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Biomedical subjects

M Timón

Publications and source records attributed to M Timón.

18 recordsLinked to original sources
PubMed2004

DNA immunisation with minimalistic expression constructs.

The low efficacy obtained in large animals makes plasmid-based DNA vaccines commercially unviable. Another concern is the presence of antibiotic resistance markers on virtually all conventional plasmids. Here we describe the use of minimalistic, immunogenically defined gene expression (MIDGE) vectors for DNA vaccination. MIDGE are linear, covalently-closed vectors containing all the essential information for gene expression and none of the non-essential and potentially dangerous plasmid backbone sequences. MIDGE vectors can also be chemically modified on both ends at defined positions allowing targeting of the DNA to specific cell types or cellular compartments. Immunisation of mice with simple and end-modified MIDGE vectors showed that they are efficacious tools to generate and/or manipulate antigen-specific immune responses.

Animals↗
PubMed2002

DNA vaccination with linear minimalistic (MIDGE) vectors confers protection against Leishmania major infection in mice.

Immunization protocols based on priming with plasmid DNA and boosting with recombinants of vaccinia virus (rVV) encoding the same antigen offer great promise for the prevention and treatment of many parasitic and viral infections for which conventional vaccination has little or no effect. To overcome some of the potential problems associated to the use of plasmids, we have developed minimalistic, immunogenically defined, gene expression (MIDGE((R))) vectors. These linear vectors contain only the minimum sequence required for gene expression and can be chemically modified to increase the immune response. Here, we demonstrate that MIDGE vectors coding for the LACK antigen confer a highly effective protection against Leishmania infection in susceptible Balb/c mice. Protection is achieved at lower doses of vector compared to conventional plasmids. This efficacy could be greatly improved by the addition of a nuclear localization signal (NLS) peptide to the end of the MIDGE vector. In fact, immunization with two doses of NLS-modified MIDGE conferred similar or even better protection than that achieved by priming with plasmid DNA followed by boosting with rVV. These results demonstrate that MIDGE vectors are a good alternative to plasmid and rVV for immunization.

Animals↗
PubMed2001

Structural and functional analysis of the human CD45 gene (PTPRC) upstream region: evidence for a functional promoter within the first intron of the gene.

Expression of the leucocyte common antigen (CD45) in mammals is restricted to the nucleated lineages of haematopoietic cells. It appears in early progenitors in the bone marrow and is expressed at the surface of these cells throughout their differentiation. However, at least in T cells, the pattern of expression switches between different isoforms during the successive stages of differentiation in the thymus and after activation in the periphery. In order to understand the mechanisms controlling the transcription of the human CD45 gene, 2.7 kbp of the 5'-flanking region were sequenced and analysed for their ability to direct expression of a reporter gene. The only region with promoter activity was localized within the first intron of the gene. This promoter shows no tissue specificity but could be enhanced by a heterologous enhancer. Mobility shift assays showed complex but specific protein binding. The sequence in this region lacks similarity with known promoters or initiators but is highly conserved in evolution. No transcription initiation could be detected within or downstream of this region, suggesting that this might be a new type of RNA polymerase II promoter able to drive transcription from an upstream sequence. An additional exon was also found upstream of exon 1. The two exons 1 (1a and 1b) are mutually exclusive and both are spliced to exon 2. This makes the structure of the 5' region of the human CD45 gene identical to its mouse homologue.

Animals↗
PubMed2000

Genomic structure and sequence of the leukocyte common antigen (CD45) from the pufferfish Fugu rubripes and comparison with its mammalian homologue.

The leukocyte common antigen (CD45) is a transmembrane protein tyrosine phosphatase expressed only in nucleated hematopoietic cells. It can be expressed as different isoforms depending on the cell type and the state of activation or differentiation and it is known to play a crucial role in the maturation and differentiation of B and T lymphocytes. However, the regulation of CD45 expression and function has been difficult to study due to the complexity of the gene in mammals. In this paper, we report the isolation and characterization of a CD45 orthologue gene from the Japanese pufferfish Fugu rubripes (Fugu). The Fugu CD45 cDNA sequence contains an open reading frame of 1,246 amino acids with a variable extracellular region as a result of the alternative splicing of two exons. The intracellular region is organized into two highly conserved tyrosine phosphatase domains. The extracellular region is not conserved except in some structural domains. The Fugu CD45 gene has a similar exon/intron organization to that of mammals except in the 5' end where some exons are missing or fused together. By contrast, the gene is ten times smaller in Fugu due to the small size of the introns. These studies show a greater flexibility to evolve at the 5' end of the gene and provide clues to the functionally important domains of the molecule. In addition, the lower complexity of this gene in Fugu should allow easier mapping of its regulatory sequences.

Amino Acid Sequence↗
PubMed1998

A point mutation within CD45 exon A is the cause of variant CD45RA splicing in humans.

The leukocyte common antigen (CD45) is alternatively spliced, generating various isoforms expressed on hemopoietic cells. The splicing pattern of CD45 in T cells is altered in some individuals who show abnormal expression of high molecular weight isoforms containing exon A. The variant splicing pattern was shown to be associated with heterozygosity for a silent point mutation within CD45 exon A. This C to G transition is located 77 nucleotides downstream of the splice acceptor junction of exon A (198 bp total length). Here we report that this mutation is the cause of abnormal splicing. To isolate the mutant gene, somatic cell hybrids of lymphocytes with a CD45 splicing defect and a mouse lymphoid line were produced and clones expressing different isoforms of CD45 were isolated. Expression of the high molecular weight isoform containing exon A was associated with the mutation within exon A. All hybrids expressing the low molecular weight isoforms lacking exon A contained the normal allele of CD45 only. In addition, minigenes including this mutation were constructed and transfected into various cell lines (COS-7, HeLa, CHO). Semi-quantitative reverse transcription polymerase chain reaction showed an increase of more than tenfold in splicing to CD45RA (concomitant with a decrease in splicing to CD45RO) when compared with the normal minigene. Taken together, these results demonstrate a causal relationship between the mutation in CD45 exon A and the variant splicing pattern observed. The involvement of trans-acting splicing factors that interact with this region of CD45 pre-mRNA is currently under investigation.

Alleles↗
PubMed1996

Herpes virus saimiri transformation of T cells in CD3 gamma immunodeficiency: phenotypic and functional characterization.

The characterization of T cell immunodeficiencies could in part be supported by using stable cell lines in which biochemical and molecular studies of the defect could be carried out thereby omitting frequent bleeding of patients. First attempts to obtain such cell lines included HTLV-I transformation and exogenous IL-2 administration, but both models have important disadvantages. Recently, a virus isolated from the squirrel monkey, Herpes virus saimiri (HVS), has been reported to have the ability to transform T cells. A stable IL-2-dependent HVS-transformed T cell line from a CD3 gamma deficient patient has been obtained; and this cell line displays both the phenotypic and the functional characteristics of the patient's lymphocytes. Moreover, the line down-modulates TCR/CD3 surface expression upon CD3 engagement, as do the patient's lymphocytes, showing that CD3 gamma and its phosphorylation are not necessary for TCR/CD3 internalization. In addition, the abnormal staining pattern of different anti-TCR/CD3 monoclonal antibodies is preserved in the HVS-patient line. Since HVS is capable of transforming CD3 gamma- T cells, the CD3 gamma chain does not seem to be involved in the HVS receptor process. The fact that it is not possible to obtain a CD8+ HVS line from the CD3 gamma- patient supports the existence of a functional anomaly in his scanty CD8+ peripheral lymphocytes. Thus, HVS transformation is a suitable model for T cell immunodeficiency studies and characterization. It may also be used in the future in cellular models for in vitro gene therapy trials.

CD3 Complex↗
PubMed1996

Diploid expression of human leukocyte antigen class I and class II molecules on spermatozoa and their cyclic inverse correlation with inhibin concentration.

A diploid expression of class I and class II human leukocyte antigens (HLA) has been found in purified spermatozoa by using double fluorescence labeling cytofluorometry and relevant monoclonal antibodies; this expression has been confirmed for the first time by the analysis of specific HLA mRNA and metabolic 35S labeling followed by immunoprecipitation, which demonstrates an active ongoing translation of HLA proteins in germinal cells. Long-living mRNA coming from diploid germinal cells may be translated to HLA molecules in spermatozoa. This translation is controlled (or at least inversely correlated) by a testicular hormone (inhibin) in a cyclic fashion. Remarkably, serum levels of inhibin, synthesized by Leydig and Sertoli cells, follow a 12- to 13-day cycle, with a peak level at Day 6; this is probably controlled by FSH (not cyclic in males) and other testicular and/or unknown hormones. Peak levels of inhibin concur with the lower density and percentage of spermatozoa expressing both HLA class I and II molecules (close to 3% by cytofluorometry); lowest levels of inhibin coincide with the highest numbers (35-40%) of spermatozoa positive for both HLA molecules and a higher surface density. These observations could put to an end a disconcerting and long-lasting controversy on the expression/non-expression of HLA antigens on spermatozoa. The possibility that HLA-bearing spermatozoa are more capacitated for fertilization than those that do not bear HLA, and the implications of our results on male fertility control are also discussed.

Antibodies, Monoclonal↗
PubMed1994

Peripheral blood reduction of memory (CD29+, CD45RO+, and "bright" CD2+ and LFA-1+) T lymphocytes in Papillon-Lefèvre syndrome.

A Papillon-Lefèvre patient with characteristic chronic periodontal disease and palmoplantar keratoderma was studied over a 4-year period. An abnormal T-cell phenotype was steadily observed in peripheral blood; both low numbers of CD29+ and CD45RO+ cells and a low density surface expression of CD2 and LFA-1 molecules were found. T-cell activation through CD3, CD2 and ConA, PWM and IL-2 receptors was normal; however, there was impairment in the activation via CD28. CD2, LFA-1 and CD45 molecules were normal in charge and molecular weight. There was no tissue sequestering of T lymphocytes in periodontal lesions, but rather a relative T-cell reduction. It is suggested that an important decrease of the so-called "memory/hyperreactive" (CD45RO-positive) T cells does exist; therefore, hyperreactive T cells would not be available in sufficient numbers to leave the bloodstream through blood vessel endothelium, and the periodontium would be left without these important defenses and thus exposed to chronic infections. A disregulated factor affecting the transition from "naive" to "memory" T cells and the increase in certain surface molecules expression (i.e., CD2, LFA-1, CD29, and CD45RO) or the reversion from memory to naive T cells may be responsible for the disease pathogenesis. CD2 and LFA-1 molecule synthesis might be conjointly regulated on T lymphocytes.

Adolescent↗
PubMed1994

Primary T lymphocyte immunodeficiency associated with a selective impairment of CD2, CD3, CD43 (but not CD28)-mediated signal transduction.

A 2-year-old female with important signs of immune response failure against virus, bacteria, fungi and protozoa and no obvious humoral or lymphocyte phenotypical defect was studied. Both peripheral blood mononuclear cells and IL-2-dependent T cell lines derived from the patient showed a severe selective T cell activation impairment via CD2, CD3 and CD43; however, this defect was reversible with the addition of either IL-2, or phorbol myristate acetate (PMA) or anti-CD28 antibodies. Concordantly, the induction of IL-2 (and, in part, IL-3 and IL-4) messenger RNA was severely reduced in stimulated T cells, but that of other cytokines was either normal (IL-5) or only slightly diminished (interferon-gamma (IFN-gamma)). It is concluded that an activation T cell defect exists previous to protein kinase C (PKC) and between membrane receptors and the activation pathway of certain response genes encoding for interleukins involved in proliferation (i.e. IL-2, IL-3 and IL-4), but not of others (i.e. IL-5). The use of T cell lines from human T lymphocyte activation deficiencies allows dissection of T cell pathology and the corresponding physiological pathways. In the present description, there is an evident independence of the CD28 T cell activation pathway from those induced through CD2 or CD3, and the differential gene regulation of the different interleukins.

Antigens, CD↗
PubMed1993

Selective disbalances of peripheral blood T lymphocyte subsets in human CD3 gamma deficiency.

The selection of T lymphocytes in the thymus and their activation upon the encounter with foreign antigens in the periphery require the aggregation and signals of the T cell receptor (TcR)/CD3 complex and several surface molecules termed coreceptors (notably CD4 or CD8 and CD45). The spatial arrangement and interactions of the different molecules in the resulting multimolecular recognition structure are mostly unknown. Here we report, from studies on a healthy human CD3 gamma deficiency, that the lack of the CD3 gamma component of the TcR/CD3 complex is associated with a long-term severe defect of peripheral blood CD4+ CD45RA+ and CD8+ lymphocytes, whereas CD4+CD45RO+, B and natural killer lymphocytes are unaffected. These results suggest that the CD3 gamma site of the TcR/CD3 complex is required for the peripheral representation of certain T cell types.

CD3 Complex↗
PubMed1993

Selective impairment of T lymphocyte activation through the T cell receptor/CD3 complex after cytomegalovirus infection.

Cytomegalovirus (CMV) infection is reported to cause transient immunosuppression in man. In this study we have analysed the effect of CMV on T lymphocyte function in 29 children diagnosed for acute CMV infection. Peripheral blood mononuclear cells (PBMC) obtained from the patients showed a significant specific impairment in their proliferative response to enterotoxins A and C1, to concanavalin A and to the anti-CD3 MoAb OKT3. The impaired responses were corrected with exogenous IL-2. In contrast, stimulation using phytohaemagglutinin, as well as activation signals delivered through the surface molecules CD26 or CD28, elicited normal proliferative responses in CMV PBMC. The results indicate that the T cell anergy associated with CMV infection is restricted to the T cell receptor/CD3 activation pathway.

Antigens, CD↗
PubMed1992

Human T-cell activation deficiencies.

The increasing understanding of T-cell activation is paralleled by the recognition of a growing range of 'experiments of nature' that cause T-cell activation deficiencies. Analysis of these deficiencies is, in turn, contributing to the understanding of T-cell function in vivo. Here, José Regueiro, Antonio Arnaiz-Villena and colleagues review current knowledge of structural and functional T-cell defects and the implications of these for T-cell biology.

Adenosine Deaminase↗
PubMed1992

From pathology to physiology of the human T-lymphocyte receptor.

The recent description of a selective human CD3 gamma deficiency and other T-cell receptor (TCR)/CD3 structural and functional defects, together with previous biochemical data on the structure and interactions of the TCR/CD3 complex, may aid in elucidating the physiology of this multi-subunit membrane ensemble. CD3 gamma seemed to be required for the commitment and thymic maturation of an important fraction of T lymphocytes to the CD8 (but not CD4) lineage, perhaps by participating with the CD8 co-receptor in the instructive signal delivered through the alpha beta TCR during intrathymic positive selection by HLA class I molecules. The homologous CD3 delta component would, in contrast, be necessary for the selection of CD4 lymphocytes by HLA class II molecules. The interaction of CD4 and CD8 with the TCR/CD3 complex during antigen recognition may thus be asymmetrical, taking place through CD3 delta and gamma, respectively. Also, the existence of in vivo functional TCR/CD3 hemireceptors (lacking either CD3 gamma or CD3 delta) is suggested, and defects in their relative amount on the T-cell surface may disrupt unresponsiveness to self antigens and generate autoimmunity.

Antigens, Differentiation, T-Lymphocyte↗
PubMed1991

Expression and function of a variant T cell receptor complex lacking CD3-gamma.

A T cell line termed DIL2 has been derived from an infant with a polyclonal T cell receptor (TCR)/CD3 cell surface expression defect. Indirect immunofluorescence showed that the expression of certain TCR/CD3 epitopes (like those detected by WT31 and BMA031 monoclonals) was strongly reduced (around five-fold) on DIL2, whereas other epitopes (like those detected by SP34 and Leu4) were only around two-fold lower than in normal T cell lines. Specific immunoprecipitates of surface-radioiodinated DIL2 cells contained TCR-alpha, TCR-beta, CD3-delta, CD3-epsilon and TCR-zeta chains, but lacked CD3-gamma. This structural TCR/CD3 variant was, however, capable of transducing certain activation signals, since normal proliferation and a low but significant calcium flux was observed in DIL2 cells after engagement with specific antibodies. Our data suggest that a functional TCR/CD3 complex can be expressed on the surface of T cells in the absence of CD3-gamma.

Antibodies, Monoclonal↗
PubMed1991

A diallelic RFLP of the CD3-epsilon chain of the clonotypic T-lymphocyte receptor is not associated with certain autoimmune diseases.

A diallelic restriction fragment length polymorphism of the CD3-epsilon (epsilon) gene, which encodes for an invariant component of the human T-lymphocyte receptor, is observed when using genomic DNA TaqI digests probed with a CD3-epsilon chain cDNA probe. This combination shows two alleles of 9.1 kb and 8.4 kb with a frequency of 0.66 and 0.34, respectively, in the Spanish population. None of these alleles is associated with susceptibility to juvenile rheumatoid arthritis (JRA) or insulin-dependent diabetes mellitus (IDDM).

Alleles↗