Biomedical subjects
Publications and source records attributed to J L Vicario.
A novel A*6836 allele was completely characterized by sequence-based typing (SBT) in a cord blood sample from an Ecuadorian donor. A*6836 discloses six clustered amino acid residue changes at the alpha-1 domain, codons 76-83, regarding its closer A*680102 allele. Therefore, A*6836 would be a new human leukocyte antigen (HLA)-A molecule showing a Bw4-epitope.
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The new human leukocyte antigen (HLA)-C allele Cw*0742 was identified by sequence-based typing in a Caucasian bone marrow donor. HLA-Cw*0742 differs from Cw*070201 by a single nucleotide position at codon 113 (TAT --> TTT), giving rise to an amino acid substitution from Tyr to Phe.
We present here the characterization of the complete coding sequences, previously unavailable, of the human leukocyte antigen (HLA) alleles B*0707, B*1524, B*4405, B*4802, DRB1*0409, DRB1*0411, DRB1*1115, DRB1*1305, and that of a new allele, DRB1*0709. For the isolation of cDNA from the DRB1 gene, we designed a novel set of polymerase chain reaction (PCR) primers that makes it possible to amplify separately the groups of DRB1 alleles associated to each of the DRB3 and DRB4 loci. The primary structures, functional features, evolutionary relationships, haplotypic associations, and population distributions of each of the nine HLA-B and -DRB1 alleles reported here are reviewed.
Four new HLA classical class I alleles in the three loci are described in Caucasian individuals. A*3012 was first suspected by an abnormal serologic pattern that would be explained by the single amino acid substitution at the A30-specific Ser17. B*270505 differs from B*270502 in a silent substitution at an up to now constant position in the B locus. B*3541 encodes for a new Cys at position 118 that has not been encountered in neither human nor primate alleles. Cw*0716 seems to be originated by a large-scale interallelic recombination event between Cw*0701/*0706/*0718 and Cw*020202, giving rise to a new antigen-binding cleft conformation.
The insertion of suicide genes in donor T lymphocytes constitutes the basis of new approaches aiming at the treatment of the graft-versus-host disease (GVHD), a frequent complication in recipients of allogeneic haematopoietic grafts. In this study we investigated the impact that the ex vivo manipulation required for the retroviral transduction of T cells had on the functionality and differentiation of these cells. Compared to fresh T cells, samples that had been subjected to standard activation (1 microg/ml of both anti-CD3i and anti-CD28i MoAbs) followed by transduction with vectors encoding for the HSV-tk and tNGFR genes maintained the proliferative response to an allogeneic stimulus. These cells, however, had a significantly lower cytotoxic response to allogeneic cells compared to fresh samples. When the concentration of anti-CD3i was reduced to up to 1000-fold (1 ng/ml), similar T-cell transductions were obtained, while the cytotoxicity of the ex vivo manipulated samples was significantly recovered, when assessed either at 7 or 14 days of culture. In all instances, a similar functionality was observed in transduced samples not subjected to immunomagnetic cell sorting, compared to purified fractions enriched in NGFR(+) and NFGR(-) cells. The analysis of CD45RA and CCR7 markers in samples transduced under standard stimulatory conditions showed a differentiation of fresh CD8(+) CD45RA(+)/CCR7(+) naive cells to cells having a predominant central CD45RA(-)/CCR7(+) and effector CD45RA(-)/CCR7(-) memory phenotype. However, when samples were activated with low doses of anti-CD3i, a significant population of naive cells became apparent. Although activation with high doses of anti-CD3i/anti-CD28i resulted in a similar phenotype in both NGFR(+) and NFGR(-) populations, the naive population observed in samples activated with low concentrations of anti-CD3i was almost restricted to the NGFR(-) population. These results show that reducing the stimulation mediated by anti-CD3i in protocols of T-cell retroviral gene transfer significantly helps to preserve the cytotoxic capacity of these cells to allogeneic cells, without affecting the susceptibility of these cells to the retroviral vector. In addition, we observed that modulating the activation of transduced T cells implies the generation of changes in the differentiation of CD8(+) cells, although we could not establish a direct relationship between the CD45RA/CCR7 phenotype of these cells and their cytotoxic reactivity to an allogeneic stimulus.
Two novel human leukocyte antigen (HLA) class II alleles for DRB3 and DQB1 genes detected in Caucasoid Spanish individuals are described: DRB3*0218 and DQB1*030202. Both alleles have been found during routine high-resolution typing by sequencing. DRB3*0218 shows a novel DRB3 gene polymorphic position, located at amino acid residue 58, alanine to glutamic acid. This residue is shared by several DRB1 alleles, including all described DRB1*11 subtypes. DQB1*030202 differs from DQB1*030201 by a point mutation at position 319 (T to C). This nucleotide change generates a new codon at amino acid position 75 that is not shared by any other DQB1 allele.
The hyperferritinemia-cataract syndrome, inherited as a Mendelian dominant trait, is due to mutations in the 5' non-coding region of the ferritin light chain gene that modifies the shape of the IRE (iron responsive element) region, which loses its normal function of regulating the synthesis of ferritin light chains. Excess of light chains results in complexes that accumulate into the lens giving rise to early cataracts. We present a Spanish family with seven affected members through three generations. A genetic study reveals a substitution of a single base (C-->T) at position 33 in the IRE sequence in the index case and in one affected brother, whereas a non-affected sister shows the normal sequence. The hyperferritinemia-cataract syndrome was identified in 1995 and is still poorly understood. Clinicians should suspect it when treating any subject with early cataracts, even more if they are familial, or in patients with very high levels of ferritinemia without evidence of iron overload. There are no known consequences of the syndrome other than cataracts, and its proper diagnosis carries a favorable prognosis and eliminates the risk of unnecessary phlebotomies.
Two new HLA class I alleles have been recognised by molecular-based typing. B*3805 was initially identified by polymerase chain reaction using sequence-specific primers (PCR-SSP) and afterwards confirmed by sequencing based typing (SBT) studies in a Spanish Caucasian blood cord unit. A unique nucleotide change throughout exons 2, 3 and 4, leading to the amino acid replacement Ser11Ala, differentiates B*3801 and *3805. This position behaves as a dimorphic residue in HLA-B and -C loci, and seems to be structurally unrelated to peptide and TcR recognition. Cw*0408 was first detected by SBT in two African American bone marrow donors in combination with its most structurally related allele, Cw*04011. The single amino acid change found between Cw*04011 and Cw*0408 was Thr163Leu, a residue involved in pocket A of the peptide-binding cleft. This new allele could be the result of a gene conversion event between Cw*04011 and any of the Cw*03 alleles.
The development of molecular techniques for HLA typing has allowed the identification of genes previously assigned as serologic blank alleles. Lack or poor cell surface expression has been found for molecules coded by HLA-A, -B, -DRB4, -DRB5, and -DPB1 genes. In this report we describe the first HLA-C gene encoding for a null cell surface molecule. HLA-Cw*0409 N shows a point deletion at position 1095 within exon 7. This mutation provokes a codon reading shift, generating a new translation stop codon 97 bp downstream to that described in alleles normally expressed. This new stop codon location implies the presence of 32 extra amino acid residues in the cytoplasmic domain. Transfection experiments suggest that elongation of the cytoplasmic domain in Cw*0409 N would be the cause of cell surface expression failure, although Cw*0409 N heavy chain is able to create stable complexes with beta2-microglobulin. HLA-C fragment length analysis in a small selected group of samples with B44-Cblk haplotypic associations allowed us to identify two additional subjects showing both a serologic silent Cw*04 allele and a point base deletion at the 3' end of the HLA-C gene. This finding indicates that the allele frequency of Cw*0409 N within serologic C blank alleles would be appreciable, although basically restricted to the (A23)-Cw*0409 N-B*4403-DR7-DQ2 haplotype.
OBJECTIVE: To determine whether the presence of radiographic erosions at disease onset in patients with early rheumatoid arthritis (RA) is associated with clinical, serological, or genetic factors of poor outcome and whether patients with erosions only in the feet have a different pattern of presentation. METHODS: Sixty one patients with early RA (<6 months of evolution) were studied. Clinical evaluation and serological, radiological, and genetic studies were performed at disease onset and after one year. RESULTS: Forty one (67%) patients showed erosions in their hands or in their feet, or in both. Subjects with erosive RA had a higher number of swollen joints (SJN; 9 (SD 6) v. 6 (3), p=0.008), and rheumatoid factor (RF) positivity was more common (80% v. 50%, p<0.02) than those without erosions. Seven (17%) of the 41 patients in the group with erosions had erosions only in their feet. This group had a longer duration of morning stiffness (120 (60) v. 72 (52) min, p<0.005), better patient's global assessment of general health (34 (22) v. 57 (25), p< 0.05), and lower erythrocyte sedimentation rate (32 (22) v. 60 (30) mm/1st h, p <0.05) than the rest of the subjects with erosions, and none of them was in remission after one year. Remission after one year was related to a lack of cortical damage at onset and RF negativity. CONCLUSIONS: Radiological damage at disease onset is associated with a worse clinical presentation and RF positivity, which are markers of poor outcome. There is a subgroup of patients, with erosions only in their feet, whose clinical presentation is less aggressive. To identify these cases of early erosive RA, radiographs of the feet should be obtained routinely.
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A procedure for performing stereocontrolled aziridine ring-opening reactions with chiral enolates derived from (S,S)-(+)-pseudoephedrine amides has been developed leading to gamma-aminoamides in good yields. The diastereoselectivity of the reaction becomes controlled by the presence of the chiral auxiliary on the enolate, although the stereogenic center contained in the structure of the aziridine has a striking influence on the stereochemical course of the reaction which results in the presence of the corresponding matched and mismatched combinations. Besides, the sense of the asymmetric induction of the chiral auxiliary has resulted to be the opposite to the one found with other type of electrophiles, although it is in good agreement with the trend observed in the reaction of the same kind of enolates with epoxides. Finally, the obtained gamma-aminoamide adducts were converted into enantiopure gamma-amino acids, gamma-aminoesters, and pyrrolidin-2-ones using easy to perform and high yielding reactions.
[reaction: see text]. The reaction of an (S,S)-(+)-pseudoephedrine acetamide based enolate with several imines afforded smoothly and with full stereochemical control a series of beta-substituted alpha-methyl-beta-aminoamides that upon hydrolysis/esterification afforded the corresponding beta-aminoester derivatives in good yields and in almost enantiomerically pure form.
HLA-C has been described as a transplantation locus in the unrelated bone marrow transplantation setting, and noticeably the number of mismatches between HLA-A,-B,-DRB1 compatible pairs is considerably high. Sequencing based typing (SBT) is an accurate and efficient methodology utilised in the HLA class I and II allele level of resolution. SBT for HLA-C locus was applied on a sample of 40 HLA-A,B,DRB1,DRB3/4/5,DQB1-compatible bone marrow recipient-donor pairs, and 3 new HLA-C alleles have been found. Cw*0307, well defined by serology as Cw3, showed two amino acid changes at the NK motif 77-80 regarding all described Cw*03 alleles, N77K80 instead of S77N80. Two new Cw*05 alleles were described, Cw*0502 properly typed by serology, and Cw*0504 that behaves as a short antigen. Cw*0502 differed from Cw*0501 by only one nucleotide at exon 3, that generated an amino acid replacement at codon 177, K to E. Cw*0504 differs from Cw*0501 by two clustered amino acid positions (114 and 116) placed at the peptide binding site. The rate of new HLA-C alleles found in this small series evidences a high grade of hidden HLA-C diversity in the Spanish population, particularly in the well-defined serologic specificities.
Between February 1995 and July 1999 25 pediatric patients (8 months to 14 years old) underwent peripheral blood stem cell transplantation (PBSCT) from an HLA-identical sibling donor. Diagnoses included ALL (17), non-ALL (6), and non-malignant disease (2). GVHD prophylaxis consisted of cyclosporine plus methotrexate (15), only cyclosporine (8), cyclosporine plus prednisone (1), or nothing (1). All donors (6 months to 41 years old) received G-CSF at 10 microg/kg/day subcutaneously for 4-5 days and on day 5 underwent large volume leukapheresis. Median number of CD34(+) and CD3(+) cells collected and infused was 6.9 x 10(6) (range 2.5-32.8) and 4.5 x 10(8) (0.5-22.1) per kg of recipient body weight respectively. Median time to achieve ANC >0.5 x 10(9)/l and platelets >20 x 10(9)/l was 10 and 12 days, respectively. Acute GVHD grade > or =II developed in 10 of 24 evaluable patients (42%). Probability of acute GVHD was 62%. Median time to discharge was 25 days (range 14-52). Among 20 evaluable patients, five (25%) developed chronic GVHD at day 100. Probability of chronic GVHD was 29% after 1 year post PBSC. At a median follow-up of 558 (9-2071) days, overall survival for the whole group is 68%. Probabilities of event-free survival, overall survival and relapse for patients with malignant hematological diseases are 53%, 59% and 24% at 5 years, respectively. This study has confirmed the feasibility and safety of mobilization and collection of PBSC products and the applicability of this procedure to the pediatric population, both donors and recipients. Studies including larger numbers of pediatric patients undergoing allogeneic PBSCT are warranted to determine the long-term outcomes of such procedures.
In this paper we describe the clinical and molecular features of a new case (GOR) of homozygous human TAP2 deficiency, analysing the phenotype and function of NK cells. The patient presented from infancy with recurrent sinopulmonary infections; a selective IgG2 deficiency, negative antibody response to polysaccharide vaccination and low level of cell surface expression of HLA class I antigens were found. The sequence of TAP2 gene identified a single mutation, a C to T substitution changing the CGA arg codon at amino acid 220 into TGA stop codon in exon 3. By using MoAbs for KIRs, CD94, CD94/NKG2A and ILT2 we observed, in agreement with others, that the latter two receptors were overexpressed on TAP2-deficient NK cells. The inhibitory CD94/NKG2A and triggering CD94/NKG2C NK receptors, specific for HLA-E, appeared to be functional in a limited number of NK clones that could be expanded in vitro. Expression of HLA-E was virtually undetectable in GOR B-LCL and very faint in PBMC, further supporting that interactions of class I leader sequence nonamers with HLA-E in the ER depend on a functional TAP complex.