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Biomedical subjects

M Pla

Publications and source records attributed to M Pla.

At least 37 records · Page 2Linked to original sources
PubMed1999

Transgene expression, but not gene delivery, is improved by adhesion-assisted lipofection of hematopoietic cells.

In contrast to adherent cells, cells growing in suspension and particularly hematopoietic cells, are notoriously difficult to transfect in vitro using nonviral approaches. In the present study, the effect of cell adhesion on gene transfer efficacy was investigated by allowing hematopoietic cells to bind to an adherent cell monolayer (ACM) before being subjected to cationic liposome-mediated DNA transfer. Human CD34 and T CD4 cell lines were cultivated on an ACM constituted of murine fibroblast NIH3T3 cells and transfected with a plasmid carrying the beta-galactosidase gene. X-gal staining showed that up to 27% of the cells expressed the transgene. In contrast, less than 0.1% of these cells were positively transfected in suspension. This adhesion-assisted lipofection (AAL) procedure was also successfully tested on blood lymphocytes, since it resulted in up to 30% of transfected human primary T lymphocytes. Flow cytometry analysis performed on T lymphocyte subsets revealed that 8 and 9%, respectively, of CD4 and CD8 cells could be transfected with a plasmid carrying the green fluorescent protein gene. Other adherent cells, such as MS5 murine stromal cells or HeLa epithelial cells, were also a compatible matrix for AAL. Moreover, the pCMV beta plasmid was present in similar amounts in the nuclei of TF1 cells transfected in suspension or with the AAL procedure. These data raise the possibility that cell matrix/hematopoietic cell interactions might govern expression of the transgene in hematopoietic cells growing usually in suspension, but not endocytosis of liposome/DNA particles and plasmid migration ot the cell nucleus.

3T3 Cells↗
PubMed1998

Strong alloantigenicity of the alpha-helices residues of the MHC class I molecule.

To evaluate the role of single residues of a MHC class I molecule in the induction of a primary allogeneic response, we have tested the ability of various point mutants (of the alpha-helices or beta-sheet of the alpha1 and alpha2 domains) of the Kd molecule to induce a primary cytotoxic T cell response in mice carrying the wild-type molecule. For that, we have used an in vivo model in which cells expressing mutant molecules were injected into the hind footpads of mice carrying wild-type Kd, and the recipient graft-draining popliteal lymph nodes were tested for the presence of alloreactive CTL. Under these experimental conditions, only 7 of the 25 mutant Kd molecules induced a primary allogeneic response. All of these mutations (positions 62, 65, 69, 72, 155, 163, 166) concern residues of the alpha-helices, demonstrating that very small variances from self in a class I molecule, located outside the peptide-binding groove, can be antigenic. To determine the peptide requirements for the generation of a primary allogeneic response, we have analyzed the repertoire of peptides selected by individual mutant molecules shown to be able or unable to induce a CTL response. No correlation was observed between the peptidic make-up presented by a given mutant and its capacity to induce a primary allogeneic response. On the whole, our data point to the alloantigenicity of potentially TCR-contacting surface residues of the MHC class I molecules.

Amino Acid Substitution↗
PubMed1998

Differential in vitro and in vivo effects of all-trans retinoic acid on the growth of human myeloma cells.

The effects of all trans retinoic acid (ATRA) on human myeloma cells growth were studied in vitro and in vivo using immunodeficient mice engrafted with malignant plasma cells. ATRA inhibited the in vitro proliferation of plasma cells originating from two patients with multiple myeloma whereas it had no effect on the in vivo growth of plasma cell grafts as assessed by the serial study of human Ig levels in mouse serum. Thus, the efficacy of ATRA for the treatment of human multiple myeloma remains to be ascertained.

Animals↗
PubMed1997

Elf-1 contributes to the function of the complex interleukin (IL)-2-responsive enhancer in the mouse IL-2 receptor alpha gene.

Lymphocytes regulate their responsiveness to IL-2 through the transcriptional control of the IL-2R alpha gene, which encodes a component of the high affinity IL-2 receptor. In the mouse IL-2R alpha gene this control is exerted via two regulatable elements, a promoter proximal region, and an IL-2-responsive enhancer (IL-2rE) 1.3 kb upstream. In vitro and in vivo functional analysis of the IL-2rE in the rodent thymic lymphoma-derived, CD4- CD8- cell line PC60 demonstrated that three separate elements, sites I, II, and III, were necessary for IL-2 responsiveness; these three sites demonstrate functional cooperation. Site III contains a consensus binding motif for members of the Ets family of transcription factors. Here we demonstrate that Elf-1, an Ets-like protein, binds to site III and participates in IL-2 responsiveness. In vitro site III forms a complex with a protein constitutively present in nuclear extracts from PC60 cells as well as from normal CD4- CD8- thymocytes. We have identified this molecule as Elf-1 according to a number of criteria. The complex possesses an identical electrophoretic mobility to that formed by recombinant Elf-1 protein and is super-shifted by anti-Elf-1 antibodies. Biotinylated IL-2rE probes precipitate Elf-1 from PC60 extracts provided site III is intact and both recombinant and PC60-derived proteins bind with the same relative affinities to different mutants of site III. In addition, by introducing mutations into the core of the site III Ets-like motif and comparing the corresponding effects on the in vitro binding of Elf-1 and the in vivo IL-2rE activity, we provide strong evidence that Elf-1 is directly involved in IL-2 responsiveness. The nature of the functional cooperativity observed between Elf-1 and the factors binding sites I and II remains unresolved; experiments presented here however suggest that this effect may not require direct interactions between the proteins binding these three elements.

Animals↗
PubMed1997

Th1 response in Salmonella typhimurium-infected mice with a high or low rate of bacterial clearance.

Previous studies have shown that the capacity to clear an attenuated strain of Salmonella typhimurium after the second week of infection varies widely among mouse strains. Bacterial clearance is mediated by CD4+ T cells and is regulated in part by the H-2 complex. The aim of the present study was to compare the patterns of cytokine mRNA expression in the spleens of C57BL/6 (H-2b) and CBA (H-2k) mice, which exhibit a low and a high rate of bacterial clearance, respectively. A transient increase in interleukin-12 (IL-12) mRNA levels was found in both mouse strains. Gamma interferon (IFN-gamma) gene expression was higher and more sustained in C57BL/6 than in CBA mice. No increase in IL-4 mRNA was detected. A transient increase in IL-10 mRNA was found in C57BL/6 mice. Separation of spleen cells into CD4+ and CD4- fractions showed that CD4+ T cells produced the bulk of IFN-gamma in both mouse strains and of IL-10 in C57BL/6 mice. Infection of H-2 congenic mice induced a higher level of IFN-gamma mRNA expression by CD4+ T cells in mice with a low rate of clearance (H-2b) than in mice with a high rate of clearance (H-2q). Treatment of infected C57BL/6 mice with anti-IFN-gamma or anti-CD4 monoclonal antibodies indicated that IFN-gamma participates in resistance in the early phase of infection, but not in bacterial clearance, and that CD4+ T cells mediate bacterial clearance during the 3rd week of infection. Taken together, these results suggest that defective bacterial clearance in H-2b mice is not linked to defective IFN-gamma production and that CD4+ T cells mediate bacterial clearance by an IFN-gamma-independent mechanism.

Animals↗
PubMed1997

Relationships of meat characteristics of two lines of rabbits selected for litter size and growth rate.

We measured meat characteristics of 46 commercial rabbit carcasses from two synthetic breeds selected for litter size or growth rate. Color measurements (CIELAB) were made on several muscle surfaces. Color measured on the muscular aponeurosis indicated a pale carcass at most of the points; Chroma (C*) and hue (H*) varied from 3.71 and 31.1, respectively, for longissimus at the 7th lumbar vertebra to 14.3 for C* of trapezius and 56.1 for H* of biceps femoris. Color of the longissimus cut surfaces differed substantially from the exterior surface color. The 1st lumbar vertebra cut also differed from the 7th lumbar vertebra cut. The pH of the biceps femoris was 5.80 and that of the longissimus was 5.75. Meat fat content of half a carcass was 5.28%. The first four principal components of an analysis with color and pH measurements of the longissimus and biceps femoris and meat fat content explained 68% of the variance. The entire set of variables was well summarized by the lightness (L*) and Chroma of the longissimus muscle exterior surface measured at the 4th lumbar vertebra, longissimus pH, and meat fat content. When the data were projected on the plane defined by the first two principal components, two separate groups of points appeared, corresponding to the animals of each breed.

Animals↗
PubMed1996

Alloreactive monoclonal antibodies select Kd molecules with different peptide profiles.

As a part of our continuing effort to study the antigenic structure of class I molecules, we have undertaken two types of studies. First, we have studied the capacity of five different Kd-reactive mAbs to recognize a panel of 25 site-directed mutants of the H-2Kd molecule. Both the gain and the decrease in Ab binding resulted from a single amino acid substitution at different positions. All mutations that increase the binding of the tested mAbs are located on the alpha-helices, indicating that the replacement of an Ig-contacting surface residue with a charged or polar side chain by a short one generally favors Ab binding. Mutation of two alpha-helix-situated residues, 58 and 166, completely abolished the binding of one mAb (Tu191.7.1), indicating that these two residues contribute to the antigenic determinant defined by this mAb. The overwhelming majority of mutations that diminished Ab binding concerns residues buried within the Ag binding groove, suggesting the possibility of peptide contribution to serologic epitopes defined by alloreactive Abs. We have addressed this issue by comparison of the repertoire of peptides eluted from Kd molecules precipitated by different Kd-reactive mAbs. The results reveal that the two-dimensional profile obtained with one (F35.119.18) of the alloreactive mAbs is clearly different. The use of 21 single amino acid variants of a Kd-restricted 10-mer peptide allowed us to identify the residue of the bound peptide contributing to the epitope recognized by this mAb. Thus, we have shown that at least in some instances, changes induced in the MHC molecules by the binding of distinct peptides can be recognized as alterations in serologic determinants expressed on the class I molecules.

Animals↗
PubMed1996

The absence of human beta 2-microglobulin increases the occurrence of ankylosing enthesopathy in HLA-B27 transgenic mice.

HLA-B27 transgenic mice develop a spontaneous ankylosing enthesopathy (ANKENT). We have investigated the occurrence of ANKENT in transgenic mice carrying transgenes for human beta 2-microglobulin (M TGM), HLA-B27-heavy chain (27 TGM), or both (27M TGM). An unexpected finding was the increase in ANKENT occurrence among the HLA-B27 transgenic mice lacking the human beta 2-microglobulin transgene (27 TGM): 33% of such mice were found to develop ANKENT, whereas 19% of 27M mice were diseased. In addition, the expression of HLA-B27 molecules in individual 27 TGM was highly variable, ranging from no expression to a level similar to that observed in 27M TGM. Our results confirm that in mice the HLA-B27 transgene is a relative risk factor for ANKENT. The increase of ANKENT occurrence is HLA-B27 transgenics in the absence of human beta 2-microglobulin suggests a possible role for impaired cell surface expression of HLA-B27. The absence of human beta 2-microglobulin might entail an accumulation of unassembled HLA-B27 heavy chains. Exposure of these mice to an environmental trigger could then lead to an inappropriate immune response which might result in disease development.

Animals↗
PubMed1995

Deletion of the last two exons of the mitochondrial nad7 gene results in lack of the NAD7 polypeptide in a Nicotiana sylvestris CMS mutant.

In Nicotiana sylvestris, two cytoplasmic male sterile (CMS) mutants obtained by protoplast culture show abnormal developmental features of both vegetative and reproductive organs, and mitochondrial gene reorganization following homologous recombination between 65 bp repeated sequences. A mitochondrial region of 16.2 kb deleted from both CMS mutants was found to contain the last two exons of the nad7 gene coding for a subunit of the mitochondrial respiratory chain complex I, which is encoded in the nucleus in fungi and animals but was recently found to be encoded by the mitochondrial genome in wheat. Although the N. sylvestris nad7 gene shows strong homology with its wheat counterpart, it contains only three introns instead of four. Polymerase chain reaction (PCR) experiments indicated that the parental gene organization, including the complete nad7 gene, is probably maintained at a substoichiometric level in the CMS mutants, but this proportion is too low to have a significant physiological role, as confirmed by expression studies showing the lack of detectable amounts of the NAD7 polypeptide. Consequently, absence of NAD7 is not lethal to plant cells but a deficiency of complex I could be involved in the abnormal CMS phenotype.

Amino Acid Sequence↗
PubMed1995

The 8 kD product of the putative oncogene MTCP-1 is a mitochondrial protein.

An unusually small (8 kD) protein (p8MTCP-1) is coded by the putative oncogene MTCP-1 (also called c6.1B), involved in the translocation t(X;14)(q28;q11) associated with some mature T-cell proliferations. Here, we show by subcellular fractionation and by confocal microscopy that this protein is located in the mitochondria. This localization orientates toward a role of p8MTCP-1 in the mitochondrial metabolism which may be relevant for the oncogenic process.

Amino Acid Sequence↗
PubMed1995

Mouse interleukin-2 receptor alpha gene expression. Delimitation of cis-acting regulatory elements in transgenic mice and by mapping of DNase-I hypersensitive sites.

The alpha chain of the interleukin-2 receptor (IL-2R alpha) is a key regulator of lymphocyte proliferation. To analyze the mechanisms controlling its expression in normal cells, we used the 5'-flanking region (base pairs -2539/+93) of the mouse gene to drive chloramphenicol acetyltransferase expression in four transgenic mouse lines. Constitutive transgene activity was restricted to lymphoid organs. In mature T lymphocytes, transgene and endogenous IL-2R alpha gene expression was stimulated by concanavalin A and up-regulated by IL-2 with very similar kinetics. In thymic T cell precursors, IL-1 and IL-2 cooperatively induced transgene and IL-2R alpha gene expression. These results show that regulation of the endogenous IL-2R alpha gene occurs mainly at the transcriptional level. They demonstrate that cis-acting elements in the 5'-flanking region present in the transgene confer correct tissue specificity and inducible expression in mature T cells and their precursors in response to antigen, IL-1, and IL-2. In a complementary approach, we screened the 5' end of the endogenous IL-2R alpha gene for DNase-I hypersensitive sites. We found three lymphocyte specific DNase-I hypersensitive sites. Two, at -0.05 and -5.3 kilobase pairs, are present in resting T cells. A third site appears at -1.35 kilobase pairs in activated T cells. It co-localizes with IL-2-responsive elements identified by transient transfection experiments.

Animals↗
PubMed1995

Mouse interleukin-2 receptor alpha gene expression. Interleukin-1 and interleukin-2 control transcription via distinct cis-acting elements.

We have shown that interleukin-1 (IL-1) and IL-2 control IL-2 receptor alpha (IL-2R alpha) gene transcription in CD4-CD8- murine T lymphocyte precursors. Here we map the cis-acting elements that mediate interleukin responsiveness of the mouse IL-2R alpha gene using a thymic lymphoma-derived hybridoma (PC60). The transcriptional response of the IL-2R alpha gene to stimulation by IL-1 + IL-2 is biphasic. IL-1 induces a rapid, protein synthesis-independent appearance of IL-2R alpha mRNA that is blocked by inhibitors of NF-kappa B activation. It also primes cells to become IL-2 responsive and thereby prepares the second phase, in which IL-2 induces a 100-fold further increase in IL-2R alpha transcripts. Transient transfection experiments show that several elements in the promoter-proximal region of the IL-2R alpha gene contribute to IL-1 responsiveness, most importantly an NF-kappa B site conserved in the human and mouse gene. IL-2 responsiveness, on the other hand, depends on a 78-nucleotide segment 1.3 kilobases upstream of the major transcription start site. This segment functions as an IL-2-inducible enhancer and lies within a region that becomes DNase I hypersensitive in normal T cells in which IL-2R alpha expression has been induced. IL-2 responsiveness requires three distinct elements within the enhancer. Two of these are potential binding sites for STAT proteins.

Animals↗
PubMed1995

Recombinant-human luteinizing hormone (r-hLH) as ovulatory stimulus in superovulated does.

PURPOSE: To study the effects of r-hLH as ovulatory stimulus in does. METHOD: New Zealand does, 18 wk old, in estrus, received 25 IU of pregnant mare serum gonadotropin (PMSG) followed at 48 h either by 50 IU of r-hLH (n = 20) or hCG (n = 20) to induce follicular growth and ovulation. All does were previously artificially inseminated to avoid endogenous LH surge. Half of the animals receiving r-hLH (n = 10) or hCG (n = 10) were killed at 72 h after the hormone administration, and the remaining half were killed at 14 days. At 72 h the number of corpora lutea and preovulatory follicles was determined, and fertilization rate, embryo quality, degree of embryonic development, and oviductal transit were all assessed. On day 14 the number corpora lutea and implanted embryos were counted, and implantation rate was determined. Median and interquartile ranges were calculated for each parameter. RESULTS: At 72 h the median for corpora lutea was 8 (7-10) in the r-hLH group vs 13 (10-14) in the hCG group (P = 0.009); preopvulatory follicles were 7 (6-10) vs 0 (0-0) (P = 0.0007); the percentage of good-quality embryos was 71.4% (54.5-75) vs 33.3% (25-37.5) (P = 0.001), for intermediate-quality embryos it was 25% (14.3-36.4) vs 33.3% (25-38.5), and the percentage of degenerated embryos was 0% (0-12.5) vs 33.3% (25-37.5) (P = 0.015), respectively. Fertilization rates were similar in both groups. Embryonic development was more homogeneous in the animals receiving r-hLH (8 to > or = 16 cells) compared to those receiving hCG (2 to > or = 16 cells). The median of embryos still in oviducts at 72 h was significantly higher in the hCG group [6 (4-13)] than in the r-hLH group [0 (0-4)] (P = 0.41). At 14 days the median of corpora lutea was higher in the hCG [12 (11-16)] than in the r-hLH group [10 (7-13)] (P = 0.008), but no differences were noted in the number of implanted embryos. Implantation rate was higher in the r-hLH group [100 (92.3-100)] than in the hCG group [87.5 (83.3-94.1)] (P = 0.056). CONCLUSIONS: At the studied dose an ovulatory stimulus with r-hLH induced fewer follicles to ovulate than hCG. Recombinant-hLH produced superior embryo morphological quality, a more homogeneous degree of embryo development, and more synchronous embryo transit than hCG. In spite of the larger number of ovulations following hCG, subsequent events essential for pregnancy were higher with r-hLH, offsetting differences in terms of implanted embryos at 14 days of pregnancy.

Animals↗
PubMed1995

HLA-B27 as a relative risk factor in ankylosing enthesopathy in transgenic mice.

HLA-B27 is a risk factor for several human diseases through a mechanism that is not yet understood. This article describes a naturally occurring joint disease in laboratory mice, ANKENT. ANKENT begins with mild inflammation and culminates in irreversible stiffening of the ankle and/or tarsal joints in one or both hind paws. The macroscopic and histologic features of ANKENT, its relationship to age, gender, and environment, and some immunologic aspects are considered. With respect to genetics, it is demonstrated that an HLA-B27 transgene is a relative risk factor for ANKENT. Its impact depends on the H-2 haplotype, reaching a relative risk value of 9.4 for C57Bl/10, H-2b males (p < 0.025). Several features of ANKENT are reminiscent of human AS: joint pathology, age and gender distribution, the presence of non-MHC as well as MHC risk factors (including HLA-B27), and the suspicion that environmental factors are involved.

Age Factors↗
PubMed1995

Effect of recombinant human luteinizing hormone versus human chorionic gonadotrophin: effects on ovulation, embryo quality and transport, steroid balance and implantation in rabbits.

A total of 40 New Zealand female rabbits which had been given follicular stimulation and artificial insemination received 50 IU of either recombinant human luteinizing hormone (rhLH; n = 20) or human chorionic gonadotrophin (HCG; n = 20) to induce ovulation. In each hormone group, 10 animals were killed 72 h later to study the ovulatory process and the number, location, morphological quality and variation in the degree of development of recovered embryos. Pre-ovulatory and post-ovulatory oestradiol and progesterone concentrations were determined in these 10 animals; the remaining 10 animals of each group were killed at 14 days to study implantation up to day 14. At 72 h the number of luteinized follicles and the total number of embryos in the rhLH group were lower than in the HCG group, and the number of pre-ovulatory follicles was higher. The percentage of good quality embryos was higher with rhLH, whereas the percentage of degenerated embryos was lower. Oviductal transit of the embryos was slower and variation in the degree of embryo development greater after HCG. Progesterone concentrations were comparable with pre-ovulatory concentrations at 24 h in the rhLH group, but not in the HCG group where they increased. In the study performed at 14 days, the implantation rate was significantly higher with rhLH versus HCG. These observations suggest that rhLH induces a lower number of follicles to ovulate than does HCG, probably due to its shorter half-life; however, the better embryo quality produced by rhLH may ultimately lead to a better implantation rate; rhLH may mimic the physiological endogenous LH surge more closely than HCG.

Animals↗
PubMed1994

Role of H-2 and non-H-2-related genes in mouse susceptibility to Chlamydia psittaci.

The genetic control of mouse susceptibility to Chlamydia psittaci was investigated after intravenous inoculation of 2 x 10(5) plaque-forming units (pfu). Splenic counts 6 days after inoculation gave the level of susceptibility. Results from inoculation of mice from H-2 congenic strains with three different genetic backgrounds (B10, BALB and C3H) suggested that both the non-H-2 genes and the genes of H-2 complex or those closely associated with it, were responsible for the observed differences in the innate capacity of various inbred lines of mice to control bacterial load in their infected organs following a challenge with C. psittaci.

Animals↗