Detection of antibodies specific to an antigenic cell wall galactomannoprotein for serodiagnosis of Aspergillus fumigatus aspergillosis

Aspergilloma and invasive aspergillosis are important opportunistic infections caused by Aspergillus species, among which Aspergillus fumigatus is the most common species associated with human disease. We developed an enzyme-linked immunosorbent assay (ELISA)-based antibody assay with Afmp1p, a purified recombinant antigenic cell wall galactomannoprotein of A. fumigatus. Evaluation of the test with guinea pig sera against A. fumigatus and other pathogenic fungi indicated that this assay was specific for A. fumigatus. Clinical evaluation revealed that the assay was 100% sensitive for patients with aspergilloma and 33.3% sensitive for patients with invasive aspergillosis. No false-positive results were found for serum samples from 80 healthy blood donors, 6 patients with typhoid fever, 4 patients with melioidosis, 20 patients with penicilliosis marneffei, 5 patients with candidiasis, and 4 patients with cryptococcosis, indicating a high specificity of the test. Thus, this ELISA-based test for the detection of anti-Afmp1p antibody can be of significant value as a diagnostic for aspergillosis.     

Detection of cell wall mannoprotein Mp1p in culture supernatants of Penicillium marneffei and in sera of penicilliosis patients

Mannoproteins are important and abundant structural components of fungal cell walls. The MP1 gene encodes a cell wall mannoprotein of the pathogenic fungus Penicillium marneffei. In the present study, we show that Mp1p is secreted into the cell culture supernatant at a level that can be detected by Western blotting. A sensitive enzyme-linked immunosorbent assay (ELISA) developed with antibodies against Mp1p was capable of detecting this protein from the cell culture supernatant of P. marneffei at 10(4) cells/ml. The anti-Mp1p antibody is specific since it fails to react with any protein-form lysates of Candida albicans, Histoplasma capsulatum, or Cryptococcus neoformans by Western blotting. In addition, this Mp1p antigen-based ELISA is also specific for P. marneffei since the cell culture supernatants of the other three fungi gave negative results. Finally, a clinical evaluation of sera from penicilliosis patients indicates that 17 of 26 (65%) patients are Mp1p antigen test positive. Furthermore, a Mp1p antibody test was performed with these serum specimens. The combined antibody and antigen tests for P. marneffei carry a sensitive of 88% (23 of 26), with a positive predictive value of 100% and a negative predictive value of 96%. The specificities of the tests are high since none of the 85 control sera was positive by either test.     

Aspergillus fumigatus and aspergillosis

Aspergillus fumigatus is one of the most ubiquitous of the airborne saprophytic fungi. Humans and animals constantly inhale numerous conidia of this fungus. The conidia are normally eliminated in the immunocompetent host by innate immune mechanisms, and aspergilloma and allergic bronchopulmonary aspergillosis, uncommon clinical syndromes, are the only infections observed in such hosts. Thus, A. fumigatus was considered for years to be a weak pathogen. With increases in the number of immunosuppressed patients, however, there has been a dramatic increase in severe and usually fatal invasive aspergillosis, now the most common mold infection worldwide. In this review, the focus is on the biology of A. fumigatus and the diseases it causes. Included are discussions of (i) genomic and molecular characterization of the organism, (ii) clinical and laboratory methods available for the diagnosis of aspergillosis in immunocompetent and immunocompromised hosts, (iii) identification of host and fungal factors that play a role in the establishment of the fungus in vivo, and (iv) problems associated with antifungal therapy.     

Monoclonal immunoglobulin G1 directed against Aspergillus fumigatus cell wall glycoprotein protects against experimental murine aspergillosis

Most of the biological functions related to pathogenicity and virulence reside in the fungal cell wall, which, being the outermost part of the cell, mediates the host-fungus interplay. For these reasons much effort has focused on the discovery of useful inhibitors of cell wall glucan, chitin, and mannoprotein biosynthesis. In the absence of a wide-spectrum, safe, and potent antifungal agent, a new strategy for antifungal therapy is directed towards the development of monoclonal antibodies (MAbs). In the present study the MAb A9 (immunoglobulin G1 [IgG1]) was identified from hybridomas raised in BALB/c mice immunized with cell wall antigen of Aspergillus fumigatus. The immunoreactive epitopes for this IgG1 MAb appeared to be associated with a peptide moiety, and indirect immunofluorescence microscopy revealed its binding to the cell wall surface of hyphae as well as with swollen conidia. MAb A9 inhibited hyphal development as observed by MTT [3-(4,5-dimethythiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay (25.76%), reduced the duration of spore germination, and exerted an in vitro cidal effect against Aspergillus fumigatus. The in vivo protective efficacy of MAb A9 was also evaluated in a murine model of invasive aspergillosis, where a reduction in CFU (>4 log(10) units) was observed in kidney tissue of BALB/c mice challenged with A. fumigatus (2 x 10(5) CFU/ml) and where enhanced mean survival times (19.5 days) compared to the control (7.1 days) and an irrelevant MAb (6.1 days) were also observed.     

Development of monoclonal antibody-based galactomannoprotein antigen-capture ELISAs to detect Aspergillus fumigatus infection in the invasive aspergillosis rabbit models

Aspergillus fumigatus is one of the most prominent opportunistic fungal pathogens in immunocompromised hosts. Early recognition of this infection along with prompt antifungal therapy may increase the survival rate. We expressed two potential bio-markers of A. fumigatus infection—galactomannoprotein Afmp1p and Afmp4p in Pichia pastoris. We generated 33 monoclonal antibodies (MAbs), 20 against recombinant Afmp1p (rAfmp1p) and the other 13 against recombinant Afmp4p (rAfmp4p). Subsequently, we developed two antigen-capture enzyme-linked immunosorbent assays (ELISAs) which employed MAbs as both the capture and the detection antibodies for rAfmp1p and rAfmp4p. The two antigen-capture ELISAs specifically detected Afmp1p/Afmp4p in cultures of A. fumigatus and had no cross-reaction with other tested pathogenic fungi, including Penicillium marneffei and other pathogenic Aspergillus species. The Afmp1p-captured ELISA would be positive even when the culture supernatant of A. fumigatus had been diluted to 128-fold of its original concentration. The two antigen ELISAs could capture circulating or excreted antigens during the acute phase of invasive aspergillosis (IA) in the animal model, and had no cross-reactivity to other Aspergillus-challenged animal models. We developed two antigen-capture ELISAs for the laboratory diagnosis of A. fumigatus infection. These two antigen-capture ELISAs may be useful in the clinical diagnosis of aspergillosis.     

Catalases of Aspergillus fumigatus and inflammation in aspergillosis.

The article describes various features of aspergillosis and a discussed the role of calatases produced by Aspergillus fumigatus during infection. Since a large body of invasive Aspergillus infection occurs as an opportunistic infection in variously impaired defense mechanisms, there is a wide spectrum of histopathological features of lesions demonstrated at the site of infection. Accordingly, histopathology of the lesions can be understood as a phenotypical representation of interaction between differently impaired functions of neutrophils and macrophages and virulence factors of invading Aspergilli. Consideration of previous pathological knowledge regarding infection and inflammation provides much important information to predict the pathophysiology of a patient. Meanwhile, detoxification of hydrogen peroxide by catalases has been proposed as a way to overcome this host response. A. fumigatus produces three active catalases, one from conidia and two from mycelia. CatAp, a spore specific monofunctional catalase, is resistant to heat and metal ions. In spite of their increased sensitivity to H(2)O(2), killing of catA conidia by alveolar macrophages, virulence in animals was similar to wild type conidia. In contrast to mycelial Cat1p, and CatAp catalases, the mycelial Cat2p is a bifunctional catalase-peroxidase enzyme and is also sensitive to heat, metal ions and detergent. Surprisingly, the mycelium of the double cat1 cat2 mutant with no catalase activity has only a slightly increased sensitivity to H(2)O(2) and was as sensitive to the killing of polymorphonuclear neutrophils as the wild type strain. However, it showed a delayed infection in the rat model of aspergillosis compared to the wild type strain. Consequently, it should be emphasized that conidial catalase is not a virulence factor but that mycelial catalases transiently protect the fungus from the host defence reactions.     

Identification and characterization of an immunodominant 58-kilodalton antigen of Aspergillus fumigatus recognized by sera of patients with invasive aspergillosis.

Sera from 38 patients with invasive aspergillosis were tested by Western immunoblotting for the presence of antibodies to antigens of Aspergillus fumigatus present in a mycelial extract of the organism. All of the sera contained antibodies to an antigen of molecular weight 58,000, which was which was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. It was the only antigen recognized in approximately 90% of the sera tested. The 58-kDa antigen is an abundant component of mycelial extracts composing approximately 50% of the Coomassie blue-stained protein. The antigen also contains carbohydrate, since it is stained by the carbohydrate stain periodic acid-Schiff and it binds to the lectin concanavalin A. It was purified by immunoaffinity chromatography employing a monoclonal antibody directed against an epitope on the 58-kDa antigen. Analysis of the purified antigen by gas-liquid chromatography revealed the presence of mannose, galactose, and glucose residues in a 2:1:2 ratio. The ratio of protein to carbohydrate is 1.16:1. The protein is slightly acidic, containing relatively high quantities of glutamic and aspartic acids, glycine, alanine, serine, and threonine. The 58-kDa antigen also contains phosphate groups as part of its structure. Serological activity was totally destroyed after treatment with sodium metaperiodate and was partially destroyed after treatment with pronase. The 58-kDa antigen was not able to hydrolyze protein.     

Use of immunoblotting to detect Aspergillus fumigatus antigen in sera and urines of rats with experimental invasive aspergillosis.

Immunoblotting was used to detect Aspergillus fumigatus antigen in sera and urines of immunosuppressed rats experimentally infected with A. fumigatus. Organisms were administered by both intravenous and intratracheal injections. Intravenously infected rats developed disseminated aspergillosis, but intratracheally infected rats developed pulmonary disease only. Fungal cultures of blood and urine samples from infected rats were negative. In the urines of intravenously infected rats, antigen was detected 24 to 48 h after infection; in the urines of intratracheally infected animals, antigen was detected on days 4 to 5 after infection. Antigen in serum was detected later than antigen in urine was. Following sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting of serum and urine samples, the most strongly reacting antigenic materials were found in the 88-, 40-, 27-, and 20-kilodalton regions. These dominant antigens appeared to be the same as those of control antigens prepared from A. fumigatus grown in vitro. Rabbit antiserum to Aspergillus filtrate antigen was found to be more immunoreactive than antiserum to mycelial or conidial antigen. No mycelium-specific antigens were detected.     

IgA antibody against Aspergillus fumigatus antigen in patients with allergic bronchopulmonary aspergillosis.

IgA antibodies against Aspergillus fumigatus (Af) antigen were measured by a radioimmunoassay in sera from patients with allergic bronchopulmonary aspergillosis (ABPA) and compared with IgA antibody levels in asthmatics who had positive prick tests to Af and normal controls. The assay for IgA antibody discriminated between both control groups and the ABPA group at two serum dilutions (10(-2) and 10(-3)) but did not discriminate all individual ABPA sera from the peak levels of antibody in the control groups. IgA antibody against Af may participate in the immunopathogenetics of pulmonary inflammation in some cases of ABPA and may be of some diagnostic value in some, but not all, cases.     

Aspergillus Fumigatus antigen detection in sera from patients at risk for invasive aspergillosis

We have developed an inhibition enzyme immunoassay (inhibition-EIA) to monitor for the occurrence of invasive aspergillosis (IA) in sera from 45 immunocompromised (IC) patients. The test uses rabbit polyclonal antibodies and a mixture of components from Aspergillus fumigatus, containing three predominant antigens with molecular weights of 18,000, 33,000, and 56,000. Circulating antigens were found in five of seven proven cases of IA due to A. fumigatus. In two of the five positive cases, antigenemia was detected with inhibition-EIA earlier than with X ray or other biological methods. No antigens were detected in the sera from two patients with proven IA due to Aspergillus flavus and Aspergillus terreus nor in the sera from four patients with probable IA. Circulating antigens were not detected in the control group, composed of 30 healthy adult blood donors. Four of the 32 at-risk patients examined, though they displayed no definite evidence of IA, gave a positive result in this test. The sensitivity, specificity, and positive predictive value of inhibition-EIA were 71.4, 94.4, and 71.2%, respectively. The data were compared with those obtained by a latex agglutination assay of galactomannan (GM) that was positive in only one patient with probable IA. The higher sensitivity obtained by inhibition-EIA may well be due to its ability to detect circulating antigens other than GM in the sera of IC patients with IA. Detecting these antigens may improve the diagnosis of IA, as they may serve as markers of this infection.     

Production and characterization of monoclonal antibodies to cell wall antigens of Aspergillus fumigatus

Two murine monoclonal antibodies (MAbs) against Aspergillus fumigatus were produced and characterized. Splenocytes from cell wall-immunized BALB/c mice were fused with SP2/0 myeloma cells. The hybridomas were screened with a cold alkali (CA) extract of mycelium containing protein, mannose, and galactose, and two MAbs of the immunoglobulin M class were purified from ascites fluid. MAbs 1 and 40 were characterized by double immunodiffusion against CA antigen, indirect enzyme immunoassay with mannans of Candida albicans serotypes A or B or Candida tropicalis, indirect immunofluorescence with C. albicans- or A. fumigatus-infected tissues, indirect immunofluorescence with smears of other pathogenic fungi, Western blotting (immunoblotting) with the lectin concanavalin A or BS-1 from the seeds of Bandeirea simplicifolia, and immunoelectron microscopy. MAb 1 did not cross-react with Candida mannan and recognized a periodate-sensitive, pronase- and heat-resistant epitope in CA antigen and three mannose- and galactose-containing components (80, 62, and 49 kilodaltons) of a mycelial homogenate. Immunoelectron microscopy demonstrated binding of MAb 1 to the inner cell wall and intracellular membranes of hyphae and conidia of A. fumigatus. Circulating antigen was detected in experimental invasive aspergillosis by inhibition enzyme immunoassay with MAb 1 and CA antigen. MAb 40 was a nonprecipitating antibody cross-reactive with Candida species, and competition for an epitope located diffusely in the cell wall of A. fumigatus hyphae was demonstrated by incubating MAb 40 with mannan of C. albicans serotype A. These results suggest that MAb 1 recognizes immunodominant oligogalactoside side chains of A. fumigatus galactomannan, while MAb 40 binds to mannopyranosyl side chains common to A. fumigatus galactomannan and C. albicans mannan.     

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.     

Detection of circulating Aspergillus fumigatus galactomannan: value and limits of the Platelia test for diagnosing invasive aspergillosis

The effectiveness of galactomannan detection with the Platelia test was evaluated in a prospective study of 3,327 sera from 807 patients. The specificity was 99.6% (748 of 751 cases). For the groups of patients with proven and probable invasive aspergillosis, the sensitivity was 50.0% (17 of 34 cases). The disappointing sensitivity associated with the presence of rare false-positive cases underlines the limits of this test.     

Allergic bronchopulmonary aspergillosis and sensitization to Aspergillus fumigatus in chronic bronchiectasis in adults

Fifty adult subjects for whom a diagnosis of idiopathic bronchiectasis (excluding those secondary to tuberculosis or hypogarnmaglobulinaemia) had been confirmed previously were investigated by: questionnaire; blood eosinophil count; sputum culture for Aspergillus fumigatus and eosinophil count; chest radiography; skin-prick tests with several aeroallergens and four preparations of A. fumigatus, including a reference extract; measurement of specific IgE antibodies; precipitin testing and self-crossed immunoelectrophoresis with A. fumigatus. Five subjects were possible cases of allergic bronchopulmonary aspergillosis in whom the condition had been previously misdiagnosed or in whom sensitization to A. fumigatus had occurred after the onset of bronchiectasis. These five subjects had positive immediate skin reactions to A. fumigatus and a history of recurrent pneumonias. Four had a previous history of asthma and the others showed increased bronchial responsiveness to inhaled methacholine. At the time of the survey, A. fumigatus grew in the sputum of one out of five subjects. These subjects had increased levels of specific IgE. Two had precipitins by double diffusion and three subjects were positive on self-crossed immunoelectrophoresis. It is concluded that allergic bronchopulmonary aspergillosis or evidence of sensitization to A. fumigatus can be identified in a significant proportion of adult subjects with so-called idiopathic bronchiectasis.     

The uptake of Aspergillus fumigatus protein by serum IgG antibody from patients with pulmonary aspergillosis

A method is presented for measuring the uptake of Aspergillus fumigatus protein by IgG antibodies from human serum. The human IgG is first isolated with an anti-IgG conjugated immunosorbent and then incubated with radio-iodinated A. fumigatus protein. A group of twenty-three control sera gave the same background levels as tests without sera. The highest uptake of A. fumigatus protein was given by the sera of patients with aspergilloma, and lower values were obtained with six out of thirteen sera from patients with asthma and twenty-five out of twenty-eight sera from patients with asthma and pulmonary eosinophilia, i.e. allergic bronchopulmonary aspergillosis. These results were in agreement with precipitin tests. Inhibition studies with unrelated antigens showed that the reactions were specific for the A. fumigatus protein, of which more was bound by test sera than with a crude extract.     

Differential IgE recognition of recombinant Aspergillus fumigatus allergens by cystic fibrosis patients with allergic bronchopulmonary aspergillosis or Aspergillus allergy

Allergic bronchopulmonary aspergillosis (ABPA), an intense inflammatory reaction to Aspergillus in the lung, is recognized as a severe complication in patients with cystic fibrosis (CF). The diagnosis of ABPA in CF patients sensitized to Aspergillus fumigatus is complicated by interfering laboratory and clinical findings shared by the diseases. We have used cDNA encoding A. fumigatus allergens which were cloned from a cDNA library displayed on phage surface to produce recombinant proteins in Escherichia coli. Differential IgE responses to the allergens in A. fumigatus-sensitized CF patients with or without ABPA and CF controls without sensitization to A. fumigatus were demonstrated. A secreted ribotoxin (rAsp f 1) and a peroxisomal protein (rAsp f 3) were recognized by sera from A. fumigatus-sensitized CF-patients with or without ABPA. An intracellular manganese superoxide dismutase (rAsp f 6) and rAsp f 4, a protein with unknown function, were recognized exclusively by IgE from sera of CF patients with ABPA. Therefore, Asp f 4 and Asp f 6 represent specific markers for ABPA and allow a sensitive, fully specific diagnosis of the disease. The data suggest distinct IgE responses to colonization of the bronchial tree in CF patients with ABPA or A. fumigatus allergy and therefore a differential recognition of the pathogen in the two IgE-related inflammatory diseases.     

Ultrastructural demonstration of specific IgG and IgE antibodies binding to Aspergillus fumigatus from patients with aspergillosis.

Aspergillus-induced diseases usually demonstrate elevated circulating antibodies belonging to different isotypes. The antigens currently used to detect antibodies are crude culture filtrate and mycelial extracts of A. fumigatus (Af). Most Af-associated diseases result from the inhalation of the spores of the organisms present in the environment. However, it is not known whether specific circulating antibodies directed only against spore or mycelia of Af exist in the sera of patients with Af-induced diseases. With colloidal gold we have investigated thin sections of spores and hyphae of Af for their reactivity with Af-specific IgG and IgE antibodies. The results indicate that both spores and hyphae reacted identically with IgG and IgE antibodies from patients. None of the sera from normal control subjects reacted in this system, although low levels of antibodies were detected in the sera by ELISA. Sera from both patients with allergic bronchopulmonary aspergillosis or aspergilloma reacted with cell envelope antigens, whereas sera from patients with invasive aspergillosis also bound to cell sap. This method therefore demonstrates localization of antigens binding to different isotypes in the sera from different clinical forms of aspergillosis and may be useful in purifying specific antigens for immunodiagnosis.     

In-vivo itraconazole resistance of Aspergillus fumigatus in systemic murine aspergillosis. EBGA Network. European research group on Biotypes and Genotypes of Aspergillus fumigatus

An animal model of disseminated aspergillosis was used to test the in-vivo activity of itraconazole against four isolates of Aspergillus fumigatus. Two reference isolates of A. fumigatus known to be resistant to itraconazole in vitro and in vivo were used as control isolates, and two new isolates were tested under the same conditions. For each isolate MICs for itraconazole and amphotericin B were determined by an NCCLS-based method. Mice infected intravenously were treated either with itraconazole 100 mg/ kg/day or amphotericin B 4.5 mg/kg/day for 10 days. Amphotericin B showed good in-vivo activity against all four isolates. For one strain, which had a low in-vitro MIC for itraconazole, in-vivo therapy with itraconazole prolonged the survival of mice and reduced fungal burdens in organs compared with untreated controls. In mice infected with a strain with a high MIC of >16 mg/L, itraconazole neither prolonged survival nor reduced fungal load in organs compared with controls. It is concluded that there is a relationship between MIC and treatment outcome in mice for A. fumigatus infection.     

Antibody response to low-molecular-weight antigens of Aspergillus fumigatus in allergic bronchopulmonary aspergillosis.

Sera from patients with allergic bronchopulmonary aspergillosis (ABPA) or aspergilloma and normal sera were analyzed for specific antibodies by Western (immuno-) blotting with Aspergillus fumigatus antigens transferred electrophoretically onto polyvinylidene difluoride membranes. Western blot analysis demonstrated consistent reactivity of low-molecular-weight A. fumigatus antigens against ABPA sera but not against uncomplicated aspergilloma or normal sera. None of these low-molecular-weight components had any lectin-binding activity. Sera from patients with aspergilloma, however, frequently reacted with high-molecular-weight components of A. fumigatus. The majority of these high-molecular-weight antigenic components demonstrated concanavalin A-binding activity. The low-molecular-weight bands were discernible in Western blots with sera from all ABPA patients irrespective of disease activities, such as relapse, flare, or treatment. Antibodies detected by methods such as immunodiffusion or enzyme-linked immunosorbent assays demonstrated total antibody responses to most or all antigenic components, while Western blots demonstrated the reactivities of the individual components with the specific antibodies. Western blot analysis thus provided more information for immunodiagnosis of ABPA than other methods, especially when only crude antigens were available.