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L D Vailes

Publications and source records attributed to L D Vailes.

32 records · Page 2Linked to original sources
PubMed1995

Cloning of cockroach allergen, Bla g 4, identifies ligand binding proteins (or calycins) as a cause of IgE antibody responses.

An allergen cloned from a Blattella germanica (German cockroach) cDNA library, encoded a 182-amino acid protein of 20,904 Da. This protein, designated B. germanica allergen 4 (Bla g 4), was expressed as a glutathione S-transferase fusion protein in Escherichia coli and purified by affinity chromatography and high-performance liquid chromatography. The prevalence of serum IgE antibody to recombinant Bla g 4 in 73 cockroach allergic patients with asthma ranged from 40% (antigen binding radioimmunoassay) to 60% (plaque immunoassay). Cockroach allergic patients gave positive intradermal skin tests to recombinant Bla g 4 at concentrations of 10(-3)-10(-5) micrograms/ml, whereas non-allergic controls, or cockroach allergic patients with no detectable serum IgE antibody to Bla g 4, gave negative skin tests to 1 microgram/ml. Polymerase chain reaction and Southern analysis identified a 523-base pair DNA encoding Bla g 4 in both B. germanica and Periplaneta americana (American cockroach). However, Northern analysis showed that mRNA encoding Bla g 4 was transcribed in B. germanica but not in P. americana, suggesting that allergen expression was species specific. Sequence similarity searches showed that Bla g 4 was a ligand binding protein or calycin and unexpectedly revealed that this family contained several important allergens: beta-lactoglobulin, from cow milk, and rat and mouse urinary proteins. Although the overall sequence homology between these proteins was low (approximately 20%), macromolecular modeling techniques were used to generate two models of the tertiary structure of Bla g 4, based on comparisons with the x-ray crystal coordinates of bilin binding protein and rodent urinary proteins. The results show that members of the calycin protein family can cause IgE antibody responses by inhalation or ingestion and are associated with asthma and food hypersensitivity.

Allergens↗
PubMed1995

Molecular cloning of a major cockroach (Blattella germanica) allergen, Bla g 2. Sequence homology to the aspartic proteases.

Inhalation of allergens produced by the German cockroach (Blattella germanica) elicits IgE antibody formation and the development of asthma in genetically predisposed individuals. We compared the allergenic importance of two cockroach (CR) allergens, Bla g 1 and Bla g 2, and determined the complete amino acid sequence of the major 36-kDa allergen, Bla g2. A survey of 106 sera from CR allergic patients showed the prevalence of IgE antibodies to Bla g 1 and Bla g 2 to be 30.2% and 57.6%, respectively. Immediate skin tests on 7 selected patients gave positive reactions using 10(-3) micrograms/ml either allergen, whereas controls showed no response to 10 micrograms/ml. Natural Bla g 2 was purified and the sequence of the NH2 terminus and tryptic peptides, comprising 36% of the molecule, was determined. The cDNA for Bla g 2 was cloned from a B. germanica expression library and encoded a 24-amino acid signal peptide and a 328-amino acid mature protein, which showed the highest degree of identity to mosquito (Aedes aegypti) lysosomal aspartic protease (30.8%), with similar identity to pepsin, cathepsins D and E, renin, and chymosin. Bla g 2 mRNA and protein were detected in B. germanica, but not in Periplaneta americana, the other principal domiciliary CR species in the U.S. High concentrations of Bla g 2 were found in CR digestive organs (esophagus, gut, and proventriculus). The results show that Bla g 2 is a major species-specific allergen of B. germanica and suggest that the allergen functions as a digestive enzyme in the cockroach.

Allergens↗
PubMed1994

Monoclonal antibodies to group II Dermatophagoides spp. allergens: murine immune response, epitope analysis, and development of a two-site ELISA.

BACKGROUND: Group II allergens are a major cause of sensitization in patients allergic to mites. To facilitate the antigenic analysis of group II allergens and to develop improved methods of allergen detection, we compared IgG anti-group II antibody responses in inbred mouse strains and raised a panel of monoclonal antibodies (mAbs). METHODS: IgE antibody responses were compared by antigen-binding radioimmunoassay. Epitope specificity of the mAbs was analyzed by two-site binding assays and by cross-inhibition radioimmunoassays. RESULTS: Comparison of polyclonal IgG antibody responses in five BALB congenic strains showed that H-2d mice had poor responses, whereas H-2b and H-2k mice had strong, cross-reactive, IgG anti-group II responses. The specificities of nine anti-Der p II IgE mAbs raised in A/J mice were compared with specificities of seven mAbs produced previously. Most mAbs (11 of 16) recognized common epitopes on Der p II and Der f II: three were specific to Der p II, and two showed high binding to Der f II. Epitope analysis showed that the mAbs defined four cross-reactive, nonoverlapping sites on the group II allergens. Binding of several combinations of mAbs was compared, and a two-site ELISA for group II antigens was developed. Linear regression analysis showed an excellent correlation between results of this assay and group II radioimmunoassay of house dust samples (n = 40, r = 0.85, p < 0.001). CONCLUSIONS: There are multiple cross-reactive B-cell epitopes on group II allergens. The group II ELISA has several important applications, including assessment of environmental allergen exposure, monitoring of the efficacy of avoidance procedures, and standardization of commercial mite allergen extracts.

Allergens↗
PubMed1994

Fine specificity of B-cell epitopes on Felis domesticus allergen I (Fel d I): effect of reduction and alkylation or deglycosylation on Fel d I structure and antibody binding.

The repertoire of B-cell epitopes on the major cat allergen, Fel d I, was analyzed with monoclonal antibodies (MoAbs) in topographic mapping studies and in immunoassays with antigen derived from other cat (Felidae) species. Four essentially nonoverlapping epitopes on Fel d I, designated Fd1A to D, were defined by use of 15 anti Fel d I MoAbs in cross-inhibition radioimmunoassay. Only MoAbs directed against epitope Fd1B bound to putative Fel d I homologues in hair and dander extracts from seven other feline species (Panthera species, [n = 5], Leptailurus serval, and Leopardus pardalus). Quantitative monosaccharide analysis showed that Fel d I was a glycoprotein, containing high levels of fucose, as well as glucosamine, galactose, and mannose. Binding of MoAbs and human IgG or IgE antibody to native, reduced and alkylated or deglycosylated Fel d I was compared by means of immunoprecipitation and immunoassay, and the effects of these treatments on the structure of Fel d I were analyzed by sodium dodecylsulfate-polyacrylamide gel electrophoresis. On reduction and alkylation, Fel d I dissociated into 14 kd and 3.2 kd peptides, and deglycosylation with trifluoromethane sulfonic acid produced a 12 to 14 kd peptide. These procedures resulted in a 100- to 1000-fold loss in murine or human antibody binding activity and caused significant loss of secondary structure, as judged by circular dichroism spectroscopy. Treatment with potassium hydroxide also caused a marked loss in antigenic reactivity. In contrast, enzymatic deglycosylation generated a 9 kd peptide, which showed strong reactivity with murine and human antibodies, comparable to native Fel d I. The results show that MoAbs define a broad repertoire of B-cell epitopes on Fel d I, one of which is expressed by other cat species. These epitopes are conformational and do not appear to involve oligosaccharide residues.

Alkylation↗
PubMed1992

Effects of various steroid milieus or physiological states on sexual behavior of Holstein cows.

Two experiments were conducted to determine how steroid milieus and pregnancy affect sexual behavior. Experiment 1 was arranged as a Latin square with five ovariectomized cows and five steroid milieus: no steroid (N); progesterone (P4); estradiol benzoate (EB); P4 + EB; and P4 followed by EB (P4:EB). Progesterone was administered via pessary (2 g of P4) for 5 d and EB was injected (1 mg i.m.) on the day before a test day. On a test day, cows were exposed for four 30-min periods, twice each with a tied or a loose estrual test cow (prepared using P4:EB). Sexual behaviors recorded were attempted mounts, successful mounts, front mounts, stands, head butts, chin rests, and vulvar sniffs. Cows exhibited more (P less than .05) sexual behavior during periods with the loose estrual test cow than with the tied estrual test cow. Cows receiving P4 alone ranked lowest among treatments for each behavior, whereas cows receiving EB or P4:EB ranked highest or second-highest. Progesterone prevented stands in cows given P4 + EB, but these cows displayed mounting behaviors similar to those of cows given EB and P4:EB. Cows given P4 + EB were similar to those given N for most behaviors. In Exp. 2, 118 intact, lactating cows were observed in groups of four or five for mounting of estrual test cows during 24, 30-min observation periods on 8 d over 2 yr. The design was an incomplete block with physiological state, parity, estradiol, progesterone, and a calculated estrogen:progesterone ratio included in the model. Each block included one or two cows at 23 +/- .8 d after insemination, divided retrospectively into one pregnant and two non-pregnant groups (low [less than 1 ng/mL] vs high progesterone), and other cows at 89 +/- 1.0, 152 +/- 1.2, and 234 +/- 1.7 d of gestation (six physiological groups). Most cows were observed once, but 27 cows were included twice during 2 yr. Only 60% of the 118 cows made attempted or successful mounts even though estrual test cows were always receptive. Physiological state was not associated with amount of mounting because very active (greater than or equal to five attempts) and inactive cows were represented in all physiological groups. The estrogen:progesterone ratio on test day accounted for small, but significant, variation in mounting behavior. For cows observed on two different days, correlations between successive observations were .46 for attempted mounts, .78 for successful mounts, and .71 for total mounts.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1991

Identification, quantitation, and purification of cockroach allergens using monoclonal antibodies.

A panel of murine IgG monoclonal antibodies (MAbs) was raised against German cockroach (CR) (Blattella germanica) extract and selectively screened to identify MAb directed against allergen(s) recognized by IgE antibodies. Sera from 28 CR-allergic patients were used as sources of IgE antibodies to detect allergens "presented" by the MAb. Four clones (10A6, 3G12, 8F4, and 1D4) produced MAb to allergen(s) that bound IgE antibodies. Quantitative radioimmunoassays were used to compare levels of the MAb-defined allergens in CR extracts. MAb 10A6 reacted with a cross-reacting allergen that was detected in 9/14 CR species, including Blattella, Periplaneta, Blatta, Leucophea, and Supella spp, at concentrations of 100 to 10,000 U/ml. In contrast, MAb 3G12, 8F4, and 1D4 were Blattella specific. The allergen defined by MAb 8F4 was purified by MAb affinity chromatography and size-exclusion by high-performance liquid chromatography. It is a 36 kd heat-sensitive protein, isoelectric point, 5.2 to 5.4. Allergen 10A6 was partially purified by isoelectric focusing and high-performance liquid chromatography. It is a heat-stable, acidic protein (isoelectric point 3.15). Based on comparison of their properties with properties of previously described CR allergens, the allergens defined by MAb 10A6 and 8F4 have been provisionally designated Blattella germanica allergen I (Bla g I) and Blattella germanica allergen II (Bla g II), respectively. Assays of six commercial CR skin test extracts demonstrated a 200-fold difference in Bla g I levels (4.7 to 1085 U/ml) and only two extracts that contained detectable Bla g II (248 and 324 U/ml). The results demonstrate that MAb can be used to identify and define CR allergens and that the strategy of the use of MAb as a first step in allergen analysis and purification can be very effective, especially for poorly characterized allergen extracts.

Allergens↗
PubMed1990

Early testicular changes after vasectomy and vasovasostomy in Lewis rats.

The testes of Lewis rats were studied at intervals from 2 weeks to 3 months after bilateral vasectomy, vasectomy followed 1 month later by vasovasostomy, or sham operations. Aims were to determine the nature of early alterations after vasectomy, and to determine whether vasovasostomy after 1 month would result in reversal of vasectomy-induced changes. Approximately one-fourth of the testes in the vasectomy and vasovasostomy groups displayed histological changes, which consisted mainly of depletion of germ cells. The extent of the depletion varied greatly in different seminiferous tubules. In testes altered in this way, no abnormal infiltrations of lymphocytes, macrophages, or other cells were observed in the seminiferous epithelium or in the interstitium. The rete testis and straight tubules were normal in testes with altered seminiferous epithelium. A few testes in the vasectomy and vasovasostomy groups had necrotic centers. The results suggest that depletion of germ cells occurred as a result of shedding from the seminiferous epithelium into the lumen of the tubules. A cellular immune response, such as occurs in experimental allergic orchitis in other species, did not appear to be responsible for the observed loss of germ cells. This suggests a possible role for humoral antibody in this model, since there is an association between testicular changes and serum antisperm antibodies at longer intervals after vasectomy. Testicular alterations were not reversed by performance of a vasovasostomy 1 month after vasectomy.

Animals↗
PubMed1990

Influence of footing surface on mounting and other sexual behaviors of estrual Holstein cows.

Seven ovariectomized Holstein cows, treated sequentially with progesterone and estradiol benzoate to induce estrus, were used to determine whether cows showed a preference for mounting and displaying other sexual behaviors toward estrual or nonestrual cows tied on dirt or concrete surfaces. Preference tests were conducted in a test area that consisted of equal-sized concrete and dirt surfaces; two cows, designated A and B, were tied on the two surfaces, one on either side. Cows A and B were treated so that on each of four test days both were estrual, only A was estrual, only B was estrual or neither A nor B were estrual. On each test day, five estrual test cows were introduced individually into the test area for two 30-min test periods. During the first test period, tied cow A was on concrete and tied cow B was on dirt, but during the second test period, their positions were switched. Test cows were able to move freely from surface to surface and to interact with tied cow A or B. Estrual test cows spent 21.6 +/- 1.4 min of each 30-min test period on dirt, regardless of the estrual status of the cow tied on concrete or dirt. Mounting activity was 3- to 15-fold greater on dirt than on concrete when there was an estrual cow tied on dirt, regardless of the estrual status of the cow on concrete. Mounting activity was fourfold greater on concrete than on dirt when there was a tied estrual cow on concrete and a tied nonestrual cow on dirt.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMedDate not supplied

Identification of a novel cat allergen--cystatin.

BACKGROUND: Cat allergen is an important cause of sensitization among children with asthma in Japan. Although there is good evidence that cats produce other allergens, only one major allergen, Fel d 1, has been studied in detail. AIMS: To identify and define the molecular structure of the other potential cat allergens. METHODS: A cat skin cDNA library was screened using IgE antibodies to cat dander and selected clones were sequenced and expressed. RESULTS: One cDNA clone contained an open reading frame encoding a 98-amino acid residue protein. Sequence homology searches revealed a high degree of identity with bovine and human cystatin A, 79 and 75%, respectively. This cat cystatin clone contained the conserved cysteine protease motif and two of three lipocalin motifs. By plaque immunoassay, 60-90% of cat allergic sera had IgE Ab to cat cystatin. This cysteine protease inhibitor motif was partially conserved in dog allergens, Can f 1 and Can f 2, which are lipocalins. Recombinant cystatin was produced in Escherichia coli cells and purified as an 11-kD protein, corresponding to the predicted MW of cystatin. The structure of cat cystatin was modeled on human cystatin B using the SWISS-MODEL. CONCLUSION: A newly identified allergen, cystatin, has been cloned from cat skin and is a member of the cysteine protease inhibitor family.

Allergens↗
PubMedDate not supplied

Molecular cloning of German cockroach (Blattella germanica) allergens.

Allergens produced by cockroaches (CRs) are an important cause of IgE antibody responses and asthma. Using molecular cloning and nucleic acid hybridization techniques, we have identified and sequenced several important allergens produced by the German CR (Blattella germanica) and studied their expression in the American CR (Periplaneta americana). Principal allergens include Bla g 2 (36-kD protein) and Bla g 4 (21-kD protein), to which 60-70% of CR-allergic patients make IgE antibodies. Bla g 2 is only expressed by B. germanica, whereas DNA encoding Bla g 4 is present in P. americana, but is not transcribed into mRNA. Sequence homology searches have identified Bla g 2 as an aspartic protease and Bla g 4 as a calycin. Other CR allergens that have been cloned include a glutathione transferase and a troponin. These studies will enable recombinant allergens to be expressed and used to investigate the role of CR allergens in asthma.

Allergens↗
PubMedDate not supplied

Defined epitopes: in vivo and in vitro studies using recombinant allergens.

BACKGROUND: The aim of the studies was to investigate the biologic activity of recombinant cockroach and mite allergens and their potential for use in diagnosis and treatment of allergic disease. METHODS: Cockroach allergens Bla g 2, Bla g 4 and Bla g 5 and mite group 5 allergens were produced in bacterial expression vectors and used for immediate skin and serum IgE antibody tests. RESULTS: The cockroach allergens showed very good skin test reactivity in allergic patients, giving positive reactions at 10(-2)-10(-5) microg/ml; controls were negative at 10(0) microg/ml. These reactions correlated with serum IgE antibody results. The prevalence of reactivity to group 5 mite allergens varied with exposure. There was a high prevalence (70%) of sensitization to Blomia tropicalis allergen, Blo t 5, among patients from Brazil and Singapore, whereas < 20% of patients from Charlottesville, US and Manchester, UK gave positive skin tests to Blo t 5 (p < 0.001). CONCLUSIONS: The results show that recombinant allergens retain biologic activity and suggest that cocktails of two to four recombinant allergens could be used for diagnostic or therapeutic purposes. The phased introduction of recombinant allergens should improve the management of allergic disease.

Allergens↗