We have investigated the delivery and the pathway in tumoral MCF7 cells of DNA carried by liposomes prepared from (trimethyl aminoethane carbamoyl cholesterol iodide (TMAE-Chol), a cholesterol-based cationic lipid with a quaternary ammonium on the polar head. The structure of DNA-liposome complexes depends on the length of DNA and on the lipid-DNA charge ratio X. Spherical beads constitute fine structures of the observed complexes even when they appear as aggregates. For oligonucleotide transfer, dissociation from liposomes after transfection, penetration of the oligonucleotides into nuclei, and a long resident time were observed. For plasmid transfer, a correlation between the variation in the transfection level and the ultrastructure of complexes was demonstrated. The results showed a cellular route of lipid/plasmid complexes from the beginning by endocytosis, entrapped into endosomes, released by the latter until entry in the perinuclear area, and then penetration of plasmids inside the nuclei resulting in the observed expression of the beta-galactosidase gene.

We recently described a novel nonviral/viral vector for gene transfer, the plasmovirus (Noguiez-Hellin P, Robert-le Meur M, Laune S, et al. C R Acad Sci Paris, Sciences de la Vie. 1996;319:45-50; Noguiez-Hellin P, Robert-le Meur M, Salzmann J-L, et al. Proc Natl Acad Sci USA. 1996;93:4175-4180). Plasmoviruses are plasmids capable of expressing all the viral genes required for generating infectious particles and packaging a defective genome containing a transgene. Transfected as plasmids, plasmoviruses transform the transduced cells into packaging cells that release infectious replication-defective retrovirus vectors (RV) containing a transgene, which are capable of infecting nearby cells. We previously showed that such a vector can efficiently "propagate" the transgene after transfection. Here we examine in greater detail the different steps of plasmovirus replication in vitro in human (143 B TK-) and murine (NIH 3T3 TK-) cells. Molecular-biological analysis revealed plasmovirus-coded protein expression starting from 24 hours post-transfection, followed by the detection of infectious RV 48 hours post-transfection. The gag proteins were correctly processed in the released particles. Electron microscopic analysis revealed typical type C particles. Nonintegrated plasmovirus DNA was not toxic for the cells and could be detected for at least 14 days post-transfection. While the transfected gag gene and the transgene could also be detected throughout this period, we observed that env-coded proteins decreased after 72 hours post-transfection. Nevertheless, the production of RV resulted in the propagation of the transgene in the culture, with stable integration of plasmovirus proviral DNA into the host genome of infected cells. We show that this propagation results in a major improvement in therapeutic efficacy using an HSV1-TK transgene and ganciclovir treatment, when compared to that of plasmovirus constructs that cannot propagate. Altogether, these results demonstrate the functionality of this gene transfer method and suggest that improvements in the vector design enhance its efficacy.

Lymphokine-activated killer (LAK) cells were cocultured in the presence of interleukin-2 (IL-2), with pulmonary surfactant secreting A549 lung carcinoma nodules and maintained in continuous three-dimensional culture for 2-6 days in an attempt to test the response of tumor cells which produce LAK cell inhibitory substances. The A549 nodules secrete mucus which envelops them. This mucus is also secreted inside pseudoalveolar structures characteristic of these nodules. The mucus contains the pulmonary surfactant and sialomucins, both LAK cell inhibitory substances. The spontaneous infiltration into the neoplastic tissue and the membrane contacts established between the two cell types were studied by means of histological, immunohistochemical and electron-microscopic methods. Free-floating LAK cells were allowed to sediment and adhere freely to the nodule surface. The cytostatic and cytolytic effects of LAK cells were tested using thymidine incorporation into DNA and flow cytometry. Despite the presence of a mucus envelope, LAK cells adhered to the A549 nodule surface and penetrated spontaneously into them in the presence of IL-2; they settled mainly in the pseudoalveolar structures where they became apoptotic. According to electron-microscopic observations performed on the second day of coculture, the LAK cells, which remained between the cancer cells, established mostly pinpoint contacts with the carcinoma cells, forming cytoplasmic fusions. These fusions indicate the induction of pores in both the cancer cell and the LAK cell membranes. Electron-microscopic observation also displayed LAK-cell-associated apoptotic and necrotic carcinoma cells. However, at this stage of the coculture (day 2), the DNA synthesis rate of the A549 nodules still remained unchanged; it diminished by approximately 3 times on day 4 and almost stopped on day 6: nodule disintegration was then complete. In the free-floating LAK cell component of the cocultures, DNA synthesis was already strongly inhibited (26x) by the second day. Nevertheless, their cytolytic effect remained unaltered, as was tested on A549 monolayer cells. The presence of tumor necrosis factor (TNF) in the coculture supernatant has been demonstrated, and when coculturing took place in the presence of monoclonal TNF antibody, nodule proliferation was significantly enhanced (up to 145%). Our results indicate that, despite the presence of pulmonary surfactant and sialomucins containing mucus, LAK cells were capable of killing lung carcinoma cells in three-dimensional culture at an early stage of coculture (day 2) by direct cell-to-cell contact. Total nodule disintegration, however, was complete much later (on day 6), and taking into account the low amount of LAK cells in the cancer tissue, this seemed to be the result of an indirect effect implying, in particular, the presence of soluble TNF.

Lymphokine activated killer (LAK) cells were cocultivated from 2 to 6 days with WM266 metastatic melanoma cells maintained as nodules in organotypic culture. The LAK cells in suspension were allowed to deposit freely on the nodule surface from where they could infiltrate spontaneously into the nodules. Immunohistochemical studies were done to localize the LAK cells as well as electron microscopical observations for effector/target membrane contacts. Proliferation of the nodules was tested and also that of the LAK cells after coculturing using tritiated thymidine incorporation into DNA. Cell death was determined by arrest of thymidine incorporation and total nodule disintegration. Infiltration rate of LAK cells into the nodules was low: after coculturing 5% of the nodule cells were LAK cells. Although close membrane contacts and cytoplasmic fusions between effector and target cells leading to tumor cell apoptosis were observed, this direct cytolytic process seemed to be too infrequent for the induction of total nodule disintegration at day 6. Therefore, the indirect pathway to cytolysis might be predominant implying, among other cytokines, soluble TNF. On the other hand, LAK cell proliferation diminished strongly after coculturing (down to 11%) but the cytotoxicity was significantly enhanced (18% higher) suggesting an enhancement of differentiation. This might account for the peculiar efficacy of LAK cells on melanomas in vivo and it would be of interest to study this phenomenon further.

In order to better understand the interaction between activated lymphocytes and breast carcinoma cells, we studied the degree of infiltration, the membrane contacts established and their cytostatic and cytolytic effects in MCF-7 nodules maintained in three-dimensional culture. A comparison was made with nodules of a nonmalignant, immortalized mastosis cell line. Histological, immunohistochemical and electron microscopical observations were performed as well as DNA synthesis measurements in the two components of the coculture. The lymphokine-activated killer (LAK) cells adhered more frequently to the carcinoma nodules than to the mastosis nodules. They actively penetrated into both of them. The penetration remained peripheral, and only a few cells migrated more deeply. The LAK cells established close cell-to-cell contacts with the two types of nodules, and intercellular gaps were formed: damaged cells could be seen near the activated killer cells. In MCF-7 nodules, a 5-fold inhibition of proliferation occurred, and extensive necrotic zones developed; this was accompanied by a general tendency for glandular redifferentiation. In mastosis nodules, necrosis also developed but no cell differentiation occurred and proliferation was less inhibited (2 times). Interleukin-2 alone enhanced DNA synthesis in mastosis nodules but had no effect on MCF-7 nodules, and no extending necrosis could be seen in both types of nodules. The cytolytic effects of LAK cells combined with their redifferentiating effect in MCF-7 breast carcinoma nodules may be a useful indication for further breast cancer therapy research.

A 12-year-old girl presents with optic atrophy, pale papilla, amblyopia and microcephaly (-3 s.d.) with mild mental retardation and facial dysmorphism. She had mitral insufficiency with mitral prolapse and moderate short stature (-2.5 d.s.). She had normal flash visual evoked potentials, normal electroretinograms and electrooculograms and normal cranial CT scan as well as other lab tests to rule out malformations, tumors or multiple sclerosis. Her lymphocyte karyotype showed a variegated mosaicism with: 2 cells with 49, XX, +mar,+mar,+mar; 21 cells with 48, XX, +mar,+mar; 57 cells, with 47, XX,+mar; 20 cells with 46,XX; while parental karyotypes were normal. This syndrome therefore associates optic atrophy, mental retardation and microcephaly and short stature with chromosomal instability in the form of variegated mosaicism.

We have cultured cells from normal and benign mastopathy tissues under conditions favoring epithelial cell proliferation. However, such primary cells rapidly lose their ability to grow in vitro. Immortal lines were established after transfection with SV 40 T gene and several cell clones isolated from Normal Breast Adjacent to a tumor (NBAT 32 T2 to T7) and two benign mastopathies (NPM 21 T1 to T14 and NPM 14 T4). This immortalization of epithelial cells reduced the doubling time of cultured cells and increased the densities of the cultured cell populations. Among there cell lines we have characterized five clones and found differences in a number of aspects. Southern blot analysis showed that the SV 40 T gene was stably integrated in the genome of all the established cell lines. Two of the clones (NPM 21 T2 and NPM 21 T4) were nearly diploid showing a high degree of stability. Two other clones (NPM 14 T4 and NPM 21 T1) had near-tetraploid karyotypes, although quite heterogeneous. Comparison of the ultrastructural phenotypes shows that the two near-diploid cell lines were more differentiated than the two others. Estradiol receptors measured by Scatchard analysis and by transactivation of vitellogenin promotor were absent from all the cell lines. Progesterone receptors measured by Scatchard analysis of hormone binding were present in the NPM 14 T4 and NBAT 32 T4 cell lines. The NPM 21 T1 cell line did not contain such steroid receptors. These cell lines are persistent in long-term culture, thus providing a useful in vitro model system for studying factors involved in the proliferation and the transformation of human mammary epithelial cells.

Lymphokine Activated Killer (LAK) cells, stimulated by interleukin 2 (IL-2) have a pronounced antitumor effect in the therapy of melanoma and renal cancers. LAK cells were cultivated in presence of the nodules of the human breast adenocarcinoma cell line MCF-7 maintained in organotypic culture to study the interactions between lymphocytes and breast tumor cells. After two days of co-culture, the proliferation of MCF-7 nodules and that of LAK cells was diminished about five folds. The cytotoxic effect of the latter, appreciated by Chrome 51 release was unchanged after the coculture. In histological sections, the penetration of the LAK cells into the MCF-7 nodules was accompanied by an increase of tumor necrosis but also by a glandular differentiation of cancerous tissue. Polarized epithelial cell formations bording neoplasic lumens with intracytoplasmic vacuoles filled with mucus, appeared in the nodules. The immunohistochemistry underlines the presence of T lymphocytes marked by UCHL1 and CD3 antibodies and of Natural Killer (NK) cells marked by IOT10, located between the MCF-7 cancer cells. In electron microscopy, the membrane contacts were tight and were accompanied by the appearance of secondary lysosomes and nuclear alterations. The relatively low infiltration level of the nodules may lead to the supposition that an indirect mechanism will intervene in this dual action of a LAK cells: increase of necrosis, although partially, and development of glandular and functional differentiation.

Biology of the CellVolume 75, Issue 3 p. 260-260 Arguments Ultrastructuraux En Faveur D'un Syndrome De Dissociation De La Croissance Des Doublets Flagellaires Dans Le Spermatozoide Humain E Tamboise, E Tamboise Laboratoire d'Histologie-Embryologie-Cytogénétique-Biologie de la Reproduction, U.F.R., S.M.B.H., 93012 BOBIGNYSearch for more papers by this authorA Tamboise, A Tamboise Laboratoire d'Histologie-Embryologie-Cytogénétique-Biologie de la Reproduction, U.F.R., S.M.B.H., 93012 BOBIGNYSearch for more papers by this authorB Martin-Pont, B Martin-Pont Laboratoire d'Histologie-Embryologie-Cytogénétique-Biologie de la Reproduction, U.F.R., S.M.B.H., 93012 BOBIGNYSearch for more papers by this authorM.C Pinatel, M.C Pinatel Laboratoire de Biologie de la Reproduction, hópital Edouard Herrlot, LYONSearch for more papers by this authorN Lievre, N Lievre Laboratoire d'Histologie-Embryologie-Cytogénétique-Biologie de la Reproduction, U.F.R., S.M.B.H., 93012 BOBIGNYSearch for more papers by this authorJ.N Hugues, J.N Hugues Centre de PMA. Hôpital Jean Verdier, 93140, BONDY.Search for more papers by this author E Tamboise, E Tamboise Laboratoire d'Histologie-Embryologie-Cytogénétique-Biologie de la Reproduction, U.F.R., S.M.B.H., 93012 BOBIGNYSearch for more papers by this authorA Tamboise, A Tamboise Laboratoire d'Histologie-Embryologie-Cytogénétique-Biologie de la Reproduction, U.F.R., S.M.B.H., 93012 BOBIGNYSearch for more papers by this authorB Martin-Pont, B Martin-Pont Laboratoire d'Histologie-Embryologie-Cytogénétique-Biologie de la Reproduction, U.F.R., S.M.B.H., 93012 BOBIGNYSearch for more papers by this authorM.C Pinatel, M.C Pinatel Laboratoire de Biologie de la Reproduction, hópital Edouard Herrlot, LYONSearch for more papers by this authorN Lievre, N Lievre Laboratoire d'Histologie-Embryologie-Cytogénétique-Biologie de la Reproduction, U.F.R., S.M.B.H., 93012 BOBIGNYSearch for more papers by this authorJ.N Hugues, J.N Hugues Centre de PMA. Hôpital Jean Verdier, 93140, BONDY.Search for more papers by this author First published: 1992 https://doi.org/10.1016/0248-4900(92)90157-VAboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat No abstract is available for this article. Volume75, Issue31992Pages 260-260 RelatedInformation

In order to study human sperm membrane proteins, a radioiodination technique using iodogen (Pierce, Interchim, Paris, France) has been tested. Iodogen was dissolved in redistilled chloroform and added in 50-μg quantities to nitric-acid-washed pyrex tubes. The chloroform was evaporated under a jet of nitrogen and the tubes rinsed with PBS shortly before use. The spermatozoa were obtained by a swim-up method in Locke buffer (0.15 M NaCl; 5.6 mM KC1; 24 mM NaHCO3; 0.25% dextrose; 2.25 mM CaCl2). Mobility of migrated sperm, evaluated in optic microscopy, was ≥85%. Migrated cells were then surface-labeled by adding 300 μCi carrier-free Na125 I (Amersham France, Les Ulis, France) to 1 ml cell suspension (50 × 107 cells) in the iodogen-coated tubes. After incubation at room temperature for 10 min, the reaction was terminated by diluting the spermatozoal suspensions with large volumes of PBS. The spermatozoa were then washed three times with PBS.

The 11q;22q translocations, whatever the breakpoints may be, are of particular interest because of their propensity to 3:1 segregation of the chromosomes at meiosis I. Until now, no unbalanced karyotype resulting from 2:2 adjacent segregation was published among offspring of 11q;22q translocation carriers. The authors report the case of an unbalanced karyotype due to adjacent 1 segregation of a maternal translocation (11;22)(q23.3;q13.2). The proband's karyotype was 46,XX,-22,+der(22)(11;22)(q23.3;q13.2)mat. This finding demonstrates that adjacent 1 segregation is possible in t(11;22) with breakpoints at 11q23 and 22q13, and can lead to birth of viable infants.

Chromosome analysis of oocytes uncleaved after IVF allows the cause of the failure of cleavage to be determined and shows the incidence of chromosome disorders among human oocytes. A total of 198 uncleaved oocytes fixed 40 h after insemination were successfully analysed according to Tarkowski's air-drying method: 78.3% were unfertilized and arrested in metaphase II. Among them, 70% were normal (23,X) and 30% aneuploid (16% were hypohaploid, 14% were hyperhaploid). The incidence of chromosome breaks was 18%. In 12.1% of the oocytes, sperm chromosome condensation appeared premature usually in the G1 phase. This was especially observed in idiopathic infertility (7% of fertilized oocytes versus 2% in tubal infertility cases). In 8.1% of the cases, chromosome analysis showed diploidy which may be interpreted by either an absence of extrusion or a reintrusion of the polar body or by first cleavage failure during mitosis. In 1% of the cases triploidy was observed. Our results show that the main reason for failure of cleavage is related to failure of fertilization (78.3%). However, premature condensation of sperm chromosomes at the G1 phase appears to be quite frequent. This may be involved in the aetiology of some cases of idiopathic infertility. Finally, the high rate of chromosomal disorders (30%) in human oocytes may explain the high rate of chromosomal abnormalities in preimplantation embryos.

Cells were isolated from post‐radiation fibrosis biopsies of patients with recurrent breast carcinoma. These cells were identified as fibroblasts and compared with fibroblasts from normal breast tissues for their proliferative activities, chromosome number and for the presence of various components of the extracellular matrix and cytoskeleton. The proliferative activity of the fibrosis‐derived fibroblasts did not significantly differ from that of normal breast fibroblasts. Both cell types required serum to grow and did not form colonies in soft agar. Cells from 2 of the 3 fibroses analyzed displayed aneuploid karyotypes with multiple structural abnormalities. All of the fibroblastic cells produced types I, III and V collagen, fibronectin and vimentin. However, in contrast to normal breast fibroblasts, fibrosis‐derived cells produced high amounts of oncofetal fibronectin. In addition, fibrosis fibroblasts also expressed the a‐actin isoform which is specific for smooth‐muscle cells. These results suggest that post‐radiation fibrosis in malignant breast contains atypical fibroblasts with fetal and myofibroblastic characteristics.

Quantitative changes in hepatocyte ultrastructures during normal gestation were studied in Wistar rats with morphometric methods. On the 18th day of the gestation, variations in nuclearcytoplasmic ratio, size of R.E.R., mitochondria, lysosomes and microbodies were observed, with variations according to the localisation of hepatocyte inside the lobule. We report the most obvious effects as follows: An increase of R.E.R. in the central and perilobular zones. Mitochondria are larger and the rounded forms are more numerous inside the two lobular zones. The number of lysosomes and microbodies are only elevated in the perilobular cells. In conclusion, it is suggested that the hepatocyte organelles are significantly increased during gestation. This is probably due to the metabolic activation. These cellular modifications are only one aspect of the liver increase.

Observations carried out on a large sample of pregnant rats, their fetuses and placentas, lead to the following findings: continuous increase of liver weight along pregnancy from 2nd to 20th days, mainly within the 2nd week (organs making), to reach limit value on the 18th day. This liver weight increases roughly along with embryos and placentas weights specially from 14th to 20th days. At the 18th day the mother's liver weight depends on embryos and placentas weights. This weight of the mother's liver increased with embryos and placentas number but does not depend on resorption number. The overall mother's weight is proportional to liver weight and embryos number. All these facts underline metabolic relations between mother and fetus during pregnancy.