PubMed Health⌕ Search

Biomedical subjects

A Louise

Publications and source records attributed to A Louise.

17 recordsLinked to original sources
PubMed2000

Accurate quantitation of Leishmania infection in cultured cells by flow cytometry.

BACKGROUND: Leishmaniases are major parasitic diseases caused by protozoans that are obligate intracellular parasites during the mammalian phase of their life cycle. Quantitation of experimental mammalian cell infections is usually performed by time-consuming microscopic examination. In this report a flow cytometry (FCM)-based assay suitable for studying in vitro infections by L.amazonensis is presented. METHODS: Intense fluorescence staining of the amastigote forms with a stage- and species-specific monoclonal antibody was obtained after permeabilization of both the host-cell cytoplasmic membrane and the parasitophorous vacuole membrane by saponin treatment. RESULTS: Upon flow cytometry (FCM) analysis, parasitized cells separated sharply from the auto-fluorescence of the mammalian host cells, giving the assay a high degree of sensitivity and specificity. Ninety to 98% of cells in the more fluorescent population harbored parasites visible by phase-contrast and UV-light microscopy, while no parasites were observed in more than 95% of the cells in the population with background fluorescence. Comparisons of the FCM results with those from microscope counting and analysis of various dilutions of parasitized cells confirmed the reliability of the method. CONCLUSIONS: The FCM assay provided rapid quantitation of Leishmania infection either in mouse macrophages, the natural host cell in murine leishmaniasis, or in Chinese hamster ovary (CHO) cells, a non-macrophage cell line proposed as an in vitro model for studying host-parasite interactions. The protocol described here should be adaptable to studies involving other parasites residing in nucleated cells.

Animals↗
PubMed2000

Liver CD4-CD8- NK1.1+ TCR alpha beta intermediate cells increase during experimental malaria infection and are able to exhibit inhibitory activity against the parasite liver stage in vitro.

Experimental infection of C57BL/6 mice by Plasmodium yoelii sporozoites induced an increase of CD4-CD8- NK1.1+ TCR alpha beta int cells and a down-regulation of CD4+ NK1.1+ TCR alpha beta int cells in the liver during the acute phase of the infection. These cells showed an activated CD69+, CD122+, CD44high, and CD62Lhigh surface phenotype. Analysis of the expressed TCRV beta segment repertoire revealed that most of the expanded CD4-CD8- (double-negative) T cells presented a skewed TCRV beta repertoire and preferentially used V beta 2 and V beta 7 rather than V beta 8. To get an insight into the function of expanded NK1.1+ T cells, experiments were designed in vitro to study their activity against P. yoelii liver stage development. P. yoelii-primed CD3+ NK1.1+ intrahepatic lymphocytes inhibited parasite growth within the hepatocyte. The antiplasmodial effector function of the parasite-induced NK1.1+ liver T cells was almost totally reversed with an anti-CD3 Ab. Moreover, IFN-gamma was in part involved in this antiparasite activity. These results suggest that up-regulation of CD4-CD8- NK1.1+ alpha beta T cells and down-regulation of CD4+ NK1.1+ TCR alpha beta int cells may contribute to the early immune response induced by the Plasmodium during the prime infection.

Animals↗
PubMed1997

Surface antigen expression in spleen cells of C57B1/6 mice during ageing: influence of sex and parity.

So far all studies on the murine ageing process have been conducted on virgin mice. Immune ageing may be influenced by sex hormone differences related to sex or pregnancies. The aim of this study was to investigate whether pregnancies and gender influence the cell changes observed during ageing in a peripheral lymphoid compartment of C57B1/6 mice. Using flow cytometry, changes in (Thy1.2+) T cell, (B220+) B cell and (CD 11b/Mac-1) macrophage spleen populations were monitored in 2, 8 (3 months after last pregnancy) 15 and 23-month-old mice including males, virgin and multiparous females. The development of naive (CD44(low)), memory (CD44(high)), activated/memory (MEL-14, CD62L) cells were investigated in CD4+ and CD8+ T cell subsets. Both short term (at 8 months) and long term (at 15 and 23 months) effects of multiparity were obvious in the lymphocyte/macrophage population changes associated with the ageing process. Short-term effects included delayed appearance of CD4+CD44(high) memory lymphocytes and increased numbers of both CD4+MEL-14(1ow) activated/memory cells and Mac-1+ macrophages when compared with virgin control mice. Later effects of multiparity were increased CD8alpha(dull) populations and increased T/B cell ratios and the ratio of memory to naive CD4+ cells (CD44+(high)/CD44+(low). A sex effect was noticed: males exhibited lower Mac-1+ levels and memory/naive ratio in CD4+ subset than virgin females throughout life. These results suggest that gender and/or pregnancies affect the age-related distribution of lymphoid and macrophage cell populations in the spleen of C57B1/6 mice.

Aging↗
PubMed1997

Sex and parity modulate cytokine production during murine ageing.

We have previously shown that physiological hormone differences related to pregnancy or sex affect the age-related distribution of mononuclear cell populations during murine ageing. To determine whether such changes are involved in the age-related changes in functions of T cells, we examined the secretion of major T cell immunoregulatory cytokines (IL-2, IL-4, interferon-gamma (IFN-gamma), IL-3, IL-6 and granulocyte-macrophage colony-stimulating factor (GM-CSF)) of in vitro concanavalin A-activated spleen cells of C57B1/6 mice. The study included multiparous and virgin females and males at 2, 8, 15 and 23 months of age. Short-term effects of parity (8 months) were evidenced by the decrease of IFN-gamma and the preserved IL-2 production in multiparous females (8 months), while IFN-gamma was unchanged and IL-2 decreased in virgin mice. The increase in IL-4 production appeared earlier in multiparous females (15 months) than in virgin mice (23 months). The increase in IL-4/IFN-gamma and IL-4/IL-2 ratios at 8 and 15 months, respectively, in multiparous females, suggests that pregnancy modifies the Th1/Th2 equilibrium. In late adulthood (15 months), IL-6 and GM-CSF production was higher in multiparous females than in virgin males or females. Sex differences were also noticed: IFN-gamma secretion capacity was lower in males than in females during ageing. This study underlines that the onset, magnitude and kinetics of the age-related changes in cytokine production are parity- and sex-dependent. These changes probably influence the incidence of age-related diseases and may explain the greater longevity of females.

Aging↗
PubMed1995

Quantitative and qualitative changes in CD44 and MEL-14 expression by T cells in C57BL/6 mice during aging.

Aging is associated with a decrease in the functional activity of T cells. We have explored age-related alterations in CD44 and MEL-14 expression by spleen cells bearing the Thy1.2, CD4 or CD8 antigens in C57BL/6 mice at 2, 8, 15 and 23 months of age. The membrane expression of CD44 and MEL-14 molecules can be used to distinguish naive (CD44low, MEL-14high) from preactivated/memory (CD44high, MEL-14low) T cells. Our results show that the proportion of CD4+ splenic cells begins to decrease at an intermediate age (8-month-old mice), whereas the proportion of CD8+ cells remains unaltered. The proportion of CD4+ and CD8+ splenic cells with the CD44high memory phenotype was increased at an early stage of aging (in 8-month-old mice) without a concomitant change in MEL-14 expression. In older mice, MEL-14 expression decreased on CD4+ but not on CD8+ subsets. Recent studies have reported that following activation, the expression of CD44 molecules containing additional, so-called variable exons can be detected. By PCR, we observed an increase in CD44 transcripts containing the v6 or v7 variable exons in murine lymph nodes at the age of 15 months. Our results suggest that v6- or v7-containing variants of CD44 may be involved in the development of memory cells. Taken together, these results suggest that the trafficking of memory T cells in aging may be altered by quantitative and/or qualitative differences in the expression of molecules involved in lymphocyte recirculation.

Aging↗
PubMed1995

Fibronectin of human liver sinusoids binds hepatitis B virus: identification by an anti-idiotypic antibody bearing the internal image of the pre-S2 domain.

Anti-idiotypic antibodies (anti-Ids) have been successfully used to characterize and isolate receptors of several cell ligands. To prepare an immunological probe for identification of cellular components interacting with the hepatitis B virus (HBV), polyclonal antisera against a panel of five HBV-specific monoclonal antibodies (MAbs) were produced in syngeneic BALB/c mice. MAbs to HBV used for immunization (Ab1) recognized biologically important and potentially neutralizing epitopes, located in the pre-S1, pre-S2, or S region-encoded domains of HBV proteins. All the anti-Ids (Ab2) were specific to idiotopes of the homologous Ab1 and inhibited their interaction with the corresponding viral epitopes, suggesting that they recognized unique determinants on the paratope of each immunizing Ab1. Therefore, all five generated polyclonal anti-Ids were of the Ab2 beta type and could represent internal images of viral epitopes. Ab2 raised against the pre-S2 region-specific MAb F124 bound to the extracellular matrix fibronectin of human liver sinusoids. Immunohistochemical studies demonstrated the attachment of viral and recombinant (S, M) hepatitis B surface antigen particles with the pre-S2 region-encoded epitopes to the fibronectin of human liver sinusoids. In contrast, recombinant (S, L*) hepatitis B surface antigen particles, in which the epitope recognized by F124 MAb was not expressed, did not show any binding capacity. These findings suggest that human liver fibronectin may bind HBV in vivo by the pre-S2 region-encoded epitopes in a species-restricted manner. Furthermore, binding of the circulating virus to liver sinusoids could facilitate its subsequent uptake by hepatocytes.

Animals↗
PubMed1988

Activation of c-myc by woodchuck hepatitis virus insertion in hepatocellular carcinoma.

Two hepatocellular carcinomas, induced in woodchucks chronically infected with woodchuck hepatitis virus, were characterized for viral integration near c-myc and alterations of c-myc expression. In one tumor, viral integration within the untranslated region of c-myc exon 3 resulted in overexpression of a long c-myc viral cotranscript. In the second tumor, a single insertion of highly rearranged viral sequences 600 bp upstream of c-myc exon 1 was associated with increased levels of normal c-myc mRNA. In both cases, viral enhancer insertion and disruption of normal c-myc transcriptional or posttranscriptional control appear to be involved in c-myc activation. These results demonstrate that integration of woodchuck hepatitis virus near a cellular proto-oncogene, as in several retroviral models, can contribute to the genesis of liver tumors.

Alleles↗
PubMed1982

Transcription of the hepatitis B surface antigen gene in mouse cells transformed with cloned viral DNA.

Mouse L cells transformed with recombinant plasmids carrying hepatitis B virus (HBV) DNA fragments were used to study the transcription of the viral surface antigen gene (gene S). An HBV-specific, polyadenylated, 2.3-kilobase RNA was mapped on the HBV genome. This RNA hybridized with approximately 75% of the genome and excluded the region of the HBV core antigen gene (gene C). The 2.3-kilobase RNA species was present only in cell lines that produced hepatitis B surface antigen. An HBV-specific 2.3-kilobase RNA was also detected in human hepatoma cell line PLC/PRF/5 which produced hepatitis B surface antigen. A study of gene S expression in the transformed mouse L cells allowed us to localize the regions of initiation and termination of gene S transcription. Our results strongly suggest that the 2.3-kilobase RNA molecule is the mRNA of the major polypeptide of the envelope, which carries the viral surface antigen determinants.

Animals↗
PubMed1981

Structure and expression of the hepatitis B virus genome.

By fusion of the hepatitis B virus (HBV) surface antigen (HBsAg) gene to that of the E. coli lac Z gene carried by a phage lambda derivative, expression of HBsAg antigenic determinants was obtained and carried by a 138,000 dalton fusion polypeptide. Such a protein could be ultimately useful for second generation vaccine production. HBsAg gene expression was studied in eukaryotic cells using the mouse L cell (tk-) system. Cotransformation using a plasmid carrying two copies of the HBV genome in a tandem, head-to-tail arrangement (pCP10) and the cloned HSV-1 tk gene resulted in the excretion of 22 nm HBsAg particles in the supernatant. No other HBV markers were detected. These particles possess the same characteristics as the human serum particles (morphology, diameter, density, antigenicity). The purified HBsAg particles from L cells were found to be highly immunogenic in mice. HBV mRNA transcripts from these cells were analysed by Northern blotting. A major species of 2,300 bases was detected. This was mapped on the genome by hybridization with subgenomic fragments and in the L cell system using a series of plasmid derivatives carrying insertions at specific sites in the HBV genome and assaying for HBsAg expression. Thus the HBsAg gene promotor was localized between positions 2,400-2,800. Indeed there is only one TATA like sequence in this region, starting at position 2,776.

Animals↗
PubMed1980

Biosynthesis of hepatitis B virus surface antigen in Escherichia coli.

Hepatitis B is a widespread viral disease. In the absence of cell cultures capable of propagating the virus (HBV) an efficient vaccine has been prepared from viral envelopes isolated from the plasma of chronic carriers. The major polypeptide of the envelope is one of molecular weight 25,000 which carries the surface antigen (HBsAg). Therefore, the biosynthesis of this polypeptide in Escherichia coli may offer an alternative procedure to produce HbsAg free from human proteins. Recently, the HBV genome has been cloned in E.coli. Determination of its primary structure allowed the localization of the gene (called gene S) coding for HBsAg and the synthesis of the core antigen in E.coli has been reported. We have constructed a derivative of bacteriophage lambda carrying a fusion between the beta-galactosidase gene (lacZ) and the HBsAg coding sequence (lambdalacHBs-1). Infection of E.coli with lambdalacHBs-1 leads to the biosynthesis of a polypeptide of molecular weitht 138,000 carrying antigenic determinants of HBV surface antigen.

DNA, Recombinant↗
PubMed1980

Presence of integrated hepatitis B virus DNA sequences in cellular DNA of human hepatocellular carcinoma.

Hepatitis B virus (HBV) may be one of the agents involved in the aetiology of human primary liver cancer. This hypothesis is supported by (1) the similarity between the geographical distribution of chronic carriers of the viral surface antigen (HBsAg) and that of hepatocellular carcinoma (HCC); (2) the increase in the prevalence of HBV markers in serum of patients with primary liver cancer when compared with the general population; (3) the observation that HBV infection precedes the development of the tumour. Moreover, these epidemiological indications of an association between HBV infecton and hepatocellular carcinoma are supported by the detection of HBV markers such as HBsAg or viral DNA sequences, although in a non-integrated form in tumour tissue. To study the relationship between HBV and primary liver cancer further, we looked for the presence of free or integrated viral DNA in tumour tissue of human hepatocellular carcinomas and in a HBsAg-producing human hepatoma cell line. Using the blot-transfer hybridization technique and cloned HBV DNA as a probe, we have now demonstrated that the viral DNA is integrated in the cellular genome both in tumour tissue and in a hepatoma cell line.

Carcinoma, Hepatocellular↗
PubMed1979

Bacteriophage lambda-E. coli K12 vector-host system for gene cloning and expression under lactose promoter control: I. DNA fragment insertion at the lacZ EcoRI restriction site.

Bacteriophage lambda vectors, derived from lambda plac5 were constructed. Their genomes have only one EcoRI restriction site, located near the end of the beta-galactosidase gene. Recombinants, constructed in vitro, having a DNA fragment inserted in the EcoRI site, are lac- and can be easily recognized. Expression of such foreign genes is then under the control of the lac promoter. Mutations Qam73 and Sam7 greatly increase the amount of beta-galactosidase synthesized by the vector bacteriophage. The lambda ZEQS vector has been certified B2 (EK2) by the French control commission "Recombinaisons génétiques in vitro".

Coliphages↗
PubMed1979

Bacteriophage lambda-E. coli K12 vector-host system for gene cloning and expression under lactose promoter control. II. DNA fragment insertion at the vicinity of the lac UV5 promoter.

Bacteriophage vectors derived from lambda plac5 have been constructed. Their genomes have one EcoRI restriction site which is located at the very beginning of the lac Z gene. The major part of this gene was deleted by an in vivo intramolecular recombination. These vectors allow the fusion of a gene or an operon with the beginning of the lac Z gene, placing them under the control of the lac promoter, which carries the UV5 mutation. Some of these vectors (lambda Y) also include the lac Y gene and it too is under the control of the lac promoter. The lambda YEQS, which carries the Qam73 and Sam7 mutations, as safety mutations, has been certified as a B2 (EK2) vector by the French control commission "recombinaison génétique in vitro".

Coliphages↗
PubMed1979

Cloning in Escherichia coli and physical structure of hepatitis B virion DNA.

A restriction map of hepatitis B virion DNA was established after cloning of the whole viral genome in Escherichia coli. By use of EcoRI, Xho I, Bgl II, Xba I, BamHI, HincII, and Hae III endonucleases, a total of 28 restriction sites were mapped. The single-stranded region was localized on the restriction map and 5' end of the short strand was mapped at a fixed position.

Chromosome Mapping↗