Panax ginseng ameliorates airway inflammation in an ovalbumin-sensitized mouse allergic asthma model.

ETHNOPHARMACOLOGICAL RELEVANCE: Panax ginseng (PG) is a medicinal herb that has been used to treat various immune diseases including asthma and COPD. In this study, we investigated the inhibitory mechanism of PG on asthma parameters in mice. MATERIALS AND METHODS: BALB/c mice were sensitized with 20 µg/200 µl OVA adsorbed on 1.0mg/50 µl aluminum hydroxide gel adjuvant by i.p. injection on days 0 and 14. Mice were then challenged with 5% OVA in PBS to the nose for 30 min once a day for 3 days, from day 20 until day 22, using a nebulizer. PG (20mg/kg) or vehicle was administrated by i.p. injection once a day 10 min before every OVA challenge for 3 days. The recruitment of inflammatory cells into bronchoalveolar lavage fluid or lung tissues was measured. The expression of EMBP, Muc5ac, CD40, and CD40 ligand (CD40L) in lung tissues was investigated. In addition, the cytokines and mitogen activated protein (MAP) kinases were measured by RT-PCR and Western blot. RESULTS AND CONCLUSIONS: PG restored the expression of EMBP, Muc5ac, CD40, and CD40L, as well as the mRNA and protein levels of interleukin (IL)-1ß, IL-4, IL-5, and tumor necrosis factor (TNF)-α. In addition, PG inhibited the numbers of goblet cells and further small G proteins and MAP kinases in bronchoalveolar lavage cells and lung tissues increased in ovalbumin-induced allergic asthma in mice. These results suggest that PG may be used as a therapeutic agent in asthma, based on reductions of various allergic responses.

Chronic pancreatitis in mice by treatment with choline-deficient ethionine-supplemented diet.

Although chronic pancreatitis is a risk factor for pancreatic ductal adenocarcinoma (PDA), the relationship between chronic pancreatitis and PDA remains obscure. A critical obstacle to understanding the role of chronic pancreatitis is the lack of animal models. To develop one such model, mice were fed long-term with a choline deficient ethionine-supplemented (CDE) diet. Histological evaluation revealed that chronic pancreatitis, characterized by acinar atrophy, fibrosis and well-developed tubular complexes (TCs), was observed after 24 weeks of CDE diet treatment. Furthermore, expression of epidermal growth factor receptor (EGFR) and its ligands; serine protease inhibitor Kazal type 3 (Spink3) and transforming growth factor alpha (TGF alpha) and activation of K-Ras (GTP-Ras formation), which are frequently observed in human PDA, were indeed observed in parallel with TCs formation. Neoplastic lesions were not found after 54 weeks of treatment, suggesting that a continuation of CDE diet or another insult is required for the development of PDA.

Relationship of strain-dependent susceptibility to experimentally induced acute pancreatitis with regulation of Prss1 and Spink3 expression.

To analyze susceptibility to acute pancreatitis, five mouse strains including Japanese Fancy Mouse 1 (JF1), C57BL/6J, BALB/c, CBA/J, and C3H/HeJ were treated with either a cholecystokinin analog, cerulein, or a choline-deficient, ethionine-supplemented (CDE) diet. The severity of acute pancreatitis induced by cerulein was highest in C3H/HeJ and CBA/J, moderate in BALB/c, and mildest in C57BL/6J and JF1. Basal protein expression levels of the serine protease inhibitor, Kazal type 3 (Spink3) were higher in JF1 and C57BL/6J mice than those of the other three strains under normal feeding conditions. After treatment with cerulein, expression level of Spink3 increased remarkably in JF1 and mildly in C57BL/6J, BALB/c, CBA/J, and C3H/HeJ strains. Increased proteinase, serine, 1 (Prss1) protein expression accompanied by increased trypsin activity with cerulein treatment was observed in susceptible strains such as CBA/J and C3H/HeJ. Similar results were obtained with a CDE diet. In the 3 kb Spink3 promoter region, 92 or 8 nucleotide changes were found in JF1 or C3H vs C57BL/6J, respectively, whereas in the Prss1 promoter region 39 or 46 nucleotide changes were found in JF1 or C3H vs C57BL/6J, respectively. These results suggest that regulation of Prss1 and Spink3 expression is involved in the susceptibility to experimentally induced pancreatitis. The JF1 strain, which is derived from the Japanese wild mouse, will be useful to examine new mechanisms that may not be found in other laboratory mouse strains.

Identification of novel serum proteins in a Japanese viper: homologs of mammalian PSP94.

Three small serum proteins (SSP-1, -2, and -3), with molecular masses of 6.5-10kDa, were isolated from Habu (Trimeresurus flavoviridis) serum, and the amino acid sequences were determined by protein and cDNA analysis. Despite only limited sequence identity to any mammalian prostatic secretory protein of 94 amino acids (PSP94), all of the Cys residues in these SSPs were well conserved. SSPs are the first PSP94 family proteins to be identified in reptiles. SSP-1 and -3 weakly inhibited the proteolytic activity of a snake venom metalloproteinase. On the other hand, SSP-2 formed a tight complex with triflin, a snake venom-derived Ca(2+) channel blocker that suppresses the smooth muscle contraction. This suggests a role for SSP-2 in the self defense system of venomous snakes.

[Inhibitive effect of soybean isoflavone on prostate hyperplasia in rats].

OBJECTIVE: To explore the inhibitive effect of soybean isoflavone on benign prostatic hyperplasia in rats. METHODS: By subcutaneously injecting testosterone propionate to induce prostate hyperplasia in rats, the changes of prostate wet weight, prostatic index, morphological change, prostate-specific acid phosphatase (PAP), acid phosphatase in the control, the model, low, moderate, high dose of soybean isoflavone groups were observed. RESULTS: The ventral prostate wet weight, prostatic index, and PAP in the low, moderate, and high dose groups were significantly lower than those in the models (P < 0.05). The ventral prostate wet weight, prostatic index, and PAP in the moderate, and high dose groups were significantly lower than those in the low dose group (P < 0.05). CONCLUSION: Soybean isoflavone inhibits prostate hyperplasia and the increase of acid phosphatase and PAP in a dose-dependent manner in rats. Soybean isoflavone may serve as supplementary therapy and prevent benign prostatic hyperplasia.

Identification of four novel potato (Solanum tuberosum) allergens belonging to the family of soybean trypsin inhibitors.

BACKGROUND: We have previously identified patatin (Sol t 1) of potato tubers as a major food allergen among atopic children. In addition to Sol t 1, concomitant IgE binding to other, then unidentified, potato proteins was observed. METHODS: Purification and identification of the putative allergens were done by both standard and advanced methods of protein chemistry. The patient series comprised 39 children with positive skin prick test (SPT) to raw potato. Immunoblotting and ELISA were used to examine IgE-binding ability and skin prick testing to assess in vivo reactivity of the purified potato proteins. RESULTS: Four IgE-binding potato proteins with molecular masses ranging from 16 to 20 kDa were purified and identified as cathepsin D-, cysteine-, and aspartic protease inhibitors belonging to the family of soybean trypsin inhibitors (Kunitz type). The proteins were designated Sol t 2, Sol t 3.0101, Sol t 3.0102, and Sol t 4. In ELISA, 51% of the sera of the 39 atopic children showed specific IgE to Sol t 2, 43% to Sol t 3.0101, 58% to Sol t 3.0102, and 67% to Sol t 4, respectively. All these four allergens were able to produce positive wheal-and-flare responses in SPT. CONCLUSION: In addition to Sol t 1, potato tubers contain several proteins belonging to the family of soybean trypsin inhibitors against which atopic children with positive SPT responses to raw potato have in vitro and in vivo reactive IgE antibodies.

Characterization of cross-reacting allergens in mango fruit.

BACKGROUND: Allergic reactions to mango fruit have become increasingly important. A cross-reaction between mango fruit, various other foods, and respiratory allergens has been assumed but not investigated until now. METHODS: The sera of nine patients were used to characterize cross-reacting allergens in mango fruits by EAST inhibition and immunoblot inhibition. RESULTS AND CONCLUSIONS: EAST inhibition and immunoblot inhibition demonstrated that cross-reactions between mango fruits, mugwort pollen, birch pollen, celery, and carrot are based on allergens related to Bet v 1 and Art v 1, the major allergens of birch and mugwort pollen, respectively.

cDNA, genomic cloning, and gene expression analysis of mouse PSP94 (prostate secretory protein of 94 amino acids).

The potential use of prostate secretory protein of 94 amino acids (PSP94) as a diagnostic biomarker or a therapeutic agent for prostate cancer has been reported. In order to establish an animal model to further elucidate on its biological role, we cloned the mouse PSP94 cDNA (approximately 500 bp) by reverse transcriptase-polymerase chain reaction (RT-PCR) and disclosed its genomic structure. The whole mouse PSP94 gene (approximately 23 kb) was amplified by long and accurate-PCR and also cloned by screening of a mouse embryo stem-cell genomic library. Computational and statistical analyses have demonstrated several highly conserved characteristics of PSP94 among different species. Comparison of PSP94 from human, two primates, pig, and rodents revealed that the most significant feature is that PSP94 is rich in cysteines (10% of the total sequence) and their positions are highly conserved. The three intron-four exon structure of the human PSP94 gene and the consensus sequence (....GT-intron-AG...) for mRNA splicing are also strongly conserved. A high divergence in cDNA sequence in the protein-coding region and also in the genomic sequence of PSP94 was also observed among these species. Comparing with alpha-globin, a typical evolutionally conserved gene, with the PSP94 gene, the rate of nonsynonymous changes per site per year (kN) is 2 to 6 times higher, indicating that PSP94 gene has been under far fewer evolutionary constraints than other genes and has a potential role as a species barrier in reproductive biology. In order to test this hypothesis, we investigated the gene expression of PSP94 and its tissue distribution in various rodent tissues by RT-PCR and in situ hybridization (ISH). Gene expression was found only in the prostate, suggesting that PSP94 is probably more tissue specific in the prostate of rodents than in mammals. The ISH analysis also revealed a prostate lobe-specific expression of the PSP94 gene in both mice and rats. It was strongly expressed in the lateral prostate, but the findings were negative in the dorsal and ventral lobe. Therefore, it is hypothesized that one of the primary functions of rodent PSP94, as a major prostate secretory protein, is related to reproductive biology.

Effects of short-term high-dose testosterone propionate administration on medium molecular-weight proteins of human seminal plasma.

Effects of short-term high-dose testosterone propionate treatment on medium molecular-weight proteins (lactoferrin, albumin, prostatic acid phosphatase, prostate specific antigen) and on zinc and fructose levels were investigated in the seminal plasma of seven normal volunteers. A significant reduction in levels of prostatic-acid phosphatase, zinc and, to a lesser degree, prostate-specific antigen, lactoferrin and fructose was observed on the 14th day of androgen treatment, concomitantly with the maximal increase in free androgen-circulating levels. The data obtained suggest that testosterone administration may induce a reduction in the sex accessory-gland secretion. Indeed, this effect tends to disappear with withdrawal of hormone treatment. Therefore, the authors suggest a close follow-up of prostatic and vesicular function during the long-term high-dose testosterone intake, used frequently as anabolic treatment by athletes and body builders.

cDNA cloning identified a calmodulin-binding protein in bovine seminal plasma as bovine C-type natriuretic peptide.

A basic protein of apparent molecular weight 15 kD, designated bSVSP15, was purified from bovine seminal vesicle secretion to homogeneity, employing affinity absorption to calmodulin-Sepharose and reverse-phase HPLC. Immunoblotting identified bSVSP15 in bovine seminal plasma and seminal vesicle secretion, but it was not present either in extracts of bovine ampulla, epididymis, and testis or in serum or follicular fluid. When added to cAMP phosphodiesterase, bSVSP15 inhibited the activation of enzymatic activity by calmodulin in a reversible manner. Immunoscreening of a lambda gt11 expression cDNA library from bovine seminal vesicle tissue yielded two positive clones, pSVS4 and pSVS5, which were characterized by sequencing. Both sequences are identical, except for the 3' region. Because the derived amino acid sequence comprises, with an identity of 81%, the amino-terminal 21 residues of bSVSP15, cDNA clones pSVS4 and pSVS5 represent bSVSP15-specific mRNAs. The mature protein bSVSP15 contains 101 residues and is preceded by 25 residues of a signal sequence, characteristic for secretory proteins. Northern analysis identified two bSVSP15-specific mRNAs of 900 bp and 1200 bp, respectively. Sequence comparison yielded high homologies to human C-type natriuretic peptide. We conclude from this result that bSVSP15 is identical with the hitherto unknown bovine C-type natriuretic peptide.

Studies on the allergenicity of the amino-terminal epitope (Bet v I 23-38) from birch pollen allergen.

An N-terminal peptide of the major allergen of birch (Bet v I 23-38) was selected for studying the activity of this segment on the basis of optimal hydrophilicity as it was tentatively suggested to be a surface exposed epitope. In addition two control peptides in the region 1-38 were similarly used for comparative assignment of the allergenicity. Peptide analogues from the amino acid terminal region, amino acid residues No. 23-38 of Bet v I, were synthesized by semiautomatic solid-phase peptide synthesis. In vitro and in vivo biological activity studies were performed on these analogous peptides. The IgE-binding capacity of the synthetic peptide 23-38 was examined using the following tests: specific IgE inhibition, skin prick test, nasal provocation and Prausnitz-Küstner inhibition. The results of these investigations suggested that the region 23-38 from the birch and hazel major allergen encompassed a single haptenic epitope.

Identification and partial purification of pollen allergens from Artemisia princeps.

The pollen of Artemisia has been considered as the main late summer-autumn allergen source in this country. To identify its allergenic components, Artemisia princeps pollen extracts were separated by 10% sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE), and transferred to nitrocellulose membrane, where IgE binding components were detected by the reaction with sera of twenty Artemisia-allergic patients and 125I-anti-human IgE, sixteen components in the molecular range of 10,000 and 85,000 daltons were detected. Twelve bands bound to IgE from 50% of the sera tested, and two bands (37,000, 23,000 daltons) showed the highest (85%) frequency of IgE-binding in twenty sera tested. When the gel of SDS-PAGE with Artemisia pollen extracts was sliced into 11 allergenic groups (AG) and the protein of each AG was obtained by the gel elution method, the wormwool-RAST inhibition test showed that the AG 10 demonstrated to be the most potent, and the AG 7 was the next. Six AGs showed significant responses (more than 100% of wheal size to histamine, 1 mg/ml) on the skin prick test in more than 50% of the patients tested. It is suggested that electrophoretic transfer analysis with SDS-PAGE may be a valuable method for Artemisia allergen identification, and the possibility of partial purification of allergens by employing gel elution is discussed.

Identification and localization of allergenic determinants on grass group I antigens using monoclonal antibodies.

Epitopes recognized by five mAb which block the binding of human IgE antibodies to grass group I (GpI) Ag were characterized and partially mapped. Site specificity studies defined four apparently non-overlapping blocking antibody binding sites on the meadow fescue GpI molecule, Fes e I. One of these sites (site A) was localized to a 14,000 m.w. fragment designated P3 generated by CNBr cleavage of purified Fes e I. The P3 peptide possessed human IgE binding sites as well as other epitopes (non-site A) defined by 19 other anti-GpI mAb. All of the P3 reactive antibodies recognized cross-reactive determinants found on GpI Ag isolated from five different grasses suggesting that P3 is a conserved portion of grass GpI molecules. The P3 fragment from Fes e I was used to immunize mice and induced antibodies which reacted with intact GpI Ag from all 5 different grasses currently being studied in this laboratory.

A comparison of the antigenic and allergenic components of birch and alder pollens in Scandinavia and Australia.

Allergens in birch (Betula) pollens from B. pendula grown in Australia and Norway, B. davurica and B. populofolia and from alder (Alnus incana) were identified by electroblotting, following separation by SDS-PAGE, transfer to nitrocellulose membranes and incubation with sera from birch pollen-allergic subjects. Of 42 antigenic components detected by protein staining in the pollen extract from B. pendula grown in Norway, 17 bound IgE. The allergenic components included those already reported in the literature at MWs of 40, 29, 25, 17 and 10-12 kd, as well as previously undescribed components at MWs of 90, 79, 60, 50, 38, 35, 31, 27, 23, 16, 15 and 14 kd. The major IgE-binding components were located in the low MW region 10-17 kd for all species of birch and alder pollen proteins studied. Results reported here provide the first evidence of birch and alder pollen allergies in Australia. Extensive heterogeneity was observed amongst the sera from birch pollen-allergic subjects in both Norway and Australia. Cross-reactivity appears to exist among the proteins present in pollen from the various birch species and from alder.

The androgen-dependent mouse seminal vesicle secretory protein IV: characterization and complementary deoxyribonucleic acid cloning.

Seminal vesicle secretory protein IV of a mouse has been isolated, and the cDNA coding for its mRNA has been cloned and sequenced. The 556-nucleotides encode 16 amino acid signal peptides and 92 residues of mature protein. Considerable homology between mouse and rat SVS IV cDNA was found. In the leader peptide and 3'-noncoding region there is 92% and 85% homology, respectively. The other regional homologies are 86% for the first 12, 68.5% for the last 35, and 40% for the middle 44 amino acids. The expression of mouse SVS IV mRNA is under the control of androgen. Administration of testosterone to castrated mice resulted in induction of the mRNA level to 50% of the mature male in 96 h of hormone treatment. Secretion of the protein after testosterone injection follows a similar pattern.

Presence of IgE suppressor factors in human colostrum.

In spite of intensive investigations, the ability of breast feeding to delay and to attenuate atopic diseases in children remains debatable. This study documents a mechanism whereby breast feeding might interfere with the synthesis of IgE by breast-fed infants. Indeed, we show that colostrum contains IgE-binding factors (IgE-BF) capable of suppressing the in vitro synthesis of human IgE. Colostrum obtained from 15 donors was successively depleted of lipids and casein, filtered through Amicon XM50 membrane (mol. mass cut-off 50 kDa) and lyophilized. IgE-BF was demonstrated in such preparations by two different approaches, i.e. a classical rosette inhibition assay and Western blot analysis. In the first instance, lyophilized preparations of colostrum inhibited the binding of IgE-coated bovine erythrocytes to IgE recovered on the surface of RPMI 8866 lymphoblastoid cells. The rosette-inhibiting activity could be absorbed on IgE- but not on IgG-Sepharose 4B and it could be recovered in the eluate of IgE-Sepharose 4B. The molecular mass of IgE-BF was comprised between 10 to 20 kDa as estimated by gel filtration through a calibrated Sephadex G-75 column. After fractionation on 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transfer to nitrocellulose membrane, colostrum displayed one band of 14 kDa and reacted with radiolabeled IgE but not with IgG nor IgM. This 14-kDa band could be removed by absorbing colostrum with IgE- but not with IgG-Sepharose 4B. Most importantly, the colostrum IgE-BF suppressed the spontaneous in vitro synthesis of IgE by B lymphocytes derived from allergic donors without altering the production of IgM.

Production and characterization of rabbit anti-idiotypic antibodies specific to mouse monoclonal anti-human IgE and reacting with IgE-binding factors and lymphocyte receptors for IgE.

A mouse monoclonal antibody specific to human IgE (mAb 75) was employed to immunize a rabbit to obtain anti-idiotypes (aId) bearing the internal image of human IgE determinants and reacting with IgE-binding factors (IgE-BF) and/or lymphocyte receptors for IgE (Fc epsilon R). mAb 75 was selected on the basis of inhibition assays where the binding of mAb 75 to radiolabeled IgE was blocked by IgE-BF. The latter were produced by a lymphoblastoid cell line (RPMI 8866) expressing Fc epsilon R. Sequential samples of rabbit serum, collected during the immunization period, were extensively absorbed on mouse and human Ig-Sepharose 4B. The IgG fractions of the rabbit serum displayed the following activities: (a) they reacted with 125I-labeled mAb 75 but not with other labeled mouse Ig including mAb-aIgE, (b) this binding was inhibited in a dose-dependent fashion by human IgE but not by other human Ig classes nor by heat-inactivated IgE, (c) they reacted with a polyclonal rabbit anti-human IgE and (d) they blocked the binding of 125I-labeled IgE to mAb 75. It was concluded that the rabbit IgG contained aId (RaId) bearing the internal image of heat labile determinants of human IgE. The rosetting of IgE-coated bovine erythrocytes with Fc epsilon R-bearing cells was inhibited by preincubating the receptor-bearing cells with IgG RaId or its F(ab')2 but not with normal rabbit IgG. The ability of RaId to react with IgE-BF as well as with Fc epsilon R was also shown in inhibition experiments where IgE-BF and solubilized Fc epsilon R blocked the binding of mAb 75 to RaId. Finally, Western blot analysis of human colostrum, known to contain IgE-BF, indicated that radioiodinated RaId and IgE identified the same 12-16-kDa molecules corresponding to IgE-BF. It is concluded that RaId expresses the internal image of a heat-labile determinant of IgE which is involved in the binding of IgE to IgE-BF and Fc epsilon R. An alternative interpretation is that RaId reacts with an idiotypic determinant of mAb 75 which is shared by IgE-BF and Fc epsilon R.

Jacalin: an IgA-binding lectin.

We previously reported that seeds of Artocarpus integrifolia (jackfruit) contain a lectin, which we call jacalin, that is both a potent T cell mitogen and an apparently T cell-independent activator of human B cells for the secretion of immunoglobulins. During the above experiments we noted a massive precipitation in cell cultures stimulated with greater than or equal to 100 micrograms of lectin. In this paper, we show that the precipitate is formed after the interaction of jacalin and the serum protein added to the culture medium. More importantly, we demonstrate that IgA is probably the major serum constituent precipitated by the lectin and that no IgG or IgM can be detected in the precipitates. In secretions such as colostrum, IgA is the only protein precipitated by jacalin. On the basis of this specificity we describe a simple and reliable affinity chromatography procedure for the purification of both human serum and colostrum IgA. Jacalin is a D-Gal binding lectin and should be a useful tool for studying of serum and secretory IgA.