Mercedes Pérez-Blas

Generation of the HLA-B35, -B5, -B16, and B15 groups of alleles studied by intron 1 and 2 sequence analysis

Abstract HLA-B is the most polymorphic of the major histocompatibility complex classical class I loci. This polymorphism is mainly in exons 2 and 3, which code for the molecule’s α1 and α2 domains and include the antigenic peptide binding site. Recent studies have indicated that not only exons but also the intron 2 region may be involved in the generation of certain HLA-B alleles such as B*3906 and B*1522. To study the degree of intron 2 participation and the mechanisms that generate polymorphism at the HLA-B locus, intron 1 and 2 sequences from the HLA-B35, -B5, -B16 and -B15 groups of alleles were obtained. A group-specific intronic polymorphism was found: namely, B*5301 shows intron 1 and 2 sequences identical to those found in all B35 alleles studied. On the other hand, B*5101 and B*52012 show the same intron 1 and 2 sequences and their intron 1 is the same as that found in the B35 group. This suggests that B5 and B35 groups of alleles may have arisen from a common ancestor. All known B16 alleles show the same introns 1 and 2, with the exception of B*39061 and B*39062, and all B15 alleles also bear the same introns 1 and 2, with the exception of B*1522. Variability at intron 1 is more restricted than at intron 2, and the use of intron 1 for HLA-B allele phylogenetic analysis is better for grouping alleles of a postulated common origin. In conclusion, there is a remarkable conservation of intronic sequences within related HLA-B alleles, which probably reflects a common origin and perhaps a selective force avoiding DNA changes. Intronic sequences are also potentially useful to design DNA typing strategies.

Impaired T cell signal transduction through CD28 in a patient with idiopathic thrombocytopenia

We describe an infant whose peripheral blood mononuclear cells were unable to proliferate or synthesize IL‐2 in response to a mitogenic combination of antibodies directed against CD2 and CD28. This peculiar defect, which has been stable to dale, was attributed to an impairment in CD28‐mediated T cell activation, because further comitogenic combinations containing anti‐CD28 monoclonals also failed to induce normal proliferation of the patients T cells. In contrast, proliferation after membrane stimulation (with anti‐CD2, recombinant IL‐2, or certain lectins) or transmembrane activation (with phorbol ester and calcium ionophore) was normal, suggesting that his lymphocytes did not have a general membrane or intracellular signalling impairment. A T cell line derived from the patient confirmed the existence of a severe defect in CD28‐mediatcd T cell proliferation, but also showed a profound impairment in CD3‐induced T cell proliferation. Other cell surface molecules like CD2 and CD25 were, in contrast, capable of transducing normal proliferation signals. As all relevant molecules were detectable by cytofluorography and immunoprecipilalion, we conclude that the patients lymphocytes had an intrinsic defect in the delivery of CD28‐mediated signals which, in the absence of monocytes, also affected CD3‐mediated proliferation. The study of this novel kind of immunodeficiency may help to unravel the complex interactions that take place among CD2. CD3 and CD28 during T cell activation. The presence of an idiopathic thrombocytopenia in the patient suggests the intriguing possibility of a role for CD28 in the maintenance of peripheral blood platelets levels, although alternative interpretations are not ruled out.

T lymphocyte anergy during acute infectious mononucleosis is restricted to the clonotypic receptor activation pathway

The transient T cell anergy associated with acute infectious mononucleosis (IM) caused by the Epstein Barr virus has been analysed in a sample of 14 IM children. Peripheral blood mononuclear cells (PBMC) obtained from IM patients showed a significant specific impairment in their proliferative response to both phytohaemagglutinin (PHA; P <0.05) and to an anti‐CD3 MoAb (P <0.001), although both responses reached normal control levels by addition of a submitogenic dose of either phorbol myristate acetate (PMA)or recombinant IL‐2 (rIL‐2). In contrast, activation signals delivered through other surface molecules (CD2, CD28) or other transmembrane pathways (PMA plus a calcium ionophore) elicited normal or high proliferative responses in most IM PBMC. In a group of live patients tested, the synthesis of IL‐2 by IM PBMC in the presence of PMA was impaired when PHA or anti‐CD3 was used as stimulus, but it reached normal levels with anti‐CD2 or ionophore. Lastly, PHA failed to induce IL‐2α receptor (IL‐2Rα) expression in IM PBMC from four tested patients, but the presence of PMA completely corrected this defect. Taken together, these results strongly suggest that the T cell anergy associated with acute IM is due lo a T cell receptor (TCR)‐specific impairment in the induction of genes involved in T cell proliferation (including those coding for IL ‐2 and IL‐2R5α) upon membrane signalling to otherwise normal T lymphocytes, since CD2, CD28 and certain transmembrane activation pathways are uncoupled from CD3 in these particular pathological conditions (and perhaps in most in vivo situations). This and other similar experimental approaches to transient secondary immunodeficiencies may help to unravel the physiopathological role of different surface molecules in T cell activation.

Coeliac- and enteropathy-associated autoantibodies in Spanish insulin-dependent diabetes mellitus patients and their relation to HLA antigens

The frequency of reticulin (ARA), endomysium (EmA), and gut epithelial cell (GECA) autoantibodies, and gliadin antibodies (AGA), was investigated in 86 Spanish diabetic patients by indirect immunofluorescence (IFI) and ELISA, along with their HLA phenotype. Four patients (5%) showed ARA-IgG (R1 pattern), eight (9%) showed AGA-IgG, and eight (9%) showed AGA-IgA. No EmA or GECA-positive patients were found. In diabetic patients, HLA-DR7 is increased in ARA-IgG+ vs. ARA-IgG- (though not significantly), and HLA-DR6 and HLA-DQ1 are significantly increased in the AGA-IgG+ group vs. the AGA-IgG- group. Comparison with a non-diabetic coeliac group showed that HLA-DR4 and HLA-DQ3 are significantly increased in the AGA-IgA+ group, whereas HLA-DQ2 shows a significant decrease in the AGA-IgG+ and AGA-IgA+ patients. Finally, when compared to the healthy group, HLA-DR7 frequency is decreased in the ARA-IgG- group, while HLA-DQ3 is significantly increased and HLA-DR6 and HLA-DQ1 significantly decreased in the AGA-IgG- group.Altogether, these data suggest that the genetic background leading to the appearance of coeliac-specific autoantibodies in Spanish diabetic patients differ depending on the autoantibody produced and is also different to the genetic background leading to diabetes in Spain.

Description of HLA-A*6803 and A*68N in Mazatecan Indians from Mexico.

HLA-A2 and A28 are two of the most frequent HLA-A serologically defined antigens in the Mestizo and Indian Mexican populations (Weckmann et al. 1997, and unpublished data). In the present study, two new HLA-A*68 subtypes present in two unrelated individuals from the Mazatecan ethnic group were characterized and seen to belong to the A2/A28 family of antigens. They were also compared with other A*68 subtypes in order to define the possible mechanisms involved in the generation of these alleles. The Mazatecan Indians who inhabit Northern Oaxaca State in Mexico and some areas of Veracruz State are linguistically classified within the Macro-Mixtecan family, and they came originally from Eastern Mexico, as was recorded in 890 AD. Genomic DNA was isolated from peripheral blood lymphocytes using standard methods. A 5 9 HLA-A-specific primer (59AE1c: CAGACGCCGAGGATGGC) and a 3 9 HLA-A-specific primer (39Ai3c: GATCAGGGAGGCGCCCCG) were used in a polymerase chain reaction in order to obtain intron 1, exon 2, intron 2, and exon 3 sequences. DNA sequences were obtained as detailed by ArnaizVillena and co-workers (1992). The primers used for the sequencing process were ASEQ3 (Blasczyk et al. 1996) and ABSEQ5: CACAGTCTCCGGGTCCGA (local designation). Complete sequences of intron 1, exon 2, intron 2, and exon 3 from A*6803 and A*68N are shown in Fig. 1 together with those of HLAA*0204 and HLA-A*68012. HLA-A*0204 and HLA-A*68012 from Mazatecan individuals lymphocytes were also sequenced in our laboratory. TheHLA-A*68012allele has a one-base difference at codon 70 (CAG?CAC) when compared withHLA-A*6803(Fig. 1); this change results in an amino acid substitution (Gln ?His) with the corresponding neutral to positive change in polarity . Position 70 is situated at the α-helix of the first domain in the HLA molecule peptide binding site (Bjorkman et al. 1987). The residue interacts with the P2-binding pocket (B pocket) forming a salt bridge with Asp-74 (Madden et al. 1992). Analysis of non-coding regions (introns 1 and 2) of A*68012 andA*6803shows they are identical (Fig. 1). This suggests that one of them could have been generated from the other by a point mutation or by a gene conversion event that involved alleles like A2 or A24 (histidine at codon 70) (Arnett et al. 1995), which are highly frequent in Amerindians. On the other hand, theHLA-A*68N allele presents an identical intron 1, exon 2, intron 2, and exon 3 to those of HLA-A*6803except for a DNA stretch that includes the 3 9 end of intron 1 and the 5 9 end of exon 2 (from base 124 at intron 1 to codon 2 at exon 2) (see Fig. 1). A C?T change at position 124 is observed, whereas at exon 2-codon 2 a TCC?TCT synonymous change (Ser) is shown (Fig. 1). Serine at position 2 is placed on the β-sheet at the bottom of the peptide binding site. This residue is invariant in class I molecules and is involved in the α1 (position 2) andα2 (position 104) domain interactions (Bjorkman et al. 1987). Interestingly, the fragment inserted in A*68N is present in certainHLA-A2 alleles (A*0201, Cereb et al. 1996; A*0204, present work; A*0205 and A*0206, unpublished results). This fact suggests that A*68N may have been originated from A*6803which could have received a donation from this segment belonging to an HLA-A2 allele (perhapsA*0204 which is also detected in Mazatecans). Pathogen-driven polymorphism may explain the evolutive force generatingA*6803, because the change that is indicated with respect to A*68012 is productive and placed in the peptide binding region; the new AmerindianHLA-B proteins most probably diversified in the century after 1492, when 60–80 000 000 Amerindians were killed by measles, small pox, and influenza viruses introduced by European invaders (Dobbins 1993). However, A*68N andA*6803, which present a synonymous change at codon 2-exon 2, may have similar peptide presenting repertoires and the generation of one from the other may be better explained by a recombination or conversion event that involved intron 1 and exon 2 segments; this would be the first recorded example amongHLA class I or II genes not involving intron 2 and exon 3 segments (Martinez-Laso et al. 1995; Vargas-Alarco ́n et al. 1997).

Blau syndrome-related CARD15/NOD2 mutations are not linked to idiopathic uveitis in Spanish patients.

Uveitis is a clinical feature of the Blau syndrome, a disease linked to CARD15 (also referred to as NOD2) mutations. Three main mutations in this gene (R334W, R334Q and L469F) have been reported as Blau syndrome risk factors, a disease that manifests uveitis as one of its clinical features. However, little is known on the involvement of this gene in idiopathic uveitis. We thus sought to determine the frequency of these Blau-related CARD15 mutations in a cohort of Spanish patients with idiopathic uveitis. To this aim, 110 patients with idiopathic uveitis, followed at the Department of Ophtalmology of a tertiary hospital (Hospital Universitario Alcalá de Henares, Madrid. Spain) were enrolled. As a control population, 104 healthy subjects were used. DNA was extracted from blood samples and the Blau-related CARD15 mutations were analysed either by PCR-RFLP or direct DNA sequencing. None of the mutations studied was found in any of the individuals tested, whether diseased or healthy. It seems thus that Blau syndrome-related CARD15 mutations are not involved in idiopathic uveitis, a finding which allows us to suggest that the genetic aetiology of the idiopathic uveitis or the Blau-associated uveitis is different.

Increased levels of bovine serum albumin antibodies in patients with type 1 diabetes and celiac disease-related antibodies.

Objectives To detect the presence of antibodies against bovine serum albumin in a cohort of Spanish patients with type 1 insulin-dependent diabetes. Methods Antibodies were measured using an in-house enzyme-linked immunosorbent assay test in 80 patients with type 1 diabetes, subdivided according to the presence or absence in their serum of celiac disease-related antibodies. For comparison, 30 patients with celiac disease (nondiabetic), 13 patients with autoimmune thyroiditis, and 45 healthy volunteers were used. Results Thirty-one percent of patients with diabetes yielded a positive result, with a mean value of 26.1 ± 21.8 arbitrary units (AU). If the group was split into those with celiac disease-related antibodies and those lacking them, the percentages were 53% and 25%, respectively, with a mean value of 39.6 ± 28.4 AU and 22.4 ± 18.3 AU (P = 0.003), respectively. Seventy-three percent of celiac patients showed bovine serum albumin antibodies with a mean level of 38.8 ± 27.7 AU, comparable to that of patients with diabetes with celiac antibodies, but higher than the group lacking them (P = 0.001). Although 46% of patients with autoimmune thyroiditis had positive results, the level detected (22.1 ± 8.7 AU) was significantly lower than that recorded in patients with type 1 diabetes who had celiac disease antibodies (P = 0.04) and celiac patients (P = 0.04). Healthy volunteers showed no antibodies against bovine serum albumin. Conclusions These data suggest that bovine serum albumin antibodies appears in patients with a compromised epithelial permeability, and they reflect a general defect in the process of immunologic tolerance associated with a predisposition to autoimmunity, rather than immunity specific to &bgr; cells.

Higher proliferative capacity of T lymphocytes from patients with Crohn disease than from ulcerative colitis is disclosed by use of Herpesvirus saimiri-transformed T-cell lines

Background: T lymphocytes play a crucial role in the pathogenesis of inflammatory bowel disease. Achieving stable T-cell lines, rather than continuous bleeding of patients, is desirable in order to dissect their implication in the disease. Methods: Long-lasting T-cell lines from patients with Crohn disease and ulcerative colitis and from healthy volunteers have been obtained by transformation of T lymphocytes using the lymphotropic Herpesvirus saimiri. Lines were subjected to phenotypic and functional analyses, and the results compared with freshly isolated peripheral blood mononuclear cells. Results: Fresh cells revealed only minor differences between patients and controls, with regard to phenotype and proliferative capacity. In contrast, the use of T-cell lines showed that cells from Crohn disease patients, but not ulcerative colitis patients, over-responded to several membrane or cytoplasmic stimuli when compared to control T-cell lines. Thus, higher responses were found when stimulated with αCD3 and IL2, αCD3 and αCD28, IL2 alone, phorbol esters (PMA) and αCD3 and, finally, PMA and αCD2 (P < 0.05 in all instances). Further, lines from patients with Crohn disease responded more vigorously to αCD3 and αCD28 or αCD3 and PMA when compared to ulcerative colitis (P < 0.05 in both instances). Conclusions: The data obtained with these lines suggest that T cells from patients with Crohn disease differ in vivo in their proliferative capacity, as compared with those from ulcerative colitis patients, a finding that may reflect the clear Th-1 phenotype found in the former and absent in the latter.

Herpesvirus saimiri (HVS)-transformed T-cell lines: A method to study mucosal T cells in inflammatory bowel disease

Alterations in the mechanisms regulating the immune response are key elements in inflammatory bowel disease (IBD, comprising Crohn’s disease (CD) and ulcerative colitis (UC)). Although the precise aetiology remains unknown, T lymphocytes play a crucial role in its pathogenesis. Initially, descriptions of the immunological features in IBD focused on peripheral blood lymphocytes. Since these data do not necessarily reflect the situation at the mucosal milieu, it is important to assess the phenotypic profile and activation state of immune cells at this level. We have used a common lymphotropic virus of squirrel monkeys, Herpesvirus saimiri (HVS), to immortalize T cells of intestinal mucosal origin from patients and healthy individuals as controls. HVS-transformed T lymphocytes become antigenand mitogen-independent for their continuous growth and upon stimulation with membrane or transmembrane stimuli, these cells show normal downstream functional responses [1,2].

Herpesvirus saimiri transformation may help disclose inherent functional defects of mucosal T lymphocytes in patients with gastric adenocarcinoma

To dissect the phenotypic and functional features of mucosal T lymphocytes in patients with gastric adenocarcinoma, we have used the Herpesvirus saimiri transformation procedure to achieve T‐cell lines from gastric origin. Once achieved, cell function was assessed by in vitro stimulation with mitogens. CD2‐specific monoclonal antibodies (α‐CD2), alone or in combination with interleukin (IL)‐2, rendered fewer counts in patients (34 408±3965 and 52 157±6473 c.p.m., respectively) than in controls (67 471±11 755 c.p.m., P<0.01 and 77 864±12 545 c.p.m., P<0.05, respectively). Likewise, CD3‐based responses were defective in cancer cell lines: α‐CD3 (54 794±9269 vs 86 104±10 341 c.p.m., P<0.01), α‐CD3+IL‐2 (57 789±8590 vs 88855±8516 c.p.m., P<0.01) and α‐CD3+α‐CD2 (52 130±7559 vs 120 852±16 552 c.p.m., P<0.01). Finally, IL‐2 failed to adequately stimulate patient cell lines (39 310±4023 vs 60 945±9463 c.p.m., P<0.05). These results suggest that mucosal T lymphocytes in cancer patients are inherently impaired in their proliferative ability. This may be crucial in the control of tumour growth.