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Biomedical subjects

R Palacios

Publications and source records attributed to R Palacios.

At least 73 records · Page 4Linked to original sources
PubMed1997

Mercurialis annua: characterization of main allergens and cross-reactivity with other species.

A multicentric study was conducted to evaluate the frequency of Mercurialis annua pollen sensitization in several areas of Spain and to select a population sample to characterize the main allergenic components in M. annua pollen. Patients were recruited from six hospitals in Spain. Out of 420 patients sensitized to pollens, 195 (46.4%) showed positive skin tests to M. annua, thus evidencing the high level of sensitization to the pollen of this plant in Spain. Thirty-seven sera with RAST class values to M. annua > or = 3 were selected for SDS-PAGE immunoblotting analysis. Two main allergenic components with molecular weights of 15.8 and 14.1 kD were detected in 59 and 51% of the sera, respectively, and they were identified as profilins. Isolation of the relevant allergens was made by affinity chromatography on a poly-L-proline-Sepharose column, followed by gel filtration and anion exchange chromatography in the micropreparative SMARTs System. A significant but low antigenic cross-reactivity between M. annua and Olea europaea, Fraxinus elatior, Ricinus communis, Salsola kali, Parietaria judaica and Artemisia vulgaris was demonstrated by several in vitro techniques.

Adolescent↗
PubMed1997

Occupational allergy in greenhouse workers: sensitization to Tetranychus urticae.

BACKGROUND: Tetranychus urticae (TU) is a macroscopic mite which is found infesting a large number of plants of economic interest. It has rarely been described as a cause of occupational allergic disease in agricultural workers. OBJECTIVE: To describe TU sensitization in greenhouse workers attending the outpatient allergy unit and its clinical associations, and to characterize the allergens involved. MATERIALS AND METHODS: Twenty-four consecutive carnation greenhouse workers with allergy-related symptoms, referred to our outpatient clinic during a 6-month period, were included. We made the diagnostic extract from carnation leaves heavily infested with TU. Skin-prick test, specific IgE measurement and bronchial provocation test with TU extract were carried out in all subjects. Allergen characterization was achieved by SDS-PAGE (sodium dodecylsulfate-polyacrylamide gel electrophoresis) and immunoblotting. RESULTS: Sixteen patients (66%) presented positive skin-prick test and specific IgE and were diagnosed allergic to TU. Fifteen patients suffered from bronchial asthma, 14 rhinitis and five urticaria. Twelve exhibited positive bronchial provocation test to the TU extract. On RAST-inhibition studies, there was no evidence of crossreactivity between TU extract and D. pteronyssinus. An allergen at 19 kDa was determined in the TU extract by SDS-PAGE immunoblotting studies. CONCLUSION: TU could be an important occupational allergen in greenhouse workers showing allergic symptomatology. There is no crossreactivity between this mite and the house dust mite D. pteronyssinus.

Adolescent↗
PubMed1997

Cloning and high level expression of Cynodon dactylon (Bermuda grass) pollen profilin (Cyn d 12) in Escherichia coli: purification and characterization of the allergen.

BACKGROUND: Profilin, an actin-binding protein, was previously described as a panallergen which is involved in about 20% of the crossreactivity found among pollen and food allergic patients. This allergen is usually under-represented in natural extracts used for allergy diagnosis. OBJECTIVES: To obtain an immunologically active and soluble recombinant profilin from Cynodon dactylon pollen which could be used for diagnostic and therapy. METHODS: Isolation of cDNA clones was performed by polymerase chain reaction amplification using degenerate primers. Expression in Escherichia coli BL21 (DE3) was carried out using vector pKN172, and the expressed product was isolated by affinity chromatography on poly L-proline-Sepharose. RESULTS: Four cDNA inserts coding for Cynodon dactylon (Bermuda grass) pollen profilin (Cyn d 12) were cloned and sequenced. Full-length C. dactylon profilin gene was expressed in Escherichia coli as non fusion protein. Induced cells could produce high amounts of recombinant Cyn d 12, and after a single purification step on poly (L-proline)-Sepharose, up to 45 mg of pure allergen per litre culture could be obtained. The reactivity of recombinant Cyn d 12 with IgE antibodies present in sera from Bermuda grass-allergic patients is comparable to that of the natural Bermuda grass allergen. Recombinant Bermuda grass pollen profilin was shown to share B-epitopes with sunflower profilin. CONCLUSIONS: Our results showed that this heterologous expression system and purification procedure are suitable for the production of large amounts of pure allergen which can be used for the characterization of allergenic epitopes recognized by T and B cells and finally for diagnostic and therapeutic purposes.

Allergens↗
PubMed1996

Primitive lymphohematopoietic precursor cell lines generated in culture from day 7 early-mid-primitive streak stage mouse embryo.

During mouse development, the first lymphohematopoietic precursor cells and myeloid or erythroid cell lineage-determined cells can be detected in the yolk sac at days 8-8.5 of gestation. The characteristics of the cells that give rise to these yolk sac primitive lymphohematopoietic cells and the molecular events controlling this process remain poorly defined. We show here that cell suspensions from day 7 early-mid-primitive streak stage embryo proper generated early immature PgP-1+ Joro 177+ Lin- hematopoietic cells and some Mac-1+ myeloid and TER 119+ erythroid cells after co-culture with the yolk sac-derived stromal cell line YS6 without addition of exogenous cytokines. Purified Lin- hematopoietic cells generated in these cultures did not express genes known to be transcribed at early stages of lymphoid, myeloid or erythroid cell differentiation and were able to give rise to T and B lymphocytes, myeloid cells and erythroid cells after appropriate further induction in vitro. Several cell lines were established in culture with a mixture of four cytokines from the PgP-1+ Joro 177+ Lin- cell population. The cell lines shared phenotypic and genotypic characteristics with the PgP-1+ Joro 177+ Lin- cell population generated in culture from day 7 embryo proper and they were able to reconstitute the lymphohematopoietic system of irradiated mice. Taken together these results support a model of lymphohematopoiesis in which cells from day 7 early-mid-primitive streak mouse embryo proper migrate and colonize the visceral yolk sac. There they generate primitive lymphohematopoietic precursor cells and the first erythroid and myeloid hematopoietic cells under the influence of yolk sac stromal cells like the YS6 cells described here.

Animals↗
PubMed1996

Long-term culture of lymphohematopoietic stem cells.

Pluripotent hematopoietic stem cells (PHSCs) show self-renewal and give rise to all blood cell types. The extremely low number of these cells in primary hematopoietic organs and the lack of culture systems that support proliferation of undifferentiated PHSCs have precluded the study of both the biology of these cells and their clinical application. We describe here cell lines and clones derived from PHSCs that were established from hematopoietic cells from the fetal liver or bone marrow of normal and p53-deficient mice with a combination of four growth factors. Most cell lines were Sca-1+, c-Kit+, PgP-1+, HSA+, and Lin- (B-220-, Joro 75-, 8C5-, F4/80-, CD4-, CD8-, CD3-, IgM-, and TER 119-negative) and expressed three new surface markers: Joro 177, Joro 184, and Joro 96. They did not synthesize RNA transcripts for several genes expressed at early stages of lymphocyte and myeloid/erythroid cell development. The clones were able to generate lymphoid, myeloid, and erythroid hematopoietic cells and to reconstitute the hematopoietic system of irradiated mice for a long time. The availability of lymphohematopoietic stem cell lines should facilitate the analysis of the molecular mechanisms that control self-renewal and differentiation and the development of efficient protocols for somatic gene therapy.

Animals↗
PubMed1996

CD98: a type II transmembrane glycoprotein expressed from the beginning of primitive and definitive hematopoiesis may play a critical role in the development of hematopoietic cells.

In our search for cell surface markers expressed on hematopoietic stem cells and/or very early progenitor cells we found that the Joro 177 monoclonal antibody (MoAb) bound to most hematopoietic cells in day 8/8.5 yolk sac, day 12 fetal liver, and day 13 fetal thymocytes; it stained hematopoietic stem cells and less immature lymphoid, myeloid, and erythroid-lineage cells, but not most thymocytes and splenic lymphocytes in adult mice. Joro 177 MoAb stimulated tyrosine phosphorylation of an integral of 124-kD protein and induced homotypic aggregation of lymphoid progenitor cells. Importantly, Joro 177 MoAb inhibited cell survival/growth and consequently the generation of lymphoid, myeloid, and erythroid lineage cells in vitro from early Lin- hematopoietic precursors. Joro 177 MoAb induced apoptosis of hematopoietic progenitor cells. Molecular cloning and expression indicated that Joro 177 MoAb recognizes a type II transmembrane protein, which is the mouse homologue of the human CD98 heavy chain gene. We suggest that CD98 is a cell membrane receptor involved in the control of cell survival/death of hematopoietic cells.

Amino Acid Sequence↗
PubMed1996

Distinct patterns of Fas cell surface expression during development of T- or B-lymphocyte lineages in normal, scid, and mutant mice lacking or overexpressing p53, bcl-2, or rag-2 genes.

Fas is a cell membrane protein involved in programmed cell death. In normal young mice, Fas was expressed on pluripotent stem cells, multipotent progenitors, pro-T and pre-T cells, most thymocytes, and a subset of CD4 and CD8 mature T lymphocytes. In contrast, Fas expression was switched off in B-cell and myelocytic progenitors and most pro-B and a proportion of pre-B cells and was switched on again later, but this occurred only in a subset of mature B lymphocytes. A lack of bcl-2 increased the proportion of Fas+ B-lymphocyte lineage cells and Fas+ CD4+ cells and decreased the percentage of Fas- CD8+ mature T-cell subsets. Overexpression of bcl-2 reversed this pattern of Fas cell surface expression. Interestingly, lack of p53 increased the proportions of Fas-expressing CD4 and CD8 mature T-cell subsets and of Fas- B-cell precursors but decreased that of Fas- mature B-lymphocyte populations. We conclude that the expression of Fas is regulated distinctly during the development of T and B lymphocytes. Although the products of neither bcl-2 nor p53 genes are essential for Fas cell surface expression on hematopoietic cells, these repressor and effector genes, respectively, of programmed cell death affect distinct subsets of lymphoid lineage cells at different stages of lymphopoiesis. Our results suggest that distinct combinations of effector and suppressor genes of programmed cell death act on distinct cell populations and at different stages of differentiation within the same cell lineage in the hematopoietic system.

Animals↗
PubMed1995

In vitro generation of hematopoietic stem cells from an embryonic stem cell line.

Hematopoietic stem cells (HSC) are unique in that they give rise both to new stem cells (self-renewal) and to all blood cell types. The cellular and molecular events responsible for the formation of HSC remain unknown mainly because no system exists to study it. Embryonic stem (ES) cells were induced to differentiate by coculture with the stromal cell line RP010 and the combination of interleukin (IL) 3, IL-6, and F (cell-free supernatants from cultures of the FLS4.1 fetal liver stromal cell line). Cell cytometry analysis of the mononuclear cells produced in the cultures was consistent with the presence of PgP-1+ Lin- early hematopoietic (B-220- Mac-1- JORO 75- TER 119-) cells and of fewer B-220+ IgM- B-cell progenitors and JORO 75+ T-lymphocyte progenitors. The cell-sorter-purified PgP-1+ Lin- cells produced by induced ES cells could repopulate the lymphoid, myeloid, and erythroid lineages of irradiated mice. The ES-derived PgP-1+ Lin- cells must possess extensive self-renewal potential, as they were able to produce hematopoietic repopulation of secondary mice recipients. Indeed, marrow cells from irradiated mice reconstituted (15-18 weeks before) with PgP-1+ Lin- cell-sorter-purified cells generated by induced ES cells repopulated the lymphoid, myeloid, and erythroid lineages of secondary mouse recipients assessed 16-20 weeks after their transfer into irradiated secondary mice. The results show that the culture conditions described here support differentiation of ES cells into hematopoietic cells with functional properties of HSC. It should now be possible to unravel the molecular events leading to the formation of HSC.

Animals↗
PubMed1995

Discrete amplifiable regions (amplicons) in the symbiotic plasmid of Rhizobium etli CFN42.

Frequent tandem amplification of defined regions of the genome, called amplicons, is a common characteristic in the genomes of some Rhizobium species, such as Rhizobium etli. In order to map these zones in a model Rhizobium replicon, we undertook an analysis of the plasticity patterns fostered by amplicons in the pSym (390 kb) of R. etli CFN42. Data presented in this article indicate the presence of four amplicons in pSym, used for the generation of tandem amplifications and deletions. The amplicons are large, ranging from 90 to 175 kb, and they are overlapping. Each amplicon is usually flanked by specific reiterated sequences. Formation of amplifications and deletions requires an active recA gene. All the amplicons detected are concentrated in a zone of roughly one-third of pSym, covering most of the symbiotic genes detected in this plasmid. No amplicons were detected in the remaining two-thirds of pSym. These data support the idea that most of the known symbiotic genes in this plasmid are located in a genomic region that is prone to the formation of frequent tandem amplification.

Crossing Over, Genetic↗
PubMed1994

Cloning and expression of a new mammalian chaperonin gene from a multipotent hematopoietic progenitor clone.

Molecular chaperones assist in the folding and assembly of proteins in cells. Although chaperonins have been shown in prokaryotes, mitochondria, and chloroplasts long-ago, a cytoplasmic heteromeric chaperonin complex was isolated only recently and found to contain at least five to six polypeptides, one of which was identified as the product of the T complex polypeptide-1 (TCP-1) gene. We have isolated and cloned a novel gene called A45 from a cDNA library constructed from poly (A)+ RNA of a multipotent hematopoietic progenitor clone. The A45 cDNA encodes a predicted polypeptide of M(r) 58,118 that exhibits 32% overall amino acid sequence identity to TCP-1 and contains the putative adenosine triphosphate-binding domain and two characteristic consensus regions that are conserved in all chaperonins. The A45 gene is expressed in hematopoietic precursors cells at a much higher level than in nonhematopoietic cells and tissues. We conclude that A45 represents a new member of the mammalian chaperonins that is involved in the folding and assembly of polypeptides.

Amino Acid Sequence↗
PubMed1994

[An analysis of domiciliary medical care in 7 health centers of Alicante Province].

OBJECTIVE: To know the characteristics of medical care at home done by the doctor and to what extent it is justified. DESIGN: An observational, descriptive-prospective study. Form filled out by the doctor after completing the visit. SETTING: Outpatients, 7 hospitals in area 17 of the Valencian Community. PATIENTS: 188 different patients attended in February 1993 by 15 family doctors. The statistical tests used were ji-squared test, Student's t-test and Anova. MEASUREMENTS AND MAIN RESULTS: An average of 0.4 visits/doctor/day were made. Less than 2 hours/week/doctor were needed. 80.5% of the cases were resolved using usual means available in the doctors emergency briefcase. 64.5% of the patients attended were over the age of 65.66% of the visits were considered to be justified. CONCLUSIONS: 66% of medical care at home was considered justified and did not require excessive time. Elderly patients are the ones who most require medical care at home.

Adult↗
PubMed1994

Fetal liver and bone marrow JORO 75+ lymphocyte progenitors are precursors of CD4+8- TCR/CD3- early thymocytes.

We show here that cell sorter purified JORO 75+ lymphocyte progenitors from fetal liver or bone marrow of adult mice give rise in vitro to CD4+8- T cell receptor (TCR)/CD3- early thymocytes and CD4+8- TCR/CD3+ thymocyte subsets after coculture with the EH6 subcapsular thymic epithelial cell line, recombinant interleukin 7 (rIL-7) and F (supernatants from the FLS4.1 fetal liver stromal cell line). We find that in cultures that had additionally received rIL-2, CD4-8+ TCR/CD3+ cells were also generated. The results strongly suggest that fetal liver and marrow JORO 75+ lymphocyte progenitors are precursors to the early CD4+8- TCR/CD3- intrathymic population previously identified in the adult mouse. The EH6 subcapsular thymic epithelial cell line should facilitate the study of the molecular events responsible for very early stages of T cell development including T lymphocyte-lineage commitment.

Animals↗
PubMed1994

Dander is the best epithelial source for dog allergenic extract preparations.

Four different commercially available raw materials of dog epithelia (dander, hair and skin scraping, skin scraping, and whole skin) were compared by means of biochemical methods such as SDS-PAGE and chromatography with the SMART System, immunochemical methods such as RAST and SDS-PAGE immunoblotting, and cutaneous tests. Dander extract clearly exhibited the highest IgE binding and in vivo allergenic activity. The dog major allergens Can fI and the 19-kDa protein were adequately detected only in dander extract. Thus, it is concluded that dander is the most suitable epithelial source for preparing dog allergenic extracts.

Allergens↗
PubMed1994

Sensitization to sunflower pollen: only an occupational allergy?

Sunflower (Helianthus annuus) pollen sensitization has been reported as an occupational allergy. In this report, the sensitization of the general population living in sunflower-growing areas to Helianthus pollen was studied. Both RAST results in 32 adults with summer symptoms previously diagnosed as allergic to Artemisia pollen, and cross-reactivity studies between H. annuus and other Compositae suggested that H. annuus pollen was the main allergen involved in the hypersensitivity reaction of those patients. Good correlation was found between RAST and SPT to Helianthus and between RAST and conjunctival provocation test to Helianthus. Bronchial challenge tests performed on 8 of the 32 patients confirmed the clinical implication of Helianthus pollen in suspected subjects. Five workers, handling sunflower pollen, who suffered from related symptoms were subjected to the same study, showing lesser wheal areas and lesser specific IgE levels than a non-worker group. Thirteen patients with RAST values > or = class 2 showed 2 IgE-binding fractions at 34.0 and 42.8 kD in 65% of sera and 3 IgE-binding fractions at pI 4.9, 9.6 and 10.2 in 54% of sera. By means of micropreparative high-resolution chromatography, it was possible to purify a 34-kD major allergen. Analysis performed by RAST inhibition with sera from atopic patients and ELISA inhibition with experimental anti-Helianthus rabbit sera demonstrated a cross-reactivity between Helianthus and other Compositae, but low affinity of specific anti-Helianthus antibodies for heterologous antigens. Taking into account the above-mentioned data, and the high prevalence of Helianthus pollen in the atmosphere during harvesting (in spite of its entomophilous character), Helianthus pollen should be considered as an allergenic source to be investigated in the general population living in sunflower-growing regions suffering from seasonal summer allergy.

Agricultural Workers' Diseases↗
PubMed1993

A novel disulfide-linked heterodimer on pre-T cells consists of the T cell receptor beta chain and a 33 kd glycoprotein.

We describe a novel signal-transducing protein complex, which consists of the T cell receptor (TCR) beta chain that is disulfide linked to a 33 kd glycoprotein and noncovalently associated with proteins of the CD3 complex on the surface of the pre-T cell line SCB.29. This 33 kd glycoprotein, provisionally designated gp33, represents neither of the known TCR chains and has escaped previous detection because it labels poorly by surface iodination. This glycoprotein is absent from the surface of mature T cell lines. A TCR beta complex with identical molecular masses before and after reduction can be immunoprecipitated from surface-iodinated large thymocytes of TCR alpha-deficient mice. The novel gp33-TCR beta complex may be entirely or partly responsible for control of early T cell development exerted by the TCR beta protein.

Animals↗
PubMed1993

At day 8-8.5 of mouse development the yolk sac, not the embryo proper, has lymphoid precursor potential in vivo and in vitro.

We have studied both in vitro and in vivo the formation of lymphocyte progenitors before blood circulation (day 9 of gestation) has started in the mouse embryo, and we have determined the tissue where this occurs. The results demonstrate that the yolk sac of embryos at day 8 and day 8.5 of gestation contains precursor cells that can give rise, in vivo and in vitro, to mature T and B lymphocytes. No lymphoid precursors were found in the embryo proper at this stage of mouse development. The yolk sac cells with lymphocyte precursor potential are most likely multipotent stem cells rather than cell-lineage-determined T- and/or B-lymphocyte progenitors. The defined in vitro assays described here that support differentiation of yolk sac stem cells along the T- or B-lymphocyte pathways also may now facilitate the study of the molecular events leading to cell-lineage commitment of lymphocyte progenitors in the mouse embryo.

Animals↗
PubMed1993

Gene amplification in Rhizobium: identification and in vivo cloning of discrete amplifiable DNA regions (amplicons) from Rhizobium leguminosarum biovar phaseoli.

A genetic element that allows the positive selection of different genomic rearrangements was used to analyze DNA amplification in Rhizobium leguminosarum biovar phaseoli. Discrete amplifiable DNA regions (amplicons) were detected in different regions of the genome of the model strain CFN42, including the chromosome and several large plasmids. Amplicons were mobilized into Escherichia coli using a genetic approach that involves the introduction of an origin of replication active in E. coli and an origin of conjugal transfer into the amplifiable DNA regions of the Rhizobium genome. The strategy can be a valuable tool for studies on genome organization and function. We propose that amplicons define a structural characteristic of the genome that may play an important biological role.

Blotting, Southern↗