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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. [ABSTRACT FROM AUTHOR]

The male-specific region of the Y chromosome (MSY) is paternally inherited and enables to trace patrilines through genetic analysis. MSY markers and haplotypes in combination with pedigree information provide precise insights into genealogical systems. While the use of the Y chromosomal genetic genealogy is well established in humans, we recently could develop an equine MSY marker system from NGS data capable to perform a genetic fine-scale analysis of sire lines in horses. This approach has been successfully applied to solve genealogical questions in the English Thoroughbred. In this work, we study sire line genealogies in Lipizzan horses by combining MSY and pedigree information. The Lipizzan breed is characterized by deep pedigree records that trace back to founder sires born in the 18th and early 19th century. We inferred Lipizzan specific MSY haplotypes based on NGS data from 15 Lipizzan males mapped to a 5.8 Mb horse MSY draft reference (LipY764). Haplotype distribution was then studied by screening 52 selected haplotype determining variants in 132 stallions representing the 8 existing Lipizzan sire lines. Samples were derived from 7 European Lipizzan state stud farms. Genomic DNA was isolated from blood and genotyping was performed with the KASP genotyping method. MSY haplotype spectra in Lipizzans were compared with other breeds, and the results confirmed a presumed Arabian and Spanish origin in 2 Lipizzan sire lines. Surprisingly, horses from the remaining 6 sire lines grouped into haplogroup Tb. This haplogroup was recently characterized as a signature of the Turkoman horse, and the allocation of Lipizzans into Tb points to a possible Turkish influence in the Lipizzans. In addition, we detected 6 haplotypes that arose via de novo mutation in the timeframe of pedigree documentation. In 2 sire lines the MSY pattern in sub-lines did not accord with the paternal lineage documentation. In 5 horses belonging to a side-branch of the Favory line, a unique haplotype was detected. This finding indicates another foundation sire in the Lipizzan horse. In conclusion, MSY haplotyping confirmed historic breed documentation and offered new insights on the male breed ancestry. Furthermore, we demonstrated that our approach is suitable to study sire lines on a genealogical scale. MSY analysis can be used for studying the paternal ancestry and as a forensic tool, to solve open questions in the paternal lineages, even if they occurred multiple generations back in time. [ABSTRACT FROM AUTHOR]

Multiple sclerosis is an autoimmune disease of the central nervous system in which T cell reactivity to several myelin proteins, including myelin basic protein (MBP), proteolipid protein, and myelin oligodendrocyte glycoprotein (MOG), has been implicated in the perpetuation of the disease state. Experimental autoimmune encephalomyelitis (EAE) is used commonly as a model in which potential therapies for multiple sclerosis are evaluated. The ability of T cell epitope-containing peptides to down-regulate the disease course is well documented for both MBP- and proteolipid protein-induced EAE, and recently has been shown for MOG-induced EAE. In this study, we describe a novel EAE model, in which development of severe disease symptoms in (PL/J x SJL)F1 mice is dependent on reactivity to two different immunizing Ags, MBP and MOG. The disease is often fatal, with a relapsing/progressive course in survivors, and is more severe than would be predicted by immunization with either Ag alone. The MOG plus MBP disease can be treated postinduction with a combination of the MOG 41-60 peptide (identified as the major therapeutic MOG epitope for this strain) and the MBP Ac1-11[4Y] peptide. A significant treatment effect can also be obtained by administration of the MBP peptide alone, but this effect is strictly dose dependent. This MBP peptide does not treat the disease induced only with MOG. These results suggest that peptide immunotherapy can provide an effective means of mitigating disease in this model, even when the treatment is targeted to only one component epitope or one component protein Ag of a diverse autoimmune response.

The interaction of T cell costimulatory molecules with their ligands is required for optimal T cell activation. Interference with such interactions can induce antigen unresponsiveness and delay xeno- and allograft rejection. We have previously shown that LFA3TIP, a soluble human lymphocyte function-associated antigen (LFA)-3 construct, binds CD2 and inhibits responses of human T cells in vitro. This study reports the first use of a human fusion protein, LFA3TIP, to significantly prolong primate cardiac allograft survival. Based on our observations that LFA3TIP inhibits baboon allogeneic mixed lymphocyte reactions, we gave baboon recipients of heterotopic cardiac allografts injections of LFA3TIP, 3 mg/kg i.v., for 12 consecutive days, starting 2 days before transplantation. This regimen delayed graft rejection from an average of 10.6 +/- 2.3 days for human IgG-treated controls (n = 5) to an average of 18.0 +/- 5.3 days for LFA3TIP-injected animals (n = 7; P < or = 0.01). Grafts from LFA3TIP-treated animals showed markedly diminished coronary endothelialitis as compared with control animals. LFA3TIP reached peak serum levels of approximately 100 micrograms/ml after 7-9 injections and persisted in the 10-micrograms/ml range for 1 to 2 weeks after the final injection. Despite these blood levels, circulating antibodies to LFA3TIP were not detected in the serum. No renal or hepatic toxicity was noted. The possible mechanism by which LFA3TIP acts to inhibit graft rejection is discussed; success in prolonging graft survival when LFA3TIP is used as a single-agent therapy suggests great potential for this novel therapeutic agent.

Aminopeptidases catalyze the hydrolysis of amino acid residues from the amino terminus of peptide substrates. Leucine aminopeptidase (LAP) from bovine lens is the best characterized aminopeptidase and the only LAP for which the amino acid sequence was determined by protein sequencing. Using this sequence information, we isolated a bovine kidney LAP cDNA and compared its deduced amino acid sequence to the published amino acid sequence for bovine lens LAP. Overall, the sequences are highly conserved. However, several differences are observed. The kidney LAP cDNA indicates a 26 amino acid extension at the amino terminus which is not found in the mature purified lens LAP. The cDNA also indicates an additional octapeptide in the C-terminal region which was not indicated in the published lens LAP amino acid sequence but which was required for best fit of crystallographic data regarding bovine lens LAP. Several other single amino acid changes were also noted. Levels of LAP transcripts were examined in bovine lens and kidney tissue as well as in cultured lens cells. Lens epithelial tissue showed only one LAP transcript (2.4 kb) whereas two transcripts (2.0 and 2.4 kb) were observed in cultured lens cells derived from epithelial tissue and in kidney tissue. Using Northern blot analysis, we correlated LAP mRNA levels with previously determined changes of LAP activity in aging lens tissue and in progressively passaged lens epithelial cells which were used to simulate aging in vitro. No differences were found in LAP mRNA levels in epithelial tissue from old and young lenses.(ABSTRACT TRUNCATED AT 250 WORDS)

Our previous studies have demonstrated that cultured human endothelial cells (EC) provide costimulation to PHA-activated CD4+ T cells, measured as augmentation of IL-2 synthesis, through a cell contact-department pathway. Here we show that fixed and living EC provide comparable degrees of costimulation to CD4+ T cell populations, indicating that EC costimulation does not depend upon active metabolism. EC achieve these effects in part by utilizing lymphocyte function-associated antigen-3 (LFA-3) to interact with T cell CD2 as shown by observations that EC augmentation of IL-2 is partially (50-70%) blocked by eight of eight mAb tested which recognize LFA-3; that purified phosphatidylinositol-linked LFA-3 (PI-LFA-3) can also provide costimulation to CD4+ T cells; and that there is a delay of the EC effect on CD4+ T cells which express low levels of CD2 compared to those which express high levels of CD2. However, three lines of evidence suggest that EC also utilize at least one additional ligand. First, there is incomplete replacement of the EC effect by PI-LFA-3 such that the costimulatory ability of EC combined with PI-LFA-3 is additive at all concentrations of PI-LFA-3 tested. Second, costimulation by PI-LFA-3, but not by EC, is fully inhibited by anti-CD2 or anti-LFA-3 mAb. Finally, costimulation by PI-LFA-3, but not by EC, is completely suppressed by cyclosporine A. We have not formally identified the second ligand but it does not appear to be intercellular adhesion molecule-1, vascular cell adhesion molecule-1, CD44, or B7/BB1.

We have used chemically cross-linked dimers, trimers, and tetramers of lymphocyte function-associated antigen-3 (LFA-3) to study the role of multivalency in the interaction of the protein with its receptor, CD2. The cross-linked adducts showed enhanced activity in systems where LFA-3 has been shown to (i) block LFA-3/CD2 interactions in a rosetting assay and (ii) provide through the CD2 on peripheral blood lymphocytes a trigger for T-cell proliferation. The level of increase was directly related to the valency state of the multimers. In the rosetting assay, the dimers, trimers, and tetramers, by weight, exhibited 15-, 150-, and 430-fold increases in activity over monomeric LFA-3. In the proliferation assay, the tetramer produced a 6-fold increase in thymidine incorporation at 0.06 micrograms/ml, the trimer was 100 times less active than the tetramer, and the dimer and monomer were inactive. The LFA-3 multimers were generated using a three-step cross-linking chemistry that was targeted at the carbohydrates on LFA-3. With this procedure over 60% of the starting protein was converted into multimers with no effect on function. The cross-linking approach should be applicable to other surface antigens, providing a simple method for analyzing multivalent interactions.

Lipocortins (annexins) are a family of calcium-dependent phospholipid-binding proteins with phospholipase A2 inhibitory activity. The characteristic primary structure of members of this family consists of a core structure of four or eight repeated domains, which have been implicated in calcium-dependent phospholipid binding. In two lipocortins (I and II) a short amino-terminal sequence distinct from the core structure has potential regulatory functions which are dependent on its phosphorylation state. We have isolated the rat and the human lipocortin I genes and found that they both consist of 13 exons with a striking conservation of their exon-intron structure and their promoter and amino acid sequences. Both lipocortin I genes are at least 19 kbp in length with exons ranging from 57 to 123 bp interrupted by introns as large as 5 kbp. Each of the four repeat units of lipocortin I are encoded by two consecutive exons while individual exons code for the highly conserved putative calcium-binding domains. The promoter sequences in the rat and in human genes are highly conserved and contain nucleotide sequences characterized as enhancer sequences in other genes. The structure of the lipocortin I gene lends support to the hypothesis that the lipocortin genes arose by a duplication of a single domain.

The influence of T cell receptor (TcR) triggering on T cell adhesion function has been systematically investigated in the present studies; we show that the adhesion function of LFA-1 is minimal in non-activated T cells but is augmented within minutes following TcR-mediated activation. In contrast, CD2 function is essentially optimal in non-activated T cells and undergoes no detectable modification within 12 h of TcR stimulation. Protein kinase C activation augments LFA-1 but not CD2 adhesion function and cyclic AMP reduces LFA-1 adhesion without affecting CD2-LFA-3 interactions. Up-regulation of the LFA-1 pathway occurs in the absence of any detectable surface redistribution of this molecule, suggesting an activation dependent modification leading to a high-affinity ICAM-1 binding state. The TcR independence of CD2 adhesion function implies a critical role of the CD2 pathway in initiating cell-cell interactions prior to TcR engagement and LFA-1-ICAM-1 binding and underscores the complementary nature of the CD2 and LFA-1 adhesion pathways during the immune response.

Lipocortin-1 protein synthesis in resting monocytes is under the control of glucocorticoid steroids. This induction occurs at reasonable dexamethasone concentrations, may require concomitant synthesis of transcriptional factors, appears to be cell type specific, and has been observed only in primary tissues in our hands. Variability in the magnitude of the induction suggests that the regulation is complex, involving either additional factors or particular differentiation states. In addition to the induction of intracellular lipocortin-1, steroids cause the appearance of labelled lipocortin-1 on the outer surface of the cells. Whether cell breakage can account for this effect is unclear. Considerable microheterogeneity was found in preparations of recombinant-lipocortin-1. Aspects of N-terminal post-translational processing, N-terminal proteolysis, conformational states and the existence of an air-denatured form lacking alpha-helical structure contributed to this heterogeneity. We believe that these aspects are responsible for the variable biological potency of different preparations. It remains unclear whether this protein actually plays a physiological role in the regulation of the inflammatory response or achieves its effects through membrane binding and subsequent non-physiological perturbation of the cells.

The CD2 T lymphocyte-surface glycoprotein serves to mediate adhesion between T lymphocytes and their cognate cellular partners which express the specific ligand LFA-3. In addition, CD2 by itself or in conjunction with T-cell receptor stimulation, transduces signals resulting in T-lymphocyte activation. One or both of these functions seems to be physiologically important, given that certain anti-CD2 monoclonal antibodies block T-cell activation and that antigen-responsive memory T cells express a high level of CD2 relative to virgin T cells, which are largely antigen-unresponsive. Nevertheless, the contribution of the individual CD2 functions in T-cell responses has not been independently examined. To this end, human CD2 complementary DNAs encoding an intact LFA-3-binding adhesion domain, but lacking a functional cytoplasmic signal transduction element (CD2trans-), were introduced into an ovalbumin-specific, I-Ad restricted murine T-cell hybridoma. The antigen-specific response of T hybridoma cells expressing human CD2trans- protein was enhanced up to 400% when the human LFA-3 ligand was introduced into the I-Ad expressing murine antigen-presenting cells. In contrast, no augmentation was observed if human LFA-3 was absent or expressed on a third-party cell lacking the I-Ad restriction element. These results directly demonstrate the functional significance of adhesion events mediated between CD2 on the antigen-responsive T lymphocyte and LFA-3 on the presenting cell in optimizing antigen-specific T-cell activation.

The anti-inflammatory action of glucocorticoids has been attributed to the induction of a group of phospholipase A2 inhibitory proteins, collectively called lipocortin. These proteins are thought to control the biosynthesis of the potent mediators of inflammation, prostaglandins and leukotrienes, by inhibiting the release of their common precursor, arachidonic acid, a process that requires phospholipase A2 hydrolysis of phospholipids. Lipocortin-like proteins have been isolated from various cell types, including monocytes, neutrophils and renal medullary cell preparations. The predominant active form is a protein with an apparent relative molecular mass (Mr) of 40,000 (40K). These partially purified preparations of lipocortin mimic the effect of steroids, and mediate anti-inflammatory activity in various in vivo model systems. Using amino-acid sequence information obtained from purified rat lipocortin, we have now cloned human lipocortin complementary DNA and expressed the gene in Escherichia coli. Our studies confirm that lipocortin is a potent inhibitor of phospholipase A2 activity.

The human genes which code for Lipocortin I and Lipocortin II, proteins that inhibit phospholipase A2 (PLA2) activity, have been regionally localized in the human genome by chromosomal in situ hybridization and segregation analysis in somatic cell hybrids using cDNA clones for Lipocortin I and II. Lipocortin I, the 35 kd substrate for the epidermal growth factor (EGF) receptor/kinase, maps to chromosome region 9q11- greater than q22. The Lipocortin II cDNA probe detects at least four independently segregating loci which map to human chromosome regions 4q21-q31.1, 9pter-q34 proximal to c-abl, 10q proximal to 10q24 and 15q21-q22 proximal to the 15q22 translocation breakpoint characteristic of acute promyelocytic leukemia (APL). Thus, Lipocortin I and one locus detected by Lipocortin II cDNA are syntenic on chromosome 9; one Lipocortin II locus is perhaps not far from the genes for EGF and IL-2 on 4q; and another of the Lipocortin II loci is on 15q, perhaps not far from the APL breakpoint.

We have purified two 35 kd phospholipase A2 inhibitors from human placenta, which we refer to as lipocortin I and II. Both proteins exhibit similar biochemical properties and occur in placenta at about 0.2% of the total protein. By peptide mapping, sequence, and immunological analyses, we show that lipocortin I and the 35 kd substrate for the EGF-receptor/kinase from A431 cells are the same protein. By similar criteria, we determine that lipocortin II is the human analogue of pp36, a major substrate for pp60src, which has been characterized in chicken embryo fibroblasts and in bovine brush border preparations. The amino acid sequences of lipocortin I and II that we deduced from cDNA clones share 50% homology, indicating that they probably evolved from a common gene.