Biomedical subjects

Douglas F Antczak

Publications and source records attributed to Douglas F Antczak.

15 recordsLinked to original sources

PubMed2006

IgE and IgG antibodies in skin allergy of the horse.

In horses, allergies have been characterized by clinical signs and/or intradermal (i.d.) allergen testing. Our aim was to find the first direct evidence that immunoglobulin E (IgE) mediates equine allergy. In addition, we tested the hypothesis that immediate skin reactions in horses can also be mediated by IgG. Anti-IgE affinity columns were used to purify IgE from serum of one healthy horse and three horses affected with summer eczema, an allergic dermatitis which is believed to be induced by Culicoides midges. A modified Prausnitz-Küstner experiment was performed in four clinical healthy horses by i.d. injection of the purified serum IgE antibodies. The following day, Culicoides allergen was injected at the same sites. Skin reactions were not observed in response to allergen alone, and in two horses after stimulation at any previous IgE injection site. However, the other two horses showed an immediate skin reaction at the previous injection sites of IgE obtained from allergic horses. In addition, purified monoclonal antibodies to various equine immunoglobulin isotypes were injected i.d. into six healthy horses. Immediate skin reactions were observed in response to anti-IgE (6/6 horses) and anti-IgG(T) injections (5/6 horses). The specificities of both antibodies for IgE and IgG(T), respectively, were confirmed by enzyme linked immunosorbent assays. The results provide the first direct evidence that IgE mediates classical Type-I allergy in horses and plays a major role in the pathogenesis of summer eczema. The data also suggest that IgG(T) can bind to skin mast cells and might contribute to clinical allergy.

Allergens↗

PubMed2006

A monoclonal antibody to equine interleukin-4.

Interleukin-4 (IL-4) is secreted by T helper type 2 cells, mast cells, basophils and eosinophils. Detection of IL-4 can contribute the evaluation of cellular immune responses during infectious diseases, immunological disorders or vaccination. We used recombinant equine IL-4 to generate a monoclonal antibody (mAb) to equine IL-4. The mAb detected recombinant IL-4 in mammalian cells transfected with different plasmids containing IL-4 cDNA. After mitogen stimulation of equine peripheral blood mononuclear cells, an intracellular protein was recognized by the new mAb in 1-2% of lymphocytes using flow cytometric analysis. In the presence of the secretion blocker Brefeldin A, the protein accumulated and was detected in 4-8% of lymphocytes stimulated with phorbol 12-myristate 13-acetate and ionomycin. Double staining with the new mAb and T-cell or B-cell markers identified a subpopulation of CD4+ T-cells expressing the protein recognized by the mAb. In addition, the protein was detectable in cell culture supernatants of mitogen stimulated cells by ELISA when using the new mAb for coating of the plates and a polyclonal antiserum to equine IL-4 for detection. In conclusion, the new mAb detects equine IL-4 and can be used for intracellular staining and ELISA to measure this important cytokine.

Animals↗

PubMed2005

Occurrence of IgE in foals: evidence for transfer of maternal IgE by the colostrum and late onset of endogenous IgE production in the horse.

IgE is the key antibody involved in type I allergies. Allergen mediated crosslinking of IgE bound to high affinity Fcepsilon-receptors on mast cells and basophils stimulates cellular degranulation and release of inflammatory mediators and cytokines. In this report, we demonstrate that IgE antibodies can be transferred from the mother to offspring in horses via the colostrum. We found a clear correlation between the IgE concentration in colostrum and the total IgE concentration in foal sera on day 2 after birth (r(sp)=0.83). Maternal IgE was detected in foal sera by ELISA and on peripheral blood leukocytes of foals by flow cytometry. Both serum and cell membrane-bound IgE were undetectable in newborn foals before colostrum uptake and peaked on days 2-5 after birth. Cell-bound IgE became undetectable at 2 months after birth. Serum IgE disappeared from the circulation within the first 3-4 months of age. These kinetics suggest that the IgE antibodies which are detectable in foals during the first 4 months after birth are of maternal origin only. The endogenous IgE production was found to begin at 9-11 months of age, when IgE could be detected on peripheral blood leukocytes and in foal sera again. After 18 months of life, the total IgE concentrations in foal sera were comparable to those detected in their dams. The late onset of endogenous IgE production offers an explanation for observations that IgE mediated allergies are generally not observed in horses before puberty. The roles of the passively transferred maternal IgE in newborn foals are not yet known, but could be manifold, ranging from passive immunity and induction of immunoregulatory functions to determinative influences of maternal IgE on the antibody repertoire in the offspring.

Aging↗

PubMed2005

Genomic characterization of MHC class I genes of the horse.

The availability of a contig of bacterial artificial chromosome (BAC) clones spanning the equine major histocompatibility complex (MHC) made possible a detailed analysis of horse MHC class I genes. Prior to this study, only a single horse MHC class I gene had been sequenced at the genomic level. Although many ( approximately 60) MHC class I cDNA sequences had been determined and published, from this information, it was not possible to determine how many class I loci are expressed in horses or to assign individual sequences to allelic series. In this study, 15 MHC class I genes were identified in BAC subclones and fully sequenced. Because the BAC library donor horse had been bred for homozygosity at the MHC, these 15 genomic clones represent distinct MHC class I genes and pseudogenes and not alleles at a smaller number of loci. For five of the genes, cDNA sequences from these loci had previously been identified. Two additional expressed class I genes were discovered, bringing the known total of different equine MHC class I genes (loci) expressed as mRNA to seven. Expression of all seven loci was detected by reverse transcriptase-polymerase chain reaction in adult, fetal, and placental tissues. The remaining eight genes were designated as pseudogenes. This work resulted in moderate expansion of the horse MHC BAC contig length, and the remaining gap was shortened. The information contained in these equine MHC class I sequences will permit comparison of MHC class I genes expressed across different horse MHC haplotypes and between horses and other mammalian species.

Alleles↗

PubMed2005

Horse cytokine/IgG fusion proteins--mammalian expression of biologically active cytokines and a system to verify antibody specificity to equine cytokines.

Recombinant cytokines are valuable tools for functional studies and candidates for vaccine additives or therapeutic use in various diseases. They can also be used to generate specific antibodies to analyze the roles of different cytokines during immune responses. We generated a mammalian expression system for recombinant cytokines using the equine IgG1 heavy chain constant region as a tag for detection and purification of the expressed cytokine, demonstrated here using equine interferon-gamma (IFN-gamma), interleukin-2 (IL-2), interleukin-4 (IL4) and transforming growth factor-beta1 (TGF-beta1). The resulting IgG1 fusion proteins were composed of the C-terminal heavy chain constant region of the IgG1 (IgGa), and the N-terminal cytokine replacing the immunoglobulin heavy chain variable domain. The fusion proteins were expressed in CHO cells as dimers and their structures had similarity to that of IgG heavy chain antibodies. In contrast to other tags, the IgG1 heavy chain constant region allowed the selection for clones secreting high levels of the recombinant protein by a sensitive ELISA. In addition, the IgG1 heavy chain constant region facilitated identification of stable transfectants by flow cytometry and the secreted recombinant fusion protein by SDS-PAGE and Western blotting. To recover the cytokine from the IgG1 fusion partner, an enterokinase cleavage site was cloned between the cytokine gene and the immunoglobulin heavy chain constant region gene. The purification of the fusion protein by protein G affinity columns, the enterokinase digestion of the cytokine from the IgG1 heavy chain region after or during purification, and the biological activity of the cytokine within the fusion protein or after its isolation was demonstrated in detail for equine IFN-gamma/IgG1 by up-regulation of major histocompatibility complex (MHC) class II expression on horse lymphocytes. Biological activity could also be confirmed for the IL-2 and IL-4/IgG1 fusion proteins. To test the crossreactivity and specificity of anti-human TGF-beta1, and anti-bovine and anti-canine IFN-gamma antibodies to respective horse cytokines, the four cytokine/IgG1 fusion proteins were successfully used in ELISA, flow cytometry and/or Western blotting. In summary, equine IgG1 fusion proteins provide a source of recombinant proteins with high structural and functional homology to their native counterparts, including a convenient system for selection of stable, high expressing transfectants, and a means for monitoring specificity of antibodies to equine cytokines.

Animals↗

PubMed2004

The proliferation inhibitory proteins p27(Kip1) and retinoblastoma are involved in the control of equine lymphocyte proliferation.

Observations in early equine pregnancy clearly reveal maternal immune recognition of and response to the presence of the conceptus. Nevertheless, both maternal cellular and humoral responses appear ineffective in destroying the developing placenta and fetus in early pregnancy. Our previous studies had shown that the pre-conditioned medium generated from the culture of equine invasive trophoblast inhibited mitogen-induced lymphocyte proliferation and the expression of cytokine messenger RNA in vitro. Those findings also suggested that lymphocytes might have been halted in the G0/G1 phase of the cell cycle. To characterize the cell cycle and the intracellular mechanisms involved in the inhibition of lymphocyte proliferation, equine peripheral blood lymphocytes were cultured in the presence or absence of pokeweed mitogen (PWM) in fresh medium, or in medium pre-conditioned through cell culture of invasive trophoblast cells or fetal fibroblasts. Two-color flow cytometric analysis for bromodeoxyuridine (BrdU) incorporation by stimulated lymphocytes, and concomitant DNA staining with 7-amino-actinomycin D (7-AAD), indicated that a greater proportion of lymphocytes were found in the G0/G1 phase of the cell cycle when cultured in the invasive trophoblast cell pre-conditioned medium compared to controls. Analysis using carboxyfluorescein diacetate succinimidyl ester (CFSE) fluorescence intensity demonstrated that lymphocytes cultured in the presence of invasive trophoblast cell pre-conditioned medium had fewer cells going through division, but that those fewer cells sustained similar numbers of cell divisions as in control cultures. Hypophosphorylated retinoblastoma (Rb) protein expression was increased and p27Kip1 expression was maintained at higher levels in lymphocytes cultured in invasive trophoblast pre-conditioned medium compared to fresh medium. In agreement with these data, flow cytometric measurement of the Ki-67 protein expression in lymphocytes cultured in invasive trophoblast pre-conditioned medium was lower in comparison to controls. These findings suggest that the equine lymphocyte proliferation is at least partially regulated by the expression of proliferation inhibitory proteins such as p27Kip1 and hypophosphorylated Rb. These proteins seem to be important regulators of cell cycle transition between G1 and S phase in equine lymphocytes.

Animals↗

PubMed2004

The complete map of the Ig heavy chain constant gene region reveals evidence for seven IgG isotypes and for IgD in the horse.

This report contains the first map of the complete Ig H chain constant (IGHC) gene region of the horse (Equus caballus), represented by 34 overlapping clones from a new bacterial artificial chromosome library. The different bacterial artificial chromosome inserts containing IGHC genes were identified and arranged by hybridization using overgo probes specific for individual equine IGHC genes. The analysis of these IGHC clones identified two previously undetected IGHC genes of the horse. The newly found IGHG7 gene, which has a high homology to the equine IGHG4 gene, is located between the IGHG3 and IGHG4 genes. The high degree of conservation shared between the nucleotide sequences of the IGHG7 and IGHG4 genes is unusual for the IGHG genes of the horse and suggests that these two genes duplicated most recently during evolution of the equine IGHG genes. Second, we present the genomic nucleotide sequence of the equine IGHD gene, which is located downstream of the IGHM gene. Both the IGHG7 and IGHD genes were found to be expressed at the mRNA level. The order of the 11 IGHC genes in the IGH-locus of the horse was determined to be 5'-M-D-G1-G2-G3-G7-G4-G6-G5-E-A-3', confirming previous studies using lambda phage clones, with the exception that the IGHG5 gene was found to be the most downstream-located IGHG gene. Fluorescence in situ hybridization was used to localize the IGHC region to Equus caballus (ECA) 24qter, the horse chromosome corresponding to human chromosome 14, where the human IGH locus is found.

Amino Acid Sequence↗

PubMed2004

Natural killer cell receptors in the horse: evidence for the existence of multiple transcribed LY49 genes.

In rodents, the Ly49 family encodes natural killer (NK) receptors interacting with classical MHC class I molecules, whereas the corresponding receptors in primates are members of the killer cell immunoglobulin-like receptor (KIR) family. Recent evidence indicates that the cattle, domestic cat, dog, and pig have a single LY49 and multiple KIR genes, suggesting that predominant NK receptors in most non-rodent mammals might be KIR. Here, we show that the horse has at least six LY49 genes, five with an immunoreceptor tyrosine-based inhibition motif (ITIM) and one with arginine in the transmembrane region. Interestingly, none of the horse KIR-like cDNA clones isolated by library screening encoded molecules likely to function asNK receptors; four types of clones were KIR-Ig-like transcript (KIR-ILT) hybrids and contained premature stop codons and/or frameshift mutations, and two putative allelic sequences predicting KIR3DL molecules had mutated ITIM. To our knowledge, this is the first report suggesting that non-rodent mammals may use LY49 as NK receptors for classical MHC class I. We also show that horse spleen expresses ILT-like genes with unique domain organizations. Radiation hybrid mapping and fluorescence in situ hybridization localized horse LY49 and KIR/ILT genes to chromosomes 6q13 and 10p12, respectively.

Amino Acid Sequence↗

PubMed2003

Recurrent airway obstruction (RAO) in horses is characterized by IFN-gamma and IL-8 production in bronchoalveolar lavage cells.

In horses prone to developing recurrent airway obstruction (RAO), we tested the hypotheses that the cytokine profile in the bronchoalveolar lavage (BAL) cells of affected horses would reflect a polarized Th-2 response; that cytokine and chemokine alterations would occur within 24 h of allergen exposure; and that allergen exposure would induce alterations in the expression of the transcription factor t-bet (t-box-expressed in T-cells). The expression levels of interleukin-4 (IL-4), IL-13, Interferon-gamma (IFN-gamma), t-bet, IL-8 and granulocyte-macrophage colony stimulating factor (GM-CSF) were measured in BAL cells obtained from control and RAO-susceptible horses during an asymptomatic phase and at 24 h and 5 weeks post-stabling and hay exposure. At each sampling time, BAL neutrophil percentages in the RAO-group exceeded controls. In the RAO-group, only IL-13 expression was decreased 2-fold during the asymptomatic phase. No differences in cytokine or chemokine expression were detected during the acute exposure phase. During the chronic phase, IFN-gamma and IL-8 expression levels were 2.5- and 3-fold greater, respectively, in the RAO-group. No other differences in gene expression were detected. We conclude that the cytokine profile of the airway cells does not reflect a polarized Th-2 response; that increases in IFN-gamma result from a t-bet independent pathway and that chemokines from epithelial or interstitial cells may contribute to early neutrophil influx.

Allergens↗

PubMed2003

Characterization of the horse (Equus caballus) IGHA gene.

Nucleotide sequences of the immunoglobulin constant heavy chain genes of the horse have been described for IGHM, IGHG and IGHE genes, but not for IGHA. Here, we provide the nucleotide sequence of the genomic IGHA gene of the horse ( Equus caballus), including its secretion region and the transmembrane exon. The equine IGHA gene shows the typical structure of a mammalian IGHA gene, with only three exons, separated by two introns of similar size. The hinge exon is located at the 5' end of the CH2 exon and encodes a hinge region of 11 amino acids, which contains five proline residues. The coding nucleotide sequence of the secreted form of the equine IGHA gene shares around 72% identity with the human IGHA1 and IGHA2 genes, as well as the bovine, ovine, porcine and canine IGHA genes, without distinct preference for any of these species. The same species also cluster together in a phylogenetic tree of the IGHA coding regions of various mammals, whereas rodent, rabbit, marsupial and monotreme IGHA genes each build a separate cluster.

Amino Acid Sequence↗

PubMed2003

The effect of strenuous exercise on mRNA concentrations of interleukin-12, interferon-gamma and interleukin-4 in equine pulmonary and peripheral blood mononuclear cells.

The effect of strenuous exercise on the mRNA concentrations of interleukin-12p35 subunit (IL-12p35), interferon-gamma (IFN-gamma) and interleukin-4 (IL-4) in equine pulmonary and peripheral blood mononuclear cells (PBMCs) was investigated. We hypothesized that strenuous exercise would suppress the expression of IL-12p35, IFN-gamma and augment the expression of IL-4. Eleven horses were randomly divided into two groups, a stall-confined control group (n=5) and an exercise-conditioned treatment group (n=6). Bronchoalveolar and PBMCs were obtained from horses in the treatment group prior to the commencement of a 9-week conditioning program and 24h after the completion of a maximum exercise test conducted in week 12. Samples were obtained simultaneously from control horses. Differential counts were performed on the bronchoalveolar lavage cells. Real-time PCR was performed on the pulmonary and PBMCs to quantitate cytokine expression using equine-specific primers and Taqman probes. Target gene expression was normalized to 18s rRNA expression. With the exception of IL-4 in the BALF cells, mRNA for the three cytokines was detected in the mononuclear cells from all horses at both sampling times. There were no significant differences in the cytokine mRNA concentrations between the two groups of horses at either of the sampling times. These findings demonstrate that strenuous treadmill exercise does not exert a deleterious effect on gene expression for IL-12p35, IFN-gamma or IL-4 when assessed in horses 24h following the intense physical activity.

Animals↗

PubMed2003

The first-generation whole-genome radiation hybrid map in the horse identifies conserved segments in human and mouse genomes.

A first-generation radiation hybrid (RH) map of the equine (Equus caballus) genome was assembled using 92 horse x hamster hybrid cell lines and 730 equine markers. The map is the first comprehensive framework map of the horse that (1) incorporates type I as well as type II markers, (2) integrates synteny, cytogenetic, and meiotic maps into a consensus map, and (3) provides the most detailed genome-wide information to date on the organization and comparative status of the equine genome. The 730 loci (258 type I and 472 type II) included in the final map are clustered in 101 RH groups distributed over all equine autosomes and the X chromosome. The overall marker retention frequency in the panel is approximately 21%, and the possibility of adding any new marker to the map is approximately 90%. On average, the mapped markers are distributed every 19 cR (4 Mb) of the equine genome--a significant improvement in resolution over previous maps. With 69 new FISH assignments, a total of 253 cytogenetically mapped loci physically anchor the RH map to various chromosomal segments. Synteny assignments of 39 gene loci complemented the RH mapping of 27 genes. The results added 12 new loci to the horse gene map. Lastly, comparison of the assembly of 447 equine genes (256 linearly ordered RH-mapped and additional 191 FISH-mapped) with the location of draft sequences of their human and mouse orthologs provides the most extensive horse-human and horse-mouse comparative map to date. We expect that the foundation established through this map will significantly facilitate rapid targeted expansion of the horse gene map and consequently, mapping and positional cloning of genes governing traits significant to the equine industry.

Animals↗

PubMed2002

Characterization of the beta2-microglobulin gene of the horse.

A clone containing beta(2)-microglobulin (beta(2)-m), the light chain of the major histocompatibility complex class I cell surface molecule, was isolated from an equine bacterial artificial chromosome library. This clone was used as a template for polymerase chain reaction (PCR) and unidirectional sequencing to elucidate the genomic sequence and intron/exon boundaries. We obtained 7,000 bases of sequence, extending from 1,100 nucleotides (nt) upstream of the coding region start through 1,698 nt downstream of the stop codon. The sequence contained regulatory elements in the region upstream of the coding sequence similar to those of the beta(2)-m gene of other species. The beta(2)-m gene was localized to horse chromosome ECA1q23-q25 by fluorescent in situ hybridization. This was confirmed by synteny mapping on a (horse x mouse) somatic cell hybrid panel. The sequence and intron/exon boundaries determined were used to design PCR primers to amplify and sequence the coding region of the beta(2)-m gene in other equids, including five breeds of domestic horse, one Przewalski's horse, five domestic donkeys and five zebras. A high degree of conservation was found among equids, illustrated by >98% (349/354) identity at the nucleotide level and 95% (113/118) at the amino acid level, because of non-synonymous nucleotide substitutions. The promoter detected in the region upstream of the coding sequence was subcloned and used in chloramphenicol acetyl transferase (CAT) assays to demonstrate the presence of a functional promoter. This study provides tools for the analysis of regulation of not only the horse beta(2)-m gene, but also for any genes dependent upon beta(2)-m for expression.

Amino Acid Sequence↗

PubMed2002

Common variable immunodeficiency in a horse.

A 12-year-old Quarter Horse mare that was nonresponsive to medical treatment was evaluated for chronic respiratory disease and hepatobiliary disease. Serum immunoglobulin concentrations were measured by use of radial immunodiffusion that revealed trace to nondetectable concentrations of IgG, IgG(T), IgM, and IgA. Use of serum protein electrophoresis confirmed agammaglobulinemia by the absence of the expected peak in the gamma region. In addition, vaccination with tetanus toxoid did not result in specific immunoglobulin production. Flow cytometric analysis of blood lymphocyte subpopulations revealed the absence of B cells in blood. Immunohistochemical analysis of tissue sections revealed the absence of B lymphocytes in bone marrow and spleen, with occasional B cells in the peripheral lymph nodes. Blood lymphocyte proliferation assays revealed weak responses to pokeweed mitogen and no response to stimulation with lipopolysaccharide. Considering the age and sex of the horse, results of the immunologic tests suggested a diagnosis of common variable immunodeficiency.

Agammaglobulinemia↗

PubMed2002

IgG antibody responses to an inhaled antigen in horses with "heaves" (recurrent airway obstruction).

A controlled experimental system for the evaluation of pulmonary immune responses in horses with "heaves" (recurrent airway obstruction) has been developed. We hypothesized that the humoral immune response to an inhaled antigen in diseased horses would be different from that of healthy horses and that chronic pulmonary inflammation would bias the production of IgG isotypes in diseased horses as compared to healthy horses. Healthy and affected horses were housed in a natural challenge environment (stabled, fed dusty hay) and exposed by inhalation, to a nebulized solution of keyhole limpet hemocyanin (KLH). Sera and bronchoalveolar lavage fluids (BALFs) were collected from horses prior to and following their inhalation exposure to the antigen. Differential cell counts were performed on the cells in the BALF. An enzyme-linked immunosorbent assay (ELISA) was used to determine the concentrations of IgGa, IgGb, IgG(T) and combined IgG specific for KLH in the sera and BALF. The percentages of neutrophils in the BALF of diseased horses were increased 4-6-fold over healthy horses. Combined IgG specific for KLH was significantly greater in BALF and serum from healthy compared to diseased horses. Differences in isotypes were also evident; however, only IgGb specific for KLH in the BALF was significantly increased in healthy versus diseased horses. Possible explanations for this difference include: (1) increased destruction of antigen before it could interact with lymphocytes, (2) down-regulation of IgGb production by inhibitory cytokines in diseased horses, or (3) binding of IgGb to Fc receptors on the large numbers of neutrophils in the lungs of diseased horses. In contrast to the prevailing notion that horses with heaves have exaggerated immune responses, our data suggest that diseased horses exposed to an aerosolized protein mount weaker IgG responses compared to healthy horses.

Administration, Inhalation↗