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Biomedical subjects

D Gerlier

Publications and source records attributed to D Gerlier.

At least 73 records · Page 4Linked to original sources
PubMed1987

In vivo activation of nude mouse macrophages by human melanoma cells.

Human melanoma cell lines inoculated ip in outbred nude mice were found to activate locally macrophages, which became tumoricidal for the EL 4 target cells in a 48-hour [3H]thymidine cytotoxicity assay. However, the kinetics of this activation largely depended on the tumorigenicity of the cell line used. One week after inoculation with a poorly tumorigenic cell line (PTCL), peritoneal macrophages showed a maximal tumoricidal activity, which then slowly declined to disappear on the 4th week. Macrophages obtained after inoculation of a highly tumorigenic cell line (HTCL) were also activated, but the level of their tumoricidal activity was somewhat lower and decreased more rapidly. Irradiated melanoma cells were also able to activate peritoneal macrophages. The inoculation of a higher number of melanoma cells (less than or equal to 8 X 10(7) cells) resulted in a parallel increase in the cytotoxicity of peritoneal macrophages when activated by PTCL and in a parallel decrease when activated by HTCL. Activated macrophages taken 1 week after tumor cell inoculation and further kept in vitro without additional stimulation progressively lost their tumoricidal activity, within 48 hours after being harvested from PTCL-inoculated mice and within 24 hours after being collected from HTCL-inoculated animals. These data allied to the in vivo capacity of peritoneal cells rich in activated macrophages to prevent the growth of HTCL in nude mice strongly leaned toward the idea that macrophages are involved in the tumor growth control in the absence of a specific immune response. In addition, tumor-macrophage interactions are likely to vary from tumor to tumor and may contribute to the expression of the xenografting capacity of human tumor cells.

Animals↗
PubMed1986

Use of MTT colorimetric assay to measure cell activation.

The MTT tetrazolium salt colorimetric assay previously described by Mosmann (1983, J. Immunol. Methods 65, 55) to measure cytotoxicity and cell proliferation was further explored to extend its application to the measurement of cell activation. The level of MTT cleavage by viable cells of various origins was found to be directly proportional to the number of cells but to increase as a non-linear function of time. This non-linear relationship was related to a time-linear cell death during MTT incubation. The cleavage of MTT by viable cells was found to follow first order kinetics and could be fitted to Michaelis' kinetics. Different cell types exhibited similar apparent Km values (1949 microM) and different apparent maximal velocities (V). The apparent V values determined for a given cell type under different experimental conditions were rigorously similar. This analysis of MTT cleavage by viable cells suggests that the colorimetric MTT test can be useful to quantify the activation level of cells, independently of proliferation.

Animals↗
PubMed1986

Interactions with host macrophages and ability of human melanoma cell lines to grow in nude mice.

The interactions of nude mouse macrophages with five human melanoma cell lines, characterized by their resistance to mouse NK activity and varying in their ability to grow s.c. in nude mice, were investigated. These lines were equally susceptible in vitro to both cytostatic and tumoricidal activities of activated peritoneal macrophages collected from nude mice inoculated 3 days previously with Brucella abortus B19R strains. I.p. injection of a poorly tumorigenic melanoma cell line (PTCL) in nude mice was followed by the local appearance of macrophages able to kill these cells in a 48-hr 3H-thymidine cytotoxicity assay. The level of tumoricidal macrophages was maximum for the first week and then slowly declined to disappear by the 4th week following PTCL inoculation. The use of an HTCL instead of a PTCL also induced macrophages able to kill HTCL cells, but the cytotoxicity level was lower and the activity disappeared more rapidly. In cross-experiments using PTCL-activated macrophages as effectors on HTCL targets, these cells were found to be less sensitive than PTCL cells when macrophages were taken at weeks 2 and 3 following PTCL inoculation. To investigate whether tumoricidal macrophages activated in vivo with human melanoma cells could also act in vivo, we inoculated these s.c. into nude mice, simultaneously with live HTCL cells. Peritoneal cells rich in melanoma-activated macrophages prevented HTCL growth in most recipients, whereas spleen cells from the same donor mice did not modify the tumor take. These data indicate that xenogeneic tumors could activate nude mouse macrophages in vivo and suggest that the ability of human tumors to grow in nude mice could be related to their capacity to activate host macrophages locally and to the susceptibility of human tumor cells to the tumoricidal activity of activated macrophages.

Animals↗
PubMed1986

Sustained IL-2 production by the EL4 subline during continuous phorbol diester stimulation is related to an increase of IL-2-mRNA.

The murine T lymphoma EL4 subline produces large amounts of interleukin 2 (IL-2) upon stimulation with phorbol myristate acetate (PMA). It was found to survive continuous PMA stimulation (10 ng/ml, renewed daily, at 10(6) cells/ml), and to maintain its IL-2 production for several days in a culture medium low in protein. IL-2 production varied from day to day with a peak within the first 2 days of stimulation. PMA stimulation was accompanied by morphologic changes (enlargement and adhesiveness), arrest in cell division and DNA replication, continued RNA synthesis and increased mitochondrial activity. These effects were reversible upon removal of PMA, with a decrease of IL-2 secretion to the background level and a progressive recovery of cell growth. The reverted EL4 cells were again able to produce large amounts of IL-2 upon restimulation with PMA. Growth of EL4 cells was shown not to depend on autologous production of IL-2, and their DNA synthesis could be arrested by aphidicolin without a concomitant accumulation of IL-2 in the supernatant. This indicates that the increase in the amount of free IL-2 is not simply due to reduced consumption by the EL4 cells themselves. PMA appears to act at the level of the expression of the IL-2 gene, since IL-2 mRNA was much more abundant in the IL-2-secreting PMA-treated cells than in untreated controls. Our observations show that considerable amounts of IL-2 can be obtained from a single EL4 cell inoculum and may represent an original model to study the effects of PMA on the differentiation and maturation processes of cells.

Animals↗
PubMed1986

Phase separation of miscible phospholipids by sonication of bilayer vesicles.

Sonication of phospholipid vesicles may result, according to their liquid or solid crystal state, in the generation of unilamellar vesicles or structural defects within their bilayers, respectively. The transition temperature Tm of the phospholipid bilayer is usually the threshold temperature delineating the physical effects of ultrasound. However, for vesicles made from a mixture of two miscible phospholipids, this threshold temperature was not found to be the intermediate Tm of the phospholipid mixture bilayers, but the Tm of the lowest melting component. This was due to a simultaneous lateral phase separation of the two phospholipids induced by the sonication as demonstrated by differential scanning calorimetry analysis.

Carbon Radioisotopes↗
PubMed1986

Enhancement of immunogenicity of tumour virus antigen by liposomes: the effect of lipid composition.

The secondary humoral response evoked in W/Fu rats by the weakly immunogenic soluble Gross cell surface antigen (GCSAa) extracted from the syngeneic (C58NT)D lymphoma can be enhanced when GCSAa is presented in liposomes, and this requires the antigen to be strongly associated with the phospholipid bilayers. In order to investigate further the role of the phospholipid microenvironment in the membrane presentation of this antigen, the relationship between the phospholipid composition and the immunogenic potency of GCSAa liposomes was explored. For a given neutral phospholipid component, optimal immunogenicity was obtained when 20% cholesterol was present and when a negatively charged phospholipid was included as a minor component. When phosphatidylcholines (PC) were used as the major neutral component, the immunogenicity of GCSAa liposomes from optimal for distearoyl PC (DSPC) decreased when decreasing the PC acyl chain length down to the background level for dilauroyl PC (DLPC). Similarly, the use of PC with acyl chain of increasing unsaturation was followed by a decline in the immunogenicity of the GCSAa liposomes up to the background level for dilinoleoyl PC (DLiPC). Replacing PC headgroups by phosphatidylethanolamine (PE) headgroups abolished the enhancing effect of the liposome presentation on GCSAa immunogenicity. Three groups of phospholipids unable to promote the expression of the GCSAa immunogenicity could be distinguished: the DLPC, DLiPC group, unable to prime the animals but, contrary to the soluble antigen, able to boost the animal after an appropriate priming with GCSAa-DSPC-liposomes, the dimyristoyl PE group, unable to prime or to boost and non-toxic in vitro for the macrophages, and the dipalmitoyl PE group, acutely toxic for macrophages.

Animals↗
PubMed1986

Presentation of an MuLV-related tumour antigen in liposomes as a potent tertiary immunogen after adoptive transfer.

The immunogenicity of Gross virus cell surface antigen (GCSAa) extracted from rat lymphoma cells can be dramatically increased by its presentation into liposomes, probably by mimicking the cell membrane presentation. Induction of an anti-GCSAa secondary antibody response has been found to require the use of liposomes as GCSAa vehicle for both the priming and the boosting immunizations. In order to investigate the sensitivity of highly immune cells to the liposome presentation, immune spleen cells were stimulated in vitro with either soluble GCSAa or GCSAa-liposomes and transferred together with the immunogen into syngeneic animals. Only spleen cells from high responders, which have been immunized twice with GCSAa-liposomes, were able to generate an antibody response in naive recipients after their restimulation with the GCSAa-liposome preparation. Their restimulation with soluble antigen was ineffective unless 10% peritoneal exudate cells (PEC) from naive rats were added during the in vitro incubation. Stimulation with GCSAa-liposomes was further improved by the addition of 10% PEC. Macrophages were found to play a central role in the induction of antibody response in the recipients after stimulation with GCSAa-liposomes. Treatment of immune spleen cells with the macrophage-killing agent leucine methyl ester prior their restimulation in vitro with GCSA-liposomes in the absence of PEC, or depletion of macrophage after their in vitro incubation with this immunogen, completely abolished the induction of anti-GCSAa antibodies in the recipients.

AKR murine leukemia virus↗
PubMed1985

Tumourigenic phenotypes of human melanoma cell lines in nude mice determined by an active antitumour mechanism.

Ten human melanoma cell lines (HMCL) were tested for their ability to grow subcutaneously in nude mice. Using a standard inoculum, the HMCL could be characterized by their highly, fairly or poorly xenografting phenotype. These phenotypes were stable and the phenotype of one HMCL was recovered within cell clones derived from it. The role of nude mice natural defences in the expression of HMCL xenografting phenotypes was studied. Sublethal whole body irradiation and silica pretreatment of recipients enabled poorly tumourigenic HMCL to grow in most animals without affecting their splenic NK activity. Admixture of BCG or MDP encapsulated in liposomes with highly tumourigenic HMCL resulted in the abrogation of tumour growth in naive nude mice. The long lasting abrogating of NK activity in vivo by treatment with anti-asialo-GM1 anti-serum did not enhance the growth of a poorly tumourigenic HMCL. The HMCL were found to be resistant to in vitro murine NK activity. These results showed that the expression of the HMCL xenografting phenotypes could be controlled by the nude mice natural defences. NK cells did not seem to be largely involved whereas macrophages might be good candidates as anti-xenograft effectors.

Acetylmuramyl-Alanyl-Isoglutamine↗
PubMed1984

Use of an automatic cell harvester in a cellular radioimmunoassay.

An automatic cell harvester was used in the final step of a cellular radioimmunoassay to collect cell bound anti-rat IgG 125I-F(ab')2. Studies on the reliability of this collection method were performed with antibodies directed against cell surface antigens induced by the Gross murine leukemia virus and produced by immunization of W/Fu rats with the syngeneic (C58NT)D lymphoma. Glutaraldehyde-fixed as well as untreated Gross virus induced lymphoma cells could be used. Similar and specific antibody binding curves were obtained when the cells were incubated with the anti-(C58NT)D serum and anti-rat IgG 125I-F(ab')2 in the presence of 0.1% NaN3. Background levels of non-specific binding of anti-rat 125I-F(ab')2 to mouse lymphoma cells or rat thymocytes were only a few cpm above the background of the gamma-counter. This allowed detection of surface immunoglobulin positive lymphocytes among as few as 30,000 rat splenocytes. In addition, this cellular radioimmunoassay was found to be suitable for the measurement of solubilized cell surface antigen by its capacity to inhibit the binding of the specific antibodies to the target cells.

AKR murine leukemia virus↗
PubMed1983

Physical separation of the aqueous phase and lipoidal lamellae from multilamellar liposomes: an analytical and preparative procedure.

An efficient and rapid method to separate the aqueous phase from lipoidal lamellae after mechanical disruption of multilamellar liposomes with preservation of the phospholipid bilayer organization has been designed. Liposome suspensions were subjected to a few short bursts of sonication at a temperature below the transition temperature (Tc) of the lowest melting phospholipid component. This was followed by ultracentrifugation. The aqueous supernatant contained more than 90% of the encapsulated aqueous elements (6-carboxyfluorescein, bovine serum albumin) and less than 1% of the lipids. The washed pellet consisted of lipoidal lamellae devoid of any encapsulated aqueous phase. The lipoidal lamellae contained more than 99% phospholipids and were contaminated with less than 0.1% aqueous components. Such a method should allow the localization of any component within the aqueous phase or lipoidal lamellae of the liposomes.

Chemical Phenomena↗
PubMed1981

Non-immunogenicity of enucleated rat hepatoma cells in syngeneic animals.

Cytoplasts and karyoplasts were obtained by ultracentrifugation of Hepatoma D23 cells on a Ficoll gradient containing cytochalasin B. Their nuclear and protein content and their metabolic activity were determined. Three i.p. injections of 2.3 x 10(7) cytoplasts were unable to protect syngeneic WAB/Not rats against an s.c. challenge of 10(4) D23 cells, whereas a similar amount of karyoplasts, or 3 injections of 10(6) irradiated D23 cells, were fully protective. Ability of cytoplasts to act as primary or secondary immunogen were also studied, and compared to that of 0.01% glutaraldehyde-treated cells, 43 degrees C heat-treated cells and 3M KCl-soluble extracts, these preparations also being of weak immunogenicity. Only heat-treated cells behaved as a primary immunogen, whereas none of the preparations provided a secondary stimulation. Moreover, when these preparations were fed in vitro to peritoneal-exudate cells before their injection into rats, cytoplasts and glutaraldehyde-treated cells showed no immunogenicity, whereas heat-treated cells induced full protection against tumour challenge. Therefore, in this tumour model, the in vivo persistence of immunogen and the presence of a nucleus are likely to be crucial in inducing transplantation resistance to tumour.

Animals↗
PubMed1981

Measurement of Gross cell-surface antigen and p30 level in murine retrovirus-infected cell lines.

The level of Gross cell-surface antigen (GCSAa) expression at the surface of murine retrovirus-infected fibroblasts was determined by quantitative absorption of the anti-GCSAa activity of a serum produced in syngeneic W/Fu rats immunized against (C58NT)D lymphoma, and tested in a cytotoxicity assay against E male G2 lymphoma cells. While GCSAa was specifically expressed on Gross-type virus (G-MuLV)-induced lymphoma cells, and while G-MuLV and G-related MuLV induced a high level of GCSAa expression on murine fibroblasts, the Friend-Moloney-Rauscher (FMR) group viruses (FMR MuLV) and xenotropic isolates were also able to induce a high or intermediate level of GCSAa. Since GCSAa has been shown to be borne by glycosylated precursors of the viral nucleocapside (gp95gag and gp85gag), the amount of GCSAa expressed on these cells was compared to the level of cytoplasmic p30. In G- and G-related MuLV-infected cell lines, a significant relationship was found between the amount of GCSAa and the level of p30, whereas in FMR-MuLV or xenotropic virus-infected cells the amount of GCSAa varied independently of the p30 level. These results could explain the discrepancy in the specificity of expression of GCSAa in vivo and in vitro.

AKR murine leukemia virus↗
PubMed1981

Cultured T lymphocytes cytotoxic for a syngeneic lymphoma: derivation in Con A-conditioned medium and in vivo activity.

Cultures of murine T lymphocytes with cytotoxic activity towards syngeneic RBL-5 lymphoma cells were obtained from spleen cells of immunized animals after co-culture in vivo with irradiated RBL-5 cells. At different times after initiation, these mixed tumour-lymphocyte cultures (MTLC) were multiplied by transfer to conditioned medium (CM) containing T cell growth factor (TCGF) activity, produced by the stimulation of rat spleen cells with Con A. The effect of residual Con A was investigated by the addition of specific blocking sugar, alpha-methyl mannoside (alpha MM), to the CM in some experiments. This procedure did not reduce the growth potential of the cells, and resulted in a dramatic increase in the cytotoxic activity of the cultures as measured by a 4-hr 51Cr-release assay. The cultures multiplied 1 X 10(3)-fold over a 3-week period with retention of cytotoxicity for RBL-5 cells at levels up to 70-fold greater than those of the MTLCs from which they were derived. The cultured cells, when injected i.p. together with RBL-5 cells into normal mice, were shown to mediate a significant prolongation of the survival of the treated animals. This effect was, however, less dramatic than had been expected from the in vitro results. It would therefore appear that, while cells grown in tissue culture using Con A-conditioned medium may fulfill some theoretical requirements for the immunotherapy of experimental tumours, other factor(s) are required for full protection.

Animals↗
PubMed1980

Association of gross virus-associated cell-surface antigen with liposomes.

Gross Cell-Surface Antigen (GCSAa) was obtained from W/Fu (C58NT)D lymphoma cells by Nonidet P40(NP40) or 3M KCl extraction and further purified by Sephadex G200 filtration. GCSAa was associated with lipids (dipalmitoylphosphatidycholine, cholesterol and dicetylphosphate, in molar ratios of 7:2:1) to form multilamellar liposomes. The amount of protein associated with liposomes was found to be proportional to the protein concentration of the sensitizing cellular extract and to the amount of phospholipids used and, under defined conditions, 22-55% of the protein of the cellular extract could be associated with liposomes. Analysis of disrupted sensitized liposomes showed that the GCSAa-specific activity of the liposome-associated proteins was quite similar to that of the proteins of the sensitizing cellular extract. Ultracentrifugation of disrupted liposomes showed that about 75% of the liposome-associated GCSAa activity was firmly associated with lipids and that little GCSAa was trapped within aqueous compartments between lipidic lamellae. 1.8--8.0% of the liposome-associated GCSAa was expressed at the liposome surface. No striking differences in degree of GCSAa association were found between liposomes sensitized by NP40 or by 3M KCl extracts. Storage experiments at +4 degrees C showed that GCSAa-sensitized liposomes were fairly stable.

AKR murine leukemia virus↗
PubMed1980

Induction of antibody response to liposome-associated Gross-virus cell-surface antigen (GCSAa).

The immunogenicity of a soluble fraction containing Gross-virus-associated cell-surface antigen (GCSAa) obtained from (C58NT)D lymphoma cells either by detergent (NP40) solubilization or by 3M KCl extraction, was studied in syngeneic W/Fu rats. Rats immunized by 2 s.c. injections of soluble antigen or soluble antigen mixed with empty liposomes and emulsified in complete Freund's adjuvant (CFA) failed to produce significant levels of cytotoxic antibodies to GCSAa. On the other hand, rats similarly immunized by negatively charged liposomes containing NP40-solubilized GCSAa, and emulsified in CFA, developed high and persistent levels of cytotoxic antibodies, and their response could even mimic that induced by viable (C58NT)D cells. A similar response could also be obtained in rats immunized with liposome-associated NP40-solubilized GCSAa, but without CFA. Rats immunized by comparable amounts of liposome-assocated 3M KCl-extracted GCSAa developed only low levels of cytotoxic antibodies, and their response was of shorter duration. These results strongly suggest that inclusion into liposomes of a solubilized proteic tumour-associated cell-surface antigen can provide an immunogen as potent as viable tumour cells in inducing an antibody response, and that the solubilization method may be critical.

AKR murine leukemia virus↗