Occupational allergy caused by cow dander: detection and identification of the allergenic fractions

We describe two cases involving cow farmers, both males, one aged 28 and the other 45, who attended our center because his presented symptoms of rhinoconjunctivitis and asthma with 18 months and 4 year of evolution respectively, related to this laboral environment. The study included the following tests: skin tests (prick tests) to inhalant allergens (mites, pollens, moulds, dog and cat epithelium), foods, cow epithelium-dander, cow serum, beef and milk proteins. We determined the total seric IgE, specific IgE (RAST-CAP System) to cow meat and cow dander. Nasal provocation test with freeze-dried biological standardized extract of cow epithelium-dander were carried out. We observed the symptoms and realized control with a previous active computerized rhinomanometry. Cow dander proteins used for the provocation test were separated by means of SDS-PAGE or isoelectric focusing (IEF). The allergenic component were identified by immunoblotting with the patients' serum. The skin tests were positive to cow dander, and negative to the other allergens tested, including cow serum, cow milk and beef. The seric IgE were 383 and 477 kU/L, and the RAST was positive to cow dander, 20.10 and 36.4 kU/L (class 4). The provocation test were positive with a concentration of 500 SBU. We observed that the IgE of the two patients reacted with the same allergens: 3 major bands were identified with MW of 11, 15, 62.3 kDa. All these bands correspond to protein with acid pl.     

Purification and some properties of low molecular weight crystallins from camel lens (Camelus dromedarius)

1. The use of an Ultrogel AcA 54 gel-filtration column separates camel lens cortex low molecular weight proteins into four peaks containing beta s-, gamma 1-, gamma 2- and gamma 3-crystallins. 2. The molecular weight of beta s-crystallin corresponded to 29 kDa on SDS-PAGE and showed three major bands between pH 5.85 and 8.45 on isoelectric focusing. In addition, as compared to gamma-crystallins it has a lower degree of homology in amino acid composition, a low sulfhydryl content and a blocked N-terminal amino acid. 3. gamma 1-, gamma 2- and gamma 3-crystallins appeared homogenous on SDS-PAGE and their molecular weights were recorded as 23, 22 and 21 kDa. The isoelectric points of the gamma-crystallin fractions ranged from pH 6.55 to 8.60 and they were found to have an unmodified glycine at the N-terminal end. 4. The three camel gamma-crystallin fractions were similar in molecular weight, isoelectric points, amino acid composition, sulfhydryl concentration and N-terminal amino acid.     

Recurrence of blackwater fever: triggering of relapses by different antimalarials

A monoclonal antibody that reacts with a 150-kDa protein of Entamoeba histolytica on Western immunoblotting under nonreducing conditions inhibits the adherence and cytotoxicity of the ameba to mammalian cells in vitro. Affinity purification of solubilized trophozoites using the monoclonal antibody and electrophoresis yielded three glycoproteins with molecular masses of 150, 170, and 260 kDa, suggesting the existence of either a common epitope or the close association of these proteins. The 260-kDa fraction was identified as the well-known galactose (Gal)- and N-acetyl-D-galactosamine (GalNAc)-inhibitable lectin. The 150- and 170-kDa fractions seemed to exist as part of a 380-kDa native protein with an isoelectric point of pH 6.9. The N-terminal amino acid sequence of the 150-kDa protein was unique, indicating that the protein was not a degraded product of the 260-kDa lectin. By gel filtration, the 260-kDa lectin and the 150/170-kDa protein could be separated. When Chinese hamster ovary cells were pretreated with the fraction consisting of the 150/170-kDa protein the adherence of trophozoites to Chinese hamster ovary cells was competitively inhibited to a level equivalent to that observed for the 260-kDa lectin. The inhibitory effect was lost in the presence of Gal and GalNAc but was not influenced by the presence of glucose. These results demonstrate that the 150/170-kDa protein is a Gal/GalNAc-inhibitable lectin. The existence of a sugar-binding domain in the protein was confirmed by Gal-affinity chromatography.     

Proteins encoded by open reading frames 3 and 4 of the genome of Lelystad virus (Arteriviridae) are structural proteins of the virion

Four structural proteins of Lelystad virus (Arteriviridae) were recognized by monoclonal antibodies in a Western immunoblotting experiment with purified virus. In addition to the 18-kDa integral membrane protein M and the 15-kDa nucleocapsid protein N, two new structural proteins with molecular masses of 45 to 50 kDa and 31 to 35 kDa, respectively, were detected. Monoclonal antibodies that recognized proteins of 45 to 50 kDa and 31 to 35 kDa immunoprecipitated similar proteins expressed from open reading frames (ORFs) 3 and 4 in baculovirus recombinants, respectively. Therefore, the 45- to 50-kDa protein is encoded by ORF3 and the 31- to 35-kDa protein is encoded by ORF4. Peptide-N-glycosidase F digestion of purified virus reduced the 45- to 50-kDa and 31- to 35-kDa proteins to core proteins of 29 and 16 kDa, respectively, which indicates N glycosylation of these proteins in the virion. Monoclonal antibodies specific for the 31- to 35-kDa protein neutralized Lelystad virus, which indicates that at least part of this protein is exposed at the virion surface. We propose that the 45- to 50-kDa and 31- to 35-kDa structural proteins of Lelystad virus be named GP3 and GP4, to reflect their glycosylation and the ORFs from which they are expressed. Antibodies specific for GP3 and GP4 were detected by a Western immunoblotting assay in swine serum after an infection with Lelystad virus.     

Purification of collagen-binding proteins of Lactobacillus reuteri NCIB 11951

Collagen type-I-binding proteins of Lactobacillus reuteri NCIB 11951 were purified. The cell surface proteins were affinity purified on collagen Sepharose and eluted with an NaCl gradient. Two protein bands were eluted from the column (29 kDa and 31 kDa), and both bound radio-labeled collagen type I. Rabbit antisera raised against the 29 kDa and 31 kDa protein reacted with the affinity-purified proteins in a Western blot with whole-cell extract used as antigen. The N-terminal sequence of the 29-kDa and 31-kDa proteins demonstrated the closest homologies with internal sequences from an Escherichia coli trigger factor protein (TIG.ECOLI). Out of nine other lactobacilli, the antisera reacted only with the L. reuteri and not with the other species tested.     

Latex allergy: allergen identification in Hevea braziliensis fractions by immunoblotting

BACKGROUND: Latex is the cause of several clinical symptoms of allergy, but the identification of allergens is not completely known. OBJECTIVE: The aim of this report was to study the immunoreactivity of purified stable latex fractions from Hevea braziliensis. METHODS: We purified the cytoplasm of Hevea braziliensis and obtained three fractions: latex particles (LP), lutoids (L) and cytosolic serum (CS). Using Western blot, specific IgE directed to latex allergens was found in 80 patients with latex allergy. RESULTS: Five major groups of allergens migrating as 14, 25, 29, 37-45 and 50 kDa were recognized. They were unequally distributed with the latex fractions: 37-45 kDa proteins were essentially recognized in CS and LP, whereas 14 and 29 kDa proteins were mainly labelled in the L fraction. As a control, aqueous glove extracts exhibited a more restricted pattern of reactivity, because only 14 and 29 kDa proteins were recognized by patient sera. The pattern of reactivity was not correlated specifically with IgE levels, but sera from patients suffering from spina bifida reacted specifically with the minor protein of 25 kDa located in LP. CONCLUSIONS: The present results show that latex allergic patients recognize several allergens which are differently distributed in subcellular fractions extracted from H. braziliensis and aqueous GE. The L fraction and GE were enriched in low molecular weight proteins and apparently contained the same allergens.     

In vitro viability of cryopreserved equine embryos following different freezing protocols

Aspergillus fumigatus grown in submerged and surface cultures was extracted, and the extracts were analyzed separately. The submerged extract contained 31.9% protein and 8.3% carbohydrate, while the corresponding values were 17.0% and 33.3% for the surface material. With individual sera from patients with allergic asthma, SDS-PAGE combined with immunoblotting revealed that the submerged extract contained at least six strong IgE-binding components (20, 30, 38, 50, 68, and 90 kDa) in addition to several weak to medium IgE-binding components. The surface extract contained about the same number of IgE-binding components, but only one gave a strong reaction (20 kDa). The allergens present were shown to have pI between 4.5 and 5.6 as demonstrated by isoelectric focusing (IEF) combined with immunoblotting. For identification of A. fumigatus glycoprotein allergens, both extracts were treated with periodate under mild conditions. Two allergens of the submerged extract (90 and 38 kDa) partly lost their IgE-binding ability by this treatment, indicating that these components are glycoproteins and that the carbohydrate moiety is involved in the IgE binding. The IgE-binding ability of the 20-kDa allergen was not influenced by periodate. For assessment of the stability of the two allergen extracts, aqueous solutions were kept at 4 degrees C for 2, 7, and 21 d and then analyzed by SDS-PAGE and immunoblotting. The results showed that most allergens of the submerged extract were partly inactivated after 2 d. After 21 d, only the 20-kDa and 30-kDa components were still able to bind IgE. Similar results were obtained by analyzing the surface extract.(ABSTRACT TRUNCATED AT 250 WORDS)     

The major dog allergens, Can f 1 and Can f 2, are salivary lipocalin proteins: cloning and immunological characterization of the recombinant forms

Canis familiaris allergen 1 (Can f 1) and Canis familiaris allergen 2 (Can f 2) are the two major allergens present in dog dander extracts. We now report the isolation of cDNAs encoding both proteins and present their nucleotide and deduced amino acid sequences. Can f 1, produced by tongue epithelial tissue, has homology with the von Ebner's gland (VEG) protein, a salivary protein not previously thought to have allergenic properties. Can f 2, produced by tongue and parotid gland, has homology with mouse urinary protein (MUP), a known allergen. Both VEG protein and MUP are members of the lipocalin family of small ligand-binding proteins. Recombinant forms of Can f 1 and Can f 2 were produced and tested for immunoglobulin E (IgE) reactivity. Among dog-allergic subjects, 45% had IgE directed exclusively to rCan f 1, and 25% had IgE to both rCan f 1 and rCan f 2. In addition, both recombinant proteins were able to cross-link IgE and elicit histamine release from peripheral blood leucocytes in vitro. These findings confirm that Can f 1 and Can f 2 are major and minor dog allergens, respectively, and demonstrate that recombinant forms of dog allergens retain at least some IgE-binding epitopes.     

Paget''s bone disease

Fusarium equiseti is one of the most important species in the class Deuteromycetes (Fungi Imperfecti). For proper diagnosis and immunotherapy, isolation and characterization of allergens of F. equiseti are necessary. In the present study, culture filtrate (CF) extract of F. equiseti was resolved into 35-37 bands on isoelectric focusing pI (3-9) and SDS-PAGE (mol. wt. 10-100 kDa). Most of them were glycoproteins, as identified by PAS staining. F. equiseti CF revealed 15 allergenic proteins on immunoblot with an allergic serum pool. It was fractionated into nine fractions (I-IX) on a Superose-12 column by FPLC. Fraction IV (65 kDa) and fraction VI (25 kDa) were found to be highly allergenic by IgE ELISA. A 65-kDa protein was observed as a major allergen because it was recognized by most of the patient sera on immunoblot. After elution from SDS-PAGE gel, it gave two bands of pI 7.4 and 6.0. Inhibition in IgE-binding components of F. equiseti CF with CF extracts of F. solani and F. moniliforme by immunoprint inhibition assay indicated the allergenicity shared between the extracts of Fusarium species. Data suggested that the 65-kDa is the major allergen in the Fusarium species and can be used for the treatment of allergic patients.     

Characterization of the major proteins of bovine seminal fluid by two-dimensional polyacrylamide gel electrophoresis

Recently, we demonstrated that the major proteins from bovine seminal plasma BSP-A1, -A2, -A3 and -30-kDa (collectively called BSP proteins) specifically interact with choline phospholipids. These proteins coat the surface of the spermatozoa after ejaculation and are believed to play an important role in membrane modifications occurring during capacitation. In this study we determined the isoelectric point (pl) and analysed the molecular heterogeneity of BSP proteins. Total protein from bovine seminal plasma (CBSP) and purified BSP proteins were iodinated using chloramine T. Samples were reduced, denatured, separated by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE), and visualized by autoradiography. Analysis of CBSP proteins demonstrated the presence of polypeptides migrating in the pH range of 3.5-7.8 and at molecular weights (M(r)) between 6 and 100 kDa. isoforms of each BSP protein were found when purified iodinated proteins were analysed by 2D-PAGE. BSP-A1 was found at a M(r) of 16.5 kDa and in the range of pl of 4.7-5.0; BSP-A2 at 16 kDa and at a pl of 4.9-5.2; BSP-A3 at 15 kDa and at a pl of 4.8-5.2, and BSP-30-kDa at 28 kDa and at a pl of 3.9-4.6. Similar results were obtained with immunolocalization of BSP proteins after Western blot using specific antibodies. The treatment of purified iodinated BSP proteins with neuraminidase increased the pl of BSP-30-kDa to 4.8-5.0 and decreased its M(r) to 25 kDa, but no change was observed for BSP-A1, -A2 and -A3. The treatment of BSP proteins with sulfatase or acid phosphatase modified neither their M(r) nor their pl. Furthermore, when CBSP proteins were separated in 2D-PAGE and the gels stained for glycoproteins with dansyl hydrazine, BSP proteins were among the major glycoproteins found in the bovine seminal plasma. In conclusion, BSP proteins are acidic and have several isoforms. Furthermore, the heterogeneity of BSP-30-kDa is mainly due to its sialic acid content.     

Identification of three isoforms for the Na+-dependent phosphate cotransporter (NaPi-2) in rat kidney

We have isolated three unique NaPi-2-related protein cDNAs (NaPi-2alpha, NaPi-2beta, and NaPi-2gamma) from a rat kidney library. NaPi-2alpha cDNA encodes 337 amino acids which have high homology to the N-terminal half of NaPi-2 containing 3 transmembrane domains. NaPi-2beta encodes 327 amino acids which are identical to the N-terminal region of NaPi-2 containing 4 transmembrane domains, whereas the 146 amino acids in the C-terminal region are completely different. In contrast, NaPi-2gamma encodes 268 amino acids which are identical to the C-terminal half of NaPi-2. An analysis of phage and cosmid clones indicated that the three related proteins were produced by alternative splicing in the NaPi-2 gene. In a rabbit reticulocyte lysate system, NaPi-2 alpha, beta, and gamma were found to be 36, 36, and 29 kDa amino acid polypeptides, respectively. NaPi-2alpha and NaPi-2gamma were glycosylated and revealed to be 45- and 35-kDa proteins, respectively. In isolated brush-border membrane vesicles, an N-terminal antibody was reacted with 45- and 40-kDa, and a C-terminal antibody was reacted with 37-kDa protein. The sizes of these proteins corresponded to those in glycosylated forms. A functional analysis demonstrated that NaPi-2gamma and -2alpha markedly inhibited NaPi-2 activity in Xenopus oocytes. The results suggest that these short isoforms may function as a dominant negative inhibitor of the full-length transporter.     

Liposomes-coated hydroxyapatite and tricalcium phosphate implanted in the mandibular bony defect of miniature swine

Mutations, expected to affect the intracellular routing, i.e. additional nuclear localization sequences (NLS; the natural 23 kDa isoform and a 17D27R mutant) and/or a deletion of amino acids 26-29 (23 delta 26-29 and 17 delta 26-29), were introduced in basic fibroblast growth factor (bFGF). The mutants were assayed for their mitotic activity and their capacity to induce a tissue-specific response in human umbilical vein endothelial cells [HUVECs; induction of urokinase plasminogen activator receptor (u-PAR)], or in rat lens epithelial cells (fibre cell differentiation). In HUVECs, the 17D27R mutant had wild type activity, the 23 kDa and the delta 26-29 proteins were impaired in the induction of both mitosis and u-PAR. The delta 26-29 proteins, but not the 23 kDa protein or 17D27R mutant, were also impaired in receptor binding in that they bound only to a subset of receptors. The concentration of 17 kDa bFGF required for half maximal u-PAR response was 30 fold higher than for the half maximal 3H-thymidine incorporation. Addition of an NLS to bFGF strongly inhibited the induction of fibre cell differentiation, though it had little effect on the stimulation of DNA synthesis. The 17 delta 26-29 kDa mutant had wild type differentiation activity but was a poor mitogen for lens epithelial cells.     

Building bridges between families and nursing home staff: the Partners in Caregiving Program

Prostaglandins are natural fatty acid derivatives with diverse physiological effects, including immune function and the control of cell growth. While the action of prostaglandins in the induction of stress proteins in vertebrate cells is well documented, their functions in invertebrate cells have been poorly investigated. The purpose of the present study was to investigate the effect of prostaglandin A1 (PGA1; 0.25, 1.25 and 12.5 micrograms/ml) on protein synthesis during the growth of Aedes albopictus cells. We found that PGA1 stimulates the synthesis of several polypeptides with molecular masses of 87, 80, 70, 57, 29, 27 and 23 kDa in Aedes albopictus cells. When the proteins induced by PGA1 and those induced by heat treatment were compared by polyacrylamide gel electrophoresis, PGA1 was found to induce the stress proteins. The HSP70 family and the low-molecular weight polypeptides (29 and 27 kDa, respectively) were induced by PGA1 in the lag phase. We also observed that PGA1 is able to induce a 23-kDa polypeptide independently of the growth phase of the cell.     

Low molecular weight Cooperia oncophora antigens. Potential to discriminate between susceptible and resistant calves after infection

The recognition of low molecular weight proteins by sera obtained during a single oral (primary) infection with 100,000 3rd-stage Cooperia oncophora larvae was studied in calves. Three groups of 6 or 7 calves were selected based on different egg excretion patterns. SDS-gel electrophoresis of adult Cooperia antigen under reducing conditions, followed by Western blotting, revealed that resistance of individual calves to C. oncophora might be related with antibody responses (42 days post infection) against at least 2 protein fragments (14-16 kDa and 27 kDa). The 14-16-kDa protein complex was bound, to some extent, by individual sera from all calves. The intensity of staining was negatively correlated with egg excretion on Day 42 p.i. Calves with high egg counts on Day 21 p.i. either did not or only weakly recognized the 27-kDa band. It has to be established whether the 14-16 kDa (or recombinant 14.2 kDa) provides a tool for immunodiagnostics and whether the 27-kDa fragment can help further unravel immune-mediated resistance to Cooperia.     

Affinity purification of the major bovine allergen by a novel monoclonal antibody

A monoclonal antibody was developed to the 20 kd major allergen of cow by immunizing mice with crude dander extract. The monoclonal antibody did not exhibit cross-reactivity to cat, dog, and horse dander extracts when studied by ELISA inhibition. The antibody was used in affinity chromatography for the purification of the 20 kd allergen from cow dander extract. Purity of the allergen was estimated to be 88%, and allergenic reactivity was verified by IgE immunoblotting and skin prick tests. After further purification with size-exclusion chromatography, the allergen was almost 100% pure. The isoelectric point of the double-purified allergen was determined to be 4.1. The amino acid composition was characterized by the predominance of acidic amino acids.     

Host H-2 haplotype modulates the induction of host-versus-graft disease after the induction of neonatal tolerance to H-2 alloantigens

Infection of Nicotiana benthamiana cells with cymbidium ringspot (CymRSV) and carnation Italian ringspot (CIRV) viruses results in the formation of conspicuous membranous bodies [multivesicular bodies (MVBs)], which develop from modified peroxisomes or mitochondria, respectively. The organelle targeting signal is located in the proteins of 33 kDa (CymRSV) or 36 kDa (CIRV) encoded by ORF 1, which contain an N-terminal hydrophilic portion followed by two predicted hydrophobic transmembrane segments. Biochemical analysis showed that the 33- and 36-kDa proteins are integral membrane proteins. By exchanging small portions of the ORF 1 sequence between the infectious full-length clones of the two viruses, hybrid constructs were obtained of which the in vitro synthesized RNA was inoculated to N. benthamiana plants and protoplasts. The structure of infectious clones suggested that both the N-terminal hydrophilic region and the transmembrane segments of the ORF 1-encoded proteins specify which organelle is involved in the synthesis of MVBs. Mutational analysis of the CIRV 36-kDa protein also suggested the presence of an internal mitochondrial targeting sequence similar to that found in several normal host proteins that are synthesized in the cytoplasm and transported to mitochondria. The CymRSV 33-kDa protein did not contain the obvious consensus signals thought to be characteristic of proteins targeted to peroxisomes, and an mitochondrial targeting sequence motif was not evident.     

Immunization with purified natural and recombinant allergens induces mouse IgG1 antibodies that recognize similar epitopes as human IgE and inhibit the human IgE-allergen interaction and allergen-induced basophil degranulation

Molecular characterization of allergens by recombinant DNA technology has made rapid progress in the recent few years. In the present study we immunized mice with aluminum hydroxide-adsorbed purified recombinant major timothy grass pollen allergens (rPhl p 1, rPhl p 2, rPhl p 5), dog albumin, a major animal dander allergen, and proteins with low (beta-lactoglobulin) or no (ribulose diphosphate carboxylase) allergenic potential in humans. Allergens that bind high levels of IgE in humans (Phl p 1, Phl p 5, dog albumin) induced high IgE and IgG1 levels in mice, whereas proteins with little or no allergenic activity in humans failed to induce significant IgE and IgG1 levels in mice. Continuous immunization for a period of 27 wk resulted in the production of mouse IgG1 Abs that recognized recombinant allergen fragments/epitopes defined by IgE Abs of allergic patients. As a consequence, allergen-specific mouse Abs strongly inhibited human IgE binding to the allergens and suppressed the allergen-induced histamine release from human basophils. In summary, our data indicate that 1) the allergenic potency of a protein may be related to its overall immunogenicity and 2) prolonged immunization with single purified recombinant allergens induces protective IgG Abs. The presented experimental in vivo/in vitro system allows the evaluation of Ag preparations (e.g., recombinant allergens) to be used for immunotherapy in humans.     

Inhibition of small G proteins by clostridium sordellii lethal toxin activates cdc2 and MAP kinase in Xenopus oocytes

The lethal toxin (LT) from Clostridium sordellii is a glucosyltransferase that modifies and inhibits small G proteins of the Ras family, Ras and Rap, as well as Rac proteins. LT induces cdc2 kinase activation and germinal vesicle breakdown (GVBD) when microinjected into full-grown Xenopus oocytes. Toxin B from Clostridium difficile, that glucosylates and inactivates Rac proteins, does not induce cdc2 activation, indicating that proteins of the Ras family, Ras and/or Rap, negatively regulate cdc2 kinase activation in Xenopus oocyte. In oocyte extracts, LT catalyzes the incorporation of [14C]glucose into a group of proteins of 23 kDa and into one protein of 27 kDa. The 23-kDa proteins are recognized by anti-Rap1 and anti-Rap2 antibodies, whereas the 27-kDa protein is recognized by several anti-Ras antibodies and probably corresponds to K-Ras. Microinjection of LT into oocytes together with UDP-[14C]glucose results in a glucosylation pattern similar to the in vitro glucosylation, indicating that the 23- and 27-kDa proteins are in vivo substrates of LT. In vivo time-course analysis reveals that the 27-kDa protein glucosylation is completed within 2 h, well before cdc2 kinase activation, whereas the 23-kDa proteins are partially glucosylated at GVBD. This observation suggests that the 27-kDa Ras protein could be the in vivo target of LT allowing cdc2 kinase activation. Interestingly, inactivation of Ras proteins does not prevent the phosphorylation of c-Raf1 and the activation of MAP kinase that occurs normally around GVBD.