Affinity of human carcinoma cell nuclei for polyinosinic acid demonstrated by monoclonal antibodies.

The anti-nuclear cross reactivity of the monoclonal anti-actin antibodies M 372/809 was studied in some detail. The reactivity against a number of nuclear constituents was examined in the ELISA test and the capacities of these constituents to block the M 372/809 anti-nuclear and anti-actin reactions were evaluated in indirect immunofluorescence tests against tissue sections and monolayer cultures of fibroblasts and Vero cells. The repetitive polynucleotides polyinosinic and polyguanylic acid and their deoxyanalogues, actin and vimentin, were found to have the antigenic epitope. The epitope was covered or otherwise inactivated in the presence of polycytidylic acid. Using the M 372/809 antibodies as a reagent, carcinoma cell nuclei were found commonly to have an affinity for polyinosinic and polyguanylic acid. This was seldom noted with non-neoplastic cells.

Autoantibodies to the basal cells of squamous epithelium react with thymic epithelial cells.

Sera from carriers of hepatitis B surface antigen (17 out of 21), which reacted in immunofluorescence with the basal cell layer (BCL) of squamous epithelium, were also shown to react with a thymic stellate epithelial cell (SEC) characterized by long, dendritic-like cytoplasmic processes. Absorption of the autoantibodies against BCL of squamous epithelium (BCL-Ab) with a thymic homogenate abolished the reactivity with BCL and SEC, demonstrating that the same antigenic determinant was recognized in both cells. In the human thymus, SEC were present both in the cortex and in the medulla. In the outer cortex SEC delineated the septal spaces. SEC were also stained by anti-HLA-DR (Ia) but not by antiactin monoclonal antibodies. The morphology and distribution of SEC were similar to those of the previously described thymic epithelial cells containing alpha-1 thymosin (K. Hirokawa, J. E. McClure, and A. L. Goldstein, Thymus 4, 19, 1982). BCL-Ab were also found to react with five human epithelial thymomas. BCL-Ab seemed to be useful for further characterization of the thymic epithelial cells and for the immunodiagnosis of thymoma.

Growth-activity related variation of a nuclear antigen in monolayer-culture cells quantitated by immunofluorometry and microspectrophotometry.

Antibodies of the hybridoma clone M 372/809 react with a nuclear antigen in growing cells. Using immunofluorometry and microspectrophotometry, the antigen was found to accumulate to a maximum coincident with the marked early increase in nuclear protein contents. The following DNA increase appeared when the intensity of the reaction with M 372/809 antibodies was decreasing. The antigen increase was inhibited by alpha-amanitin. It may therefore be associated with transcription early in the cell cycle.

Reactivity of sera from patients with liver disease with glutaraldehyde-treated erythrocytes: role for false positive results in hemagglutination tests.

The use of glutaraldehyde as a coupling reagent in the passive hemagglutination test (HA) has gained wide application, especially for the coating of red blood cells (RBC) with glutaraldehyde-polymerized human serum albumin (pHSA), for studies of the albumin receptor on hepatitis B surface antigen (HBsAg) or for the detection of anti-albumin antibodies (AAA). Here we report a previously unrecognized reactivity with glutaraldehyde-treated RBC mainly with sera from patients with liver disease. The highest incidences of this reaction were found in patients with acute viral hepatitis A and B, namely 44 of 50 (88%) and 31 of 50 (62%) respectively. In 234 HBsAg carriers the frequency was low (3%). This reactivity was also observed in 19 of 50 sera from patients with chronic liver disease documented by biopsy, but not in sera from 68 healthy subjects. By immunofluorescence on glutaraldehyde-treated RBC it was shown that the corresponding antibodies belonged mainly to the IgM class. In all HBsAg-negative patients studied the HA titer against glutaraldehyde-treated RBC was in agreement with the titer against RBC coated with pHSA or pBSA (polymerized bovine serum albumin). Absorption with pHSA abolished the reaction with glutaraldehyde-treated RBC in 7 of 8 sera, suggesting a common reactivity between glutaraldehyde-polymerized HSA and glutaraldehyde-treated RBC. Apart from the possible clinical importance of these antibodies, their existence is a possible source of false positive results when glutaraldehyde is used as a coupling reagent for immunological assays, in particular with sera from patients with liver diseases.

Monoclonal antibodies to epitopes shared by actin and vimentin obtained by paramyxovirus immunization.

Three hybridoma clones producing IgM antibodies against actin were obtained from mice immunized with purified virions of paramyxoviruses. When tested on growing lung fibroblasts, ascites fluids of all clones stained in immunofluorescence cytoplasmic bundles of microfilaments, but also fibrillar networks. On colchicine-treated cells, perinuclear coils were seen in addition to microfilament bundles. In addition, one clone gave a pronounced speckled staining to the nuclei. Absorption of the ascites fluids with purified actin abolished all staining patterns. Using the Western blotting technique the antibodies reacted with both actin and vimentin polypeptides. DNase I abolished the staining of the actin filaments and of the nuclei, but left the vimentin pattern unimpaired. Thus, the monoclonal antibodies evidently reacted with epitopes common to actin and vimentin.

Replication of poliovirus and measles virus in cultures of human lymphoblastoid and of Burkitt lymphoma cell lines.

Poliovirus type 1 replicated in 4 different human lymphoblastoid cell lines (LBL) transformed in vitro by EBV virus or isolated from cases of mononucleosis. Maximum virus titers were reached 2--4 days after inoculation. There was a decrease in percentage of viable cells in the infected cultures but a considerable fraction of cells was not destroyed by virus replication. A persistent low grade replication of virus was observed and demonstrable during 56 days after inoculation in one LBL cell line. Presumably a small fraction of cells supporting virus replication is continuously recruited from refractory cells. No virus propagation was demonstrable in 4 Burkitt lymphoma (BL) cell lines. Measles virus grew efficiently in both types of cell lines. Using indirect immunofluorescent technique poliovirus and measles virus antigen could be demonstrated in the cytoplasm of LBL cells in parallel with disappearance of F-actin containing microvilli.

A radioimmunoassay for determination of anti-actin antibodies.

The reaction of spontaneously occurring human anti-human antibodies and experimentally produced rabbit anti-actin antibodies was investigated in a solid-phase radioimmunoassay (RIA). Three structurally different in vitro forms of actin monomeric G-actin filamentous F-actin and aggregated denatured actin were used as antigens. Human anti-actin antibodies reacted with F- and G-actin but not with aggregated actin, while rabbit anti-actin antibodies gave a strong reaction with all 3 forms of actin indicating differences in antibody specificities. The results of the anti-actin RIA were compared with those obtained by indirect immunofluorescence (IFL) on cryostat sections of rat stomach. The anti-actin RIA discriminated between patients' sera and control sera in most cases, although the indirect IFL test gave more conclusive results. The seemingly low sensitivity of the anti-actin RIA compared with that of indirect IFL test for detection of human anti-actin antibodies is probably due to favourable antigen distribution in tissue, not available in the solid phase. The anti-actin RIA was able to detect anti-actin antibodies in 8 out of 8 immunized rabbits although only two produced antibodies detectable by indirect IFL. The differences in reactivity between the two methods may depend on the presence of aggregated denatured actin in the antigen preparation used for immunization and exposure of the corresponding antigenic determinants of actin on the solid phase.

F-Actin-depolymerizing activity of human serum.

Non-heated human and animal sera contain a factor which exhibited an inhibiting activity on the staining of actin-containing structures by anti-actin antibodies in indirect immunofluorescence experiments. The presence of this factor lowered the viscosity of F-actin preparations and caused, as studied by electron-microscopy, a depolymerization of F-actin filaments as well as inhibition of filament formation of G-actin. The factor was, after its reaction with F-actin, liberated seemingly unaffected, indicating an enzymatic activity. The factor tentatively termed 'F-actin depolymerizing factor' was heat-sensitive and trypsin sensitive but resisted reduction. It was Ca2+ dependent and the staining inhibiting reaction was faster at 30 degrees C and 37 degrees C than at lower temperatures. Gel filtration experiments on Sephadex G-200 suggested a molecular size of the actin depolymerizing factor slightly higher than that of albumin. The electrophoretic mobility was that of gamma 2 globulin. The physiological role of the factor might be to prevent the presence of F-actin filaments within the circulation.

Anti-actin antibodies of human and rabbit origin.

The production of actin antisera in rabbits was described and their reaction in various immunological tests were compared with the results obtained with human anti-actin sera (smooth muscle antibodies). Even if the sera may react with different antigenic determinants of actin as suggested by results in immunoprecipitation and blocking experiments, the rabbit and human sera can be used for studies of cells with equal or similar results. A prerequisite was that the rabbit anti-actin titres were high. The difficulty in obtaining strong anti-actin sera in rabbits was confirmed.

Actin filaments in paramyxovirus-infected human fibroblasts studied by indirect immunofluorescence.

Fibroblasts growing on glass have microfilaments arranged in bundles. These can be demonstrated by indirect immunofluorescent technique using human antiactin serum or experimentally produced rabbit anti-actin serum. When monolayer cultures of epithelial cells and fibroblasts are infected with paramyxovirus, such as measles, mumps, Sendai and NDV, there is a striking decrease of the bundles. Rabies and adenoviruses do not seem to influence the staining of microfilaments. The microfilament decreasing effect in the cells correlates to the finding by SDS-polyacrylamide-gel-electrophoresis of actin within virions of the paramyxoviruses.