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S Alberti

Publications and source records attributed to S Alberti.

At least 73 records · Page 4Linked to original sources
PubMed1991

Immunofluorescence analysis in flow cytometry: better selection of antibody-labeled cells after fluorescence overcompensation in the red channel.

Selection of cells labeled with fluorescein isothiocyanate-conjugated (FITC) antibodies can be difficult if large autofluorescent cells are used and if the cells bind only a few molecules of antibody. We have developed a simple flow cytometric procedure that allows better selection of stained cells. When an argon ion laser emitting at 488 nm is used, the green fluorescence detected is the sum of cell autofluorescence and of the signal generated by the FITC antibody. Thus, when we subtract green signal from the red by fluorescence compensation, the signal of stained cells is on average reduced more than for the unstained counterpart. In this scenario, positive selection of cells with low red signal allows more efficient selection of stained cells. We tested the overcompensation procedure on mixtures of cells unstained and stained with a relevant FITC antibody. Cell mixtures were analyzed using normal vs increased levels of compensation in the red channel. Increased levels of compensation resulted in easier gating and higher recovery of stained cells. The efficiency of the overcompensation procedure was particularly high when using red filters with low cutoff (i.e., 560 or 570 nm), possibly because of the significant emission of fluorescein in the red channel, which caused separation between stained and unstained cells also in the red dimension. This method is useful for sorting cells expressing low levels of surface markers and facilitates selection of rare cells transfected with surface antigen genes. This technique is compatible with the use of propidium iodide for live/dead cell discrimination and with the subtraction of the cellular background of autofluorescence.

Animals↗
PubMed1991

Dimer-to-tetramer assembly of Lac repressor involves a leucine heptad repeat.

The C-terminus of Lac repressor is responsible for the formation of repressor tetramers from active dimers. If properly grafted, the C-terminus of Lac repressor (amino acids 331 to 360) converts Gal repressor dimers into tetramers. Amino acids 342 to 356 of Lac repressor contain a 4-3 hydrophobic repeat of four leucines and one valine. Systematic amino acid replacements of all residues in this region show that the protein-protein interaction between repressor dimers depends mainly on the hydrophobic residues of the 4-3 repeat, which is constitutive for coiled coils. Thus the tetramerization site of Lac repressor resembles the leucine zipper motif found in a family of eukaryotic transcription factors.

Amino Acid Sequence↗
PubMed1990

The Ca-MOv18 molecule, a cell-surface marker of human ovarian carcinomas, is anchored to the cell membrane by phosphatidylinositol.

The structure of the 38 kD cell surface glycoprotein identified by the monoclonal antibody MOv18 and specifically expressed by human ovarian carcinomas has been investigated at a molecular level. The ovarian carcinoma cell line IGROV-1, which expresses high levels of Ca-MOv18, was treated with the phosphatidylinositol-specific phospholipase C from B. thuringiensis. The phospholipase C specifically released most of the Ca-MOv18 molecules as shown by flow cytometric analysis of the treated cells and by radioimmunometric assays of the corresponding supernatants. Consistent with the known structure of other phosphatidylinositol-linked molecules, Ca-MOv18 was biosynthetically labeled by [3H]ethanolamine and the labeled molecules were immunoprecipitated from the supernatant fo the phospholipase C treated cells. Evidence that Ca-MOv18 is anchored to the cell membrane via phosphatidylinositol may prove to be relevant in current investigations regarding the biological and clinical significance of this tumor marker.

Antibodies, Monoclonal↗
PubMed1990

Higher transfection efficiency of genomic DNA purified with a guanidinium thiocyanate-based procedure.

Efficient transfection of eukaryotic cells is dependent on the purity of the transfected genomic DNA. In an attempt to obtain a more reliable method of DNA purification we have modified the widely used protocol of Blin and Stafford to include a treatment with guanidinium thiocyanate. The DNA obtained following the present protocol transfects eukaryotic cells with higher efficiency.

Animals↗
PubMed1990

Expression of 38-kD cell-surface glycoprotein in transformed human keratinocyte cell lines, basal cell carcinomas, and epithelial germs.

In this study, we attempted to identify and characterize transformation-induced cell-surface glycoproteins of human keratinocytes. Therefore, we first searched for glycoproteins which are significantly elevated in human keratinocytes after transformation and immortalization by SV40 virus and which are also found at high levels in keratinocytic cell lines derived from squamous cell carcinomas of the skin. Out of at least 80 different cell-surface antigenic systems of human tumor cells, only three glycoproteins showed elevated expression in transformed keratinocytes. Among these, a 38-kD glycoprotein (gp 38) was highly increased in all transformed keratinocyte cell lines tested, but was not elevated in transformed fibroblasts. The expression of gp 38 was further characterized in normal epidermis and in its benign and malignant hyperproliferative disorders: gp 38 was generally not expressed in normal epidermis and in benign hyperproliferative disorders. In contrast, strong and homogeneous reactivity was found in solid and fibrosing basal cell carcinomas whereas no or low reactivity was detected in squamous cell carcinomas and in those parts of BCC revealing keratotic differentiation. Interestingly, high expression of gp 38 was also found in primary epithelial germs of fetal skin, secondary germ cells of the telogenic hair follicle and secretory tubules of sweat glands. The immunohistologic data suggest that gp 38 is preferentially expressed by epidermal cells which lack squamous and pilosebaceous differentiation.

Basal Cell Carcinoma↗
PubMed1990

Transfection into mouse L cells of genes encoding two serologically and functionally distinct bovine class I MHC molecules from a MHC-homozygous animal: evidence for a second class I locus in cattle.

w10 and KN104 are distinct class I major histocompatibility complex (MHC) serological specificities present in Boran (Bos indicus) cattle. Although these specificities are commonly expressed together, they may also be expressed independently. To establish whether w10 and KN104, when expressed together, are on the same or different molecules, and whether a second class I MHC locus exists in cattle, genomic DNA from an animal homozygous for a haplotype encoding the w10 and KN104 specificities was transfected into thymidine kinase-deficient mouse L cells (Ltk- cells), and the transfected cells were screened with monoclonal antibodies (mAb) specific for the w10 or KN104 allospecificities. Two different populations of transfectants were identified: the cells of one population reacted only with w10-specific mAb, whereas those of the other population were recognized only by the KN104-specific mAb. Alloreactive cytotoxic T lymphocytes (CTL) also distinguished between the two populations. Two CTL clones, shown to be restricted by the KN104 specificity, killed only those L cells expressing molecules recognized by the KN104-reactive mAb. Of eight CTL clones which recognized class I molecules associated with the w10 specificity, four killed the L cells expressing the w10 specificity. The remaining four clones did not kill either population of transfectants. Finally, immunoprecipitation studies revealed that both populations express full-length bovine class I MHC molecules. These results demonstrate that the w10 and KN104 specificities are on distinct class I molecules. As the genes encoding these molecules were derived from a MHC-homozygous animal, the findings also provide strong evidence that there are at least two classical class I loci in cattle.

Animals↗
PubMed1988

DNA methylation prevents transfection of genes for specific surface antigens.

Sperm and trophoblast are among the few nucleated human cells that do not express HLA class I antigens. DNA methylation, which is proposed to be a tight mechanism of regulation, may be necessary to turn off these genes. We have investigated the transfectability of HLA class I genes and of the genes for the T-cell differentiation antigens Leu-1 (CD5) and Leu-2 (CD8) in mouse L cells by using human sperm cells and choriocarcinoma cell lines, tumors of trophoblastic origin, as sources of DNA. It was found that DNA from one choriocarcinoma line (JAR) does not transfect genes for HLA, Leu-1, or Leu-2 and that DNA from two other choriocarcinoma lines (BeWo and Ima) transfects only some of the surface markers. Sperm DNA transfects genes for all the surface antigens tested except Leu-1. DNA from control cells and from the line SCH transfects all the markers studied. Southern blots show that all cell types contain apparently intact genes encoding HLA, Leu-1, and Leu-2 and reveal differences in the DNA methylation patterns of genes from different sources of DNA. We treated JAR (the cell line with the lowest transfecting ability) with 5-azacytidine and obtained demethylation of its DNA. This demethylated DNA transfects genes for both HLA class I antigens and Leu-2. Further culture of JAR cells in the absence of 5-azacytidine results in remethylation of their DNA and decreased ability to transfect these surface antigens. These findings indicate that DNA methylation affects the efficiency of transfection of surface antigen genes in L cells.

Cell Line↗
PubMed1987

Autosomal dominant microcephaly without mental retardation.

We describe six Italian families affected by microcephaly with an apparently autosomal mode of inheritance (total number of microcephalic children and adults, 21). All microcephalic subjects were of normal height, with the exception of one. The head volume was measured directly in at least one adult microcephalic member from five of the six families, and lower values were obtained in these subjects than in control subjects. Psychometric tests were performed on seven children and five adults among the microcephalic subjects. Normal values were found for all but one of the subjects. In the selected families microcephaly seems to be inherited in an autosomal dominant manner. Because some families showing autosomal dominant microcephaly have normal intelligence, psychometric evaluation of microcephalic children and their microcephalic relatives is important for genetic counseling.

Adult↗
PubMed1987

A single laser method for subtraction of cell autofluorescence in flow cytometry.

In flow cytometry cell autofluorescence often interferes with efforts to measure low levels of bound fluorescent antibody. We have developed a way to correct for autofluorescence on a cell-by-cell basis. This results in improved estimates of real staining and better separation of the fluorescence histograms of stained and non-stained cells. Using a single laser, two-color fluorescence measurement system and two-color compensation electronics, autofluorescence and one fluorescent reagent are measured (rather than two fluorescent reagents). With fluorescein-conjugated antibodies the signal in the 515 to 555 nm range (green fluorescence) includes both fluorescein emission and part of the cellular autofluorescence. In the cases we have investigated, autofluorescence collected at wavelengths above 580 nm ("red") is well correlated with the green autofluorescence of the cells. A fraction of this red fluorescence is subtracted from the green fluorescence to produce an adjusted fluorescein output on which unstained cells have zero average signal. Use of this method facilitates the selection of rare cells transfected with surface antigen genes. Culture conditions affect the level of autofluorescence and the balance between red and green autofluorescence. When applied with fluorescein-conjugated reagents, the technique is compatible with the use of propidium iodide for live/dead cell discrimination.

Animals↗
PubMed1986

Metastatic growth of a murine tumor: evidence of dissemination to the lungs in the absence of subcutaneous growth.

Growth of MCA-38/B colon adenocarcinoma was detectable 30-33 days after subcutaneous (s.c.) tumor cell inoculation in mice. Seventy percent of the mice receiving 10(7) tumor cells, 50% of those receiving 10(6), and 15% of the mice given 10(5) cells developed s.c. tumors (mean of 4 experiments, total of 80 mice per group). Metastases in the presence of a primary tumor were observed in 11% of 10(7) and in 10% of 10(6) tumor-cell injected animals. Lung metastases were detected in the absence of tumor growth at the site of s.c. cell injection in 19% of 10(7), in 8% of 10(6) and in 5% of 10(5) and 10(4) tumor-cell inoculated mice. In parallel experiments an intravenous (i.v.) inoculum of tumor cells produced lung colonies in 40% of 10(6) and in 14% of 10(5) tumor-cell injected animals. Smaller inocula did not give rise to lung colonies, thus making it unlikely that accidental i.v. inoculations of tumor cells during the s.c. injections caused the observed metastatic dissemination to the lungs.

Adenocarcinoma↗
PubMed1985

Relationship between large granular lymphocytes and NK-1.2+ cells from normal and poly(inosinic:cytidylic acid) (poly(I:C]-treated mice.

The present work analyzes the relationship between large granular lymphocytes (LGL), NK-1.2+ cells, and natural killer (NK) activity of C3H/HeN mice. Different hematic cell fractions were obtained according to their nylon-wool adherence and density on Percoll gradients. NK-1.2+ cells (8% of nucleated cells) were more numerous than LGL (3% of nucleated cells) in the input blood population. Eighty-five percent of LGL were recovered from the sorted NK-1.2+ cell fraction. After incubation on nylon-wool column, 63% of LGL and 36% of NK-1.2+ were eluted in the nonadherent fraction. Eighteen percent of NK-1.2+ cells were recovered from the most adherent elutable cell fraction. After the discontinuous Percoll gradient most LGL were present in the low-density fractions while 20% of NK-1.2+ cells were recovered from the highest-density fraction. NK activity was significant both in the nylon-wool-nonadherent and -adherent fractions. After the Percoll gradient most NK activity was present in the low-density fractions. In the present experimental conditions treatment poly(inosinic:cytidylic acid) (poly(I:C] did not increase the numbers of LGL and NK-1.2+ cells either in the blood or in the spleen. However it increased significantly the NK activity of the input cell populations and of the nonadherent and low-density fractions. Similarly, exposure of specific pathogen-free (SPF) mice to non-SPF conditions stimulated NK cytotoxicity but did not alter the percentage of LGL in the blood or in the spleen. Poly(I:C) treatment induced a shift of LGL and NK-1.2+ cells toward the low-density fractions. In poly(I:C)-treated mice images of granule secretion from LGL were detected. Taken together, the present results indicate that LGL and NK-1.2+ cell populations do not totally overlap. Moreover subpopulations of LGL and NK-1.2+ cells can differ in NK activity, morphology, density, adherence to nylon wool, and response to poly(I:C).

Animals↗
PubMed1985

Large granular lymphocytes from murine blood and intestinal epithelium: comparison of surface antigens, natural killer activity, and morphology.

Large granular lymphocytes obtained from murine blood (B-LGL) and intestinal epithelium (IE-LGL) are cells associated with natural killer (NK) activity and thought to be a first line of defense against tumors and/or infectious organisms. Since B-LGL and IE-LGL represent circulating and mucosal NK effectors, respectively, we compared their surface markers, NK activity and morphology to define possible differences between NK cells in different anatomical compartments. B-LGL and IE-LGL were purified by Percoll gradient centrifugation from nude, normal, and beige C57BL/6 mice. We have defined the following surface phenotypes. B-LGL: In nude mice most of them expressed T-200 (89%), asialo-GM1 (71%), and NK-1.1 (72%); 15% possessed the Thy-1.2 antigen, few cells expressed Ly-2, and none showed Ly-1 positivity. Beige mouse B-LGL were positive for T-200 and NK-1.1. IE-LGL; Nude IE-LGL compared to nude B-LGL showed a similar expression of T-200 and Thy-1.2. Ly-1+ and Ly-2+ cells were more numerous than in B-LGL, whereas NK-1.1+ and asialo-GM1+ cells were less numerous. Interestingly, Ly-2+ IE-LGL were at least partially Thy-1.2-. In euthymic mice IE-LGL had a phenotype comparable to that of nude IE-LGL. The NK activity of B-LGL from nude and normal mice was considerably higher than that of IE-LGL from the corresponding mice. IE-LGL from nude mice possessed larger cytoplasms, and more numerous and bigger azurophilic granules than B-LGL. Similar findings were obtained in normal mice. In beige mice 95% of B-LGL showed a single granule whereas 80% of IE-LGL contained multiple granules (mean 3/cell). Giant granules were frequently found in beige IE-LGL while they were rare in beige B-LGL. Thus, clear differences exist between B-LGL and IE-LGL and they may reflect either different homing patterns of subpopulations of LGL or different stages of maturation of the same lineage of cells.

Animals↗
PubMed1984

Effect of hydrocortisone on the macrophage content, growth and metastasis of transplanted murine tumors.

Hydrocortisone (HC) reduced the macrophage content of four murine tumors to less than half of control values. Since HC causes monocytopenia and inhibits the recruitment of mononuclear phagocytes at sites of inflammation but does not affect the viability and proliferative capacity of macrophages, this finding suggests that the maintenance of macrophage levels in growing tumors is in part dependent upon the entry into the neoplasm of circulating monocytes. HC inhibited the growth of these tumors. The effect of HC was most marked on the mFS6 and MN/MCA1 sarcomas and 3LL carcinoma, with only marginal inhibition in the M109 carcinoma. HC augmented spontaneous and/or artificial (i.v.) metastasis in these tumor models. Cells from metastatic foci of the mFS6 sarcoma and M109 carcinoma inoculated i.m. were as susceptible to HC as cells from primary tumors. The effects of HC on macrophage content, growth and metastasis of the M109 carcinoma and mFS6 sarcoma were similar in mice with defective T-cell function (nude or thymectomized) or defective NK activity (beige or antiasialo GM1-treated) and in controls. The in vitro growth of the mFS6 and MN/MCA1 sarcomas was not modified by HC. Some inhibition of tumor-cell proliferative capacity was observed with the 3LL and M109 tumors. Tumor cell had high-affinity binding sites for glucocorticoid hormones, but these were not correlated to susceptibility in vivo. Thus it is unlikely that a direct interaction of HC with tumor cells accounts for the inhibition of tumor growth observed in vivo. To evaluate whether HC affected tumor growth by reducing the macrophage content of tumors, mFS6 sarcoma cells were transplanted, mixed with peritoneal macrophages into normal or HC-treated mice. Macrophages did not affect the growth of tumor cells in normal mice, whereas in HC-treated animals lesions from mixtures of macrophages and sarcoma cells appeared earlier and weighed more than those from tumor cells alone or tumor cells and thymocytes. However, macrophages did not reconstitute growth of the other sarcoma (MN/MCA1) in HC-treated mice. These results are consistent with the hypothesis that HC inhibited growth of some poorly immunogenic transplanted murine tumors, at least in part, by interfering with the macrophage levels in neoplastic tissues, tumor-associated macrophages providing the conditions for optimal tumor-cell proliferation at least in some neoplasms.

Animals↗
PubMed1984

Preservation of cells sorted individually onto microscope slides with a fluorescence-activated cell sorter.

Fluorescence-activated cell sorters permit analyses and separation of cell populations based on light scatter and surface immunofluorescence parameters. Since a sorter can deposit individually identifiable cells onto a microscope slide, it was considered of interest to combine the flow measurements with analyses available on cells adhering to a surface as in, for example, morphological studies, cytoplasmic immunofluorescent staining, and mRNA in situ hybridization. A necessary condition for these studies is the preservation of cell structures after sorting. We report here a procedure suitable for this purpose. The most important features of this procedure are A) reducing the saline content of the sorter sheath fluid to about 0.0015 M (one-hundredth that of normal saline) to prevent cell damage due to hypertonicity during drying, and B) coating the substrate with a thin layer of newborn calf serum to promote the adherence of the cells to the substrate during subsequent fixing and staining.

Animals↗
PubMed1984

Transfection and cloning of genes for membrane antigens using the FACS.

In order to facilitate cloning of genes for cell surface molecules, we cotransfected LTK- mouse fibroblasts with thymidine kinase (TK) genes and total human or mouse DNA. TK+ cells, selected by growth in HAT medium, were stained with fluorochrome conjugated monoclonal antibodies or other fluorescent ligands which bind to one or another membrane differentiation antigen or receptor. We isolated fluorescent transfectants expressing these molecules using a fluorescence activated cell sorter (FACS). For some antigens, spontaneous gene amplification occurred. By repeated cycles of FACS sorting and regrowth we obtained high expressing clones. We then isolated cDNA and genomic clones using selected cDNA probes to screen phage with cDNA inserts. DNA from virtually any tissue source transfected equally well for the various molecules except for DNA from a trophoblast derived choriocarcinoma cell line which did not transfect for Leu-2.

Animals↗
PubMed1983

BK virus-induced tumors in hamsters: a morphological, histochemical and ultrastructural study.

Macroscopic morphology, histology and ultrastructure of BK virus (BKV)-induced hamster and mouse tumors were investigated. Groups of animals were immunosuppressed to study the relationship between immune system and BKV oncogenesis. Ependymomas had the highest incidence, followed by tumors of pancreatic islets, osteosarcomas, lymphomas and sarcomas, sometimes associated in the same animal. All the tumors were found to be BKV specific. Ependymomas showed the shortest latency, infiltrated surrounding tissues but did not metastasize. Pseudo-rosettes were common and basal bodies were observed. Atypia and necrosis were more often present in immunosuppressed animals both for the ependymomas and for the other oncotypes. Pancreatic insulomas were frequently multinodular, possibly because of multifocal origin and metastasized to the liver. Hormone secretory granules were often found on electron microscopy. Osteosarcomas metastasized to lungs and peritoneum and showed the presence of osteoid, chondroblastoid and mixoid areas. Characteristic giant cells were present. Immunosuppression did not enhance tumor incidence and did not influence the latency period. However, neoplastic growth appeared to be more rapid and with more aggressive behavior in immunodepressed animals. These findings suggest an influence of the immune system in tumor development, whereas the virus oncogenic process seems unaffected.

Animals↗
PubMed1981

Effects of in vivo treatments with cyclosporin-A on mouse cell-mediated immune responses.

The fungal metabolite Cyclosporin-A (CyA) was investigated for its activity on several cell-mediated immune responses. Oral administration of 70 mg kg-1 of CyA for 5 days to C3H/HeN mice completely abolished the in vivo antigen-dependent production of a lymphokine capable of increasing macrophage cytotoxicity against tumor cells. Moreover, spleen cells from CyA treated mice were significantly depressed in their ability to produce in vitro lymphokines in response to PHA, whereas only a slight depression was observed when ConA was employed to induce lymphokine production. In parallel to the depression of proliferation-independent immune response, spleen cells from CyA treated mice showed a strongly depressed proliferative response to PHA, marginal reduction being observed in the response to ConA. B-lymphocytes did not seem to be affected by in vivo treatment with CyA, judging from the proliferative responses to LPS. Macrophage responses also remained unaltered after CyA treatment. No depressions in natural or lymphokine-induced macrophage cytotoxicity and in monokine production were in fact observed in CyA treated mice. Finally, a short-lived depression of natural killer (NK) activity was observed after CyA administration. These results indicate that in vivo CyA treatment selectively depressed cell-mediated functions of lymphocytes of the T-cell lineage. The hypothesis that T-cell lineage. The hypothesis that T-helper lymphocytes are the preferential target of CyA immunodepression is discussed.

Animals↗