P. Richiardi

A polymorphic variation in a putative regulation box of the TNFA promoter region.

The genes encoding tumor necrosis factor alpha (TNFA) and beta (TNFB) are located close to each other within the class III region of the HLA complex (Spies et al. 1986; Nedwin et al. 1985; Browning et al. 1993). TNF alpha is a cytokine showing a potent immunomodulator activity (Beutler and Cerami 1989); its expression is induced by bacterial lipopolysaccharide, mitogens, and viruses, and it is regulated both transcriptionally and postranscriptionally (Golfeld et al. 1990, 1991; Economou et al. 1990; Sung et al. 1991; Han et al. 1990, 1991). The level of TNFA expression has been correlated with the HLA genotype, in particular HLA-DR alleles (Bendtzen et al. 1988; Jacob et al. 1990) and with polymorphic sequences closely linked to the TNFB locus: namely, the Nco I restriction fragment length polymorphism (RFLP) in the first intron and microsatellites within the first intron or the upstream region of the TNFB gene (Pociot et al. 1993). These findings suggest that TNFA expression depends on polymorphic variations in linkage disequilibrium with the above HLA markers. The best candidate would be a polymorphic variation in the promoter region of the TNFA gene itself. A study by Messer and co-workers (1991) analyzing the TNFA promoter region from position -687 to -370 did not reveal any polymorphic sequence variation. More recently, a biallelic polymorphism has been described by Wilson and co-workers (1993), consisting of a G vs A transition at position -308. The aim of the present study was to find polymorphisms in the TNFA promoter region that could be interesting not only as genetic markers but also for their involvement in the regulation of TNFA expression. Among the possible polymorphisms, those inducing

Characterization of a murine monoclonal antibody specific for human early lymphohemopoietic cells

This paper summarizes the results of the characterization of A10, a murine monoclonal antibody which recognizes an epitope not restricted to cells of a definite lineage, but which seems to be specific for an early differentiation antigen present on precursors of circulating T and B cells. The structure recognized by the A10 monoclonal antibody, although strikingly structurally similar to the HLA-A,B,C complex, is immunologically different both from histocompatibility antigens and from beta 2 microglobulin. Furthermore, the distribution of the A10 antigen is analyzed in different cell and tissue samples, both in health and disease conditions.

Genetic and population structure of four Sardinian villages

1. Data on microgeographic population structure on four neighbouring villages of Sardinia island (Italy) are presented and discussed.

Fine Specificity And Idiotype Diversity Of The Murine Anti-hla-a2, A28 Monoclonal Antibodies Cr11–351 And Ks1

The monoclonal antibodies (MoAb) CR11–351 and KS1 are secreted by hybridomas generated with splenocytes from BALB/c mice immunized with the cultured human B lymphoid cells LG-2 (HLA-A2,A2,B27,B27). The 2 monoclonal antibodies immunoprecipitated components with a superimposable 2-dimensional gel electrophoretic profile from the cultured B lymphoid cells LG-2 and inhibited the cytotoxicity of the anti-HLA-A2,A28 T cell clone MI#3 to a similar extent. In crossinhibition experiments, the MoAb CR11–351 and KS1 completely inhibited the binding of each other to lymphoid cells with the appropriate HLA phenotype. Testing with a panel of HLA-typed lymphoid cells showed that the MoAb CR11–3 51 and KS1 display the same serologic specificity, since both of them react with HLA-A2 and/or A28 antigens bearing cells. The 2 monoclonal antibodies recognize distinct, although spatially close, determinants, since only the MoAb CR11–351 displays differential reactivity with HLA-A2 variant cell lines and reacts with HLA-A9 bearing B lymphoid cells. Analysis of MoAb CR11–351 and KS1 with syngeneic polyclonal and monoclonal antiidiotypic antibodies detected no sharing of idiotopes between the 2 monoclonal antibodies. In view of their reactivity with distinct determinants, these results are in agreement with the concept that the antigenic specificity of an antibody controls at least in part the expression of its idiotypes.

An intragenic polymorphism in the human tumor necrosis factor alpha (TNFA) chain-encoding gene.

A G-A transition was detected at position 4583 [according to the sequence number reported by Nedospasov and coworkers (1986)] in the first intron of the TNFA gene by analyzing a polymerase chain reaction (PCR)-amplified DNA fragment spanning positions 4422 and 4820. The variation was first detected by heteroduplex analysis of the PCR products in a non-denaturing 12% polyacrylamide gel. The presence of the polymorphic sequence was tested in the population by dot blot of the PCR-amplified DNA fragment hybridized with sequence-specific oligonucleotides (SSO). In a panel of 102 HLA-typed random healthy Italian individuals the following genotype frequencies were observed: GG (0.74), AG (0.24), AA (0.02). The tested population was in Hardy-Weinberg equilibrium at this locus with G and A gene frequencies 0.86 and 0.14, respectively. Linkage disequilibria between the A/G alleles and markers of the HLA region were calculated in the same panel. Class II, class I, and complement loci were considered plus three microsatellite loci in the TNF region (see Figure 1). All the significant associations concerned the less frequent A allele and are reported in Table 1. The strong association with the TNFalO allele suggests that all the observed A nucleotides at position 4583 derived from the same or few mutational events. Linkage disequilibrium decreases rapidly with increasing distances. Moreover, the A-positive individuals do not share any particular common combination of HLA markers. Thus, the less frequent A allele is not a marker of conserved HLA ancestral haplotypes.

Split of HLA-DRw2 into subtypic specificities closely correlated to two HLA-D products

Two B-lymphocyte-specific human alloantisera were studied, PA59 and 51.23. They identify two new HLA-DR alleles, which are both subtypic to HLA-DRw2. Moreover, they are closely correlated with two HLA-D products, Dw2 and tb24 (tb24 is a new specificity described by our group). Thus, DRw2 can be “split” into two subtypic specificities that have been named TO60 and TO61, which appear more strongly correlated with HLA-D antigens. Absorption studies demonstrated cytotoxicity-negative, absorption-positive (CYNAP) reactions, and cross-reactive groups of antibodies.

The HLA-DRβ16 allogenotype constitutes a risk factor for hypertrophic scarring

Nineteen patients that had developed hypertrophic scars subsequent to thermal injury were typed for HLA class II allogenotypes with the restriction fragment length polymorphism technique. A significant association was found with DR beta 16 (pc = 1.45 x 10(-4); relative risk = 12.25). This finding adds evidence to other data suggesting that immunologic phenomena are involved in pathologic scarring. Moreover, the results presented here have allowed an identification of a genetically determined risk factor for hypertrophic scar formation located in the HLA region.

HSP70-1 promoter region polymorphism tested in three autoimmune diseases

Many autoimmune diseases have been shown to be associated with genes in the HLA region. In some cases, e.g., coeliac disease (CD), insulin-dependent diabetes (IDDM), narcolepsis, the degree of association with polymorphism at a given locus is so strong (relative risks of 10 or more, presence of the marker in more than 90% of the patients) that the locus can plausibly be assumed to be involved in the pathogenesis. In other cases, the degree of association is much lower, as in the three diseases considered in the present study: systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), and multiple sclerosis (MS). The HLA alleles most significantly associated, DR3 in SLE, DR4 in RA, and DR2 in MS, show relative risks around 3 and are present, typically, in only 30%-50% of the patients. This allows the hypothesis that the described associations are due to linkage disequilibrium with an undetected polymorphism of a primarily involved linked locus. Consequently, the search for more strongly associated polymorphisms is actively pursued whenever new loci in the HLA region are identified, especially when these loci participate in the control of the immune response. The HLA genes controlling heat shock proteins

FOUR NEW HL-A ALLELIC FACTORS SUBTYPIC TO HL-A12 AND W15. THEIR CORRELATION WITH W4 AND W6

Several anti‐HL‐A antisera, correlated with either HL‐A12 or W15, were studied. Immunogenetic analysis was carried out by population and family studies, adsorptions, elutions and resistance to lysis induction capacity. Antibodies present in the sera allow the distinction to be made between two alternative factors subtypic to HL‐A12,‘TO 50’and‘TO 51′, and two alternative factors subtypic to W15,‘TO 52’and‘TO 53′. These subdivisions are genetically determined. Lysostrip experiments demonstrated them to be on the same molecule carrying either HL‐A 12 or W15. Thus, they enable the identification of new locus FOUR allelic products. These new HL‐A factors are strictly correlated with W4 and W6: haplotypic association has regularly been found between TO 50 and W4, TO 51 and W6, TO 52 and W4, TO 53 and W6. This subdivision of HL‐A allelic products into specificities subtypic either to W4 or to W6 is a characteristic common to most, if not all, FOUR factors. Finally, the commonly observed cross‐reaction between W15 and W17 preferably takes place with TO 52. It should be noted that both TO 52 and W17 are subtypic to W4.

A xenogeneic monoclonal antibody recognizing specificities controlled by hla-a and b alleles.

In the present paper one reagent among the many prepared has been carefully studied. It is a xenogeneic monoclonal antibody, F10.13/13, obtained by immunizing mice with human peripheral blood lymphocytes. The splenocytes of the immunized mice were fused with a murine myeloma and the supernatants of the resulting Ig-secreting hybridomas were tested against appropriate targets. — F10.13/13 behaves in a very peculiar manner from the serological point of view and we think that it reacts with maximal affinity with an epitope expressed most strongly on HLA glycoproteins controlled by genesB8, B7, andAw19.