Studies on the frequency and associations of equine leucocyte antigens in sarcoid and summer dermatitis

The equine leucocyte antigen (ELA) types and the clinical diagnosis for equine sarcoid and summer dermatitis were evaluated in 2026 horses representing five breeds. Data were analysed in unrelated animals and in family material. In the case of equine sarcoid, a strong association was observed between the ELA class II DW13 antigen and its effect on Swiss (cP < 0·001), French (cP < 0·0001) and Irish (cP < 0·01) Warmblood horses. The class I antigen A3 occurred more frequently in sarcoid-affected French horses (cP < 0·001). These results confirm our earlier findings (Gerber et al. 1988). Among Freiberger horses, which lack the ELA DW13 and A3 specificities, a breed-specific class I antigen, ABe108, displayed an increased frequency (cP < 0·05) in the affected group. Among Arabian horses, a tendency for increased frequency of the A1 antigen was observed in the affected animals, but the number of affected horses is too small for statistical significance. The Mendelian segregation in diseased half-siblings by ELA DW13 heterozygous stallions showed a strong association (P < 0·0001) between the inherited DW13 antigen and susceptibility to the sarcoid effect. In the case of summer dermatitis, previously published data (Marti et al. 1992) have been extended. The ELA types in four multiple-case families, founded by the same stallion, were analysed for an association with the effect of sarcoid. Eight out of nine ELA-typed affected offspring inherited the paternal haplotype A15, DW23 in contrast to nonaffected offspring where three out of 12 displayed these antigens (P < 0·005). Moreover, the ELA haplotypes of 11 out of 12 informative affected half-siblings sired by another stallion inherited the paternal haplotype A3, W12, DW23 (P < 0·05). Our findings demonstrate statistically significant associations between certain ELA antigens and two equine diseases. It is still unknown if the major histocompatibility complex (MHC) molecules themselves or another linked gene(s) play a role in the pathogenesis of these conditions.     

Two-color flow cytometric analysis of monocyte depleted human blood lymphocyte subsets

Peripheral blood mononuclear cells (PBMC) from normal individuals were examined using 16 pairs of FITC and phycoerythrin (PE) directly conjugated monoclonal antibodies. Each pair of reagents was used to evaluate a conventional lymphocyte gate as well as open (non) gate of monocyte depleted PBMC. Parallel studies using the same panel of monoclonal antibodies were carried out on selected, nonmonocyte depleted samples. The major findings of this analysis were that 1,000-1,200 lymphocytes in a 10,000 cell analysis are found outside the lymphocyte gate and of these approximately 2/5 are CD16 positive LGL/NK cells, 2/5 are CD3 positive T cells, and 1/5 are CD19/CD20 positive B cells. Thus, it appears that 10-15% of the lymphoid cells fall outside of the conventional lymphocyte gate, and in certain settings monocyte depletion may be useful to perform more complete evaluation of the total lymphoid cell population obtained after ficoll-hypaque separation.     

The complete amino acid sequence of the A-chain of human plasma alpha 2HS-glycoprotein

Normal human plasma alpha 2HS-glycoprotein has earlier been shown to be comprised of two polypeptide chains. Recently, the amino acid and carbohydrate sequences of the short chain were elucidated (Gejyo, F., Chang, J.-L., Bürgi, W., Schmid, K., Offner, G. D., Troxler, R.F., van Halbeck, H., Dorland, L., Gerwig, G. J., and Vliegenthart, J.F.G. (1983) J. Biol. Chem. 258, 4966-4971). In the present study, the amino acid sequence of the long chain of this protein, designated A-chain, was determined and found to consist of 282 amino acid residues. Twenty-four amino acid doublets were found; the most abundant of these are Pro-Pro and Ala-Ala which each occur five times. Of particular interest is the presence of three Gly-X-Pro and one Gly-Pro-X sequences that are characteristic of the repeating sequences of collagens. Chou-Fasman evaluation of the secondary structure suggested that the A-chain contains 29% alpha-helix, 24% beta-pleated sheet, and 26% reverse turns and, thus, approximately 80% of the polypeptide chain may display ordered structure. Four glycosylation sites were identified. The two N-glycosidic oligosaccharides were found in the center region (residues 138 and 158), whereas the two O-glycosidic heterosaccharides, both linked to threonine (residues 238 and 252), occur within the carboxyl-terminal region. The N-glycans are linked to Asn residues in beta-turns, while the O-glycans are located in short random segments. Comparison of the sequence of the amino- and carboxyl-terminal 30 residues with protein sequences in a data bank demonstrated that the A-chain is not significantly related to any known proteins. However, the proline-rich carboxyl-terminal region of the A-chain displays some sequence similarity to collagens and the collagen-like domains of complement subcomponent C1q.     

Count-dependent filter for smoothing bivariate FCM histograms

A data-smoothing filter has been developed that permits the improvement in accuracy of individual elements of a bivariate flow cytometry (FCM) histogram by making use of data from adjacent elements, a knowledge of the two-dimensional measurement system point spread function (PSF), and the local count density. For FCM data, the PSF is assumed to be a set of two-dimensional Gaussian functions with a constant coefficient of variation for each axis. A set of space variant smoothing kernels are developed from the basic PSF by adjusting the orthogonal standard deviations of each Gaussian smoothing kernel according to the local count density. This adjustment in kernel size matches the degree of smoothing to the local reliability of the data. When the count density is high, a small kernel is sufficient. When the density is low, however, a broader kernel should be used. The local count density is taken from a region defined by the measurement PSF. The smoothing algorithm permits the reduction in statistical fluctuations present in bivariate FCM histograms due to the low count densities often encountered in some elements. This reduction in high-frequency spatial noise aids in the visual interpretation of the data. Additionally, by making more efficient use of smaller samples, systematic errors due to system drift may be minimized.     

Association between coated vesicles and microtubules

In this study, a possible functional association between microtubules and coated vesicles is described. We have found that our preparations of microtubules contained coated vesicles in quantities of usually above 10%. These coated vesicles were identified both by immunological methods using anticoat antibodies and by electron microscopy of negatively stained specimens. In the immune replica, two components of coated vesicles, i.e., heavy (clathrin) and light chains, were recognized as constituents of the preparations. In the electron microscope, it was found that coated vesicles were attached predominantly along the length of microtubules. Furthermore, projections from the microtubules to the triskelion centers of the clathrin lattice were identified and thus seem to serve as linkers between the cytoskeletal structure of the organelle. A similar type of association was detected in tissue culture cells; bridges between coated vesicles and microtubules were clearly identified by electron microscopy of thin sections.     

Isolation and characterization of a large,neurite-associated glycoconjugate from neuroblastoma cells

A high molecular weight glycoconjugate has been isolated from neurite-producing neuronal tumor cells in culture and has been designated as I(0) based on its elution characteristics in gel filtration chromatography. This molecule cannot be found in a variety of nonneuronal cells. I(0) is found in the substratum-attached material or cell fraction of neurite-producing neuroblastoma cells, depending upon culture conditions. It is found in the substratum-bound fraction of B104 rat neuroblastoma cells during serum starvation and in the EGTA-detached cell fraction of B104 cells grown in chemically defined N2 medium. It occurs only in the cell fraction of the human neuroblastoma line Platt. Examination of behavioral variants of the B104 rat line further strengthens the association of I(0) with neurite production; the constitutive neurite-producing E(R)B9 variant contains I(0) while the non-neurite-producing E(R)A11 variant does not. I(0) is large, eluting in the void volume of sepharose-CL2B columns. Radioiodination of intact cells with lactoperoxidase shows I(0) to be a cell surface component. Metabolic radiolabeling studies show that it contains a high proportion of polysaccharide to protein, does not contain mannose, and is unsulfated. Alkaline borohydride reduction release two size classes of large polysaccharide chain. The alkaline reduction results, along with the mannose incorporation studies, show the presence of O-glycosidic linkages and few, if any, N-linkages. Resistance to nitrous acid deamination, insensitivity to glycosaminoglycan lyases, and the absence of sulfation, indicate that I(0) does not contain the glycosaminoglycans hyaluronic acid, chondroitin-, dermatan-, or heparin- sulfates. Affinity column chromatography reveals high binding affinity of I(0) to polyornithine and no binding to gelatin (collagen) or the glycosaminoglycans hyaluronate and heparin. These studies describe a unique high molecular weight glycoconjugate on the surface of neurite-producing neuroblastoma cell lines from two species.     

Antigenic expression of B-cell chronic lymphocytic leukemic lymphocytes

A flow cytometric analysis of lymphocytes in B-cell chronic lymphocytic leukemia (B-CLL) samples and in monocyte-depleted and T-cell-depleted normal peripheral blood (B-PBL) samples was undertaken using 129 reagents from the blind panel (BP) and 72 reagents from the cluster designation (CD) panel obtained from the Fourth International Leucocyte Differentiation Conference and Workshop, B-Cell Section. After determining the average mean channel fluorescence and the average percentage of positive cells for the B-CLL and the normal B-PBL preparations, a combined ratio and difference analysis was performed for each monoclonal antibody reactivity. This analysis confirmed the intense expression of class II antigens on B-CLL and the preferential expression of CD19, CD20, CD23 and CD24 antigens. In addition, three new clustered and three new unclustered antigens were also preferentially expressed on B-CLL lymphocytes. Cluster analysis of these differences suggests the existence of at least three overlapping immunophenotypic subpopulations, composed of CD19, CD20, CD21, CD22, CD23, CD24, CD75, CD76 and CDw78.