Mechanisms of fibrinolysis in chronic liver injury (with special emphasis on MMPs and TIMPs).

Regeneration from liver fibrosis is characterized by four essential events: 1. eradication of pathological agents, 2. apoptosis of activated hepatic stellate cells, 3. remodeling of extracellular matrix, and 4. regeneration of parenchyma and liver function. The temporal and spatial regulation of matrix metalloproteinase (MMP) activity and expression of their specific inhibitors, the tissue inhibitors of metalloproteinases (TIMPs), play a pivotal role in matrix remodeling during hepatic fibrogenesis and recovery. According to current knowledge, the main topics and mechanisms in regeneration from hepatic fibrosis with special emphasis on MMPs and TIMPs are presented. MMP and TIMP expression patterns during hepatic fibrogenesis and fibrolysis, specific characteristics like regulation, expression of cell types, gene expression (RNA/protein), and the underlying disease are summarized. Studies presenting a time course for MMP and TIMP expression during recovery from hepatic fibrosis were taken into consideration to point out a synchronizing behavior in the expression pattern.

Selected recombinant Aspergillus fumigatus allergens bind specifically to IgE in ABPA.

BACKGROUND: Allergic bronchopulmonary aspergillosis (ABPA) is a hypersensitivity lung disease resulting from exposure to Aspergillus fumigatus allergens. Patients with ABPA show elevated Aspergillus-specific serum IgE, a major criterion used in the diagnosis of the disease. Crude culture filtrate and mycelial antigens have been used widely to demonstrate IgE antibody to Aspergillus in the sera of patients. While these antigens have been useful in the diagnosis of ABPA, occasionally they present inconsistency in their reactivity and lack of specificity. Although in recent years, a number of purified A. fumigatus allergens have been produced by molecular cloning, no attempt was made to evaluate them systematically. OBJECTIVE: To evaluate the recombinant proteins from A. fumigatus for their IgE antibody binding, we studied sera from ABPA patients and controls by antigen specific enzyme linked immunosorbent assay (ELISA). METHODS: Recombinant Aspergillus allergens Asp f 1, f 2, f 3, f 4, and f 6 were studied for their specific binding to IgE in the sera of ABPA patients, A. fumigatus skin prick test positive asthmatics, and normal controls from the USA and Switzerland. The sera were blinded and studied by ELISA in two different laboratories. RESULTS: All the recombinant allergens showed IgE antibody binding with sera from patients with ABPA, whereas only fewer asthmatics and normal sera showed significant binding. The three selected recombinant allergens together reacted with all the ABPA patients studied. CONCLUSIONS: The results demonstrate that Asp f 2, f 4, and f 6 can be used in the serodiagnosis of ABPA, while IgE antibody binding to Asp f 1 and f 3 was not specific.

Skin test reactivity to 2 recombinant Aspergillus fumigatus allergens in A fumigatus-sensitized asthmatic subjects allows diagnostic separation of allergic bronchopulmonary aspergillosis from fungal sensitization.

BACKGROUND: Aspergillus fumigatus, an opportunistic pathogen, is associated with an impressive list of pulmonary complications. Among these, allergic bronchopulmonary aspergillosis (ABPA) represents a complex clinical syndrome that is difficult to diagnose. A clear distinction between allergic sensitization to A fumigatus and ABPA is essential for therapy to prevent deterioration of pulmonary function in subjects with ABPA. OBJECTIVE: This study was carried out to determine the specificity and sensitivity of 2 A fumigatus allergens for the in vivo diagnosis of ABPA. METHODS: Serologic investigations with recombinant A fumigatus allergens indicated the existence of disease-specific allergens that are useful for discrimination between ABPA and fungal sensitization. However, serologic studies fail to indicate the allergen-specific IgE levels required to elicit an allergic reaction in vivo. RESULTS: We show that the recombinant A fumigatus allergens rAsp f 4, a protein with unknown biologic function, and rAsp f 6 (manganese superoxide dismutase) are able to provoke immediate skin reactions exclusively in patients with ABPA. The reactions, which are elicited by a few nonograms of the allergens, strictly depend on the presence of allergen-specific serum IgE. The IgE cut-off values for positive skin reactions to rAsp f 4 and rAsp f 6 of 0.9 and 1.2 kU(A)/L correspond to allergen-specific serum concentrations of 2 to 3 microg/L and allow a sensitive, highly specific diagnosis of ABPA. CONCLUSIONS: In contrast to fungal extracts, rAsp f 4 and rAsp f 6 allow discrimination between ABPA and sensitization to A fumigatus. Moreover, the allergens are suitable for an automated serologic diagnosis of ABPA, facilitating their introduction in clinical practice.

Disease-specific recombinant allergens for the diagnosis of allergic bronchopulmonary aspergillosis.

Allergic bronchopulmonary aspergillosis (ABPA), a severe pulmonary complication caused by Aspergillus fumigatus, is considered a complex clinical syndrome with defined serological, pathological radiological and clinical features. The diagnosis of ABPA is often difficult because of several overlapping clinical and laboratory findings shared between asthma with sensitization to A. fumigatus and ABPA, but essential for treatment to prevent severe deterioration of pulmonary function. We have cloned A. fumigatus allergens from a cDNA library displayed on phage surface, sequenced the inserts and produced recombinant proteins in Escherichia coli. The single recombinant allergens were used to assess the immunological response in representative groups of A. fumigatus-sensitized asthmatics with or without ABPA and healthy controls. The allergens rAsp f 1, a 16.9 kDa ribotoxin, rAsp f 3, a 18.5 kDa peroxisomal protein, and rAsp f 6, a 23 kDa manganese superoxide dismutase, were identified as proteins with known biological function and rAsp f 4, a 30 kDa allergen, lacks sequence homology to known proteins. The secreted ribotoxin rAsp f 1 and rAsp f 3 are recognized by serum IgE of A. fumigatus-sensitized asthmatics with or without ABPA, whereas the non-secreted manganese superoxide dismutase rAsp f 6 and the rAsp f 4 allergen are exclusively recognized by serum IgE of ABPA patients. The dissection of IgE-mediated immune responses to single recombinant A. fumigatus allergens in asthmatic patients allow a discrimination between ABPA and A. fumigatus sensitization with high specificity (100%) and sensitivity (90%).

[Molecular aspects and diagnostic value of fungal allergens]. / Molekulare Aspekte und diagnostische Wertigkeiten von Pilzallergenen.

Cloning, sequencing and production of highly pure recombinant allergens allows to produce perfectly standardised allergen preparations. The development of a new cloning system based on filamentous phage allowed the fast isolation and characterisation of allergens from the fungus Aspergillus fumigatus. The produced recombinant allergens were tested in serological and clinical studies as well as for their performance for routine assessments in the ImmunoCAP-system. Thereby, a perfect correlation between skin test results and serology was found showing the potential of recombinant allergens for the diagnosis of allergic diseases. Moreover, the characterisation of fungal allergens substantially contributes to our understanding of the molecular nature of proteins involved in the elicitation of allergic reactions. Apart from allergenic proteins with unknown biological function, fungal allergens can be subdivided into two classes: 1. Species-specific, secreted proteins and 2. cytoplasmic, even in phylogenetically distant organisms, well conserved proteins. These fungal allergens show extended sequence similarity, a high level of IgE cross-reactivity and in some cases also cross-reactivity with homologous human proteins indicating autoimmune reactions involved in fungal allergy.

Skin-test reactivity and isotype-specific immune responses to recombinant Asp f 3, a major allergen of Aspergillus fumigatus.

BACKGROUND: The in vitro diagnostic value of recombinant allergens depends on the correlation between allergen specific IgE in serum and ability of the protein to elicit immediate type allergic reactions in sensitized individuals. Objective The study was carried out to evaluate the reliability of recombinant Asp f 3 (rAsp f 3)-based serological determinations in ELISA and ImmunoCAP compared to skin-test responses in A. fumigatus sensitized subjects. METHODS: Patients suffering from allergic bronchopulmonary aspergillosis (ABPA, n=11) or allergic asthma with A. fumigatus sensitization (n =8) and healthy control subjects (n=4) were investigated. Intradermal skin tests (IDT) and allergen-specific serology were performed using rAsp f 3 protein. RESULTS: All 11 patients with ABPA and five out of eight A. fimigatus-sensitized asthmatics without ABPA exhibited a type I skin reaction. All rAsp f 3 skin test positive subjects showed relevant rAsp f 3-specific serum IgE levels compared with IDT-negative individuals who scored below the rAsp f 3-ImmunoCAP cut-off values of < 0.35 kU(A)/L. Analyses of rAsp f 3-specific immunoglobulins revealed significantly higher serum levels of IgG, IgG1, IgG4 and IgE antibodies in patients with ABPA compared with A. fumigatus-sensitized asthmatics and healthy controls. CONCLUSION: rAsp f 3 represents a major allergen of A. fumigatus. It is recognized by 84% of asthmatic individuals sensitized to the fungus and elicits specific type I skin reactions. The skin test outcome strictly depends on the presence of rAsp f 3-specific IgE and the lack of false positive or false negative results comparing skin test outcome with specific serum IgE levels determined by ELISA or rAsp f 3-ImmunoCAP suggests the possibility to rely on serological data obtained with recombinant allergens to diagnose sensitization to A. fumigatus.

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.

Allergens of Aspergillus fumigatus and Candida boidinii share IgE-binding epitopes.

From an Aspergillus fumigatus complementary deoxyribonucleic acid (cDNA) library displayed on phage surface, an allergen formally termed rAsp f 3 was cloned. The open-reading frame of the cloned gene for the allergen encodes a protein of 168 amino acids with a predicted molecular mass of 18.5 kD, showing 36% identity and 58% similarity to two peroxisomal membrane proteins of Candida boidinii. Recombinant Asp f 3 was expressed as a [His]6-tagged fusion protein in Escherichia coil at yields of 30 mg/L, and was purified by Ni(2+)-chelate chromatography. In an enzyme-linked immunosorbent assay (ELISA), serum IgE antibody reactivity to rAsp f 3 could be detected in 72% of 89 individuals sensitized to A. fumigatus, demonstrating that the protein represents a major allergen of the mold. IgE specific to rAsp f 3 and the two recombinant Candida proteins was further demonstrated by IgE-immunoblot analysis. IgE binding to rAsp f 3 could be inhibited in the ELISA by adding either of the recombinant Candida peroxisomal proteins to sera containing IgE directed against Asp f 3. Taken together, these observations prove that the Asperigillus allergen and the two Candida proteins share IgE-binding epitopes.

Cloning, production, characterization and IgE cross-reactivity of different manganese superoxide dismutases in individuals sensitized to Aspergillus fumigatus.

BACKGROUND: Manganese superoxide dismutase (MnSOD) from Aspergillus fumigatus has been demonstrated to be an allergen showing a high degree of homology with phylogenetically distant MnSODs. We describe cloning, production and characterization of MnSODs from different species. METHODS: MnSODs were cloned by PCR, expressed as inclusion body protein in Escherichia coli, purified by Ni2+-chelate affinity chromatography and characterized. RESULTS: The MnSODs from A. fumigatus, man, yeast, Drosophila melanogaster and E. coli show about 50% identity and 70% similarity on the primary structure. All proteins were produced at a high level in E. coli and refolded to achieve enzymatic activity. The proteins were able to bind IgE from sera of individuals sensitized to the A. fumigatus MnSOD. CONCLUSIONS: MnSOD represents a novel pan-allergen restricted to individuals sensitized to A. fumigatus.

Humoral and cell-mediated autoimmunity in allergy to Aspergillus fumigatus.

A cDNA encoding an allergenic protein was isolated from an Aspergillus fumigatus (A. fumigatus) cDNA library displayed on the surface of filamentous phage. Serum immunoglobulin E (IgE) from A. fumigatus-sensitized individuals was used to enrich phage-expressing gene products binding to IgE. One of the cDNAs encoded a 26.7-kD protein that was identified as a manganese superoxide dismutase (MnSOD) sharing 51.5% identity and 67.2% homology to the corresponding human enzyme. Both human and A. fumigatus MnSOD coding sequences were expressed in Escherichia coli as [His]6-tagged fusion proteins and purified by Ni(2+)-chelate affinity chromatography. The two recombinant MnSODs were both recognized by IgE antibodies from subjects allergic to the A. fumigatus MnSOD and elicited specific immediate type allergic skin reactions in these individuals. Moreover, both human and A. fumigatus MnSOD induced proliferation in peripheral blood mononuclear cells of A. fumigatus-allergic subjects who showed specific IgE responses and reacted in skin tests to MnSOD. These observations provide evidence for autoreactivity to the human MnSOD in allergic persons sensitized to an environmental allergen from A. fumigatus who share a high degree of sequence homology to the corresponding human enzyme.

IL-8 is expressed by human peripheral blood eosinophils. Evidence for increased secretion in asthma.

Eosinophils possess the capacity to synthesize various cytokines. We demonstrate that IL-8 mRNA and protein are constitutively expressed by freshly isolated resting human eosinophils. Most of the patients with bronchial asthma or atopic dermatitis show evidence for up-regulated IL-8 protein expression in eosinophils but not in neutrophils, suggesting that an eosinophil-specific cytokine may act in these patients. To investigate whether the intracellular IL-8 can be released, eosinophils were stimulated by different cytokines and platelet-activating factor. Priming with granulocyte-macrophage CSF and a subsequent 25-min stimulation with RANTES or platelet-activating factor resulted in release of IL-8 from highly purified human eosinophils in vitro. As the eosinophil is the predominant cell in asthmatic inflammation, we determined IL-8 concentrations in bronchoalveolar lavage fluids from normal individuals and asthmatic patients. Bronchoalveolar lavage fluids from patients with bronchial asthma consistently demonstrated high IL-8 concentration compared with the controls. This suggests that IL-8 is released in vivo by inflammatory bronchial cells in asthma.