Role of Combined Sentinel Lymph Node Biopsy and Axillary Node Sampling in Clinically Node-Negative Breast Cancer.

Axillary lymph node status is a prognostic marker in breast cancer management, and axillary surgery plays an important role in staging and local control. This study aims to assess whether a combination of sentinel lymph node biopsy (SLNB) using patent blue dye and axillary node sampling (ANS) offers equivalent identification rate to dual tracer technique. Furthermore, we aim to investigate whether there are any potential benefits to this combined technique. Retrospective study of 230 clinically node-negative patients undergoing breast-conserving surgery for single T1-T3 tumours between 2006 and 2011. Axillae were staged using a combined blue dye SLNB/ANS technique. SLNs were localized in 226/230 (identification rate 98.3 %). Three of one hundred ninety-two patients with a negative SLN were found to have positive ANS nodes and 1/4 failed SLNB patients had positive ANS nodes. Thirty-four of two hundred twenty-six patients had SLN metastases and 11/34 (32.4 %) also had a positive non-sentinel lymph node on ANS. Twenty-one of twenty-four (87.5 %) node-positive T1 tumours had single node involvement. Nine of thirty-eight node-positive patients progressed to completion axillary clearance (cALND), and the rest were treated with axillary radiotherapy. Axillary recurrence was nil at median 5 year follow-up. Complementing SLNB with axillary node sampling (ANS) decreases the unavoidable false-negative rate associated with SLNB. Appropriate operator experience and technique can result in an SLN localization rate of 98 %, rivalling a dual tracer technique. The additional insight offered by ANS into the status of non-sentinel nodes has potential applications in an era of less frequent cALND.

Role of the Internet as an information resource for surgical patients: a survey of 877 patients.

BACKGROUND: This study was conducted to provide preliminary data regarding current Internet use practices for information about anaesthesia in patients undergoing elective surgical procedures at a major academic institution. METHODS: With IRB approval, 2936 patients coming for preanaesthetic evaluation at a tertiary academic hospital's preadmission testing (PAT) centre were invited to voluntarily participate in a 20-item questionnaire designed to obtain participants' characteristics and Internet use for information pertaining to their upcoming surgery. Data were analysed using statistical software SAS (Cary, NC, USA). Descriptive statistics were calculated for continuous variables using mean (sd), and for categorical data using n (%). Association analysis was performed using the Fisher's exact test. RESULTS: Eight hundred and seventy-seven patients (30%) responded. Of these, 356 (41%) looked for information about their medical condition, 321 (37%) for their surgery, 279 (32%) for surgeon, 163 (19%) for the hospital, and only 36 (4%) for information regarding anaesthesia. Of these 36 patients, 14 (39%) said the sites they used helped answer their questions regarding anaesthesia. Of the 831 patients who did not use the Internet for anaesthesia, 503 (57%) indicated that they would be receptive to being directed to specific websites for anaesthesia. CONCLUSIONS: Of the patients coming for elective surgery who responded (30%), the majority did not use the Internet to seek information regarding anaesthesia. Respondents indicated a high degree of interest in being directed to appropriate websites for further information. These results suggest that it may be beneficial to include information regarding reliable web-based resources to interested patients at preoperative visits.

The costimulatory molecules CD80, CD86 and OX40L are up-regulated in Aspergillus fumigatus sensitized mice.

Aspergillus fumigatus (Af) is a fungus associated with allergic bronchopulmonary aspergillosis (ABPA) and other allergic diseases. Immune responses in these diseases are due to T and B cell responses. T cell activation requires both Af-specific engagement of the T-cell-receptor as well as interaction of antigen independent costimulatory molecules including CD28-CD80/CD86 and OX40-OX40L interactions. Since these molecules and their interactions have been suggested to have a potential involvement in the pathogenesis of ABPA, we have investigated their role in a model of experimental allergic aspergillosis. BALB/c mice were primed and sensitized with Af allergens, with or without exogenous IL-4. Results showed up-regulation of both CD86 and CD80 molecules on lung B cells from Af-sensitized mice (79% CD86+ and 24% CD80+) and Af/rIL-4-treated mice (90% CD86+ and 24% CD80+) compared to normal controls (36% and 17%, respectively). Lung macrophages in Af-sensitized mice treated or not with IL-4 showed enhanced expression of these molecules. OX40L expression was also up-regulated on lung B cells and macrophages from both Af-sensitized and Af/rIL-4 exposed mice as compared to normal controls. All Af-sensitized animals showed peripheral blood eosinophilia, enhanced total serum IgE and allergen-specific IgG1 antibodies and characteristic lung inflammation. The up-regulation of CD80, CD86 and OX40L molecules on lung B cells and macrophages from Af-allergen exposed mice suggests a major role for these molecules in the amplification and persistence of immunological and inflammatory responses in ABPA.

Aspergillus antigens: which are important?

Aspergillus fumigatus is a ubiquitous fungus that causes a variety of diseases in man and animals. A number of protein, carbohydrate, and glycoprotein antigens have been identified from A. fumigatus. The diseases are diverse, and therefore are the antigens and their roles in causing or modulating the diseases. The induction and binding of antibodies and the interaction of antigen and various immune cells are of immense significance in the diagnosis and prognosis of the disease. In recent years, over 20 genes encoding A. fumigatus antigens have been cloned and the proteins expressed. Among these allergens, Asp f 1, f 2, f 3, f 4, and f 6 showed strong but diverse IgE binding with sera from different groups of patients. Results currently available suggest that Asp f 2, f 3, and f 6 together reacted with IgE from more patients with asthma and allergic bronchopulmonary aspergillosis (ABPA), although they are only marginally effective in demonstrating specific IgE in patients with cystic fibrosis and ABPA. The molecular structure of allergens also plays a major role in the immunological response in the allergic patients. Antigens can be engineered with less or more binding with IgE, and such antigens may have significant roles as specific reagents or as immunomodulators.

Day-case breast cancer axillary surgery.

INTRODUCTION: The standard locoregional management of breast cancer is excision of the primary tumour and axillary staging with suction drainage of the axilla. The objective of this study was to determine the safety, tolerability and efficacy of day-case surgery without suction drainage. PATIENTS AND METHODS: A review of complete, prospectively collected data was performed on all breast cancer patients (screening and symptomatic) planned to undergo day-case axillary surgery at a University Teaching Hospital between 2000 and 2002. Postoperative complications were recorded and the notes of patients not discharged on the day of their surgery were also examined to establish the reason for overnight stay. RESULTS: 165 patients underwent intended day-case axillary surgery (axillary dissection level 1/2; median age, 55 years; range, 39-76 years). Of these, 16 (9.7%) were admitted overnight usually due to over-running of theatre lists (n = 13; 81%). 29 patients (17.6%) underwent axillary dissection alone, the remainder had axillary surgery combined with wide local excision (median number of lymph nodes excised 11; range, 2-18). Complications included symptomatic seroma formation in 37 patients (22%) and wound infection in 16 patients (10%). CONCLUSIONS: Day-case axillary surgery can be performed safely with surgical morbidity comparing favourably to published work of 'traditional' axillary drainage following lymphadenectomy.

Generation of cytotoxic T cell responses directed to human leucocyte antigen Class I restricted epitopes from the Aspergillus f16 allergen.

Invasive aspergillosis (IA) is a major cause of infection-related mortality in patients with haematological malignancies, especially in recipients of haematopoietic stem cell transplants. We have prepared overlapping pentadecapeptides (11-aa overlap with previous peptide) spanning the entire 427-aa coding region of the Aspergillus allergen, Asp f16 shown previously in mice to induce Th1-type cell responses in vivo and in humans to induce proliferative and cytotoxic CD4(+) T cell responses. Mature dendritic cells (DC) pulsed with a complete pool of peptides were used to generate T cell lines. Two lines from HLA-B*3501(+) donors were found to be strongly cytotoxic to autologous Asp f16-peptide pool- and Aspergillus culture extract-pulsed targets after 4-5 weekly primings. Cytotoxic T lymphocyte (CTL) culture supernatant killed Aspergillus conidia, and cells directly killed Aspergillus hyphae. Cytotoxic activity and interferon (IFN)-gamma production were mediated exclusively by CD8(+) T cells in response to pool-pulsed targets. Interleukin (IL)-4 production was not detected. CTL activity was restricted by HLA-B*3501 and based on peptide prediction programmes was most probably directed to YFKYTAAAL (YFK), LPLCSAQTW (LPL) and GTRFPQTPM (GTR) in one donor, while only LPL was recognized by CTL from the second donor. Pool-pulsed B*3503(+) BLCL but not B*3502(+) or B*3508(+) BLCL presented peptide to donor no. 1. B*3503(+) BLCL presented YFK and to a lesser extent GTR, but not peptide LPL. Our data show that in addition to our previously identified Class II restricted peptide response, DC pulsed with a pentadecapeptide pool from Asp f16 are capable of inducing polyclonal, HLA-Class I-restricted, Aspergillus-specific T cells that may be capable of conferring immunity to IA.

Generation of Th1 T cell responses directed to a HLA Class II restricted epitope from the Aspergillus f16 allergen.

The Aspergillus allergen Asp f16 has been shown to confer protective Th1 T cell-mediated immunity against infection with Aspergillus conidia in murine models. Here, we use overlapping (11-aa overlap with preceding peptide) pentadecapeptides spanning the entire 427-aa coding region of Asp f16 presented on autologous dendritic cells (DC) to evaluate the ability of this antigen to induce Th1 responses in humans. Proliferative responses were induced in five out of five donors, and one line with a high frequency of interferon (IFN)-gamma-producing CD4(+) T cells in response to the complete peptide pool was characterized. This line was cytotoxic to autologous pool-pulsed and Aspergillus culture extract-pulsed targets. Limitation of cytotoxicity to the CD4(+) T cell subset was demonstrated by co-expression of the degranulation marker CD107a in response to peptide pool-pulsed targets. Cytotoxic T lymphocytes (CTL) killed Aspergillus hyphae and CTL culture supernatant killed Aspergillus conidia. By screening 21 smaller pools and individual peptides shared by positive pools we identified a single candidate sequence of TWSIDGAVVRT that elicited responses equal to the complete pool. The defined epitope was presented by human leucocyte antigen (HLA)-DRB1-0301. These data identify the first known Aspergillus-specific T cell epitope and support the use of Asp f16 in clinical immunotherapy protocols to prime protective immune responses to prevent or treat Aspergillus infection in immunocompromised patients.

Generation of Aspergillus- and CMV- specific T-cell responses using autologous fast DC.

BACKGROUND: Recent reports have described a new strategy for differentiation and maturation of monocyte (Mo)-derived DCs within only 48 h of in vitro culture (fast-DC). Here we assess the efficacy of the fast-DC to process and present different Aspergillus fumigatus and CMV Ag preparations to autologous T cells, compared with DCs generated in standard 7-day cultures (standard-DC). METHODS: Adherent blood Mo were treated with GM-CSF and IL-4 (1 day for fast-DC, 5 days in the standard-DC) to generate immature DCs, and then were matured for either 1-2 days (fast-DC) or 2 days (standard-DC) with inflammatory cytokines. DCs were pulsed with A. fumigatus or CMV Ag preparation immediately prior to maturation, or infected after maturation with adeno-pp65. Mature DCs were then used to prime Ag-specific proliferative and cytotoxic T lymphocytes (CTL) responses. RESULTS: Fast-DC were CD14- and expressed mature DC surface markers to the same degree as standard-DC, and maintained this phenotype after withdrawing cytokine from the cultures. Fast-DC and standard-DC were equally capable of inducing A. fumigatus and CMV-specific T-cell proliferation, as well as priming Ag-specific CTL activity. The Aspergillus- and CMV-specific CTL were of mixed CD3+/CD4+ and CD3+/CD8+ phenotype, and specifically killed autologous DC pulsed with A. fumigatus Ag and autologous CMV infected fibroblasts, respectively. DISCUSSION: Fast-DC are as effective as standard-DC in the generation of Ag-specific T-cell responses. Moreover, use of fast-DC not only reduces labor and supply cost, as well as workload and time, but also increases the number of DCs derived from adherent Mo, which may facilitate the use of DCs in clinical trials of cellular immunotherapy.

IgE antibody to Aspergillus fumigatus recombinant allergens in cystic fibrosis patients with allergic bronchopulmonary aspergillosis.

BACKGROUND: Allergic bronchopulmonary aspergillosis (ABPA) in cystic fibrosis (CF) is characterized by a heightened Th2 CD4+ T-cell response to Aspergillus fumigatus (Af) allergens and a hyper-immunoglobulin E (IgE) state compared with cystic fibrosis patients without ABPA. The IgE serologic differentiation of ABPA from atopic CF patients can be difficult. We propose as the reactivity with purified antigens varies qualitatively and quantitatively and that the antibody response is more specific than with crude Af antigen extract, the IgE responses to purified recombinant Af allergens may differentiate ABPA from atopic CF patients. METHODS: Serum IgE reactivity to seven recombinant purified allergens and to a crude extract of Af was measured in 15 ABPA, in 23 Af skin test positive (ST+), and in 19 Af skin test negative (ST-) CF patients. Four of the ABPA CF patients were studied before and after developing ABPA. Nine ABPA patients were studied during flares and remissions of ABPA. RESULTS: Allergic bronchopulmonary aspergillosis patients had significantly increased IgE reactivity to Asp f2, f3, f4, f6, and f16 compared with the Af ST+ and ST- non-ABPA CF patients. In the ABPA patients studied before and after developing ABPA, IgE reactivity also increased to Asp f2, f3, f4, and f6, and to the crude extract. In ABPA CF patients, IgE reactivity to Asp f1, f2, f3, and f6 significantly increased during periods of ABPA flares compared with periods of remission. Analysis of the receiver operating curve demonstrated that IgE reactivity to Asp f3 and f4 gave the best sensitivity and specificity and were better than IgE reactivity to a crude extract of Aspergillus. Furthermore, in ABPA patients studied during periods of remission the IgE reactivity to Asp f3 and f4 remained significantly elevated compared with Af ST+ non-ABPA patients. The IgE responses when considered either to be positive or negative to Asp f3 and f4 significantly differentiated ABPA from Af ST+ and ST- non-ABPA CF patients. In contrast, IgE reactivity was considered positive to the crude extract in 89% of ABPA, 61% of Af ST+, and 0% of Af ST- non-ABPA CF patients. CONCLUSIONS: Immunoglobulin E reactivity to a panel of purified Af allergens, especially to Asp f3 and f4, differentiates ABPA from atopic Af ST+ non-ABPA CF patients. Serial determinations of IgE reactivity to individual purified Aspergillus antigens, especially Asp f3, demonstrates that increases in IgE reactivity may provide improved distinction between stages of flares and remission compared with changes in IgE reactivity to a crude Aspergillus extract.

Increased sensitivity to IL-4 in cystic fibrosis patients with allergic bronchopulmonary aspergillosis.

BACKGROUND: Allergic bronchopulmonary aspergillosis (ABPA) is characterized by a heightened Th2 CD4+ T-cell response to Aspergillus fumigatus allergens and a hyper-immunoglobulin (Ig)E state compared with cystic fibrosis patients without ABPA. We hypothesize that one reason for this response is increased sensitivity to interleukin (IL)-4 in ABPA resulting in increased expression of CD23 and CD86 and leading to a positive amplification mechanism that increases Th2 CD4+ T cell responses. METHODS: Peripheral blood mononuclear cells (PBMC) were isolated from seven ABPA CF and 19 non-ABPA CF patients and 16 nonatopic controls and stimulated with rIL-4 (range 0.1-10 ng/ml) and rIL-13 (range 1-10 ng/ml) for 48 h. The number of CD23 molecules and percentages of CD23+ B cells were quantified by flow cytometry. Both phorbol 12-myristate 13-acetate (PMA)/ionomycin (IO) and antigen stimulated, toxoid and Asp f2/f3/f4, PBMC were examined for cytoplasmic cytokine synthesis enumerated by cytokine staining using flow cytometry to measure Th2 and Th1 CD3+ T cells. RESULTS: The numbers of CD23 molecules on B-cells were significantly elevated at time 0 in ABPA CF patients compared with both non-ABPA CF patients and nonatopic controls. Following IL-4 stimulation in vitro, the numbers and percentages of CD23 expression on B cells were significantly up-regulated in ABPA CF patients compared with non-ABPA CF patients and controls. The IL-13 stimulation up-regulated CD23 expression; however, there was no significant difference in ABPA CF patients compared with non-ABPA CF patients and controls. The percentages of interferon (IFN)-gamma+ CD3+T cells following PMA/IO stimulation were significantly decreased in both ABPA and non-ABPA CF patients compared with controls. There were no significant differences of IL-4+ and IL-13+ CD3+ T cells between ABPA and non-ABPA CF patients. When tetanus toxoid stimulated T cells were examined, both ABPA and non-ABPA CF patients had significantly decreased IFN-gamma+ CD3+ T cells compared with controls. In Asp f2/f3/f4 stimulated T cells, ABPA CF patients had significantly increased IL-4+ CD3+ T cells compared with non-ABPA CF patients and controls. CONCLUSIONS: ABPA CF patients have increased sensitivity to IL-4 but not to IL-13 up-regulation of CD23 molecules compared with non-ABPA CF patients. There were decreased percentages of IFN-gamma+ and IL-2+ Th1 T cells in CF patients compared with nonatopic controls but similar percentages of IL-4+ Th2 T cells in all three groups. However, ABPA CF patients had increased frequency of Aspergillus-stimulated Th2 T cells. This indicated that there is skewing of Th2 T cells in ABPA CF patients. Thus, in CF ABPA patients there is increased Th2 T cells and increased sensitivity to IL-4.

Immunotherapy of allergic bronchopulmonary aspergillosis: a clinical and experimental approach.

Allergic bronchopulmonary aspergillosis (ABPA) is a severe allergic pulmonary complication caused by the saprophytic fungus Aspergillus fumigatus. The present review examines the pathogenesis of this disease describing in detail the role of innate and acquired immunity in the induction of sensitivity to A.fumigatus. Different approaches in developing specific immunotherapeutic treatments such as induction of anergy, regulatory cells, a switch from Th2 to Th1 type of immune response, CpG and genetic immunization and the usage of altered peptides or modified allergens are critically examined.

Immune response modulation by recombinant peptides expressed in virus-like particles.

Aspergillus fumigatus, a ubiquitous fungus, is implicated in the pathogenesis of a number of clinically different allergic diseases in man, including allergic bronchopulmonary aspergillosis. Peptide-based immunotherapy may offer an alternative treatment strategy for the management of allergic disease. The objective of this study was to alter the allergen-specific immune response using dominant T cell epitopes of a major A. fumigatus allergen, Asp f2, expressed in yeast as virus-like particles (VLP). The T cell epitopes of Asp f2, recognized in mice with an H-2d background, were determined by producing T-cell hybridomas. Two dominant T cell epitopes, aa60--71 and aa235--249, were identified and expressed in a yeast VLP system. To induce tolerance VLP-peptides were injected subcutaneously into mice previously immunized with recombinant Asp f2. The T cell immune response was abrogated totally in 3 weeks following a single injection of VLP but was restored 2 months later following intranasal antigen exposure. T-cell depletion resulted in the reduction of 20-30% of all antigen-specific immunoglobulin classes. Thus, recombinant peptides expressed in the VLP system can be used successfully in the modulation of Asp f2-induced immune response in mice, although a single administration is not sufficient to maintain a state of tolerance for a long period of time.

Do T helpers 1 and 2 recognize different class II MHC molecules? Humoral and cellular immune responses to soluble allergen from Aspergillus fumigatus Asp f2.

Cellular signals leading to T helper (Th)1/Th2 shift are not well known. Here we demonstrate that Th1 possibly recognizes peptides presented by the IE molecule of MHC class II while Th2 is activated by the recognition of peptides presented by the IA molecule. BALB/c mice immunized with Asp f2 developed stable IA-restricted Th2 immune response to the 12th day after immunization, as analyzed by IL-2 production. On the contrary, early Th0 cells did not secrete IL-2 upon Asp f2 stimulation but did produce a high level of IL-2 if stimulated in the presence of anti-IA Abs. This effect of anti-IA Abs on early Th0 cells was both MHC IE and CD4(+) cell restricted. In vivo blocking of Asp f2 peptide presentation by the IA molecule led to the formation of antigen-specific cytotoxicity as demonstrated using immune splenocytes as effector cells and Asp f2 loaded P815 cells as targets.

T cell proliferation and cytokine secretion to T cell epitopes of Asp f 2 in ABPA patients.

The pathogenesis of allergic bronchopulmonary aspergillosis (ABPA) involves specific cytokines secreted by lymphocytes in response to Aspergillus fumigatus (Af) allergens. To gain information about the lymphoproliferative response and cytokine production against a major Af allergen, Asp f 2, we studied Asp-f-2-specific T cell clones (TCCs) from ABPA patients. TCCs were stimulated with rAsp f 2, its deletion mutants, and synthetic peptides to identify the T cell epitope(s) and to understand cytokine production. PBMCs from four of five ABPA patients showed proliferation in response to Asp f 2. Three TCCs from one patient showed higher IL-5 secretion compared to IL-4 and IFN-gamma. Two TCCs from the second patient showed a mixed Th1/Th2 response, as evidenced by production of IL-4, IL-5, and IFN-gamma. An epitope from the N-terminal region of Asp f 2 induced only IL-5 secretion. High IL-5 secretion might explain the marked eosinophilia observed in ABPA patients.

Il-13 and IFN-gamma: interactions in lung inflammation.

Chronic inflammatory diseases of the lungs, such as asthma, are frequently associated with mixed (Th2 and Th1) T cell responses. We examined the impact of critical Th1 and Th2 cytokines, IFN-gamma and IL-13, on the responses in the lungs. In a mouse model of airway inflammation induced by mixed T cell responses, the number of Th1 (IFN-gamma-positive) cells was found to be negatively correlated with airway hyperreactivity. In these mice, blockade of IL-13 partially inhibited airway hyperreactivity and goblet cell hyperplasia but not inflammation. In contrast, in mice that responded with a polarized Th2 response to the same Ag, blockade of IL-13 inhibited airway hyperreactivity, goblet cell hyperplasia, and airway inflammation. These results indicated that the presence of IFN-gamma would modulate the effects of IL-13 in the lungs. To test this hypothesis, wild-type mice were given recombinant cytokines intranasally. IFN-gamma inhibited IL-13-induced goblet cell hyperplasia and airway eosinophilia. At the same time, IFN-gamma and IL-13 potentiated each other's effects. In the airways of mice given IL-13 and IFN-gamma, levels of IL-6 were increased as well as numbers of NK cells and of CD11c-positive cells expressing MHC class II and high levels of CD86. In conclusion, IFN-gamma has double-sided effects (inhibiting some, potentiating others) on IL-13-induced changes in the lungs. This may be the reason for the ambiguous role of Th1 responses on Th2 response-induced lung injury.

Identification of a Hevea brasiliensis latex manganese superoxide dismutase (Hev b 10) as a cross-reactive allergen.

BACKGROUND: Cross-reactive allergens play an increasingly important role in latex allergy in complicating both the diagnosis and time course of allergic symptoms. Manganese superoxide dismutase (MnSOD), a ubiquitous protein of prokaryotic and eukaryotic organisms, was described as a cross-reactive allergen in Aspergillus fumigatus. Little information is available on the importance of this pan-allergen in Hevea brasiliensis latex. The aim of this study was to clone and express MnSOD from H. brasiliensis latex, and to obtain the soluble and immunologically active recombinant allergen for diagnosis of latex allergy and to investigate possible cross-reactivities with the structurally related A. fumigatus and human MnSODs. METHODS: A complementary DNA coding for Hevea latex MnSOD was amplified by PCR. The recombinant protein was produced in Escherichia coli with an N-terminal hexahistidyl tag. Enzymatic activity of the recombinant protein was determined using an enzyme assay for SODs. IgE immunoblotting and IgE inhibition assays were performed to characterize the recombinant allergen and its cross-reactivity. RESULTS: A Hevea latex MnSOD consisting of 206 amino acid residues was cloned and expressed in E. coli. The allergen was designated Hev b 10. The recombinant protein was enzymatically active, indicating the correct folding of the protein. In immunoblots, latex- as well as A. fumigatus-allergic patients revealed IgE binding to recombinant (r)Hev b 10. Cross-reactivity to Asp f 6, the MnSOD from A. fumigatus, and human MnSOD was determined by inhibition of IgE binding to these MnSODs by rHev b 10. CONCLUSIONS: Hev b 10 is a new cross-reactive allergen of H. brasiliensis which belongs to the 'latex-mold' group of latex allergens. Furthermore, it is a candidate for primary sensitization in patients allergic to the pan-allergen MnSOD.

Cloning and expression of Aspergillus fumigatus allergen Asp f 16 mediating both humoral and cell-mediated immunity in allergic bronchopulmonary aspergillosis (ABPA).

BACKGROUND: Aspergillus fumigatus, a ubiquitous fungus, is responsible for a number of lung disorders in atopic and non-atopic individuals. Standardized, pure, and relevant allergens are desirable for reliable immunodiagnosis of the disease and to understand the structural and functional properties of these allergens and the role they play in causing ABPA. OBJECTIVE: Molecular cloning and characterization of a relevant allergen from A. fumigatus cDNA library. MATERIALS AND METHODS: A cDNA library was constructed from 96 h old mycelium of A. fumigatus using lambda ZAP expression vector. A novel gene encoding an A. fumigatus allergen was identified by screening the library with sera from ABPA patients. The gene was cloned and the allergen over-expressed in Escherichia coli. This recombinant allergen, Asp f 16, was evaluated in ELISA and Western blots using sera from patients and normal subjects and peripheral blood mononuclear cells (PBMC) for antigen-induced stimulation. RESULTS: Seventy percent of the patients with ABPA demonstrated high levels of serum IgE antibodies to Asp f 16, a 43-kDa protein, whereas patients with allergic asthma, Aspergillus skin test-positive asthmatics without clinical evidence of ABPA, and normal controls failed to show Asp f 16-specific IgE binding by ELISA. The deduced amino acid sequences of Asp f 16 showed extensive sequence homology to 30.6-kDa Asp f 9 at the N-terminal region of the protein. PBMC from the majority of patients with ABPA exhibited significant proliferation with the recombinant Asp f 16 allergen. CONCLUSION: Specific humoral and cell-mediated immune responses of Af-sensitized patients against Asp f 16 suggest its usefulness in the immunodiagnosis of hypersensitivity diseases due to Af and understanding the pathophysiology of ABPA.

Modulation of inhaled antigen-induced IgE tolerance by ongoing Th2 responses in the lung.

The normal response to inhaled Ag is the absence of Ag-specific IgE and cytokine production to later Ag challenges. Although the mechanism of this aerosol-induced IgE tolerance is not completely understood, it may prevent sensitization to inhaled Ags, which could otherwise lead to allergy and asthma. We examined the consequences of ongoing Th1 and Th2 responses in the lungs of mice during OVA inhalation to mimic conditions that may subvert tolerance and lead to sensitization. We found that concurrent, secondary Th2 lung responses to keyhole limpet hemocyanin or primary responses to Nippostrongylus larvae or Asperigillus fumagatus extract prevented establishment of IgE tolerance to aerosolized OVA. Intranasal rIL-4 given before OVA aerosolization also prevented establishment of tolerance, whereas concurrent Th1 responses to influenza virus or Mycobacterium bovis bacillus Calmette-Guérin had no effect. However, once established, aerosol tolerance to OVA could not be completely broken by OVA rechallenge concurrent with a secondary Th2 response to keyhole limpet hemocyanin or A. fumagatus extract, or by intranasal rIL-4. These data suggest that the immune status of the lung of an individual may profoundly influence the initial response to inhaled Ag, and that aerosol-induced IgE tolerance may not be appropriately established in individuals undergoing concurrent, Th2-mediated responses to Ags or pathogens.

Purified recombinant A. fumigatus allergens induce different responses in mice.

Aspergillus fumigatus an opportunistic fungus is associated with a number of diseases in humans. Allergy resulting from exposure to the A. fumigatus allergens has been recognized frequently. The damage caused by the disease is very striking in patients with atopy and those with cystic fibrosis. Avoidance to exposure is not feasible because A. fumigatus spores are ubiquitously distributed in the environment. Hence, immunotherapeutic regimens in severe forms of A. fumigatus allergy may have a high potential. However, before such forms of therapy can be envisaged, it is essential to understand the immunopathogenesis. In the present study, we investigated the role of purified A. fumigatus allergens in the development of allergic asthma in mice. We have used four major recombinant A. fumigatus allergens in the murine model. Mice exposed to Asp f 1, f 3, and f 4 showed inflammatory changes in the lungs and airway hyperreactivity. The immune responses, including elevated serum IgE, enhanced eosinophils, recruitment in the peripheral blood and lungs, and expression of regulatory cytokines, are characteristic of a Th2 response. Asp f 6 demonstrated only a reduced response in these animals. The results suggest that the pathology induced by crude A. fumigatus extract results from the cumulative effects of the allergens and the individual responses varied considerably with different purified antigens.