhibited when a positive serum sample was absorbed with recombinant Pen ch 18 before immunoblotting. Both inhibition and almost no inhibition of IgE-binding to the 32 kDa major allergen of Pen ch 18 were detected when eight positive serum samples were preabsorbed individually with purified Pen ch 13 before immunoblotting. The major IgE binding region was located in a fragment (PN1) encompassing the N-terminal 102 amino acid residues of the recombinant Pen ch 18. A dominant linear IgE epitope was further mapped within residues 73-95 (peptide PN1-e) of the N-terminally processed allergen. Monoclonal antibody FUM20 that reacts with Pen ch 18 but not with Pen ch 13 binds a synthetic peptide with sequence encompassing the N-terminal 23 residues of the recombinant Pen ch 18. Monoclonal antibody PCM39 that reacts with both Pen ch 13 and Pen ch 18 recognizes a peptide containing residues 132-154 of the allergen. CONCLUSIONS: Our results confirm that the Pen ch 18 allergen is a vacuolar serine protease of P. chrysogenum that matures through N- and possibly C-terminal processing. The finding that there are cross-reactive and allergen-specific IgE epitopes for Pen ch 18 and Pen ch 13 suggests that both major allergens should be included in clinically diagnostic P. chrysogenum extracts.

Deficient transforming growth factor beta and interleukin-10 responses contribute to the septic death of burned patients.

In order to understand the roles of pro-inflammatory and anti-inflammatory cytokines in burn injury and sepsis post-burn, serial changes in serum levels of transforming growth factor beta-1 (TGF-beta-1) were determined and compared to those of IL-6 and IL-10 in 15 burned patients. Among these 15 patients, 8 recovered without sepsis. The other seven, who were septic, expired. Our results showed that an initial peak serum TGF-beta-1 response was detected within 1 day post-burn. Peak serum IL-6 and IL-10 responses were also detected within 4 days after the burn injury of these patients. Significant differences in peak serum IL-6, IL-10 and TGF-beta-1 levels were not found between patients with total body surface area (TBSA) of greater or less than 50% and between patients who survived or expired from burn injury. Afterwards, levels of circulating IL-6 and IL-10 remained low in the survivors. However, a second peak response in serum TGF-beta-1 levels was observed in all burned patients analyzed. The second peak serum TGF-beta-1 levels post-burn of the eight survivors and the seven non-survivors were from 28,542 to 76,554 pg/ml (a mean value of 51,256+/-14,264 pg/ml) and from 8616 to 40,851 pg/ml (a mean value of 24,079+/-10,399 pg/ml), respectively. A significant difference (P<0.01) in mean values of the second peak TGF-beta-1 responses between groups of survivors and non-survivors was detected. Levels of circulating IL-6 in the septic non-surviving patients showed a tendency to increase 1-2 weeks post-burn and reached high levels before the expiration of these patients. After an initial peak response, the serum IL-10 level remained low in one of the seven non-survivors, while it increased in the other six non-survivors. However, marked increases in circulating IL-10 levels were observed only just before the death of these non-survivors. In conclusion, an initial increase in serum levels of IL-6, IL-10 and TGF-beta-1 was detected post-burn. A marked increase in serum levels of IL-6 before death suggests its role in the pathophysiology of sepsis in burned patients. In addition, a low secondary TGF-beta-1 response and a lack and/or delay in the increase of circulating IL-10 in the non-survivors may all contribute to the pathophysiology of septic death in burned patients.

Identification of vacuolar serine proteinase as a major allergen of Aspergillus fumigatus by immunoblotting and N-terminal amino acid sequence analysis.

Aspergillus species are common airborne fungi that have been identified as causative agents of extrinsic bronchial asthma. More than 10 allergens from A. fumigatus have been recently characterized by cDNA cloning. The objective of this study is to identify A. fumigatus allergens through immunoblot analysis using sera from asthmatic patients. IgE-binding components of A. fumigatus and IgE cross-reactivity among allergens of different prevalent airborne fungal species were analysed by immunoblot and immunoblot inhibition, respectively, using sera from asthmatic patients. The N-terminal amino acid sequences of major allergens identified were determined by Edman degradation. Among two batches (70 and 41 sera) of asthmatic sera tested, 19 (27%) and 14 (34%), respectively, have IgE immunoblot reactivity towards components of A. fumigatus. A 34-kDa protein that reacts with IgE antibodies in 15 (79%) and 11 (79%) of the 19 and 14 positive samples, respectively, may be considered a major allergen of A. fumigatus. The N-terminal amino acid sequences of the 34 kDa major allergen and the 30.5 and 30 kDa IgE-binding components of A. fumigatus showed sequence identity to that of the vacuolar serine proteinase from A. fumigatus. The results from immunoblot inhibition show IgE cross-reactivity among major allergens of A. fumigatus, P. notatum and P. oxalicum. Results obtained suggest that the 34 kDa major allergen of A. fumigatus may be a vacuolar serine proteinase. There is IgE cross-reactivity among serine proteinase allergens of A. fumigatus, P. notatum and P. oxalicum.

cDNA cloning and immunologic characterization of Pen o 18, the vacuolar serine protease major allergen of Penicillium oxalicum.

Penicillium species are prevalent indoor airborne fungi that have been identified as causative agents of human extrinsic bronchial asthma. In the preparation of standardized diagnostic reagents, it is imperative to define the allergens of these ubiquitous fungi. Results from our previous study on P. oxalicum suggest that the 34-kd major immunoglobulin E-reacting component of this prevalent Penicillium species is probably a vacuolar serine protease. The purpose of the present study was to define this major P. oxalicum allergen (Pen o 18) through cDNA cloning and immunologic characterization. The cDNA of Pen o 18 was isolated through a combination of reverse transcriptase-polymerase chain reaction and 5'- and 3'-rapid amplification cDNA ends reactions. The primers used in these reactions were constructed according to the internal amino acid sequences of Pen o 18 and the conserved amino acid sequences of fungal serine proteases. Our results showed that a 1897-bp cDNA with an open reading frame of 503 residues was isolated for the proenzyme of Pen o 18. The encoded protein has a 16-residue signal peptide and a 119-residue prosequence. On maturation, the protein has an N-terminal glutamate that is the 136th residue encoded by the cDNA. Apparently the precursor also undergoes C-terminal processing with the cleavage of about 47 amino acids. The cDNA for Pen c 18 (the vacuolar serine protease allergen from P. citrinum ) was also isolated for comparison. Contrary to a previous report, the C-terminal region of Pen c 18 is similar to that of Pen o 18. Recombinant proteins (rPen o 18 and rPen c 18) with the putative mature N-termini and a his-tag were obtained by expressing the corresponding cDNAs in Escherichia coli. Serum samples from 7 asthmatic patients with immunoglobulin E reactivity to the 34-kd component of P. oxalicum also react to his-tagged recombinant Pen o 18. The presence of immunoglobulin E cross-reactivity between rPen o 18 and rPen c 18 was detected by immunoblot inhibition. Two monoclonal antibodies (PCM39 and FUM20) against fungal serine proteases react with rPen o 18, rPen c 18, and the 35/34-kd components in the corresponding crude fungal extracts. These components also react with immunoglobulin E antibodies in serum samples from asthmatic patients. In conclusion, results obtained confirm that the 34-kd major allergen of P. oxalicum is a vacuolar serine protease. The cDNAs of Pen o 18 and Pen c 18 encode precursor molecules that appear to undergo both N-terminal and C-terminal processing. Constructs beginning with mature N-terminal can be expressed in E. coli to produce recombinant polypeptides that are reactive to monoclonal antibodies or immunoglobulin E antibodies in serum samples from asthmatic patients. Results obtained may provide useful information and materials for preparation of standardized diagnostic reagents in clinical mold allergy.

Production and characterization of monoclonal antibodies to serine proteinase allergens in Penicillium and Aspergillus species.

BACKGROUND: Alkaline and/or vacuolar serine proteinases are major allergens in prevalent airborne Penicillium and Aspergillus species. OBJECTIVE: The object of this study is to generate and characterize monoclonal antibodies against these serine proteinase allergens. METHODS: BALB/c mice were immunized individually with the Penicillium citrinum culture medium or the crude extract and culture medium preparations of Aspergillus fumigatus. Hybridoma cells that secrete monoclonal antibodies against serine proteinase allergens were selected by immunoblotting. Antigens in three different Penicillium (P. citrinum, P. notatum and P. oxalicum) and two different Aspergillus species (A. fumigatus, and A. flavus) recognized by these monoclonal antibodies were analysed by sodium dodecyl sulphate and two-dimensional polyacrylamide gel electrophoresis immunoblotting and N-terminal amino acid sequence analysis. RESULTS: Four (PCM8, PCM10, PCM16 and PCM39) and one (FUM20) monoclonal antibodies against serine proteinase allergens were generated after fusion of NS-1 cells with spleen cells obtained from BALB/c mice immunized with antigens from P. citrinum and A. fumigatus, respectively. Immunoblotting results showed that PCM8 reacted with an alkaline serine proteinase allergen in P. citrinum and P. notatum. PCM10 and PCM39 reacted with the alkaline serine proteinase in two Penicillium (P. citrinum, P. notatum) and two Aspergillus species (A. fumigatus, and A. flavus) tested. PCM16 reacted with the alkaline serine proteinase allergen in P. citrinum, A. fumigatus and A. flavus but not with that in P. notatum. MoAb FUM20 reacted with the alkaline serine proteinase allergen in two Aspergillus species (A. fumigatus and A. flavus) but not with that in two different Penicillium species (P. citrinum, P. notatum) tested. Among these five monoclonal antibodies generated, only PCM39 and FUM20 can react with the vacuolar serine proteinase allergen in P. notatum, P. oxalicum and in A. fumigatus. The 35 kDa P. citrinum component that reacted with FUM20 has an N-terminal amino acid sequence of DSPSVEKNAP. CONCLUSION: Five monoclonal antibodies against different epitopes of the serine proteinase major allergens in prevalent Penicillium and Aspergillus species were generated in the present study. Antibodies obtained may be useful in the characterization and standardization of serine proteinase allergens in crude fungal extracts.

Respiratory fungal allergy.

Fungal allergy including allergic rhinitis, conjunctivitis, bronchial asthma, and allergic bronchopulmonary mycoses results from exposure to spores. In this review we have dealt with the common allergenic fungi and allergens, immunopathogenesis, diagnostic assays, and the possible control of allergy in the future based on epitope-specific immunotherapy and vaccination.

Cross-reactivity of T-cell responses to Dermatophagoides pteronyssinus and D. farinae. Studies with group 1 and 7 allergens.

BACKGROUND: Comparative information on T-cell responses to allergens from different Dermatophagoides species is limited even though differences in the epitypic recognition have been described. OBJECTIVE: To compare the level of T-cell proliferation and cytokine production to allergens from the mite species, D. pteronyssinus and D. farinae. METHODS: Freshly isolated peripheral blood mononuclear cells (PBMC) from house dust mite (HDM)-allergic and HDM-nonallergic donors were stimulated with the group 1 and group 7 allergens of D. pteronyssinus and D. farinae and the level of proliferation as well as IL-5 and IFNgamma production were measured. RESULTS: The proliferative response and the level of IL-5 produced after in vitro challenge with group 1 and group 7 allergens were equivalent for the allergens from both mite species even though D. farinae is not detected in the environment where the study population live. As expected the level of IL-5 production to the individual allergens was higher for the allergic donor group than for the nonallergic donors, however, there was no difference in the level of T-cell proliferation between the different donor groups. CONCLUSION: The proliferative and cytokine response to the group 1 and group 7 allergens for D. pteronyssinus and D. farinae indicates that there is a large degree of T-cell cross-reactivity between the whole purified allergens from each species. This is despite previous reports demonstrating different responses to synthetic peptides representing Der p 1 and Der f 1 in a similar study population.

Changes in circulating levels of an anti-inflammatory cytokine interleukin 10 in burned patients.

In order to understand the role of an anti-inflammatory cytokine interleukin 10 (IL-10) in the pathophysiology of burn injury, IL-10 levels in serial serum samples of 22 burned patients were analyzed. The total body surface areas (TBSA) of the burn injury ranged from 30 to 90%. Among these 22 patients, 14 recovered and the other eight, who were septic, expired. A significant difference in serum IL-10 values on admission (5-20 h postburn) was found (P<0.05) between patients who survived or died from burn injury as analyzed by the Student's t test. In addition, a significant difference in serum IL-10 on admission was also found (P<0.05) between patients with TBSA of greater or less than 50%. An initial peak serum IL-10 response was detected within 2.5 days postburn. Significant differences in the peak serum IL-10 levels were not found between patients with TBSA of greater or less than 50% and patients who survived or expired from burn injury. Afterwards, serum IL-10 remained low in the survivors, while an increase in serum IL-10 could be detected in the non-survivors with proven sepsis. Levels of circulating IL-6 in these non-surviving patients showed a tendency to increase starting from about 1-2 weeks postburn which coincided temporally with the detection of infections. However, marked increases in circulating IL-10 levels were observed just before death in four of the eight non-survivors. The serum samples of these four patients were collected at 31 h (404.8 pg/ml), 2 h (773.9 pg/ml), 5 days (150.7 pg/ml) and 12 h (177.1 pg/ml) before the expiration of these patients, respectively. IL-10 levels of 28.6, 27. 5 and 13.5 pg/ml were detected in sera of three of the remaining four non-survivors that were collected at 2.5 h, 36 h and 30 h before the expiration of these patients, respectively. There was one non-surviving patient who suffered an 80% burn (patient D4 in Table 1 and Fig. 4) and his IL-10 level at 20 days postburn was 13.4 pg/ml. The serum sample of this patient was collected 22 days before death and he was not suffering from sepsis at this stage. In conclusion, an initial increase in serum levels of IL-10 was detected postburn. A marked increase in serum levels of IL-10 was detected in four of the eight septic patients just before their death. It was considered that a lack and/or a delay in the increase of circulating IL-10 may play a significant role in the pathophysiology of sepsis in burned patients.

Complementary DNA cloning and immunologic characterization of a new Penicillium citrinum allergen (Pen c 3).

BACKGROUND: Penicillium citrinum has been identified as the most prevalent airborne Penicillium species in the Taipei area. It is important to understand the allergenic composition of this ubiquitous fungal species. OBJECTIVE: The complementary DNA (cDNA) clone of an allergen from P citrinum was isolated and expressed in Escherichia coli as a fusion protein. mAbs were prepared with the recombinant protein as antigen. The corresponding natural allergen in the fungal extracts was identified with the mAbs. METHODS: A Uni-Zap XR P citrinum cDNA library was screened with sera from asthmatic patients. An IgE-binding cDNA clone was isolated and expressed as a glutathione-S-transferase fusion protein. The frequency of IgE binding to the expressed protein was analyzed by immunoblotting. Spleen cells from BALB/c mice immunized with the recombinant protein were fused with NS-1 cells for mAb generation. RESULTS: A P citrinum cDNA library was screened with a mixture of serum samples from 4 asthmatic patients. An IgE-binding cDNA clone was obtained and designated as PCE2. PCE2 has a 694-bp insert that contains a 167 amino acids open reading frame. The deduced amino acid sequence of the encoded protein has 82.6% (138 amino acids) identity with an Aspergillus fumigatus peroxisomal membrane protein allergen (Asp f 3). PCE2 was expressed in E coli as a fusion protein and designated as Pen c 3. Sera from 13 (46%) of the 28 Penicillium-sensitized asthmatic patients demonstrated IgE binding to Pen c 3. In addition, 11 of the 13 Pen c 3-positive serum samples have IgE immunoblot reactivity to recombinant Asp f 3. The presence of IgE cross-reactivity between Pen c 3 and Asp f 3 was also detected by immunoblot inhibition. Four of the 6 mAbs generated against Pen c 3 cross-react with Asp f 3. The presence of the corresponding 18-k natural allergens in the crude extracts of P citrinum and A fumigatus were detected by immunoblot with use of the mAbs and sera from asthmatic patients. CONCLUSION: Results obtained suggest that the peroxisomal membrane protein (Pen c 3) is an important allergen of P citrinum. PCE2 is a full-length cDNA clone encoding this allergen. In addition, the mAbs generated may be useful in standardizing the diagnostic allergenic extracts.