Polymorphisms of the equine major histocompatibility complex class II DRA locus

The full extent of the polymorphism of ELA-DRA in Equidae is not yet known. Given the apparent differences in DRA polymorphisms between Equidae and other species, the aims of this study were to more fully characterize ELA-DRA, determine the extent of gene polymorphism and establish the allele-frequency distribution. An allele reference panel for the second exon of ELA-DRA was established by sequence-based typing of 69 equine DNA samples consisting of various breeds of domestic horse (Equus caballus), together with donkeys (Equus asinus), Grant's zebras (Equus boehmi) and one onager (Equus hemionus). Five of the six previously reported alleles detected using single-strand conformation polymorphism were found: ELA-DRA*0101, ELA-DRA*0201, ELA-DRA*0301, ELA-DRA*0501 (Albright-Fraser DG et al. Polymorphism of DRA among equids. Immunogenetics 1996: 43: 315-7) and ELA-DRA*0601 (GenBank accession number AF5419361). In addition to the previously reported alleles, five novel ELA-DRA alleles were detected within the ELA-DRA allele reference panel. One of these was identified in E. caballus (ELA-DRA*JBH11), one in E. boehmi and E. hemionus (ELA-DRA*JBZ185) and three in E. asinus (ELA-DRA*JBD3, ELA-DRA*JBD17 and ELA-DRA*JBH45). A total of 565 equine DNA samples were screened using reference-strand-mediated conformation analysis, a double-stranded conformation-based mutation detection system that can be used to type existing ELA-DRA alleles and identify new variants. Based on our findings, at least 11 ELA-DRA alleles are now known to exist, and this level of polymorphism at the DRA locus appears to be unique to the genus Equus. Both the previously reported alleles and the new alleles displayed a species-specific distribution.     

The nuclear pore complex protein Tpr is a common autoantigen in sera that demonstrate nuclear envelope staining by indirect immunofluorescence

We studied the autoantigen targets of 75 human sera that had antibodies to the nuclear envelope (NE) as identified by indirect immunofluorescence (IIF) on HEp‐2 cells. Several different IIF staining patterns could be identified when antibodies to different components of the nuclear membrane (NM) and nuclear pore complexes (NuPC) were identified: a smooth membrane pattern characteristic of antibodies to nuclear lamins, a punctate pattern typical of antibodies to the nuclear pore complex and more complex patterns that included antibodies to nuclear and cytoplasmic organelles. Western immunoblotting of isolated nuclear and NE proteins and immunoprecipitation of radiolabelled recombinant proteins prepared by using the full‐length cDNAs of the Translocated promoter region (Tpr), gp210 and p62 were used to identify specific autoantibody targets. Fifty‐two of the 75 (70%) sera bound to Tpr, 25 (33%) bound to lamins A, B or C, 15 (20%) reacted with gp210 and none reacted with p62. Sixteen (21%) did not react with any of the NE components tested in our assays. The clinical features of 37 patients with anti‐NE showed that there were 34 females and three males with an age range of 16–88 years (mean 59 years). The most frequent clinical diagnosis (9/37 = 24%) was autoimmune liver disease (ALD; two with primary biliary cirrhosis), followed by seven (19%) with systemic lupus erythematosus (SLE), four (11%) with a motor and/or sensory neuropathy, three (8%) with anti‐phospholipid syndrome (APS), two with systemic sclerosis (SSc), two with Sjögren's syndrome (SjS), and others with a variety of diagnoses. This report indicates that Tpr, a component of the NuPC, is a common target of human autoantibodies that react with the NE.     

Cannabinoid agonist, CP 55,940, facilitates intake of palatable foods when injected into the hindbrain

Cannabinoids have been shown to influence food intake, and until recently, the neural pathways mediating these effects have remained obscure. It has been previously shown that intracerebroventricular injection of delta-9-tetrahydrocannabinol (Δ⁹-THC) causes increased consumption of palatable foods in rats, and we postulated the involvement of the hindbrain in this cannabinoid-induced food intake. Cannulated rats (both female and male groups) trained to consume sweetened condensed milk received either lateral or fourth ventricle injections of CP 55,940 and were presented with sweetened condensed milk 15 min after injection. Rats were injected over a range of doses between 100 pg and 10 μg per rat. Milk intake was recorded for a total of 3 h. Lateral ventricle injection of CP 55,940 increased milk intake at doses in the microgram range. However, CP 55,940 was effective in increasing food intake at nanogram doses when injected into the fourth ventricle. Finally, male rats appeared to be more sensitive to CP 55,940 than female rats inasmuch as milk consumption was increased at the 1 ng dose in male rats, whereas only the 10 ng dose was effective in females. These results indicate that CP 55,940 may act in the hindbrain to influence feeding behavior in rats.     

Pemphigus Vulgaris Autoantibody Response is Linked to HLA-DQB10503 in Pakistani Patients

ABSTRACT: Pemphigus vulgaris (PV) is an autoimmune disease of the skin and mucous membranes characterized by an autoantibody response against an epidermal cadherin. We performed high resolution HLA class II typing in 19 patients with PV from Rawalpindi, Pakistan and 19 non-Jewish European PV patients from Boston by sequence-specific oligonucleotide probe hybridization. The results were compared with two separate ethnically matched control populations. We found that PV patients from Pakistan had significantly increased frequencies of DRB1*1404 ( p = 0.01), DQA1*0101 ( p = 0.02), and DQB1*0503 ( p = 0.01). Among the patients of non-Jewish European ancestry, DRB1*1401 ( p < 10⁻⁶), DQA1*0101 ( p < 10⁻⁵) and DQB1*0503 ( p < 10⁻⁶), were increased in PV patients. Formal linkage analysis between the major histocompatibility complex and the PV antibody was performed in 67 relatives of the 19 Pakistani patients. The results showed strong evidence for linkage of HLA-DRB1*1404, DQA1*0101, DQB1*0503, with the presence of PV antibody in relatives’ families with a significant logarithm of the odds score of 6.06. Based on the three dimensional structure of class II molecules, we propose that HLA-DQA1*0101 and DQB1*0503, encode a negatively charged P9 peptide binding pocket of the DQ molecule and are significantly associated with susceptibility to PV in non-Jewish populations.     

Elongated peptides,not the predicted nonapeptide stimulate a major histocompatibility complex class I-restricted cytotoxic T lymphocyte clone with specificity for a bacterial heat shock protein

The peptides recognized by an H-2D^b-restricted CD8 cytotoxic T lymphocyte (CTL) clone which is specific for the 60-kDa mycobacterial heat shock protein (hsp) and cross-reacts with stressed host cells were characterized. None of the nonapeptides from hsp60 conforming to the H-2D^b binding motif were able to sensitize target cells for lysis by this CTL clone. Sequence analysis of the stimulatory fraction from a trypsin digest of hsp60, together with synthetic peptide studies, defined a cluster of overlapping epitopes. Carboxy-terminal extension by at least one amino acid of the nonamer predicted to bind best to H-2D^b was essential for CTL recognition. Two such elongated peptides, a 10-mer and a 12-mer stimulated the clone at similarly low concentrations in the 100 pM range. We assume that these two peptides comply best with the natural epitope. In contrast, the 11-mer was inactive. The stimulatory 10-mer bound to H-2D^b with an efficacy similar to that of the nonapeptide corresponding to the H-2D^b motif, as revealed by peptide induced major histocompatibility complex (MHC) surface expression on RMA-S cells and competitive blocking of epitope recognition by the nonamer. Binding of these carboxy-terminally extended peptides to the MHC groove can be explained by anchoring through the amino acid residue Asn in position 5 of the peptide and by intrusion of the hydrophobic carboxy-terminal Ala (10-mer) or Leu (12-mer), but not Gly (11-mer), into the hydrophobic pocket of the H-2D^b cleft. Because the carboxy-terminal part is thus larger than predicted this region of the peptide may arch up from the binding groove. We assume that recognition of steric components of the MHC/peptide complex broaden the range of epitope specificity for a single T cell receptor. This flexibility not only promotes recognition of several overlapping peptides from a single antigen, but may also increase the chance of cross-reaction with similar peptides from unrelated proteins, including autoantigens. Consistent with this latter assumption, the T cell clone cross-recognizes mycobacterial hsp60 and stressed host cells.     

The major histocompatibility complex (CyLA) of the cynomolgus monkey. I. Serologic definition of 21 specificities

Thirty-seven lymphocytotoxic antisera, 27 of which were raised by immunization with skin grafts and blood from partially matched donors, were tested against cells obtained from 218 unrelated animals and 205 offspring from a colony of cynomolgus monkey (Macaca fascicularis). Evidence was obtained for the presence of at least 21 specificities defined by cluster analysis and segregation within families. Allelic relationships between 16 specificities was suggested by segregation patterns, the absence of triplets and statistical analysis of association in the unrelated population sample. The data support a two-locus model, with tentative assignment of seven specificities to the A locus and six to the B locus. That these lymphocyte alloantigens constitute the major histocompatibility complex (MHC) of the cynomolgus monkey is suggested by analogy with other known MHCs and by the increased survival times of skin grafts between paternally matched half sibs compared to haplodistinct full sibs.     

Cell-Growth Kinetics and Karyotype Analysis of Human Memory Lymphocytes Responding to Alloantigen and Mitogen

Peripheral-blood lymphocytes were primed in vitro with the mitogen phytohemagglutinin (PHA) or with allogeneic cells and their memory responses studied following sequential restimulation with either mitogen or alloantigen. Chromosome preparations were made every 12 hours following exposure to the stimulating agents. Cultures were labeled with BUdR for sister-chromatid staining of the chromosomes which provided information about the kinetics of cell growth and rates of sister chromatid exchange. Cultures containing no BUdR were used for the investigation of cell karyotypes after chromosome-banding.Following PHA as well as alloantigen restimulation, an earlier reaction of the responding cells was observed. The peak response after the first stimulation was found at 120 h with allogeneic stimulation and at 60 h with mitogen stimulation. In the second round of stimulation, the peak occurred after 48 h (allogeneic) and 36 h (PHA) and following the third stimulation after 36 h (allogeneic) and 24 h (PHA). The speed of cell growth was decreased following restimulation with either alloantigen and mitogen. In contrast to the allogeneic restimulation, the number of cells responding after PHA restimulation was decreased.No systematic numerical or structural aberration of the karyotype was detected following repeated stimulation with either alloantigen or mitogen. In this sense, the lymphocyte subpopulations selected by repeated stimulation did not differ from the starting material. On the other hand, the sister-chromatid exchange (SCE) frequency was increased following allogeneic restimulation, whereas it remained constant with PHA restimulation.     

Intraduodenal neuropeptide levels,but not plasma levels,vary in a cyclic fashion with the migrating motor complex

The neurohormonal control of the migrating motor complex (MMC) is not fully understood. The hypothesis of the present study was that neuropeptide levels might vary with the different phases of the MMC and that a similar variation might be found in the secretions of the gastrointestinal tract. Thus, plasma and intraduodenal concentrations of vasoactive intestinal peptide (VIP), somatostatin (SOM), substance P (SP) and neurokinin A (NKA) were determined by radioimmunoassay every 10 min during two complete MMC cycles in eight male subjects. For comparison, plasma motilin (MOT) concentrations were measured. Plasma concentrations of MOT (mean peak value ± SEM; 39 ± 6 pmol L^?1), but none of the neuropeptides studied, showed a cyclic variation in plasma with the different phases of the MMC. Peak intraduodenal concentrations of VIP (79 ± 23 pmol L^?1),?SOM (2437 ± 432 pmol L^?1) and SP (718 ± 326 pmol L^?1) occurred at or at the time point before the onset of phase III of the MMC. No such correlation was observed for NKA. These results demonstrate that intraduodenal but not plasma concentrations of the neuropeptides VIP, SOM and SP show an association with phase III of the MMC. The biological relevance of this finding is yet unclear, but the results raise the possibility that gut neuropeptides may regulate fasting motility through a luminal release.     

Synaptonemal complex analysis in spermatocytes and oocytes of rainbow trout,Oncorhynchus mykiss (Pisces,Salmonidae): the process of autosome and sex chromosome synapsis

The surface-spreading synaptonemal complex (SC) technique was employed to analyze spermatocytes and oocytes of rainbow trout in order to visualize the process of autosome and sex chromosome synapsis in this species. The structure of lateral elements (LEs) of the SC and the chromosome synapsis process at the stages of leptotene, zygotene and pachytene are described. Comparative analysis of SCs of spermatocytes and oocytes showed a difference in the synaptic process, i.e. in spermatocytes all LEs were synapsed before the appearance of centromeric regions in the biarmed elements, while in the oocytes some fully synapsed LEs, including the centromeric region of the biarmed elements, were found together with fully or partially unsynapsed LEs. In males the sex chromosome synapsis starts only after all autosomes have synapsed. Irregular synapses involving three or four LEs were found in 3.4% of the cells analyzed in mid or late zygotene. Multivalents were found in males and females. Some aspects of initial meiotic development and their implications in rainbow trout cytogenetics, genetics and evolution are discussed.