Serum IgG-like glycoferroprotein: identification of its final dissociation form of thermostable protein coupled with albumin.

Human serum IgG-like glycoferroprotein identical to ascitic IgG-like glycoferroprotein that binds labeled monoclonal antibodies to CA125 is a complex consisting of three proteins: IgG, human serum albumin, and unidentified thermostable protein. Final dissociation form of serum IgG-like glycoferroprotein also appears as a complex of three nonidentical polypeptides with a molecular weight of 55 kDa (PC55) migrating in the albumin zone of thermostable protein coupled with albumin and structures chemically identical to human serum albumin and IgG heavy chains. Under denaturing conditions of electrophoresis in polyacrylamide gel, IgG-like glycoferroprotein and PC55 have the same molecular weight (about 55 kDa), while under reducing conditions their weight is about 75 kDa. Transition form (form the lower to the higher molecular weight) appears as an oblique (at about ≈ 30°) protein band creating a ladder string effect. Ladder string effect was reproduced with thermostable protein coupled with albumin, PC55, IgG-like glycoferroprotein, with all commercially available human and bovine albumins, rat albumin as well as with heated and renatured albumins and can serve as electrophoretic identification sign for thermostable protein coupled with albumin. Renatured after boiling (100°C for 15 min) bovine albumin under reducing conditions appeared as bow string twisted in helix, that raises molecule in 2.5 turns from ≈ 2 to ≈ 75 kDa. These data attest to the existence of an albumin double and to its possible double structure.

[Partial reversion of the phenotype of a poorly differentiated hepatocellular carcinoma in a three-dimensional culture].

In vivo, normal tissues and organs have a three-dimensional structure and function in a three-dimensional environment. The standard two-dimensional cell culture conditions drastically differ from those in vivo. For this reason, three-dimensional cultures based on different variants of the extracellular matrix are more adequate for analyzing normal and tumor cell growth. Culturing a poorly differentiated hepatocellular carcinoma in a collagen gel yielded spheroids whose growth pattern shifted towards the epithelial phenotype. The shift was expressed in changes in the cytoskeleton, enhanced formation of extracellular matrix fibrils between cells, and formation of fibronectin fibrils on the outer surface of spheroids. Analysis of 25 genes reflecting the level of morphological and functional hepatocyte differentiation showed that the expression of the gene encoding the transforming growth factor TGFbeta2 was suppressed the most significantly.

[The purification of the protein alternative to alfa-fetoprotein].

The subject of the study was the unidentified protein Ag A2/3, which is found in some cells of rat hepatoma McA RH7777 and their clones. The feature of this protein is that its expression is alternative to alfa-fetoprotein (AFP), i.e. Ag A2/3 is not found in cells and cell clones containing AFP, and vice versa. Ag A2/3 proved to be a cell stress protein--it was induced by heavy metal salts (Pb2+ and Cd2+) in the liver of adult rats and AFP+/A2/3(-) clones of hepatomas; the attenuation of AFP synthesis occurred simultaneously. This paper describes the preparation of Ag A2/3 for sequence analysis, and the scheme of Ag A2/3 purification. When trying to obtain a blot for sequencing it proved to be impossible to reveal the protein using McAb to Ag A2/3 after the transfer of separated proteins on PVDF. The reactivity of the antigen determinant was reestablished with blot processing on PVDF membrane with methanol and Twin 80.

Inducible protein in rat hepatomas with expression alternative to alpha-fetoprotein.

The rat hepatoma cell line McA RH7777 was cloned into alpha-fetoprotein-producing (AFP+) and non-producing (AFP-) sublines. A monoclonal antibody (MAb A2/3) reacting with an antigen (Ag A2/3) present only in AFP- clones or AFP- cells in mixed clones was obtained. Ag A2/3 was absent from the liver of embryonic, fetal, newborn and adult rats, but it was present in gastric and intestinal mucosa of adult rats. Ag A2/3 was found to be a heavy metal-inducible protein: Cd2+ and Pb2+ strongly induced the expression of Ag A2/3 in vivo in the liver of adult rats, while xenobiotics and CCl4 were not active in this respect. In vitro Cd2+ and Pb2+ induced Ag A2/3 expression in several AFP+ clones, leading to a simultaneous marked decrease of AFP+ cells from such clones. The effect of Cd2+ in the induction of Ag A2/3 and suppression of AFP was reversible. SDS PAGE revealed one protein band with an m.w. close to 45,000, which was not sensitive to mercaptoethanol. Despite its inducible properties, Ag A2/3 was shown not to belong to metallothioneins, cytochrome P-450, glutathion-transferase or heat shock proteins families, well-known as being inducible cell stress proteins. Expression of Ag A2/3 could be one of the factors determining the high amplitude of AFP production by individual liver tumors. The nature of Ag A2/3 and its alternative expression with respect to AFP remain to be studied.

Antimacrophage monoclonal antibody D11 in the diagnosis of tumors of histiocytic origin.

OBJECTIVE: To investigate the possible application of the human antimacrophagal antibody D11 (MAb D11) to the diagnosis of tumors of histiocytic origin. STUDY DESIGN: Biopsy and surgical materials from 181 patients with nonepithelial tumors and tumorlike lesions were used in the study. The reactivity of MAb D11 with tumor cells was assayed as the percentage of positively reacted cells in cryostat sections and on smears of tissues determined by the immunoperoxidase method. RESULTS: According to the reaction, all tumors could be divided into three groups: group 1, 39 cases, including all malignant fibrous histiocytomas (MFH) (24 cases); all tumors were D11 antigen positive; group 2, 130 cases, tumors with the same nosologic forms revealed positive, as well as negative, reactions with MAb D11; and group 3, 12 cases, all tumors exhibited a negative reaction with MAb D11. CONCLUSION: Although MAb D11 gave a positive reaction with all tumors in the histiocyte series, its reactivity with some tumors of other histogeneses limits the application of MAb D11 for differential diagnosis of MFH. Nevertheless, MAb D11 may be used for the exclusion of tumors from the MFH group in the case of a negative reaction. The results support the view that MFH is derived from cells of the mononuclear phagocyte system.

Reactivity of anti-macrophage monoclonal antibody D11 in human leukemia and malignant lymphoma.

We have studied the reactivity patterns of a previously described pan-macrophage monoclonal antibody (MAb) D11 in 324 cases of acute leukemia and malignant lymphoma (ML). Reaction of D11 in tissue sections was restricted to histiocytes and macrophages. In non-Hodgkin's ML, D11 helped to confirm or to establish the histiocytic nature in 8 of 96 cases, i.e., in 4 of 6 histiocytic MLs; 2 of 13 anaplastic large-cell lymphomas; 1 of 4 large-cell immunoblastic clear-cell MLs; and 1 of 2 histiocytosis X cases. Positive reaction of D11 in acute lymphoblastic leukaemia (ALL) was found in 9 of 86 cases (all belonging to early B-lineage leukemia), of which 4 were CD34-positive and 5 co-expressed 1 or more myeloid/monocytic antigens. MAb D11 did not react in 42 cases of acute-myeloblastic-leukemia (AML) FAB variants M0-M5, except 1 acute mixed-lineage leukemia M1/pre-pre-B. Comparative study of the MAb D11 and a standard CD68 MAb KP- 1 showed that the antigens belong to different epitopes of different molecules.

[Anti-macrophage monoclonal antibody D-11 in the diagnosis of histiocytic tumors]. / Antimakrofagal'noe monoklonal'noe antitelo D-11 v diagnostike gistiotsitarnykh opukholei.

Anti-macrophage monoclonal antibody (Mab) D-11 was tested in surgical material and biopsies of non-epithelial tumors and tumor-like lesions from 181 patients in order to assess possibility of using this Mab for diagnosis of histiocytic tumors, malignant fibrous histiocytoma in particular. The study was performed in parallel on cryostat sections and smears by immuno-peroxidase method. It is established that D-11 reacts positively with both histiocytic tumors and tumors of other genesis this being a limiting factor in differential diagnosis of histiocytic tumors. However, taking into consideration 100% of positive results with histiocytic tumors only, this antibody can be used for exclusion of tumors studied from the group of histiocytomas in cases of negative reaction.

[Use of a new antimacrophage monoclonal antibody D11 in diagnosis of hemoblastosis]. / Primenenie novogo antimakrofagal'nogo monoklonal'nogo antitela D11 v diagnostike gemoblastozov.

Expression of new Mab D11 on blood and bone marrow cells was investigated in 85 hemoblastosis patients. In normals, antigen D11 is expressed on some monocytes and all tissue macrophages. D11 was noted on lymphoblasts of 5 out of 10 cases with B-cell ALL and of 1 case in B-lymphoid blast crisis of chronic myeloid leukemia. In T-ALL, ANLL, non-Hodgkin's lymphomas in leukemization stage, hairy cell leukemia and chronic lymphoid leukemia the cells were nonresponsive to Mab D11. Unlike D11 which have round nuclei, lymphoblasts D11+ have folded nuclei and more pronounced cytoplasmic basophilia. There were both B and myeloid antigens on D11+ blasts. ALL D11+ patients had extramedullary foci, more suppressed granulocytic and thrombocytic components of hemopoiesis, shorter remissions than those with ALL D11-.

Common antigen of oval and biliary epithelial cells (A6) is a differentiation marker of epithelial and erythroid cell lineages in early development of the mouse.

The A6 antigen--a surface-exposed component shared by mouse oval and biliary epithelial cells--was examined during prenatal development of mouse in order to elucidate its relation to liver progenitor cells. Immunohistochemical demonstration of the antigen was performed at the light and electron microscopy level beginning from the 9.5 day of gestation (26-28 somite pairs). Up to the 11.5 day of gestation A6 antigen is found only in the visceral endoderm of yolk sac and gut epithelium, while liver diverticulum and liver are A6-negative. In the liver epithelial lineages A6 antigen behaves as a strong and reliable marker of biliary epithelial cells where it is found beginning from their emergence on the 15th day of gestation. It was not revealed in immature hepatocytes beginning from the 16th day of gestation. However weak expression of the antigen was observed in hepatoblasts on 12-15 days of gestation possibly reflecting their ability to differentiate along either hepatocyte or biliary epithelial cell lineages. Surprisingly, A6 antigen turned out to be a peculiar marker of the crythroid lineage: in mouse fetuses it distinguished A6 positive liver and spleen erythroblasts from A6 negative early hemopoietic cells of yolk sac origin. Moreover in the liver, A6 antigen probably distinguishes two waves of erythropoiesis: it is found on the erythroblasts from the 11.5 day of gestation onward while first extravascular erythroblasts appear in the liver on the 10th day of gestation. Both fetal and adult erythrocytes are A6-negative. In the process of organogenesis A6 antigen was revealed in various mouse fetal organs.(ABSTRACT TRUNCATED AT 250 WORDS)

D11, a novel monoclonal antibody specific for human mature macrophages and peripheral blood monocytes.

A new monoclonal antibody designated Mab D11 is described, which shows a restricted reactivity to cells of the monocyte/macrophage system. When tested by light and electron microscopic immunoperoxidase methods, Mab D11 specifically reacts with blood monocytes and stains resident macrophages in a wide variety of human tissues; it does not mark the macrophages of other species, i.e., rat, swine and mouse. Antigen-presenting cells, e.g., Langerhans cells, are Mab D11 negative. Mab D11 reveals the antigen on cryostat and paraffin tissue sections. Ultrastructurally the antigen recognized by Mab D11 in all macrophage types studied is located on the plasma membrane and within cytoplasmic structures including lysosomes. On immunoblotting, Mab D11 detects the 125-kDa antigen in human liver and the 135-kDa protein in tumours of histiocytic origin. The similarity of Mab D11 to known "pan-macrophage" monoclonal antibodies is discussed.

[Histogenesis of experimental renal tumors in mice]. / O histogeneze éksperimental'nykh opukholei pochek myshei.

Epithelial kidney tumours induced in CBA male mice by 1,2-dimethylhydrazine were studied histochemically and immunohistochemically. Histologically, 48 tumours studied were diagnosed as clear-cell, acidophilic or mixed adenomas located in the renal cortex. Gamma-glutamyl transpeptidase (GGT) was strongly positive in normal proximal convoluted tubules, slightly positive in the cells of Bowman capsule and negative in 47 of 48 tumours examined. Antibodies against the new antigen obtained from the mouse liver oval cells and called A6 antigen were also used. This antigen in normal kidney is negative in proximal tubules but always positive in distal tubules and collective ducts. It was also positive in all 47 GGT-negative tumours. One tumour was GGT-positive and A6 antigen-negative. The conclusion is made that the majority of renal cell adenomas in mice most likely originate from the collective duct system and not from the proximal tubules.

[New differentiation markers of mouse liver epithelial cell lines]. / Novye differentsirovochnye markery épitelial'nykh kletochnykh linii pecheni myshi.

We describe two new markers of mouse liver epithelial cells detected by monoclonal antibodies. Immunomorphological localization of antigens was performed using light and electron microscopy. Antigen G7 is a marker of cholangiocytes and oval cells. Antigen A6 is present in cholangiocytes and oval cells; moreover, it is expressed in normal liver in single hepatocytes adjacent to the portal vein, in preneoplastic liver, in newly formed hepatocytes, and in certain hepatocarcinomas. Thus, antigen A6 is a marker of cholangiocytes, oval cells and of certain stages of hepatocyte differentiation. We also detected phenotypic heterogeneity of Gehring cells in terms of antigen A6 content. We have formulated problem of the relationship between A6-negative Gehring cells and liver stem cells. Both marker antigens are species-specific but are not specific for the liver. Antigen A6 is simultaneously a differentiation marker of cells belonging to the erythroid series. It is expressed in erythroblasts of fetal liver and is absent in erythroblasts of the yolk sac and erythrocytes. The relationship between antigen A6 and blood group antigens is discussed.

[Mouse hepatoblastoma: comparative aspects]. / Gepatoblastoma myshei v sravnitel'nom aspekte.

The structure of 12 spontaneous hepatoblastomas found in old (average age 26.5 months) male mice is described. There were considerable strain differences in their incidence: 0.5% (1/194), 0.5% (1/194) and 5% (10/198) in strain C57B1, CBA and F1 (CBA X C57B1), respectively. This proves the importance of genetic factor the role of which in the development of human hepatoblastoma is not established so far. Mouse hepatoblastoma develops almost invariably within or adjacent to liver cell tumours (adenoma or carcinoma). There was a correlation between the incidence of liver cell tumours within a given strain treated with different doses of carcinogen but such correlation was absent in mice of different strains. Histologically and ultrastructurally, mouse hepatoblastoma corresponds to the anaplastic variant of human hepatoblastoma. As distinct from human tumour, mouse hepatoblastoma does not contain alpha-fetoprotein. One tumour was transplanted to the syngeneic host and passed 30 transplant generations retaining the structure of a primary tumour with areas of osteoid tissue and foci of squamous cell metaplasia. Mouse hepatoblastoma may be induced by carcinogens. Likewise, according to the literature, risk of hepatoblastoma is higher in children whose mothers were exposed to the potential carcinogens before or during the pregnancy.

[Common antigens of oval cells and cholangiocytes in the mouse. Their detection by using monoclonal antibodies]. / Obshchie antigeny oval'nykh kletok i kholangiotsitov myshi. Vyiavlenie s pomoshch'iu monoklonal'nykh antitel.

Monoclonal antibodies (MAb) were produced against antigens (Ag) of oval cells isolated from the preneoplastic murine liver. To suppress the immune response to major antigens common with hepatocytes, the principle of anti-idiotype immunization was employed. Characteristics of three MAb reacting selectively with the foci of oval cell proliferation are described. MAb A6 and G7 detected two different antigens (Ag A6 and Ag G7, respectively) common for oval cells and cholangiocytes. Ag A6 was also found in normal parenchyma (in membranes of single hepatocytes adjacent to portal veins), in the preneoplastic liver (in hepatocytes formed de novo) and in some hepatoma cells. Ag G7 was not detected in hepatocytes. MAb E5 stained the matrix in the areas adjacent to oval cells and large bile ducts. All the three Ag were widely distributed in normal tissues of mice. The significance of the detected Ag as markers of murine liver epithelial cell lines and stages of their differentiation is discussed as well as the possible relationship between Ag A6 and Ag of human blood groups.

Common antigens of mouse oval and biliary epithelial cells. Expression on newly formed hepatocytes.

Two antigens - A6 and G7 - shared by mouse biliary epithelial and oval cells were revealed by monoclonal antibodies raised in rat immunized with oval-cell-enriched liver fraction. Oval cells were induced in CBA or F1 (CBA x C57BL6) mice by a combination of a single injection of the alkylating drug Dipin with partial hepatectomy. In normal liver A6 antigen was localized, using light and electron microscopy, in biliary epithelial cells of all ducts including Hering canals. Some bile ductal and Hering cells were A6-negative. Occasionally, A6 antigen was present in single hepatocytes forming the periportal ends of hepatic cords. In preneoplastic and tumorous liver A6 antigen was present in bile ductal and oval cells and in a fraction of newly formed hepatocytes and tumor cells. G7 antigen was revealed in normal, precancerous and tumorous liver in biliary epithelial and oval cells but not in hepatocytes. A6 and G7 antigens were not liver-specific: they were expressed in various normal organs and tissues, especially in epithelia. In studies of mouse liver lineages A6 antigen can be used as a common marker of biliary epithelial and oval cells and hepatocytes at certain stages of differentiation. G7 antigen is a marker of oval and biliary epithelial cells. There was a striking similarity in A6 antigen localization to that of human blood group antigens in normal liver and liver tumors. A6 antigen may thus provide a useful tool for the study of neoexpression of human blood group antigens in liver tumors.

Human alpha-fetoprotein epitopes as revealed by monoclonal antibodies.

The epitope specificity of 12 anti-human alpha-fetoprotein monoclonal antibodies (Mabs) was estimated in an enzyme-linked immunosorbent assay (ELISA). A combination of two different approaches: (i) Mabs binding to heterologous alpha-fetoprotein (AFP); and (ii) cooperative Mabs binding to human AFP (hAFP) when tested in pair mixtures; was used. This double-approach methodology was found to be more reliable for the definition of Mab specificities than either method alone. The anti-hAFP Mabs studied recognised eight unique non-repeated epitopes on hAFP. Two of the epitopes were specific for humans, whereas six were common to other species (mouse, rat, calf, dog, pig and cat) with a characteristic species distribution for each epitope. All epitopes were present on hAFP synthesised by hepatoma, yolk sac tumour and embryo.

Transplantable mouse hepatoblastoma: histologic, ultrastructural and immunohistochemical study.

Hepatoblastoma found adjacent to a liver cell adenoma in an aged (CBA x C57Bl/6)F1 male mouse was transplanted to the syngeneic host and passed through 30 generations. Histologically, tumours that grew on transplantation retained principal morphological features of the primary tumour. Transplanted tumours were negative for AFP and for antigen A6. This antigen in the normal mouse liver is found in epithelial cells lining bile ducts and ductules including the terminal Hering canals. Some of the Hering cells were consistently A6 negative under normal conditions, and the suggestion is made that these A6 negative cells might be the cells of hepatoblastoma origin.

Mixed precipitation in gel: a new method of identification and characterization of monoclonal antibodies.

A mixed precipitation in the gel (MPG) technique is suggested for detection and characterization of monoclonal antibodies (MAbs). The MPG is based on the formation of a mixed precipitate composed of an antigen, the corresponding MAb and precipitating polyclonal antiserum. MAb incorporated into the precipitate is revealed by Fab'-peroxidase conjugate added to polyclonal antiserum. The MPG technique was applied to hybridoma screening as well as for the antigen and epitope specificity analysis of different MAbs. The MPG is a one-step, simple, inexpensive technique and valuable for the study of any antigen which could be revealed by immunodiffusion.

[Mixed gel precipitation in the analysis of monoclonal antibodies against determinants of the carcinoembryonic antigen]. / Smeshannaia pretsipitatsiia v gele pri analize monoklonal'nykh antitel k raznym determinantam rakovoémbrional'nogo antitela.

Mixed gel precipitation technique was used in the study of 5 monoclonal antibodies (MA) to carcinoembryonic antigen (CEA). The method is based on specific inclusion of MA into the precipitate formed by the antigen and polyclonal antibodies to it. The test-system for the determination of nonspecific cross-reacting antigen (NCA) has demonstrated that MA 2D10 are directed to the determinant, common for CEA and NCA, whereas MA 3C12 and 2G10 give no reaction with NCA. The specificity of MA 192 and 35 correlated with the previously established one. Mixed gel precipitation technique is recommended for primary screening and study of MA specificity to any precipitating antigen.