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Biomedical subjects

A N Vallejo

Publications and source records attributed to A N Vallejo.

At least 19 recordsLinked to original sources
PubMed2001

HLA-DR modulates autoantibody repertoire, but not mortality, in a humanized mouse model of systemic lupus erythematosus.

To evaluate the disease-modulating role of HLA-DR2 and DR3 molecules, which have been associated with systemic lupus erythematosus, a humanized mouse model was examined. HLA-DR2 (DRB1*1502)- and DR3 (DRB1*0301)-transgenic mice were backcrossed to the New Zealand Mixed 2410 (NZM 2410, H2(z)) strain. Seventh generation DR2 and DR3 transgene-positive animals along with their transgene-negative littermates and the parental strain NZM2410 were monitored for proteinuria, azotemia, autoantibody production, development of nephritis, and mortality. The results showed no significant differences in proteinuria, azotemia, or mortality between the backcrosses with and without HLA-DR2 or HLA-DR3. However, the genetic analysis of different backcrosses showed that heterozygosity at the endogenous H2-E locus (E(z)/E(b)) was strongly linked with acceleration of lupus nephritis in both HLA-DR2 and HLA-DR3 transgenics. More importantly, the presence of the HLA-DR2, but not the HLA-DR3, transgene significantly enhanced the production of anti-dsDNA, but not anti-ssDNA, anti-histone-dsDNA complex, or anti-histone, Abs. In contrast, neither HLA-DR2 nor HLA-DR3 influenced the development of glomerulonephritis or the degree of immune complex deposition. Moreover, nephritic kidneys from mice with and without HLA-DR2 or HLA-DR3 transgenes showed similar patterns of cytokine expression. Collectively, these findings provide molecular evidence that the association of HLA-DR2 or HLA-DR3 with lupus susceptibility is related to the type of autoantibody rather than to disease mortality. The use of a humanized mouse model provides a way of dissecting the roles of human MHC genes in systemic lupus erythematosus pathogenesis.

Animals↗
PubMed2001

Down-regulation of CD28 expression by TNF-alpha.

Aging and chronic inflammatory syndromes, such as rheumatoid arthritis, are associated with high frequencies of CD4(+)CD28(null) T cells, which are rarely seen in healthy individuals younger than 40 years. Inasmuch as rheumatoid arthritis and aging are also associated with elevated levels of TNF-alpha, we examined whether this proinflammatory cytokine influences CD28 expression. Incubation of T cell lines and clones as well as Jurkat cells with TNF-alpha induced a reduction in the levels of cell surface expression of CD28. This effect of TNF-alpha was reversible; however, continuous culture of CD4(+)CD28(+) T cell clones in TNF-alpha resulted in the appearance of a CD28(null) subset. In reporter gene bioassays, TNF-alpha was found to inhibit the activity of the CD28 minimal promoter. Inactivation of the promoter was accompanied by a marked reduction in DNA-protein complex formation by two DNA sequence motifs corresponding to the transcriptional initiator of the CD28 gene. Indeed, in vitro transcription assays showed that nuclear extracts from TNF-alpha-treated cells failed to activate transcription of DNA templates under the control of a consensus TATA box and the CD28 initiator sequences. In contrast, similar extracts from unstimulated T cells supported transcription. These results demonstrate that TNF-alpha directly influences CD28 gene transcription. We propose that the emergence of CD4(+)CD28(null) T cells in vivo is facilitated by increased production of TNF-alpha.

Aging↗
PubMed2001

Molecular fingerprint of interferon-gamma signaling in unstable angina.

BACKGROUND: Activation of circulating monocytes in patients with acute coronary syndromes may reflect exposure to bacterial products or stimulation by cytokines such as IFN-gamma. IFN-gamma induces phosphorylation and nuclear translocation of transcription factor STAT-1, which initiates a specific program of gene induction. To explore whether monocyte activation is IFN-gamma driven, patients with unstable (UA) or stable angina (SA) were compared for nuclear translocation of STAT-1 complexes and upregulation of IFN-gamma-inducible genes CD64 and IP-10. METHODS AND RESULTS: Peripheral blood mononuclear cells were stained for expression of CD64 on CD14(+) monocytes and analyzed by PCR for transcription of IP-10. Expression of CD64 was significantly increased in patients with UA. Monocytes from UA patients remained responsive to IFN-gamma in vitro, with accelerated transcriptional competency of CD64. IP-10-specific sequences were spontaneously detectable in 82% of the UA patients and 15% of SA patients (P<0.001). Most importantly, STAT-1 complexes were found in nuclear extracts prepared from freshly isolated monocytes of patients with UA, which provides compelling evidence for IFN-gamma signaling in vivo. CONCLUSIONS: Monocytes from UA patients exhibit a molecular fingerprint of recent IFN-gamma triggering, such as nuclear translocation of STAT-1 complexes and upregulation of IFN-gamma-inducible genes CD64 and IP-10, which suggests that monocytes are activated, at least in part, by IFN-gamma. IFN-gamma may derive from stimulated T lymphocytes, which implicates specific immune responses in the pathogenesis of acute coronary syndromes.

Active Transport, Cell Nucleus↗
PubMed2000

Clonality and longevity of CD4+CD28null T cells are associated with defects in apoptotic pathways.

CD4(+)CD28(null) T cells are oligoclonal lymphocytes rarely found in healthy individuals younger than 40 yr, but are found in high frequencies in elderly individuals and in patients with chronic inflammatory diseases. Contrary to paradigm, they are functionally active and persist over many years. Such clonogenic potential and longevity suggest altered responses to apoptosis-inducing signals. In this study, we show that CD4(+)CD28(null) T cells are protected from undergoing activation-induced cell death. Whereas CD28(+) T cells underwent Fas-mediated apoptosis upon cross-linking of CD3, CD28(null) T cells were highly resistant. CD28(null) T cells were found to progress through the cell cycle, and cells at all stages of the cell cycle were resistant to apoptosis, unlike their CD28(+) counterparts. Neither the activation-induced up-regulation of the IL-2R alpha-chain (CD25) nor the addition of exogenous IL-2 renders them susceptible to Fas-mediated apoptosis. These properties of CD28(null) T cells were related to high levels of Fas-associated death domain-like IL-1-converting enzyme-like inhibitory protein, an inhibitor of Fas signaling that is normally degraded in T cells following activation in the presence of IL-2. Consistent with previous data showing protection of CD28(null) cells from spontaneous cell death, the present studies unequivocally show dysregulation of apoptotic pathways in CD4(+)CD28(null) T cells that favor their clonal outgrowth and maintenance in vivo.

Adult↗
PubMed2000

Functional disruption of the CD28 gene transcriptional initiator in senescent T cells.

We recently reported that aging is accompanied by the emergence of CD4(+)CD28(null) T cells, a functionally aberrant lymphocyte subset rarely seen in individuals younger than 40 years. Here, we directly examined whether the lack of CD28 expression is due to a defect at the level of transcriptional initiation. Molecular studies reveal that CD28 gene transcription is controlled by two sequence motifs, sites alpha and beta. In vitro transcription assays using initiator-dependent DNA templates revealed that reversed polarity or the deletion of either motif inhibited transcription, indicating that alpha/beta sequences constitute a composite initiator. Moreover, nuclear extracts from CD28(null) cells failed to activate transcription of alphabeta-initiator DNA templates. Transcription of such templates was, however, restored with the addition of extracts from CD28(+) cells. Although previously described initiator elements have been defined by a consensus sequence, the alphabeta-initiator has no homology to such sequence. These studies demonstrate that initiators have functions other than positioning elements for the basal transcription complex. Rather, initiators can have a direct role in regulating the expression of specific genes. The gain or loss of initiator activity can be an important determinant of cell phenotypes.

Amino Acid Motifs↗
PubMed2000

Central role of thrombospondin-1 in the activation and clonal expansion of inflammatory T cells.

Thrombospondin-1 (TSP) is a transiently expressed matricellular protein known to promote chemotaxis of leukocytes to inflammatory sites. However, TSP and its receptor CD36 are abundantly expressed in chronically inflamed tissues such as the rheumatoid synovium. Here, we show that TSP provides the costimulatory signal that is necessary for the activation of autoreactive T cells. Data presented reveal that TSP-mediated costimulation is achieved through its independent interaction with CD36 on APCs and with CD47 on T cells. We propose that a CD47-TSP-CD36 trimolecular complex is a novel costimulatory pathway that significantly decreases the threshold of T cell activation. Consistent with the paradigm that lesions in rheumatoid synovitis are sites of antigenic recognition, the characteristic focal expression of TSP on APCs such as macrophages and fibroblast-like synoviocytes suggest a central role of TSP in the expansion of tissue-infiltrating T cells.

Antigens, CD↗
PubMed1999

Modulation of CD28 expression: distinct regulatory pathways during activation and replicative senescence.

The costimulatory molecule CD28 has a restricted tissue distribution and is expressed on T cells and some plasmacytoma cells. Although CD28 is constitutively expressed, its expression is transiently down-regulated following T cell activation and declines progressively with in vitro senescence. In vivo, CD8+ T cells and, less frequently, CD4+ T cells may completely lose CD28 surface expression during chronic infections and with aging. This correlates with changes of nuclear protein-binding activities to two motifs, site alpha and beta, within the CD28 minimal promoter. Both alpha- and beta-bound complexes are found only in lymphoid tissues, in CD28+ T cells, and in some transformed B cells. These complexes are coordinately expressed except during replicative senescence, which is characterized by the down-modulation of site beta- but not site alpha-binding activities. In contrast, T cell activation induces a parallel decline in both site alpha- and beta-binding activities. CD4+ and CD8+ T cells differ in their beta-binding profiles, which may explain the more pronounced down-regulation of CD28 in senescent CD8+ T cells. In vivo expanded CD4+CD28null and CD8+CD28null T cells uniformly lack alpha- and beta-bound complexes, resembling the pattern seen in chronically activated cells and not of senescent cells.

Antibodies, Monoclonal↗
PubMed1998

Resistance to apoptosis and elevated expression of Bcl-2 in clonally expanded CD4+CD28- T cells from rheumatoid arthritis patients.

Patients with rheumatoid arthritis have a subset of CD4+ T lymphocytes that are characterized by a defect in CD28 expression. CD4+CD28- T cells frequently undergo clonal expansion in vivo. These clonotypes include autoreactive cells and persist over many years. The clonogenic potential and longevity of these T cells could be related to an altered response to apoptosis-inducing signals. To explore this possibility, CD4+CD28- T cell lines and clones were examined for their response pattern to stimuli inducing physiologic cell death. CD4+CD28- T cells were found to be resistant to apoptosis upon withdrawal of the growth factor, IL-2. To examine whether the altered sensitivity to this apoptotic signal was correlated with the expression of proteins of the bcl-2 family, the expression of bcl-2, bcl-x, and bax proteins was determined. CD28+ and CD28-CD4+ T cells could not be distinguished by the levels of bax or bcl-xL protein; however, CD4+CD28- T cells expressed higher amounts of bcl-2 protein than did CD4+CD28+ T cells. The increased bcl-2 expression in CD4+CD28- T cells was relatively independent of signals provided by exogenous IL-2. In CD28-deficient CD4+ T cells, bcl-2 was not significantly up-regulated by the addition of exogenous IL-2 and was maintained despite IL-2 withdrawal, as opposed to CD28-expressing CD4+ T cells. We propose that CD4+CD28- T cells are characterized by a dysregulation of the survival protein, bcl-2, which may favor the clonal outgrowth of autoreactive T cells and thus contribute to the pathogenesis of rheumatoid arthritis.

Apoptosis↗
PubMed1998

Aging-related deficiency of CD28 expression in CD4+ T cells is associated with the loss of gene-specific nuclear factor binding activity.

Changes in T cell populations and concomitant perturbation of T cell effector functions have been postulated to account for many aging-related immune dysfunctions. Here, we report that high frequencies of CD28(null) CD4+ T cells were found in elderly individuals. Because deviations in the function of these unusual CD4+ T cells might be directly related to CD28 deficiency, we examined the molecular basis for the loss of CD28 expression in CD4+ T cells. In reporter gene bioassays, the minimal promoter of the CD28 gene was mapped to the proximal 400 base pairs (bp) of the 5' untranslated region. CD28 deficiency was associated with the loss of two noncompeting binding activities within a 67-bp segment of the minimal promoter. These binding activities were not competed by consensus Ets, Elk, or AP3 motifs that were found within the sequence stretch. The DNA-protein complexes were also not recognized by antibodies to Ets-related transcription factors. Furthermore, introduction of mutations into the 67-bp segment at positions corresponding to the two DNA-protein interaction sites, i.e. nucleotides spanning -206 to -179 and -171 to -148, resulted in the loss of specific nuclear factor binding activities and the abrogation of promoter activity. These observations implicate at least two regulatory motifs in the constitutive expression of CD28. The loss of binding activity of trans-acting factors specific for these sequences may contribute to the accumulation CD4+CD28(null) T cells during aging.

Aging↗
PubMed1996

The locus-specific enhancer activity of the class I major histocompatibility complex interferon-responsive element is associated with a gamma-interferon (IFN)-inducible factor distinct from STAT1alpha, p48, and IFN regulatory factor-1.

Recent analyses of the upstream regulatory regions of the class I major histocompatibility complex genes in higher primates provided a generalized structural basis for the differential expression of A- and B-locus gene products in response to specific physiological stimulus. Among the regulatory sequences that differ between the loci is the interferon-responsive element (IRE). While the B-IRE is conserved, the A-IREs have species-specific sequence variation. We previously demonstrated that the B-IRE was an interferon (IFN)-inducible enhancer, whereas none of the A-IREs were functional. In the present study, we examined the biochemical basis for the enhancer activity of the conserved B-IRE and found that this may be attributed to a novel gamma-IFN-inducible factor. This factor accumulated in nuclei of cells within minutes of exposure to gamma-IFN. Its appearance was independent of de novo protein synthesis. However, it was not detected in nuclei of cells treated with herbimycin A, suggesting that its appearance depends on a protein kinase activation pathway. Supershift assays indicated that it was distinct from STAT1alpha, IFN regulatory factor-1, and p48, transcription factors known to bind IRE-like sequences found in regulatory regions of many non-major histocompatibility complex gamma-IFN-responsive genes. Competition assays show that this novel factor bound B-IRE with relatively high affinity, about 100-fold more than that for the A-IRE sequence. This factor was also present in STAT1alpha and p48 somatic mutants that also exhibited B-IRE enhancer activity in reporter gene bioassays in a manner similar to those seen with wild type cells. These observations indicate the existence of a novel gamma-IFN-dependent transcriptional activation pathway that correlates with the differential enhancer activity of the HLA-B IRE.

Benzoquinones↗
PubMed1995

Structure of the MHC A and B locus promoters in hominoids. Insights on the evolution of the class I MHC multigene family.

The expansion and contraction of mammalian class I multigene families raises the issue as to what determines the loss or retention of family members. We propose that accumulating changes in regulatory regions result in the loss of expression of the gene products during times critical to selection, leading to the extinction of misregulated genes. The structures of promoter regions of MHC class I genes in nonhuman primates support this view. The B promoters are more homogeneous and contain regulatory elements also found in the promoters of the homologous class I genes of more distant mammals, whereas the A locus promoters were significantly more heterogeneous, have fewer sequence motifs related to known transcription factor-binding sites and have accumulated nucleotide substitutions within one of the widely conserved class I promoter elements. These findings are consistent with the view that the more polymorphic B locus is the principal MHC locus encoding functional class I Ag-presenting molecules whereas the less polymorphic A locus is assuming a secondary role as a consequence of promoter defects.

Animals↗
PubMed1995

A common mutation in the hominoid class I A-locus IFN-responsive element results in the loss of enhancer activity.

Despite the observed coordinate expression of HLA-A and -B antigens in somatic tissues, there is growing evidence that the A and B class I genes are differentially regulated at the transcriptional level. Previous studies indicate that this may be related to locus-specific structural differences in certain enhancer elements. We have recently examined the 5' proximal regulatory regions of the A and B homologs in the higher non-human primates and found pronounced differences between the loci. Sequence analysis shows the B-promoters are more homogeneous, whereas the A-locus promoters are divergent among the various species examined. The differences in A- and B-promoters is exemplified by the regulatory element referred to as the IFN-responsive element (IRE). While the B-locus IRE is conserved among all primates examined, the A-locus IRE are divergent and reveal different sequences in the human/chimpanzee, gorilla and orang-utan. In reporter gene bioassays, the B-locus IRE exhibited an enhancer activity in response to induction with IFN-beta and IFN-gamma. In contrast, all the variants of the A-locus IRE found in hominoid primates were unresponsive to IFN. One base substitution shared by all the primate IREs proved to be inactivating. These results provide a molecular basis of how duplicated gene loci encoding structurally and functionally similar antigen-presenting molecules may become differentially responsive to physiological stimulus.

Animals↗
PubMed1992

Cellular pathway(s) of antigen processing and presentation in fish APC: endosomal involvement and cell-free antigen presentation.

Studies were conducted to address further the role(s) of antigen processing and presentation in the induction of immune responses in a phylogenetically lower vertebrate, specifically a teleost, the channel catfish. In particular, studies were aimed at determining the subcellular compartments involved in antigen degradation by channel catfish antigen-presenting cells (APC) as well as ascertaining the reexpression of immunogenic peptides on the surfaces of APC. The results showed that exogenous protein antigens were actively endocytosed by APC as detected by flow cytometry. Use of radiolabeled antigen and subcellular fractionation protocols also showed that antigen localized in endosomes/lysosomes. Furthermore, there was an apparent redistribution of antigen between these organelles and the plasma membrane during the course of antigen pulsing. Functional assays for the induction of in vitro antigen-specific proliferation of immune catfish peripheral blood leukocytes (PBL) showed that membrane preparations from antigen-pulsed autologous APC were highly stimulatory. The magnitude of responses elicited with such membrane preparations was very similar to that of PBL cultures stimulated with native antigen-pulsed and fixed intact APC or prefixed intact APC incubated with a peptide fragment of the nominal antigen. Current data further corroborate our previous findings that steps akin to antigen processing and presentation are clearly important in the induction of immune responses in lower vertebrates like fish, in a manner similar to that seen in mammalian systems. Consequently, it would appear that many immune functions among the diverse taxa of vertebrates are remarkably conserved.

Animals↗
PubMed1991

Spontaneous development of functionally active long-term monocytelike cell lines from channel catfish.

During the course of studies involving the in vitro manipulation of channel catfish peripheral blood leukocytes, spontaneous proliferation was observed with unexpectedly high frequency. Propagation of these spontaneously proliferating cells has resulted in the development of long-term (greater than 11 mo.) cell lines which stain positively for nonspecific esterase and peroxidase, are phagocytic for latex beads, and morphologically resemble mammalian monocytes or macrophages. These long-term cell lines also exhibit two important additional functional features. First, induction with lipopolysaccharide results in the secretion of relatively high levels of catfish high and low molecular weight species of interleukin-1 active on channel catfish and mouse T cells, respectively. Second, these cell lines are efficient antigen-presenting cells to autologous peripheral blood leukocytes for antigen specific in vitro proliferative and antibody responses. This antigen-presenting function is blocked by inhibitors known to prevent antigen processing and presentation by mammalian monocytes. Allogeneic mixtures of cell line (used as antigen-presenting cells) and responding peripheral blood leukocytes, however, resulted in strong mixed leukocyte reaction but not in specific antibody responses. The availability of such cell lines should facilitate further studies on accessory cell functions in fish immune responses.

Animals↗
PubMed1991

Phylogeny of immune recognition: processing and presentation of structurally defined proteins in channel catfish immune responses.

This work was undertaken to investigate whether or not antigen processing and presentation are important in channel catfish in vitro secondary immune responses elicited with structurally defined proteins, namely, pigeon heart cytochrome C (pCytC), hen egg lysozyme, and horse myoglobin. The use of in vitro antigen-pulsed and fixed B cells or monocytes as antigen presenting cells (APC) resulted in autologous peripheral blood leukocytes (PBL) responding with vigorous proliferation and antibody production in vitro. In addition, several long-term catfish monocyte lines have been found to function as efficient APC with autologous but not allogeneic responders. Subsequent separation of the responding PBL into sIg- (T-cell-enriched) and B (sIg+) cells subsets showed that both underwent proliferative responses to antigen-pulsed and fixed APC. Moreover, allogeneic cells used as APC were found to induce only strong mixed leukocyte reactions without specific in vitro antibody production. Initial attempts at identifying the immunogenic region(s) of the protein antigens for catfish indicated there are two such regions for pCytC, namely, peptides 66-80 and 81-104.

Animals↗