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Biomedical subjects

A Colombatti

Publications and source records attributed to A Colombatti.

At least 91 records · Page 5Linked to original sources
PubMed1987

Widespread codistribution of glycoprotein gp 115 and elastin in chick eye and other tissues.

Frozen sections of chick tissues were exposed to affinity-purified monoclonal antibodies raised against chick gp 115 and to affinity-purified antibodies raised against chick tropoelastin to study the distribution pattern of the corresponding antigens by the avidin-biotin immunoperoxidase technique. Laminin and fibronectin antibodies were used for comparison. Gp 115 and tropoelastin antibodies localized to the same structure in several of the tissues examined. The endothelial membrane of the cornea and Bruch's membrane in the choroid were positive, while the corneal epithelial membrane was negative. Both antibodies displayed a peculiar punctate reactivity in the corneal stroma and a very fine fibrillar pattern in the conjunctiva and at the corneal-scleral junction. Liver, heart and large vessels, striated muscle and skin showed a similar pattern both for tropoelastin and gp 115 antibodies. Few differences were seen in the distribution of the reactivity: the pericellular matrix of intestinal smooth muscle cells was stained by gp 115 but not by tropoelastin antibodies. However, the reactivity of gp 115 and tropoelastin antibodies was similarly distributed in the lung smooth muscle cell clusters. The peritubular matrix in the kidney did also not react with tropoelastin antibodies as did the brain intraparenchymal vessels; whereas gp 115 antibody reactivity was present in both sites. We interpret these lack of apparent codistribution in some tissues as a variation in the relative availability of the target antigen for the reaction with the antibody and not as a consequence of a qualitative difference in the distribution of gp 115 and tropoelastin. By the use of anti gp 115 monoclonal antibodies that do not cross-react, and presumably recognize different epitopes, it was shown that some but not all antibodies, react with brain intraparenchymal blood vessels; whereas the pattern of distribution in other tissues was the same. This suggests that in vessels with an undetectable level of elastin, certain epitopes of gp 115 molecule might not be recognized as a result of being masked by other components or by a different conformation of the molecule.

Animals↗
PubMed1987

A solid-phase receptor binding assay for 125I-hCG.

A solid-phase radioligand-receptor assay (RRA) to measure the binding of 125I-labelled human chorionic gonadotropin (125I-hCG) to target cell membranes has been developed. The binding of 125I-hCG to membranes immobilized on the wells of microtitration plates reached a maximum at about 3 hours at 37 degrees C, was saturable, displayed a high affinity (Ka = 2.4 X 10(9) M-1) and was specifically inhibited by unlabelled hCG. In comparison with RRAs carried out with membranes in suspension, the solid-phase RRA is significantly simpler and much faster to perform as it avoids centrifugation or filtration procedures. The solid-phase RRA was adapted profitably to process large numbers of samples at the same time. It proved particularly useful as a screening assay to detect anti-hCG monoclonal antibodies with high inhibitory activity for binding of 125I-hCG to its receptors.

Animals↗
PubMed1987

Multiple binding reactivities of an IgG1 mouse monoclonal antibody raised against the extracellular matrix glycoprotein Gp 115.

An IgG1 mouse monoclonal antibody that recognized five different polypeptides from chick and man is described. The antibody was raised against the elastin associated chick glycoprotein gp 115. In addition to the immunizing antigen, antibody 106D6 bound to chick smooth muscle cell myosin and to the alpha, beta and gamma chains of human fibrinogen. Cross reaction was demonstrated by radioimmunobinding, immunofluorescence and immunoblotting experiments. The immunoblotting assays were carried out using identical amounts of the various antigens to exclude the possibility that relevant quantitative differences in their concentration might result in non-specific binding.

Amino Acid Sequence↗
PubMed1987

c-fos oncogene expression in human hematopoietic malignancies is restricted to acute leukemias with monocytic phenotype and to subsets of B cell leukemias.

To evaluate relationships between c-fos proto-oncogene expression and specific lineages of hematopoietic differentiation we analyzed the constitutive and TPA-induced expression of the c-fos gene in a wide variety of fresh human leukemic cells. High constitutive c-fos expression was detected in acute leukemias with monocytic phenotype (FAB M4/M5) and in subsets of B lymphoid leukemias, some of which coexpressed B lymphocytic and monocytic markers. Conversely, low basal levels of c-fos transcripts were found in pure acute granulocytic leukemias (FAB M1/M2/M3), in erythroleukemias (FAB M6), in the great majority of B, and in all T lymphoid leukemias. TPA-induced c-fos expression seems to correlate with monocytoid differentiation only when sustained levels of transcripts (ie, detectable for at least 24 hours) were detected. Sustained c-fos expression was in fact observed only in those myeloid or lymphoid cells that acquired a stable monocyte-like phenotype in response to the phorbol ester. These results indicate that high constitutive c-fos expression may identify myelomonocytic-oriented forms of leukemia, specific subsets of B lymphoid malignancies, and at least some cells terminally differentiated in vitro to a monocyte-like phenotype. c-fos oncogene expression can therefore be regarded as an additional marker for the subclassification of human leukemias.

Acute Disease↗
PubMed1986

S-100 protein, fibronectin, and laminin immunostaining in lymphomas of follicular center cell origin.

Forty-nine paraffin-embedded biopsy specimens of involved nodal and extranodal tissue (bone marrow, spleen, and liver) from 13 patients with follicular center cell lymphomas (FCCL) and 14 with small lymphocytic lymphomas (SLL), including 11 cases with chronic lymphocytic leukemia, were tested for S-100 protein immunoreactivity. Analysis for fibronectin and laminin immunoreactivities was limited to the lymph node biopsy specimens. In FCCL, S-100-positive dendritic reticulum cells (DRCs) were found in 23 of the 26 tissue specimens examined, regardless of the involved sites and the growth pattern. Cases with completely or predominantly follicular pattern were usually associated with a spherical meshwork pattern of S-100-positive DRCs; in the FCCL specimens with a diffuse pattern (lymph nodes and bone marrow) as well as in the specimen areas with a minimally follicular tumor pattern, S-100-positive DRCs were consistently fewer in number and composed loosely aggregated nests. No S-100-positive DRCs were found in all the biopsy specimens in SLL. Concerning fibronectin and laminin immunostainings, results showed that no differences were present between areas of follicular and diffuse neoplastic growth and that the neoplastic growth of FCCL maintained for each antiserum the same distribution pattern as that seen in normal follicles. Analysis of the microenvironmental components as revealed with antisera used in the current study--particularly with anti-S-100 protein antiserum--appears to be a useful adjunct for the identification of FCCL in paraffin-embedded biopsy specimens, especially in extranodal sites.

Dendritic Cells↗
PubMed1986

Monoclonal antibodies against ricin: effects on toxin function.

Monoclonal antibodies against ricin toxin were produced and some of their properties investigated. Antibodies 196 C12 and 197 C7 raised against A-chain reacted with a CnBr fragment probably comprised between amino acid 254 and 262. Antibodies 193 A9, 196 A3, and 191 B7 recognized a 6-7 kD CnBr peptide. A second set of antibodies was raised against whole inactivated ricin. Most of them bound in a solid phase radioimmunobinding assay only to ricin and few had a low activity against purified A-chain. Different effects were noted on toxin action in cultured leukemic cells. If cells were preincubated with ricin followed by antibodies, MAb 207 E5 and 216 B3 had a strong enhancing effect on toxin action. If antibodies and toxin were mixed and then added to sensitive cells, antibody 207 E5 gave a strong protection while 216 B3 maintained its enhancing activity. The effect of antibody 216 B3 was further investigated by quantitative cloning experiments which showed that toxin had a fivefold enhancement in its activity by a preincubation with this antibody. Binding of fluoresceinated ricin to leukemic target cells was inhibited by a preincubation with antibody 207 E5 while antibody 216 B3 had no effect.

Animals↗
PubMed1985

Monoclonal antibodies against chick gp 115, a matrix glycoprotein with broad distribution.

Hybridoma cell lines were generated producing monoclonal antibodies to chick gp 115, a 115,000-dalton glycoprotein widely distributed in the connective tissue. The specificity of the antibodies was determined by indirect radioimmunobinding: the extent of binding was a function of i) antigen and ii) antibody concentration; iii) inhibition of binding of radiolabelled antibody by unlabelled antibody and iv) among many known extracellular collagenous or noncollagenous glycoproteins tested only gp 115 gave a strong positive binding reaction. The antibodies were used for indirect immunofluorescence and a strong staining reaction was detected in all blood vessels, around smooth muscle cells in several organs, and in the connective matrix of other tissues such as the liver, and the lung. Based on the competition of binding of [125I]-labeled purified antibody by unlabeled antibodies, two separate epitopes were identified on gp 115. Further analysis of the localization of the epitope was obtained by CNBr cleavage and partial digestion of gp 115 with Staphylococcus aureus V8 protease and alpha-chymotrypsin digestion. Following CNBr cleavage a major fragment of Mr = 35,000 was recognized by 4 monoclonal antibodies, and fragments of comparable Mr were detected following V8 protease and alpha-chymotrypsin digestion.

Animals↗
PubMed1985

Glycoprotein 115, a glycoprotein isolated from chick blood vessels, is widely distributed in connective tissue.

An extracellular glycoprotein (gp 115) with an apparent Mr = 115,000 isolated from chick aortas (Bressan, G. M., I. Castellani, A. Colombatti, and D. Volpin, 1983, J. Biol. Chem., 258:13262-13267), was used to immunize mice. The antisera were shown to specifically recognize gp 115 by numerous criteria: a major band around Mr = 115,000 plus minor bands of lower Mr were visible by immunoblotting on aorta extracts, and a similar pattern was observed with a monoclonal antibody; no cross-reactivity was detected by radioimmunobinding with other extracellular proteins, namely, fibronectin, laminin, and collagen types I, III, IV, V, and VI. Antigen distribution on frozen tissue sections from newborn chicks was investigated by using affinity-purified antibody. Strong immunoreactivity was always found in blood vessels. In the digestive tract, the fluorescent staining was localized both at the level of muscular layers and in the stromal matrix of the villi. Within skeletal muscle and myocardium, staining was associated with large connective tissue bundles and the matrix around each muscle fiber. Intense fluorescence was observed in the kidney, in smooth muscle cells rich areas of parabronchi, and within the portal space and along liver sinusoids. The antigen was not detected at the epidermal-dermal junction; immunoreactivity in the dermis was present as a diffuse fibrillar pattern. That the antigen detected by immunofluorescence in the various organs was indeed gp 115 was demonstrated by immunoblotting analysis: as in aorta extracts, a major band around Mr = 115,000 was detected in several tissues. Antibody-reacting material was also incorporated into the extracellular matrix produced by embryo smooth muscle cells grown in vitro and was organized as a meshwork of fine fibrils.

Animals↗
PubMed1984

Thy 1.2+ leukemia cells eradicated from in vitro leukemia-bone marrow cell mixtures by antibody-toxin conjugates.

A conjugate constituted by a monoclonal anti-Thy 1.2 antibody chemically coupled to the toxic subunit of ricin was synthesized. The conjugate was specifically cytotoxic for Thy 1.2+ (EL 4) but not for Thy 1.2- (BW5147) thymoma cells. In vitro treatment of bone marrow-leukemia cell mixtures (10:1 and 1:1 bone marrow to EL 4 cell ratios) with anti-Thy 1.2-ricin A chain totally eradicated EL 4 tumor cells, while the number of myeloid colony-forming units and pluripotent colony-forming units developed in vitro and in vivo was unaffected, which suggested that the treatment had no toxic side effects at least on the precursor cells examined.

Animals↗
PubMed1983

Isolation and characterization of a 115,000-dalton matrix-associated glycoprotein from chick aorta.

Chick aortas were extracted sequentially with phosphate-buffered saline, 6 M guanidine HCl, and 6 M guanidine HCl containing dithioerythritol. The proteins present in the guanidine HCl + dithioerythritol extract were separated by DEAE-cellulose chromatography, and the fractions recovered were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Five major glycoprotein components with apparent Mr = 205,000, 195,000, 150,000, 135,000, and 115,000 (gp 115) were identified. gp 115 was further studied since it was the only noncollagenous protein based on amino acid analysis. The protein was purified to homogeneity by preparative electrophoresis. Its amino acid composition was characterized by a high content of glutamic acid and arginine and a relatively high content of leucine, glycine, and alanine. The concentration of gp 115 in the guanidine HCl + dithioerythritol extract was about 15-fold that in the guanidine and saline extracts. Overall, about 80% of the protein was solubilized with guanidine HCl + dithioerythritol, suggesting that most of it formed large aggregates stabilized by disulfide bonds in vivo. Immunofluorescence studies with specific antibodies showed that gp 115 formed an extracellular fibrillar network in the aorta wall. One-dimensional finger printing with Staphylococcus aureus V8 protease and immunological studies indicated that the protein was unrelated to fibronectin and laminin. The data led us to conclude that gp 115 is a novel extracellular component of chick aorta.

Amino Acids↗
PubMed1983

Murine cell surface glycoproteins. Purification of the polymorphic Pgp-1 antigen and analysis of its expression on macrophages and other myeloid cells.

We previously reported the initial characterization of a polymorphic major cell surface glycoprotein of about 80,000 daltons from mouse embryo 3T3 cells. This glycoprotein has now been purified 1800-fold to apparent homogeneity by monoclonal antibody affinity chromatography. The purified molecule retained the total antigenic activity of the cell, as determined by antibody binding assays. The quantity of the glycoprotein, 0.06% of the total protein of the crude cell extract, confirmed its presence as a major constituent of the cell plasma membrane. The monoclonal antibody was also used to detect related antigens in cells and tissues of C57BL/6J mice. The antigen was present in high concentration in macrophages and subpopulations of bone marrow and blood polymorphonuclear cells. Much lower concentrations of antigen were detected in spleen cells, thymocytes, and extracts of solid tissues. The apparent Mr of the target antigen of myeloid cells was 92,000. This molecule was a major surface constituent of myeloid cells with 10(6) antibody binding sites per cell containing 10% of total 125I incorporated by the lactoperoxidase procedure. The macrophage glycoprotein labeled on the cell surface with 125I was highly sensitive to trypsin, yielding an antigenically active soluble glycopolypeptide of about 65,000 daltons, that contained all of the incorporated 125I. A similar 65,000-dalton glycopeptide was released from 3T3 cells by trypsin cleavage. These data indicate that a major cell surface constituent of mouse myeloid cells is a 92,000-dalton glycoprotein closely related to the 80,000-dalton glycoprotein of mouse embryo 3T3 cells.

Animals↗
PubMed1982

Gene for a major cell surface glycoprotein of mouse macrophages and other phagocytic cells is on chromosome 2.

A gene controlling the expression of a polymorphic 92,000-dalton glycoprotein of mouse macrophages and granulocytes has been identified. This glycoprotein was previously shown to be the major iodinated, trypsin-sensitive component of the murine phagocyte cell surface. The gene has been provisionally designated Pgp-1 for phagocyte glycoprotein 1. Expression of the glycoprotein was measured by monoclonal antibody binding to a polymorphic antigenic determinant. Antibody binding to cells of positive strains of mice was proportional to cell number, whereas binding to cells of negative strains was insignificant. The concentration of the antigen in cells of heterozygous mice was approximately 50% of that in homozygous mice. Thirteen of 33 recombinant inbred strains of mice were positive, with binding values 100-fold over background, suggesting that a single gene controlled expression of the antigen. Segregation of the antigen correlated with markers on chromosome 2. The segregation of Pgp-1, with nonagouti coat color (a) and hemolytic complement (Hc) activity among progeny of (C3H/HeJ x DBA/2J)F1 x DBA/2J mice confirmed the single gene control and the chromosomal assignment. Another gene on chromosome 2, Ly-m11, was also typed by using (BALB/cJ x C57BL/6J)F1 x C57BL/6J mice. The data from both of these crosses indicated the following gene order: Hc--Pgp-1--Ly-m11--a.

Animals↗
PubMed1981

Spontaneous lymphomas in SJL/J-(v+) mice: ecotropic and dualtropic virus expression in normal and lymphomatous tissues.

Approximately 60% of inbred SJL/J-(v+) adult mice having high levels of ecotropic endogenous XC+ virus showed virus activation within the first month of life, while the others produced virus at comparable levels later on, in an attempt to correlate the time of virus activation with the incidence and latency of lymphomas, the tails of 57 1- and 2-month-old mice were tested for virus presence, and the mice were then observed for lymphoma appearance. While all 2-month-old mice expressed ecotropic virus, only 63% of the 1-month-old mice were virus-positive. However no relationship existed between early virus production (within 1 month) or late virus production and lymphoma latency, total lymphoma incidence, and histopathology. In contrast with high titers of XC+ virus in tail tissues of diseased mice, a markedly low virus content was found in lymphomatous organs. This difference was not due to selective growth of poor virus-producer cells or to inhibitory factors possibly released by the inflammatory cell component. Viral protein content and XC+ virus titer were not closely correlated in the neoplastic organs. Search for XC- viruses revealed that only 1 of 6 aged normal and 16 of 19 lymphomatous mice produced viruses that grew on mink lung cells. By use of a standard limiting dilution cloning procedure, four isolates were obtained that showed tropism for both murine and heterologous cells. Three of these isolates induced cytopathic changes similar to those induced by MCF viruses on mink lung cells but not on mouse cells. Interference and neutralization assays performed to better characterize the virus envelope properties further indicated that SJL/J isolates had features typical of MCF dualtropic viruses.

Aging↗
PubMed1979

Identification of a mouse gene required for binding of Rauscher MuLV envelope gp70.

Mouse chromosome segregating somatic cell hybrids were established between a mouse thymic leukemai cell line (GRSL) and Chinese hamster E36 cells. The GRSL cells specifically bound purified Rauscher leukemia virus gp70 while the E36 cells exhibited no binding. The hybrids selectively bound Ruascher gp70 depending on the presence of a mouse cellular gene for the ecotropic murine luekemia gp70 receptor. A syntenic relationship was observed between the DIP-3 chromosome marker (on chromosome 5) and the gp70 receptor in primary clones and subclones of these hybrids; this was confirmed by chromosome analysis. The involvement of H-2 in the binding of Rauscher MuLV gp70 could be ruled out, because discordancies of the receptor presence and H-2 absence as well as of the receptor absence and H-2 presence type could be observed. Our results indicate that the Rec-1 (replication ecotropic MuLV) gene of Gazdar et al. (4) may well be the receptor gene for the ecotropic murine leukemia virus.

Animals↗
PubMed1979

H-2-dependent regulation of the high level of expression of ecotropic murine leukemia virus.

Adult B10.Y mice, which are congenic with C57BL/10ScSn ((B10) mice for the H-2 region, expressed a high titer of infectious ecotropic virus in the spleen. F1 hybrids between B10.Y and B10 mice were negative or had very low levels of virus expression. In (B10.Y x B10)F2 segregant mice, the high virus phenotype segregated with the H-2pa haplotype of B10.Y, whereas the virus-negative phenotype was associated with the H-2b haplotype of B10. Molecular hybridization experiments with a selected ecotropic AKR murine leukemia virus cyclic DNA probe indicated that both partner strains possessed ecotropic virus sequences and that the number of sequences present was the same in B10.Y mice as in B10 mice. This finding excluded the possibility that the H-2-related effect might be due to the presence of additional viral structural genes within or close to the H-2 region of B10.Y mice. The level of expression of this endogenous ecotropic virus was therefore affected by regulatory genes of the H-2 region.

Animals↗
PubMed1979

A radioimmunoassay for virus antibody using binding of 125I-labelled protein A.

An assay for virus antibodies using protein A from Staphylococcus aureus is described. Type B and type C RNA tumour viruses adsorbed on to polystryrene microtitre plate wells were incubated with antiserum and then with 125I-labelled protein A (I-pA) and bound radioactivity was determined. Technical details such as labelling, antigen concentration, storage of I-pA are reported. The specificity of the reaction was investigated in detail by competition experiments with purified unbound homologous viruses. This assay also proved to be sensitive for demonstration of autogenous immunity to both type B and type C RNA tumour viruses. A study using antisera against purified core and envelope virus proteins of mammary tumour and leukaemis viruses suggested that the reaction mainly involves surface antigens of the intact virions.

Animals↗
PubMed1979

Lack of T-cell mediated cytotoxicity in M-MSV system depending on H-2 haplotypes.

The H-2 restriction phenomenon was evaluated with regard to the immune response to Moloney sarcoma virus (M-MSV)-induced tumours in the mouse. Using an in vitro 51Cr release assay the lytic activity of lymphocytes from M-MSV immune strains of mice, including several H-2 recombinant strains, was determined on leukaemic cell lines originally induced in mice of different strains by neonatal infection with Moloney leukaemia virus. In analogy with other experimental models, it was observed that a compatibility between effector and target cells at the K and/or D regions is generally necessary and sufficient to obtain the cytotoxic effect. However, for the H-2d and H-2b haplotypes, identity at the K or D region respectively was not sufficient for lysis to occur. Attempts to demonstrate that this lack of activity might be associated with the absence of H-2 linked Ir responder genes, were not successful.

Animals↗