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Biomedical subjects

M L Gaspar

Publications and source records attributed to M L Gaspar.

At least 19 recordsLinked to original sources
PubMed1999

Differential involvement of the transcription factor Blimp-1 in T cell-independent and -dependent B cell differentiation to plasma cells.

Along humoral immune responses, different stimuli drive the differentiation of B lymphocytes to Ig-secreting plasma cells in discrete microenvironments. The Blimp-1 transcription factor is up-regulated early during the transition of mature B cells to IgM-secreting plasma cells. In the present study, we have examined the requirement of Blimp-1 in plasma cell formation after both T cell-independent (LPS) and -dependent (CD40 + IL-4, Th cell lines) stimulation of spleen B cells. B lymphocyte-induced maturation protein (Blimp-1) was expressed early after in vitro LPS stimulation, mainly in a population of IgM+Syndecan+CD43+ preplasma cells. In contrast, the BSAP transcription factor expressed in mature B cells was down-regulated during the differentiation to plasma cells. Treatment of these cultures with Blimp-1-specific antisense phosphorothioate oligonucleotides suppressed both Blimp-1 protein levels and the emergence of IgM+Syndecan+ cells and plasma cells. However, T-B cell cocultures of spleen B cells from C3H/HeJ (H-2k) mice and syngeneic autoreactive SR.10 Th2 cells submitted to the anti-Blimp-1 therapy did not show any significant reduction in IgM- and IgG1-secreting plasma cell formation. Spleen B cells treated with anti-CD40 mAb + IL-4 differentiated to IgG1-secreting cells without significant transcription of the Blimp-1 gene; anti-Blimp-1 treatment subsequently did not have any effect in the later cultures. Altogether, these results suggest that Blimp-1 transcription factor specifically promotes T cell-independent B cell differentiation to plasma cells, probably at preplasma cell stages. In contrast, T cell-dependent plasma cell formation likely evolves through Blimp-1-independent pathways.

Animals↗
PubMed1999

Influence of prolactin on the differentiation of mouse B-lymphoid precursors.

Development and activation of immune cells are submitted to hormonal influences, as illustrated by the roles of corticosteroids in thymus, pregnancy-related estrogens in B-cell development, or prolactin (PRL) on T-cell generation and function. We have analyzed the putative role of PRL in B lymphopoiesis and differentiation. We chose as an experimental model the interleukin (IL)-3 dependent BaF-3 pro-B cell line, which was transfected with the rat long form of the PRL receptor (PRL-R) and transferred from IL-3- to PRL-enriched media. When stimulated with PRL, the PRL-R transfectants underwent some changes characteristic of B-cell differentiation: (a) IL-2R alpha chain became positively controlled by PRL; (b) antiapoptotic Bcl-2 protein was induced by PRL in a dose-dependent manner; and (c) transcription of the pre-B cell receptor encoding the lambda5 gene was strongly up-regulated. We attempted to evaluate the differentiation-promoting activity of PRL in more physiological conditions, and the presence of PRL-R in bone marrow B-cell precursors was revealed. Furthermore, PRL promoted significant expansions of defined B-lineage cell populations in short-term bone marrow cell cultures. These findings suggest that PRL, in collaboration with other cytokines and hormonal influences, modulates B-cell development.

Animals↗
PubMed1998

Identification and characterization of a new oncogene derived from the regulatory subunit of phosphoinositide 3-kinase.

p85/p110 phosphoinositide 3-kinase (PI3K) is a heterodimer composed of a p85-regulatory and a p110-catalytic subunit, which is involved in a variety of cellular responses including cytoskeletal organization, cell survival and proliferation. We describe here the cloning and characterization of p65-PI3K, a mutant of the regulatory subunit of PI3K, which includes the initial 571 residues of the wild type p85alpha-protein linked to a region conserved in the eph tyrosine kinase receptor family. We demonstrate that this mutation, obtained from a transformed cell, unlike previously engineered mutations of the regulatory subunit, induces the constitutive activation of PI3K and contributes to cellular transformation. This report links the PI3K enzyme to mammalian tumor development for the first time.

3T3 Cells↗
PubMed1998

Developmental hematopoiesis.

Hematopoiesis is a developmental process that evolves throughout the lifespan of an individual. Most work in the field has focused on events occurring in the adult bone marrow (BM). In the embryo, blood and endothelial cell generation begins very early after gastrulation, in defined intraembryonic mesodermic sites. Recent multidisciplinary studies, taking advantage of classic embryological and gene targeting technology in various species, have provided a new image of embryofetal lymphohemopoiesis, which includes the suggestion of developmental compartmentalization or waves. The first hematopoietic stem cells (HSC) migrate further and home in an ordered sequence of supporting microenvironments depending on scarcely known molecular requirements. These early hematopoietic progenitors show important differences in their cell biology and differentiation potentialities with respect to those present in adult stages; this fact, together with specific microenvironmental influences, define a process that diverges significantly from that occurring in the BM. Here, we update the latest developments in the field, the new understanding of lymphohemopoiesis in prenatal life, and the novel questions that this emerging paradigm is producing.

Cardiovascular System↗
PubMed1997

Antigenic phenotype and gene expression pattern of lymphohemopoietic progenitors during early mouse ontogeny.

Hemopoiesis, initiated in the early embryo yolk sac (YS) (7.5-8 days postcoitum (pc) in mouse), takes place thereafter in sites successively seeded by extrinsic hemopoietic stem cells (HSC). Since the existence of intraembryonic HSC has been proven experimentally in some vertebrates, it is also likely that not all HSC originate in the YS in mammals, as previously thought. Candidate intraembryonic sites that may be active in producing HSC before liver colonization are the para-aortic splanchnopleura (P-Sp) and the aorta-gonads-mesonephros region (AGM). Here we explore these sites directly for the presence of cells with hemopoietic-specific surface molecules and gene activities. The Ags c-kit, AA4.1, Mac-1, and Sca-1 begin to be expressed on some P-Sp and AGM cells, making it possible to distinguish subpopulations that evolve according to reproducible developmental patterns. On the basis of RAG-1 gene transcription, the first lymphoid precursors in the mouse embryo appear to be present 9.5 to 10 days pc in P-Sp/AGM and YS. Starting B-cell lymphopoiesis (9-12 days pc) is characterized by nonexpression of the surrogate light chain lambda 5-encoding gene and biased usage of IgH DJ4 rearrangements. In the 12.5- to 13.5-day-pc fetal liver (FL), a switch occurs, characterized by the random use of all IgH DJ and the detection of lambda 5 gene transcripts.

Animals↗
PubMed1994

Isolation of peritoneal precursors of B-1 cells in the adult mouse.

Two weeks of daily peritoneopheresis of adult mice result in the selective depletion of B-1 cells, followed by the appearance of a population of B220+IgM-lymphocytes in the peritoneal cavity. These cells share with bone marrow (BM) pre-B cells expression of lambda 5, VpreB, and RAG-1 genes and a higher fraction of unrearranged V to DJ heavy (H) chain immunoglobulin (Ig) gene segments, when compared with mature B lymphocytes. Upon transfer to SCID recipients, sorted peritoneal B220+IgM- cells fail to colonize the BM, repopulate very few B cells in the spleen, but entirely reconstitute the B-1 cell compartment in the peritoneal and pleuropericardial cavities. In contrast, parallel transfers of sorted BM and pleuropericardial cavities. In contrast, parallel transfers of sorted BM B220+IgM- cells result in reconstitution of the BM and spleen B lineage cell compartments, but in no coelomic B cell repopulation. Both types of pre-B cells reconstitute splenic plasma cells of donor origin, but with markedly distinct efficiencies: the ratio of IgM-plasma cell/B cell numbers in the spleens of peritoneal pre-B cell recipients is more than 500-fold higher than that of recipients reconstituted by BM pre-B cells. We take these data to indicate that (1) differentiative commitment to the B-1 cell population occurs before selection events on mature cells; (2) B-1 precursors exist or may be locally produced in the adult mouse; (3) there is a lineage-related differential ability of mature B cells to undergo terminal differentiation to high-rate Ig secretion.

Animals↗
PubMed1991

A single base deletion in the Tfm androgen receptor gene creates a short-lived messenger RNA that directs internal translation initiation.

Testosterone-resistant male mice hemizygous for the X-chromosome-linked mutant gene Tfm express detectable but severely reduced levels of androgen receptor mRNA, amounting to about 10% of the level found in normal male littermates. No structural abnormality could be identified in the coding region of the messenger by a series of RNase-protection assays. However, cell-free translation of RNAs transcribed in vitro from enzymatically amplified overlapping segments of exon 1 revealed a truncated receptor protein and helped to localize the site of premature termination. Sequence analysis of the relevant DNA segment disclosed that deletion of a single nucleotide in the hexacytidine stretch at position 1107-1112 alters the reading frame of the messenger and introduces 41 missense amino acids before a premature termination codon at position 1235-1237. Separately initiated carboxyl-terminal polypeptides are synthesized in vitro, starting probably at the in-frame AUG codon 1507-1509, which lies in a favorable context for translation initiation, and at the non-AUG codon 1144-1146. Transcriptional impairments of the Tfm gene were ruled out by a quantitative analysis of enzymatically amplified nuclear RNA precursors. No other change could be identified by sequencing the complete coding region of Tfm cDNA. The finding of the unsuspected termination codon and the evidence of internally initiated carboxyl-terminal polypeptides reconcile previous conclusions and account for all known phenotypic properties of the mutation.

Amino Acid Sequence↗
PubMed1991

Selective expansion of idiotype sharing T and B cells in cyclosporin A-mediated autoimmunity.

CBA/N mice submitted to autologous bone marrow reconstitution after lethal irradiation and simultaneous Cyclosporin A (CsA) treatment develop a chronic graft-versus-host disease with autoimmune characteristics. When compared to normal controls, diseased mice show an overrepresentation of V beta 8-expressing T cells (65-80% of all CD3+ lymphocytes), together with a marked increase in the titres of serum Ig that specifically bind to F(ab')2 fragments of anti-V beta 8 F23.1 antibodies. Such 'V beta 8-like' Ig V regions are abundantly represented among the IgG2b and mAbs of an unselected collection of hybridomas derived from these mice. These mAbs are not multireactive Ig as they fail to bind to a panel of various antigens and antibodies, but often show simultaneous reactivity with anti-idiotypic mAbs to F23.1 and auto-binding. These molecules may provide the structural basis of V-region specific complementarities, driving the expansion of restricted T and B cell repertoires associated with pathological autoimmunity.

Animals↗
PubMed1991

[Structure of messenger RNA of androgen receptor in mice and molecular characterization in Tfm mutant].

Complementary DNA clones covering the complete coding region of the mouse androgen receptor were assembled by enzymatic amplification (PCR) from testicular RNA and genomic DNA and fully sequenced. The deduced amino acid sequence departs from the rat sequence at 21 positions, 20 of which are in the amino-terminal trans-activation domain. A single 10 kb long messenger RNA containing a 3' noncoding portion longer than 5 kb was detected. The murine cDNA sequence provided the basis to examine the testicular feminization (Tfm) mutation at the molecular level. The androgen receptor messenger RNA level was found reduced about 10-fold in the Tfm mice. The expression of the mutant receptor is affected at a post-transcriptional level. A single base deletion in the hexanucleotide stretch 1107-1112, not far from the 3' end of exon 1, introduces a frame-shift that leads to premature termination of the AR protein. Separately initiated polypeptides containing the DNA-binding and the steroid-binding domains of the androgen receptor are produced in vitro by using strong internal translation initiation sites. These carboxyl-terminal polypeptides have the characteristics of the shortened form of the receptor previously described in the tissues of Tfm mice.

Animals↗
PubMed1990

Structure and size distribution of the androgen receptor mRNA in wild-type and Tfm/Y mutant mice.

Complementary DNA clones covering the coding region of the mouse androgen receptor (AR) were assembled by enzymatic amplification from testicular RNA and genomic DNA. The deduced amino acid sequence consists of 899 residues and departs from the rat sequence at 21 positions, 20 of which are in the amino-terminal trans-activation domain. A notable cluster of substitutions lies in the region of the long glutamine repeat at positions 174-195. The size heterogeneity of AR messengers suggested by previous blot hybridization experiments was examined by RNase protection analysis of sucrose gradient-fractionated poly(A) RNA from mouse liver. A predominant 10-kilobase long mRNA species was found to encode the AR, and a 3' noncoding portion longer than 5 kilobases was demonstrated by internal cleavage with RNase-H, followed by blot hybridization with a 3' probe. The sensitivity afforded by the use of homologous RNA probes in solution hybridizations allowed the demonstration in Tfm/Y mutant mice of an AR mRNA that covers the entire coding region, but is present at 10- to 20-fold lower levels than in normal animals. The detection of significant amounts of receptor messenger revives earlier suggestions of an AR protein in Tfm/Y mice and indicates, at variance with other conclusions, that the expression of this mutant AR is affected at a post-transcriptional level.

Amino Acid Sequence↗
PubMed1989

Proliferation of B cells from chronic lymphocytic leukemia is selectively promoted by B cell growth factor.

B cell activation was studied in B cells from B cell chronic lymphocytic leukemia (B-CLL) patients. After in vitro stimulation, these B cells showed extensive proliferation in the presence of high-molecular-weight B cell growth factor (BCGF). In contrast, this effect was not observed upon addition of recombinant interleukin-2 (IL-2). In agreement, upon stimulation, B cells expressed Bac-1 antigen but failed to acquire the IL-2 receptor. These results demonstrate that the utilization of the BCGF pathway can be segregated from that of IL-2 in B cells from B-CLL patients.

B-Lymphocytes↗
PubMed1988

The B-cell activation pathway in human systemic lupus erythematosus: imbalanced in vitro production of lymphokines and association with serum analytical findings.

Recent knowledge of B-lymphocyte physiology has clarified the role of T cell-derived lymphokines in clonal proliferation and differentiation of B-cell responses. Lymphokine production was analyzed in 19 systemic lupus erythematosus (SLE) patients and sex- and age-matched controls in relation to clinical activity and steroid treatment. When in vitro production of interleukin-2 (IL-2) and B-cell growth factor (BCGF) was tested, both activities were found to be diminished in the group of patients (P less than 0.01), while B-cell differentiation factor (BCDF) activity was higher in this group with respect to normal controls (P less than 0.01). Interestingly enough, this in vitro BCDF synthesis was positively correlated with clinical activity regardless of low-dose steroid treatment. A correlation was also found between BCDF production and the levels of IgG (r = 0.64, P less than 0.01), anti-DNA antibodies (r = 0.52, P less than 0.05), and the IgG/IgM ratio (r = 0.7, P less than 0.01) in serum. Implications of these abnormal T-lymphocyte functions in SLE with respect to in vivo B-cell function are discussed.

Adolescent↗
PubMed1988

Role of interleukin 2 in inducing normalization of natural killer activity in systemic lupus erythematosus.

A low natural killer (NK) activity has been well documented for lymphocytes from systemic lupus erythematosus (SLE) patients. Given the defect in interferon-gamma (IFN-gamma) and interleukin 2 (IL-2) production also reported in SLE, it seemed possible that the absence of these molecules could account for some of the defective NK activity. We have tested the NK activity present in peripheral blood mononuclear cells (PBMC) from SLE patients and its in vitro modulation by different preparations of exogenous IL-2. We have found a low NK activity for PBMC from steroid-treated SLE patients, and we report an enhancement in lytic activity after incubation with mitogen-induced, IL-2-containing supernatants obtained from human tonsils. This increase did not seem to be due to recruitment of new effectors because the numbers of cells expressing NK phenotype (Leu 7+ or Leu 11b+) did not change after the incubation, nor did the target specificity of the lysis. However, a similar degree in the improvement in cytotoxicity was not obtained by incubation with a purified preparation of IFN gamma and/or recombinant IL-2 (rIL-2). A possible role for a molecule other than these lymphokines that might influence this effect is discussed.

Cell Line↗
PubMed1988

Presence of an autoantibody against a Golgi cisternal membrane protein in the serum and cerebrospinal fluid from a patient with idiopathic late onset cerebellar ataxia.

Tissue and cultured cells of different species and embryological origins incubated with serum (diluted up to 10,000-fold) or cerebrospinal fluid (CSF) (6-fold dilution) from a 48-year-old female patient with idiopathic late-onset cerebellar ataxia, exhibited a bright specific perinuclear staining when studied by indirect immunofluorescence microscopy. The pattern of the staining was that characteristic of the Golgi apparatus, consisting of a crescent-shaped juxtanuclear reticulum located in the vicinity of the microtubule organizing center. Changes in location and organization of the organelle stained by the patient's serum during mitosis or after incubation of the cells with Colcemid, taxol or monensin, resulted in a disruption of the reticulum that followed the expected patterns for Golgi apparatus. The staining was specifically absorbed with Golgi cisternae-enriched membrane fractions. Finally, dot-immunoblotting studies of membrane and soluble fractions of Golgi cisternae and vesicles showed that the anti-Golgi antibody (AGA) reacted with the cytoplasmic domain of an integral membrane protein contained in the Golgi cisternae. The presence of this unusual autoantibody in an idiopathic late-onset cerebellar ataxia-bearing patient can afford some insights into the pathogenesis of these neurological diseases.

Autoantibodies↗
PubMed1987

Development of Ly-1+ B cells in immunodeficient CBA/N mice.

Spleen cells from CBA/N mice developing a systemic autoimmune disease after daily injection of CsA during an autologous bone marrow reconstitution were transferred into unmanipulated syngeneic recipients. Adoptive transfer allowed the development of Ly-1+ B cells, which shared Mac-1 differentiation antigen expression with the myelomonocytic lineage. Interestingly, expansion of formerly absent Ly-1+ B cells was paralleled by a severe reduction in common, Ly-1-, B cell development in the recipient. We conclude that precursors for Ly-1+ B lineage do exist in CBA/N mice.

Animals↗