Biologic and chemical characterization of HLA antigens in human serum.

HLA antigens of both the A and B loci were shown to be associated with the high density lipoprotein fraction of serum prepared by ultracentrifugal flotation. HLA-A9 antigens were purified 100-fold with essentially complete recovery by a simple procedure of high density lipoprotein preparation involving precipitation with polyanions and ultracentrifugal flotation. The purified lipid-associated antigen was immunogenic since it elicited the formation of cytotoxic xenoantibodies in rabbits. Serum HLA-A9 antigens were found by immunoprecipitation and gel electrophoresis to consist of a 45,000 m.w. heavy chain associated with beta2-microglobulin. The size of the HLA-lipid complex (less than 190,000 m.w.) and of the HLA-deoxycholate complex (less than 102,000 m.w.) suggests that HLA antigens are shed into plasma as a complex of a single HLA molecule and a single beta2-microglobulin chain, associated with boundary lipid.     

Determination of HLA antigens in serum and thrombocytes using microneutralization and microabsorption tests

The microneutralization and microabsorption test is described for identifying HLA-antigens in the serum and on thrombocytes. The incubation time for neutralization and absorption amounts to 3 hours at 20-25 degrees C. Absorption is made by platelet suspension at a concentration of 300 000 to 500,000 thrombocytes per mm3. In the majority of cases satisfactory results can be achieved by a neutralizing ratio (absorption ratio) of 1: 1. Some HLA sera show a deviating ability of neutralization with those HLA-antigens contained in sera and absorption with appropriate platelets; this behaviour is not connected with the titre of HLA-cytotoxins. Depending on the case there will be a varying content of HLA-antigens in sera and on thrombocytes of individuals. Moreover there is no regular conformity concerning the content of HLA-antigens in the serum and on the thrombocytes of a single person.     

The self determinants recognized by human virus-immune T cells can be distinguished from the serologically defined HLA antigens

The self specificity of human influenza virus-immune cytotoxic T cells has been analyzed in order to clarify the relationship between the self antigens that they recognize and the serologically defined HLA-A and -B antigens. Virus-immune effectors from HLA-A2-positive donors were tested on panels of virus-infected target cells from donors who were either HLA-mismatched or matched only for HLA-A2. Virus-immune T cells from 11 out of 11 A2-positive donors lysed all A2-matched virus-infected target cells (and no HLA-mismatched targets), except that each of these effector cells consistently failed to lyse virus-infected target cells from one A2-positive donor (designated M7). Although the A2 specificity of donor M7 could also be distinguished from the A2 antigen of other donors by alloimmune cytotoxic T cells, no differences in the A2 antigen of donor M7 could be defined by extensive serologic analyses. These results indicate that there is a strong but incomplete association between a self antigen recognized by virus-immune T cells and the serologically defined HLA-A2 specificity.     

Separation and comparison of human TL-like antigens and HLA(A,B, C) antigens expressed on Cultured T cells

Beta₂-microglobulin-bound T-cell membrane components containing both human TL-like antigens and HLA(A, B, C) antigens were partially purified from Renex 30-solubilized membrane material of cells of a human T-cell-type leukemia cell line, HPB-ALL. The radioiodinated preparation was subjected to limited papain digestion; the HLA(A, B, C) antigens split, whereas a large portion of the human TL-like antigens remained intact. The antigen molecules were recovered by lentil-lectin affinity chromatography and separated by gel filtration on the basis of the induced difference in molecular size. The human TL-like-antigen preparation thus obtained was essentially free of HLA(A, B, C) antigens. The human TL-like antigens were immunospecifically precipitated and the component polypeptide, heavy and light, chains were separated by acid dissociation followed by gel filtration. The component chains were compared with the corresponding chains of HLA(A, B, C) antigens obtained similarly from the same HPB-ALL cells with respect to their fragmentation patterns on chemical or enzymatic cleavage. The results provided convincing evidence for the identity of the light chains of human TL-like antigens and HLA(A, B, C) antigens, and also evidence suggesting the presence of substantial differences in the fundamental structure of the heavy chains of human TL-like antigens and HLA(A, B, C) antigens.A unit of the New York State Department of Health.     

Distribution of HLA A,B and C antigens in an Australian population

Summary HLA A and B antigens have been determined in 2740 adult responders to a population health survey in Busselton, Western Australia. HLA A B and C antigens have been determined in 481 schoolchildren. The antigen frequencies are generally close to those obtained elsewhere for subjects of British origin, but there are some differences from the frequencies found in North American Caucasians. The frequencies were not affected by the inclusion of genetically related individuals in the sample. Seventeen HLA A-B haplotypes, six A-C haplotypes and six B-C haplotypes had frequencies above 1%. A total of 1071 distinct phenotypes were identified out of the 5069 which are theoretically possible for the HLAA-B model used in the study. The most frequent phenotype was A2, B12 which occurred in 2.5% of the sample.     

The common antigenic determinants of the H antigens of salmonellae]

Salmonella H-antigens have common and specific antigenic determinants. Studies on the determination of common antigenic determinants of H-antigens (a; b; d; i; 1,2 and nt) has been carried out. The order of the distribution of H-antigens according to the decrease of these common antigenic determinants in size is presented: d greater than a greater than 1,2 greater than b greater than nt. These data broaden our knowledge of the structure of H-antigens; they are also necessary for obtaining specific antibodies and conjugate preparations used in the enzyme immunoassay.     

Capping of HLA antigens in human lymphocytes as followed by immunogold label-fracture

We used immunogold label-fracture to follow the migration of HLA I class and HLA II class antigens during capping as induced by specific monoclonal antibodies. Capping is achieved through a process of clustering and "consolidation" of clusters into larger patches and, finally, a single cap. All receptors appear to cluster from the very start, with no "stray" molecules joining already formed patches. Characterization of exoplasmic and protoplasmic fracture-faces of capping cells fails to reveal any corresponding accumulation of intramembrane particles and/or subtler rugosities. Our results are consistent with the concepts that view the migration of capping molecules as contemporaneous with the efflux of noncapping integral membrane proteins.     

Detection of the determinants of tetracycline A, B and C resistance in Shigella and Salmonella using DNA probes

Genetic determinants of tetracycline resistance of classes A, B and C were detected with DNA probes labeled with radioactive phosphorus (32P) in Shigella (44 strains) and Salmonella (50 strains). It was shown that in the Shigella strains the frequency of the Tet A gene amounted to 66 per cent, the frequency of the Tet B gene was equal to 84 per cent, the frequency of their combination was equal to 50 per cent and the frequency of the Tet C gene was nil. In the Salmonella strains the frequency of the Tet A, Tet B and Tet C genes was equal to 0.100 and 20 per cent, respectively, and that of their combination amounted to 20%. Possible use of the DNA probes in epidemiological analysis of outbreaks of Shigella and Salmonella infections is suggested.     

Inhibition of absorption capacity of HLA antigens on in vitro hormone-treated lymphocytes.

Hydrocortisone, Agostilben, Agovirin, Neolutin and Superanabolon partially to completely inhibit the absorption capacity of HLA antigens on the lymphocytes in vitro. The effect is directly proportional to the duration of lymphocyte treatment and the type of hormone, yet unrelated to the type of antigen. The importance of this phenomenon for HLA antigen typing in hormones-treated patients is discussed.     

Regulatory role of monomorphic and polymorphic determinants of HLA class I antigens in the proliferation of T cells stimulated by autologous and allogeneic B and T lymphoid cells

Monoclonal antibodies (mAb's) to monomorphic and polymorphic determinants of HLA Class I antigens were shown to inhibit proliferation of T cells stimulated with autologous and allogeneic B and T lymphocytes. Inhibition of proliferative responses was lower when T cells were used as stimulators than when B cells were used. The inhibitory activity was similar for mAb's to monomorphic and polymorphic determinants of HLA Class I antigens, suggesting that the density of antigen-antibody complexes on the cell membrane does not play a major role in the phenomenon. The anti-HLA Class I mAb's exerted their inhibitory effect at the level of both the responding and the stimulating cells. Addition of exogenous interleukin 2 to the mixed cultures did not affect the mAb-mediated inhibition.     

Detection of bacterial antigens using immuno-PCR

E. KAKIZAKI, T. YOSHIDA, H. KAWAKAMI, M. OSETO, T. SAKAI AND M. SAKAI. 1996. A new and very sensitive antigen detection technique, immuno-polymerase chain reaction (immuno-PCR), was developed. This method is basically similar to the enzyme-linked immunosorbent assay which detects an antigen-antibody reaction, but instead of an enzyme being conjugated to an antibody, a DNA fragment is used and this DNA can be amplified by PCR. We applied this method to the detection of the fish pathogen, Pasteurella piscicida , in naturally infected yellowtail. Using immuno-PCR, 3.4 cfu ml⁻¹ of bacteria could be detected. In comparison, ELISA detected only 3.4 × 10⁴ cfu ml⁻¹. Immuno-PCR is a powerful method for detection of pathogens in host tissues.     

A genetic analysis of human minor histocompatibility antigens demonstrates Mendelian segregation independent of HLA

An analysis of the genetic traits of human minor histocompatibility (mH) antigens is, unlike with inbred mice, rather complicated. Moreover, the fact that mH antigens are recognized in the context of MHC molecules creates an additional complication for reliable segregation analysis. To gain insight into the mode of inheritance of the mH antigens, we relied upon a series of HLA-A2-restricted cytotoxic T-cell (CTL) clones specific for four mH antigens. To perform segregation analysis independent of HLA-A2 gene: we transfected HLA-A2-negative cells with the HLA-A2 gene: this results in the cell surface expression of the HLA-A2 gene product and, if present, mH antigen recognition. The mode of inheritance of the HLA-A2-restricted mH antigens HA-1, -2, -4, and -5 was analysed in 25 families whoese members either naturally expressed positive. Analysis of distribution of the mH antigens in the parent population among the mating types, together with their inheritance patterns in the families, demonstrated that the four mH antigens behaved as Mendelian traits, whereby each can be considered a product of a gene with two alleles, one expressing and one not expressing the detected specificity. We also showed that the loci encoding the HA-1 and HA-2 antigens are not closely linked to HLa (lod scores Z (0 = 0.05) <–4.0). Some indication was obtained that the HA-4- and HA-5-encoding loci may be losely linked to HLA. While we are aware of the limited results of this nonetheless comprehensive study, we feel the similarity in immunogenetic traits between human and mouse mH antigens is at least striking.     

Induction of cell surface expression of HLA antigens by human IFN-gamma encoded by recombinant vaccinia virus

A recombinant vaccinia virus (VV) encoding human IFN-gamma (VV-huIFN-gamma) was constructed and its effects on MHC Ag expression in human and murine cells in vitro analyzed by flow cytometry. At high multiplicities of infection (5 pfu/cell) the IFN-gamma expressed by vaccinia was not able to overcome the profound decrease of MHC concentration, normally associated with VV infection, in any of the cells tested. However, at successively decreasing multiplicities of infection, a gradual increase in MHC class I concentration above control levels was observed in human 143B cells but not in murine L929 cells, thus indicating that the species specificity of IFN-gamma is preserved in VV-huIFN-gamma-infected cells. We infer from these data that the IFN-gamma secreted by infected 143B cells is able to exert an MHC upregulating effect on uninfected cells in the vicinity. Antiviral activity of the IFN-gamma expressed by the virus was also assessed. Pretreatment for 24 to 48 h of human 143B cells with IFN-gamma containing supernatants had a significant antiviral effect comparable to rhuIFN-gamma. However, when added 1 h after virus infection, antiviral activity was much less evident. Also, the IFN-gamma secreted by infected 143B cells in monolayers infected at low multiplicity did not efficiently inhibit spread of infection to other cells in the vicinity.     

Defective MLR capacitation in the human: Low xenogeneic MLR capacity associated with HLA antigens AW25 B18, and DW2

In tests with 72 human donors, the outcome of the xenogeneic human-mouse MLR was influenced more by the general capacity of the human responder cells to react than by the MHS genotype of the mouse stimulating cells. While the response of the human lymphocytes of the same donor to the lymphocytes of six congenic mouse strains with differentH-2 haplotypes was generally rather similar, there were pronounced differences among individual human donors in the reactivity in these tests. These differences correlated with HLA, since the low reactivity was associated with the presence of the antigens HLA-AW25, HLA-B18, and HLA-DW2. The effect of the presence of antigens AW25, B18 was much more pronounced in males than in females. The low capacity to react in xenogeneic MLR in individuals with antigens HLA-AW25 and HLA-B18 may be the result of the general immune insufficiency associated with these antigens, which also includes C2 defficiency and association with immunopathological diorders.