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R Palacios

Publications and source records attributed to R Palacios.

At least 109 records · Page 6Linked to original sources
PubMed1990

Identification and characterization of pro-T lymphocytes and lineage-uncommitted lymphocyte precursors from mice with three novel surface markers.

The study of prethymic stages of T cell development has been limited because specific markers for mouse pro-T lymphocytes were not available. We developed a panel of rat monoclonal antibodies (mAbs) that bind to our pro-T lymphocyte clones obtained from bone marrow of young adult mice and the thymus of 14-d-old embryos. The mAbs, called Joro 30-8, Joro 37-5, and Joro 75, were found to bind to all pro-T clones tested but not to cell lines representing later stages of T cell development, B lymphocyte, or myeloid lineages. We determined the frequency and tissue distribution in normal and immunodeficient mouse strains as well as the ontogeny in liver and thymus of cells positive for these mAbs. The results were consistent with the pattern of reactivity observed with cell lines. We isolated Joro 30-8+, Joro 37-5+, and Joro 75+ bone marrow cells by cell sorter and found that: (a) phenotypically, they are Thy-1+, CD4-, CD8-, CD3-, B-220-, IgM-, F4/80-, and PgP-1+; (b) they grew in response to the combination of interleukin 3 (IL-3) + IL-4 or IL-3 + IL-4 + IL-6; and (c) Joro 37-5+ and Joro 75+ marrow cells gave rise to mature T lymphocytes but not to B lymphocytes, while Joro 30-8+ marrow cells generated both T and B lymphocytes after 8-12 wk of transfer into severe combined immunodeficient (Scid) mice. In normal mice subjected to 600 rad of irradiation to induce a wave of thymus recolonization, we found by flow fluorocytometry analysis that Joro+ cells entered the thymus 2 d after irradiation, expanded during the next 4 d, and underwent further differentiation, and from day 8 up to day 21, post-irradiation Joro+ cells were no longer detectable in the thymuses. Immunohistochemical analysis of normal thymus shows the presence of very few Joro 30-8+, Joro 37-5+, and Joro 75+ lymphoid cells in the subcapsular area and outer cortex but not in the medulla. The kinetic analysis of tissue sections from thymuses at various days post-irradiation suggests that Joro+ cells enter the thymus via blood vessels through the subcapsular and outer cortex areas; subsequently, these cells seem to migrate to the inner cortex without reaching the medulla, and give rise to Joro- thymocytes.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1990

Growth factors involved in lymphocyte differentiation.

We report here that an interleukin-3-dependent precursor B-cell line, LyD9, differentiated in vitro into mature B cells, producing immunoglobulin (Ig)M and IgG by co-culture with bone marrow stroma cells. Induced LyD9 cells underwent heterogenous immunoglobulin gene rearrangement and synthesized mRNAs encoding immunoglobulin mu (mu), gamma (gamma) and kappa (kappa) chains. LyD9 was also shown to differentiate into myeloid cells. We have established an interleukin-4-dependent derivative clone K-4 that is an intermediate between myeloid-lymphoid cells and the LyD9 clone. This differentiation required direct contact between LyD9 and stromal cells.

Animals↗
PubMed1990

Physical map and properties of a 90-MDa plasmid of Azospirillum brasilense Sp7.

Homology was previously detected between the DNA restriction fragments containing Rhizobium meliloti nodulation genes and the 90-MDa plasmid, p90, of Azospirillum brasilense Sp7. Two DNA loci from Sp7 genome that complement mutations in the exopolysaccharide synthesis genes, exoB and exoC, of R. meliloti were also shown to be present on the plasmid. A more detailed characterization of the plasmid was undertaken to establish its physical map and to localize the nod homologies and other specific regions. Six loci were mapped, the region homologous to the nodulation genes, nodPQ, of R. meliloti, the exoB and exoC mutation-correcting loci, a locus for Ap resistance, a bla homology region different from the Ap resistance locus, and a region necessary for the maintenance of p90 as an independent replicon. Mobilization into Agrobacterium tumefaciens of p90-Tn5-Mob was obtained at a frequency of 10(-4), with the plasmid helper pJB3JI. Self-transfer of p90 was not demonstrated. Fragments of p90 hybridized with a plasmid of 90 MDa present in most A. brasilense and some A. lipoferum strains, suggesting a plasmid family in Azospirillum.

Ampicillin Resistance↗
PubMed1990

LD1: a CD4-CD8- TCR alpha beta/CD3+ peripheral T cell line with helper function for B lymphocytes.

In order to learn more about the small subset of CD4-CD8- TCR alpha beta/CD3+ peripheral T lymphocytes, we firstly characterized at the cellular and molecular levels the CD4-CD8- LD1 cell line isolated from the spleen of an MRL/lpr-lpr mouse. Secondly we studied its functional properties. LD1 cells are Thy1+ CD5+ CD4-CD8- LFA-1+ PgP-1+ and do not bind the T cell precursor-specific antibodies Joro 37-5 or Joro 75. They are negative for IgM, B-220, BP-1, J11d, Lyb8, Ia, F4/80, BP-2, and Mac-1 surface markers. LD1 cells have deleted the TCR delta locus, have rearrangements at the TCR gamma gene cluster (i.e. a V gamma 1-J gamma 1-C gamma 1 and a V gamma 4.3-J gamma 4-C gamma 4) and have two rearrangements of the TCR beta gene cluster (i.e. a D beta 1-J beta 1 and V-D-J beta 2). LD1 cells produce normal sized RNA transcripts from TCR alpha and beta genes and lower levels of gamma-mRNA. These cells bind CD3- and pan-TCR beta-specific antibodies as determined by FM analysis. We conclude that LD1 cells bear a TCR alpha beta/CD3 type of receptor complex. LD1 cells fail both in vivo and in vitro to differentiate into CD4+ or CD8+ cells. These cells help B lymphocytes to mature into antibody-secreting cells, secrete IL-3 and IL-6 but not IL-2, IL-4, or IL-5, and exert no detectable cytolytic activity. These results together with recent reports of antigen-specific CD4-CD8- TCR alpha beta/CD3+ cytotoxic T cell lines show that the CD4-CD8- TCR alpha beta/CD3+ subset comprises functionally competent helper and cytotoxic T lymphocytes and thereby argue for their potential to participate in immune responses. Our results also suggest that cells like LD1 represent terminally differentiated T lymphocytes rather than cells with precursor potential for CD4+ or CD8+ TCR alpha beta/CD3+ T lymphocytes.

Animals↗
PubMed1989

Thymic epithelial cells induce in vitro differentiation of PRO-T lymphocyte clones into TCR alpha,beta/T3+ and TCR gamma,delta/T3+ cells.

PRO-T lymphocyte clones, which have the T cell receptor (TCR) alpha, beta, gamma and delta genes in germline configuration and heterogeneous T cell precursors freshly isolated from bone marrow of athymic nude mice, gave rise to single positive L3T4+ TCR alpha,beta+ and double negative (L3T4-LyT2-) TCR alpha,beta+ or TCR gamma,delta+ cells, but not to any cells expressing LyT2, when co-cultured with the thymic epithelial clone ET. The T cell progenitors were able to develop into cells expressing LyT2 only when cocultured with heterogeneous thymic epithelial cell preparations. The progeny of the induced PRO-T clones included cells bearing V beta 8, V beta 17 and V gamma 3 gene family products. The presence of cells expressing a TCR gamma, delta/T3 receptor complex in the cultures was also documented by the expression of RNA transcripts from the TCR delta and TCR gamma genes by induced PRO-T cells. The TCR/T3+ cells generated in the cultures expressed functionally competent T cell receptor complexes. Our results show that: (i) the same PRO-T clone can give rise to all major subsets of thymocytes upon interaction with the appropriate thymic epithelial cells; (ii) both TCR alpha,beta+ and TCR gamma,delta+ cells may originate from a common T cell progenitor; (iii) L3T4+ TCR alpha, beta+ and L3T4-LyT2- TCR alpha,beta+ cells do not necessarily pass through a L3T4+LyT2+ intermediate stage of development; and (iv) different types of thymic epithelial cells play an essential role in the differentiation of PRO-T cells into either L3T4+ TCR alpha,beta+ L3T4-LyT2- TCR alpha,beta+ or L3T4+LyT2+ and LyT2+ TCR alpha, beta+ cells in vitro. Finally, we have attempted to integrate our results and those of others in a suggested model of T cell development within the thymus.

Animals↗
PubMed1989

Monoclonal antibodies reactive with the mouse interleukin 5 receptor.

The rat mAbs R52.120 and R52.625 inhibit the action of IL-5 on both IL-5-sensitive cell lines and freshly isolated splenic B lymphocytes. Neither antibody inhibits the proliferative cell responses promoted by IL-2, IL-3, or IL-4. Purified R52.120+ lymphoid spleen cells contain 15-20-fold higher numbers of B lymphocytes responding to IL-5 in the form of maturation into antibody-producing cells. By immunofluorescence staining and flow fluorocytometry, the R52.120 and R52.625 antibodies bound to all 12 IL-5-sensitive cell lines tested. Both antibodies react with 2-4% cells in the spleen, 5% lymphoid cells, and 10-15% myeloid cells in the bone marrow, and 10-14% in the peritoneum of C57BL/6, DBA/2, and BALB/c adult mice. No positive cells for either antibody were detected in the thymus and lymph nodes of these mice. Both R52.120 and R52.625 antibodies specifically inhibit the binding of radiolabeled IL-5 to its receptor. Finally, R52.120 and R52.625 antibodies precipitate from 35S-methionine-labeled IL-5-R+ cell lysates three proteins with Mr 46,000, 130,000, and 140,000. Taken together from these results, we conclude that the R52.120 and R52.625 mAbs recognize epitopes on the IL-5-R complex very close or identical to the IL-5 binding sites.

Animals↗
PubMed1989

Expression of the p75 interleukin 2-binding protein on CD3+4-8-Tac- cells from autoimmune MRL/MP-lpr/lpr mice.

The recently described (Sharon, M. et al., Science 1986. 234:859) interleukin 2 (IL 2)-binding molecule p75 was detected in the CD3+4-8-Tac- "double-negative" cell population selectively expanded in lupus-like autoimmune mice MRL/MP-lpr/lpr using cross-linking studies. Scatchard analysis of the IL 2 binding revealed the existence of approximately 4700 sites per cell with an apparent Kd of 1500 pM. The cell line LD1.T3B, derived from this population, shared surface markers and the p75 presence/p55 absence of IL 2-binding proteins with its in vivo counterpart, displaying around 3100 sites per cell with a Kd of about 1300 pM. Functional studies showed that high doses of IL 2 had an inhibitory effect on the autonomous growth of this cell line in the absence of the development of killer activity. This study provides evidence of the functional abilities of p75, and shows that the use of Tac/p55 surface expression only to evaluate IL 2 receptors and T cell activation can be an oversimplification as well as misleading.

Animals↗
PubMed1989

The epigenetic influences of bone marrow and fetal liver stroma cells on the developmental potential of Ly-1+ pro-B lymphocyte clones.

The Ly-1+ Mac-1+ B-220+ CC11+ progenitor clones LyD9 and LyB9 were previously shown to give rise to Ly-1+IgM+ B lymphocytes either in vivo or in vitro by co-culture with nonlymphoid accessory cells from spleen and lipopolysaccharide. The clones did not generate T lymphocytes either in vivo or in vitro. We now find that both LyD9 and LyB9 progenitors are induced to differentiate in vitro by co-culture with the RP.0.10 bone marrow stroma clone or with heterogeneous marrow-adherent stroma cell populations obtained from adult mice into Ly-1-IgM+ B lymphocytes as well as myeloid GM1.2+ Mac-1+ cells. We could obtain evidence that a high proportion of LyD9 and LyB9 cells already switched off expression of Ly-1 and CC11 (interleukin 3 receptor) surface molecules 2 days after initiation of the cultures, and by days 8-10 of culture no detectable Ly-1+ cells and only about 20% CC11+ cells were observed. Ly-1 surface expression could not be re-induced on the progeny of LyD9 and LyB9 progenitors generated under the influence of marrow stroma cells. Remarkably, the LyD9 and LyB9 progenitors gave rise to both Ly-1+IgM+ and Ly-1-IgM+ B cells upon culture with heterogeneous stroma monolayers obtained from 18-day fetal liver. Finally, the differentiating property of the stroma cells for the LyD9 and LyB9 progenitors could not be replaced with soluble factors produced either spontaneously or after stimulation by the marrow stroma cells. Our results show the importance of epigenetic influences provided by a given microenvironment on the developmental potential of B cell progenitors. They provide direct evidence that the same pro-B lymphocyte can give rise to both Ly-1+ and Ly-1-IgM+ B cells depending on both the time of development and the tissue of origin of stroma cells with which the B cell progenitor interacts. Also, the results strongly suggest that cell contact between the stroma cell and the B cell progenitor is essential to induce rearrangement and expression of the Ig genes in pro-B lymphocytes.

Animals↗
PubMed1989

In vitro effects of recombinant interleukin 7 on growth and differentiation of bone marrow pro-B- and pro-T-lymphocyte clones and fetal thymocyte clones.

We have studied the effects of recombinant (r) interleukin 7 (IL-7) on growth and differentiation of marrow pro-B-lymphocyte clones (CB/Bm7, LyD9, LyB9), marrow pro-T-lymphocyte clones (C4-77/3, C4-86/18, C4-95/16), and fetal thymocyte clones (FTH5, FTA2, FTD5) in the presence or absence of the bone marrow stroma clone RP.0.10, which was selected for its ability to promote differentiation of the pro-B clones. rIL-7 alone stimulated some DNA synthesis (measured by [3H]thymidine uptake) but not actual growth (increase in cell number) of the pro-B clones. Antibodies against IL-4 and IL-6 or against receptors for IL-2, IL-3, and IL-5 did not inhibit this effect of rIL-7 on the pro-B clones. rIL-7 alone or in various combinations with other cytokines (from rIL-1 alpha to rIL-6) could not induce differentiation of the pro-B clones into IgM+ B cells regardless of the presence of lipopolysaccharide (LPS). The RP.0.10 marrow stroma cells by themselves do not support the growth of the pro-B clones. However, the pro-B clones grew when cultured with rIL-7 and monolayers of the RP.0.10 stroma cells. While the RP.0.10 stroma cells induced the pro-B clones to differentiate into IgM+ B cells but not T3+ T cells when cultured in the presence of LPS and rIL-3, the B-cell progenitor clones gave rise to significantly higher numbers of IgM+ B cells (up to 63%) and to many more B cells expressing higher levels of surface IgM when cocultured with rIL-7, LPS, and RP.0.10 stroma cells. The pro-B clones also generated IgM+ B cells (up to 20%) when cocultured with RP.0.10 stroma cells and rIL-7 in the absence of LPS. By using culture plates designed for testing requirements for cell-cell contact, we found that cell interactions between the pro-B cell and the marrow stroma cell are essential to induce rearrangement and expression of the immunoglobulin genes in the pro-B clones. Possible mechanisms to account for the remarkable effects of rIL-7 in the presence of RP.0.10 stroma cells on both growth and differentiation of the pro-B clones are discussed. Finally, rIL-7 alone or together with RP.0.10 stroma cells neither supported proliferation nor induced differentiation into T3+ T cells or IgM+ B cells of the marrow pro-T clones or the fetal thymocyte clones. In light of these findings, we postulate that the interaction of the pluripotential stem cell with marrow stroma cells like RP.0.10 and the availability of IL-7 could play a critical role in the commitment to develop along the B-lymphocyte pathway.

Animals↗
PubMed1989

An interleukin-4-dependent precursor clone is an intermediate of the differentiation pathway from an interleukin-3-dependent precursor clone into myeloid cells as well as B lymphocytes.

An interleukin-3-dependent progenitor clone LyD9 and its interleukin-4-dependent derivative clone K-4 were shown to differentiate into myeloid cells as well as B lymphocytes by coculture with bone marrow stroma cells. The K-4 clone is an intermediate between myeloid/lymphoid cells and the LyD9 clone that requires interleukin-4 for differentiation into B lymphocytes and myeloid cells. Granulocyte-macrophage colony stimulating factor-dependent derivatives (LS-1 and K-GM) were also established from induced LyD9 cells. LS-1 and K-GM were myeloid-committed cells.

Animals↗
PubMed1988

Thymocyte clones from 14-day mouse embryos. II. Transcription of T3 gamma gene may precede rearrangement of TcR delta and expression of T3 delta, T3 epsilon and T11 genes.

We have studied the state of TcR delta gene and the expression of T3 delta, T3 epsilon, T3 gamma and T11 (CD2) genes in the fetal thymocyte (FT) clones A2, G15, H5, E10, D5, H12, F1 and D11 obtained from a 14-day B10.BR mouse fetal thymus. The eight FT clones contain the TcR delta gene in the germ-line configuration as determined by Southern blot analysis. With the exception of clones E10 and D5, the other six FT clones express normal sized transcripts for T3 gamma gene and none of the eight FT clones produced detectable RNA transcripts for T3 delta, T3 epsilon and T11 genes as assessed by Northern blot analysis. Together with our previous work showing that all eight FT clones contain the TcR gamma and the TcR beta gene clusters in the germ-line configuration, the data indicate that the FT clones represent the earliest stage of T cell development identified within the thymus. Our results provide evidence that (a) the T3 gamma gene is the first of the genes that encode components of a TcR/T3 complex to be expressed in ontogeny within the thymus; (b) the T3 (delta, epsilon, gamma) genes are switched on asynchronously and their expression must be differentially regulated, and (c) the T11 gene product may not be involved in early stages of T cell development within the thymus.

Animals↗
PubMed1988

Analysis by in situ hybridization of cells expressing mRNA for interleukin 4 in the developing thymus and in peripheral lymphocytes from mice.

We have made use of RNA.RNA in situ hybridization to study the presence of cells producing mRNA for interleukin 4 (IL-4) in the developing thymus, spleen, and T-cell line 2.19. Approximately 1 of 300-400 spleen cells expressed detectable IL-4 mRNA 24 hr after their stimulation by the lectin concanavalin A. Spleen cells were also induced to express mRNA for IL-4 by stimulation with alloantigens. Splenocytes producing mRNA for IL-4 were detected 4 hr after stimulation by concanavalin A; the response peaked at approximately equal to 24 hr and was undetectable by 72 hr. Cyclosporin A inhibited the synthesis of IL-4 mRNA in the T-cell line 2.19, which had been induced by concanavalin A. Approximately 1 of 10 fetal thymocytes at day 14 of gestation expressed mRNA for IL-4 after their stimulation by phorbol 12-myristate 13-acetate and ionomycin. Both the frequency of fetal thymocytes expressing IL-4 mRNA and the amount of mRNA for IL-4 synthesized per cell sharply decreased at day 16 of gestation, and less than 1 of 1800 fetal thymocytes at day 18 of gestation expressed detectable IL-4 mRNA. Our results define the relative frequency of cells capable of expressing IL-4 mRNA after stimulation in vitro in the spleen and in the developing thymus. The data strongly argue for an important role of IL-4 in growth and differentiation of lymphoid cells, notably during T-cell development within the thymus.

Animals↗
PubMed1988

Differentiation of an interleukin 3-dependent precursor B-cell clone into immunoglobulin-producing cells in vitro.

Precursors to B-cell lines with immunoglobulin genes in the germ-line context have been shown to be capable of generating mature B cells in vivo. We report here that an interleukin 3-dependent precursor B-cell line, LyD9, differentiated in vitro into mature B cells, producing IgM and IgG by coculture with bone marrow accessory (or stroma) cells or with dendritic cells and T cells. Up to 50% of IgM-positive cells, but no Thy-1-positive cells, appeared after the 7- to 10-day coculture. Induced LyD9 cells underwent heterogenous immunoglobulin gene rearrangement and synthesized mRNAs encoding immunoglobulin mu, gamma, and kappa chains. However, these cells did not show any rearrangement of genes encoding the alpha and beta chains of the T-cell receptor. The induction of differentiation by coculture with bone marrow stroma cells was blocked by anti-lymphocyte function-associated antigen 1 or anti-interleukin 4 antibody. These systems are useful for molecular biological studies on regulation of differentiation of bone marrow-derived cells into the B-cell lineage.

Animals↗
PubMed1988

Narrow- and Broad-Host-Range Symbiotic Plasmids of Rhizobium spp. Strains That Nodulate Phaseolus vulgaris.

Agrobacterium transconjugants containing symbiotic plasmids from different Rhizobium spp. strains that nodulate Phaseolus vulgaris were obtained. All transconjugants conserved the parental nodulation host range. Symbiotic (Sym) plasmids of Rhizobium strains isolated originally from P. vulgaris nodules, which had a broad nodulation host range, and single-copy nitrogenase genes conferred a Fix phenotype to the Agrobacterium transconjugants. A Fix phenotype was obtained with Sym plasmids of strains isolated from P. vulgaris nodules that had a narrow host range and reiterated nif genes, as well as with Sym plasmids of strains isolated from other legumes that presented single nif genes and a broad nodulation host range. This indicates that different types of Sym plasmids can confer the ability to establish an effective symbiosis with P. vulgaris.

Journal Article↗
PubMed1988

Genomic instability in Rhizobium phaseoli.

Experience from different laboratories indicates that Rhizobium strains can generate variability in regard to some phenotypic characteristics such as colony morphology or symbiotic properties. On the other hand, several reports suggest that under certain stress conditions or genetic manipulations Rhizobium cells can present genomic rearrangements. In search of frequent genomic rearrangements, we analyzed three Rhizobium strains under laboratory conditions that are not considered to cause stress in bacterial populations. DNAs from direct descendants of a single cell were analyzed in regard to the hybridization patterns obtained, using as probes different recombinant plasmids or cosmids; while most of the probes utilized did not show differences in the hybridization patterns, some of them revealed the occurrence of frequent genomic rearrangements. The implications and possible biological significance of these observations are discussed.

Cloning, Molecular↗
PubMed1987

Ly1+ PRO-B lymphocyte clones. Phenotype, growth requirements and differentiation in vitro and in vivo.

Several clones obtained from the bone marrow of a BALB/c mouse were found to contain the heavy and light chain Ig genes in the germline configuration, to express Ly1 and to carry the B cell lineage markers B-220, Lyb8 and BP-1; these clones are Pgp-1+, LFA-1+, J11d+, Mac-1+ and Thy1-, Lyt2-, L3T4-, GM1.2- and Ia-. Three clones analyzed in detail (Lyd9, LyH7 and Lyb9) have receptors for interleukin (IL) 2 and IL3 as assessed with the 7D4 and CC11 monoclonal antibodies respectively. They grow in rIL3 but not in rIL2 or rIL1; both rIL4 and rIL5 also promote their proliferation, albeit to a much lesser extent than rIL3. None of the interleukins tested alone or in various combinations promoted the clones to differentiate in vitro along the B cell pathway. Treatment with 5-Azacytidine (5-Aza) induced cell surface Ia expression but not rearrangement or expression of Ig genes. However, 5-Aza-treated Lyd9, LyH7 and Lyb9 cells co-cultured with X-ray irradiated accessory cells and LPS gave rise to Ly1+, IgM+ B lymphocytes (range 14-51%) including mu + kappa + (78-93%), and mu + lambda + (9-25%) B lymphocytes. In vivo, the Lyd9, LyH7 and Lyb9 clones gave rise to IgM+ B lymphocytes (8.5-17%) including mu + kappa +, and mu + lambda +, but not to Lyt2+ or L3T4+ T lymphocytes after 4-6 weeks of transfer into Scid mice. Our results indicate that Ly1+ IgM+ cells comprise a subpopulation of B lymphocytes that is derived from IL3-responsive Ly1+ PRO-B lymphocytes.

Animals↗