In vitro transformation of cells from human neoplasms.

The possible involvement of RNA tumor virus genomes in human cell transformation was investigated. Forty-nine cell cultures from neoplastic, normal, or embryo tissues were examined for transformation, following inoculation of murine leukemia virus (MuLV), feline leukemia virus (FeLV) grown in human cells, or bone marrow aspirates from leukemia patients. Five cultures exhibited transformation (1 after inoculation of MuLV grown in human cells; 4 after inoculation of human leukemic bone marrow), and 4 were established as cell lines. They were derived from giant cell tumor and fibrosarcomas. The established transformed cells formed colonies in soft agar, grew progressively in immunosuppressed mice, and carried antigens common to FeLV and MuLV. Although virus particles were not seen in these cultures, 68S RNA was detected in their media. Medium from nontransformed parent cultures also contained 68S RNA but in amounts about 15 times less than in transformed cultures. Transformed human cells passaged in mice produced both type C virus particles and 68S RNA. Antigens common to MuLV and FeLV were found in these particles. However, the results of biological and serological studies indicate their difference from conventional MuLV and FeLV. The relationship of this virus and 68S RNA found in transformed cultures remains to be determined.     

Spontaneous Induction of Endogenous Murine Leukemia Virus-Related Antigen Expression During Short-Term In Vitro Incubation of Mouse Lymphocytes

Short-term lymphocyte cultures from mouse thymus, spleen, or lymph nodes were studied for the presence of murine leukemia virus group-specific antigens with an immunofluorescence test using rat immune sera against syngeneic cells infected with the radiation leukemia virus or its pseudotype of murine sarcoma virus and goat and rabbit antisera against purified murine leukemia virus group-specific antigen. Antigens reacting with these sera appeared in the cultured lymphocytes within 24 hr, and the proportion of immunofluorescent-positive cells increased to 25-80% by the second or third day of cultivation, concomitantly with a decrease in cell viability. The appearance of these antigens can be suppressed by inhibitors of DNA (mitomycin-C), RNA (actinomycin-D, cordycepin, and polyadenylic acid), and protein (cycloheximide) synthesis. No infectious virus could be detected by the immunofluorescence and XC-cell tests. The observed phenomenon appears to represent the spontaneous partial derepression of endogenous murine leukemia virus replication in lymphocytes during short-term in vitro cultivation.     

Complement-fixation-inhibition as a test for antibodies in cats and humans to C-type RNA tumor virus antigen.

The complement-fixation-inhibition (CFI) test was evaluated as a means of detecting humoral antibodies in cat sera and in human sera to mammalian C-type RNA virus interspecies antigen(s). CFI antibody titers of greater than or equal 1:2 were detected in sera from all tumor bearing (23) and normal cats (23), however, sera from most germ free cats were negative. When the same cat sera were tested for blocking antibody by the paired radioiodine labeled antibody technique the correlation between the radioimmune assay and CFI tests was 85%. Sera from 378 cancer patients and 193 normal people were tested for antibodies to the mammalian oncornavirus interspecies-specific antigen in the CFI test. This test used a rabbit antiserum prepared toward a purified feline leukemia virus (FeLV) interspecies antigen. Disrupted Rauscher murine leukemia virus (RLV) was used as source of interspecies antigen in the CFI test. A significantly (P=0.01) higher number of reactions occurred with sera from patients with lymphosarcoma (70.4%), osteosarcoma (41.0%), reticulum cell sarcoma (56.7%), and rhabdomyosarcoma (31.8%) as opposed to sera from normal individuals (6.2%). Of 51 sera from patients with acute lymphocytic leukemia 23.5% (P=0.05) were reactive. Of the sera from 88 breast cancer patients 22.7% reacted, as opposed to 7.8% of 116 normal females and 13.9% of 43 patients with benign breast disease. CFI antibody titers were shown to be dependent on RLV antigen concentration. Absorption with human A and B red blood cell (RBC) and Forssman antigen did not reduce the CFI titers in human sera whereas absorption with RLV reduced them significantly. By indirect radioimmunoelectrophoresis the antibody in selected human sera was shown to be an IgG.     

Immunity to cytopathic agents associated with Crohn's disease: a negative study.

Serum and peripheral blood lymphocytes from 10 patients with Crohn's disease and 10 healthy subjects were examined for immunological reactivity against chick embryo cell cultures displaying cytopathic effects after inoculation with 0.2 micro filtrates prepared from Crohn's disease intestinal tissues. Although the assay systems (indirect immunofluorescence, lymphocyte transformation, and cytotoxicity) yielded positive results using well-characterized cytopathic viruses (mumps, measles), neither Crohn's disease nor healthy subjects showed immune reactivity to the chick embryo cell cultures inoculated with Crohn's disease intestinal tissues in any of the assay systems. These experiments provide evidence against the hypothesis that the in vitro cytopathic effect on chick embryo cell cultures produced by Crohn's disease intestinal filtrates are caused by a replicating virus or viruses.     

Human megakaryocytes. III. Characterization in myeloproliferative disorders

Abnormal proliferation of the megakaryocytic line was observed in the marrow tissue from patients with myeloproliferative disorders. Megakaryocytes were identified by immunofluorescence using distinct platelet protein markers. Plasma factor VIII antigen (factor VIII:AGN) and platelet glycoproteins IIb and IIIa were detected in normal mature and early megakaryocytes, as well as in a morphologically heterogeneous population of low density marrow cells regarded as atypical megakaryocytes. Atypical megakaryocytes were defined as oval/round 14- 35-micron diameter blast-like mononuclear/multinucleated cells bearing platelet protein markers with distinct morphological features, including cytoplasmic vacuolation, variable nuclear/cytoplasmic ratios, and variable cytoplasmic granulation. Atypical megakaryocytes were observed in most chronic myelogenous leukemia (CML) patients and in two patients with polycythemia vera, representing between 60 and 1,840 cells/10(4) cells (less than 1.050 g Percoll/cu cm). No atypical megakaryocytes were found in (a) 20 normal controls, (b) two patients with essential thrombocythemia, (c) a patient with thrombocytosis secondary to acute bleeding, and (d) in two patients with CML. Atypical megakaryocytes appear to represent a single-cell population, as demonstrated by a series of double immunofluorescence assays using combinations of five different antiplatelet protein sera. There was a statistically significant correlation between the frequency of atypical megakaryocytes and the presence of immature forms of myeloid cells in blood. Analyses of Fc IgG receptors conducted with two different immunofluorescence systems have demonstrated that phenotypic similarities existed between atypical megakaryocytes and myeloproliferative platelet proteins and differentiation markers on megakaryocytes are useful in elucidating the pathophysiologic alterations occurring in the megakaryocytic compartment in patients with myeloproliferative disorders.     

Tissue and organ culture studies of hepatitis B virus.

Techniques have been developed for the cultivation of human and nonhuman primate liver cells in tissue and organ culture. Progressive non-cytocidal involvement of the normal cytoplasmic and nuclear components of cultured liver cells has been demonstrated by specific attachment of fluorescent antibody to hepatitis B core and surface antigens after inoculation of the cultures of human origin with known infective sera and with clinical material. Hepatitis B surface antigen may be produced, although infrequently, in inoculated liver organ cultures but serial passage has not been achieved. Serial passage of hepatitis B virus has been reported with fragments of human embryo liver cultivated on the chorioallantoic membrane of the developing chick embryo. Intranuclear virus-like particles have been localized in hepatocytes of cultured explants of liver biopsies obtained from infants with chronic hepatitis B antigenemia. Further studies are urgently required to determine whether cultivation of hepatitis B virus can be firmly established in readily available cell and organ cultures.     

Seropositivity in Dutch Crohn's disease patients against primed nude mouse lymph nodes, and the difference with lymphocytotoxic antibodies.

Sera from patients with Crohn's disease have been reported to show positive immunofluorescence with lymph nodes of nude mice primed with a filtrate of intestinal tissue affected with Crohn's disease. An indirect immunofluorescence assay was used to test sera of 63 unrelated patients with Crohn's disease, 21 with ulcerative colitis and 36 control subjects against lymph nodes of athymic nude (nu/nu) mice which had been injected with Crohn's disease and ulcerative colitis intestinal tissue filtrates. Forty nine per cent of Crohn's disease patients, 10% of ulcerative colitis patients and 3% of control sera reacted against lymph nodes of mice injected injected with ulcerative colitis intestinal tissue filtrates, 18% of Crohn's disease sera were with intestinal tissue homogenate from Dutch Crohn's patients. With the lymph nodes of mice injected with ulcerative colitis intestinal tissue filtrates, 18% of Crohn's disease sera were positive, whereas all ulcerative colitis and control sera were negative. Lymph nodes from 18 of the 19 mice injected with Crohn's disease tissue filtrates reacted with Crohn's disease sera, whereas only three of these 19 mice reacted with ulcerative colitis sera. A comparative study, carried out in parallel with Crohn's disease filtrate induced hyperplastic lymph nodes from the Bilthoven colony (W2) and from the New York colony (E671) using sera from 54 Crohn's disease patients from Leiden, showed immunoreactivity with 44 and 57% of the Crohn's disease sera against the two hyperplastic lymph nodes. Thirty six of the 54 Crohn's disease sera (67%) reacted with either or both lymph nodes. Only 11% of the Crohn's disease sera which were examined for immunofluorescence and lymphocytotoxic antibodies had lymphocytotoxic antibodies, whereas 40% and 46% of the same sera showed positive immunofluorescence against E671 and W2, respectively. Absorption studies indicated that lymphocytotoxic antibodies activity and the immunofluorescence against the primed nude mouse lymph node are mediated by different serum antibodies in Crohn's disease. The reproducibility of the nude mouse immunofluorescence test system for a preferential immunoreactivity of Crohn's disease sera against Crohn's disease tissue primed murine lymph nodes has been confirmed by the present study. Further studies are necessary to find out whether crossreactive antigen(s) as recognised by some of the Crohn's disease sera in mice injected with ulcerative colitis tissue filtrate is similar to the antigen(s) detected by Crohn's disease sera in mice injected with Crohn's disease tissue filtrates.     

Presence of complement-fixing antibodies against antigens of Gross virus-induced rat lymphoma and normal rat thymus in sera of patients with some forms of malignancies.

Presence of complement-fixing antibodies against soluble antigen(s) of Gross virus-induced rat lymphoma W/Fu(C58NT)D was determined in sera of 180 healthy donors, in sera from 100 healthy pregnant women and in sera obtained from 139 patients with acute myelosis, acute lymphadenosis, acute undifferentiated leukosis, chronic myelosis, chronic lymphadenosis and Hodgkin's disease. Antibodies against soluble complement-fixing antigen from W/Fu(C58NT)D rat lymphoma were found in 33 (5).5%) out of 64 sera of patients with acute leukemia, in 15 out of 60 sera of patients with Hodgkin's disease, in 17 (9.4%) out of 180 healthy donors and in 22 (22.0%) out of 100 sera from healthy pregnant women. None of 15 sera from patients with chronic leukemia were positive. Titers of complement-fixing antibodies in positive sera remained unchanged after absorption with pooled lymphocytes from healthy human donors. Some selected positive sera reacted also with a soluble antigen prepared from normal rat thymus. Nature of complement-fixing antibodies detected and of corresponding antigens is discussed.     

Tissue and Organ Culture Studies of Hepatitis Type B

Abstract. The morphological and functional capacity of cells maintained in organ culture may offer a system for cultivating viruses which closely simulates conditions in the intact host. Several attempts to propagate the hepatitis B virus in organ cultures have recently been reported. Hepatitis B antigen may be produced in suitable preparations of human embryo liver in organ culture. A progressive rise in the titre of antigen, as measured by several techniques, has been demonstrated with a limited number of sera; and one successful passage of harvested material from cultures on day 8 has been accomplished with two specimens. A most important and urgent need is the adaptation of the agent passaged in organ culture to growth in readily available cell cultures, because of the difficulty in obtaining suitable human fetal liver.     

The spectrum of complement-fixing antinuclear antibodies in patients with hepatocellular carcinoma

Sera from 230 hepatocellular carcinoma patients were tested for antinuclear antibodies by anticomplement immunofluorescence in 16 types of transformed, diploid or primary cells of human, monkey, chimpanzee or rat origin. As controls, we tested 85 sera from patients with chronic liver diseases, 48 sera from patients with nonhepatic cancers and 164 sera of normal controls. Exactly 11.2% of all cancer patients but only 3.6% of noncancer patients had complement-fixing antinuclear antibody that reacted with all substrates. Only sera from hepatocellular carcinoma reacted with subsets of the tumor cell substrates. These sera reacted with hepatocellular carcinoma cells and nonhepatic cancer cells (antitumor) or only with one or more of the human hepatocellular carcinoma cell lines, PLC/PRF/5, Hep3B and Mahlavu, that were derived from HBsAg-positive patients (antihepatocellular carcinoma). Three of these reacted only with hepatitis B virus DNA-positive cells (PLC/PRF/5 and Hep3B) that contained "hepatitis B-associated nuclear antigen," 1 reacted only with hepatitis B virus DNA-negative Mahlavu cells, 1 reacted with PLC/PRF/5 and Mahlavu and 3 reacted with all 3 cells. The nuclear antigen in Mahlavu was expressed as a homogeneous fluorescence that spared the nucleoli, was present in a lower percentage of cells than hepatitis B-associated nuclear antigen and was more thermostable than hepatitis B-associated nuclear antigen. However, it resembled hepatitis B-associated nuclear antigen in kinetics of expression and susceptibility to digestion with DNase, RNase and proteinase K. The nature of the nuclear antigens in the hepatocellular carcinoma cells is poorly understood but one possibility is that they may represent the expression of viral or tumor-related genes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Human Leukemia-Associated Anti-Nuclear Reactivity

A brilliant, coarsely granular nuclear antigen was detected by anti-complement immunofluorescence in the nuclei of acute myeloid leukemia myeloblasts. Designated as LANA (leukemia-associated nuclear antigen), the reactivity differs from that of the Epstein-Barr-virus-determined nuclear antigen (EBNA) in immunological specificity and morphological appearance, although it is visualized by the same method. Serum from acute myeloid leukemia patients gave positive reactions in 73% of the cases. In acute lymphatic leukemia, chronic myeloid leukemia, chronic lymphatic leukemia, and Burkitt's lymphoma the sera were positive in 35, 14, 19, and 24%, respectively. Two of five polycythemia and two of eleven myeloma sera were also positive. Among 61 healthy controls, 58 were negative, whereas three showed a diffuse nuclear staining with a different pattern. Among 24 carcinoma patients, 18 were negative, whereas six gave a nuclear staining with a different, diffuse pattern. Sera from 20 patients who had recovered from infectious mononucleosis were all negative. In addition to the blasts of acute myeloid leukemia, a similar reactivity was seen with two Epstein-Barr virus DNA and EBNA-negative African lymphoma biopsies and in a short-lived tissue culture line derived from one of them. LANA could be a fetal or tissue-specific antigen, a virally determined antigen, or a specific form of anti-nuclear reactivity.     

Expression of the microsomal antigen on the surface of continuously cultured rat thyroid cells is modulated by thyrotropin

The thyroid microsomal antigen, commonly implicated in thyroid autoimmune diseases, is not confined to cytoplasm, but is also present on the surface of human thyroid cells in primary culture. In this study, a strain of differentiated rat thyroid cells (FRTL5) grown in continuous culture was used to investigate the relationship between functional status of the thyroid cell and expression of microsomal antigen on its surface. Sera from eight patients with Hashimoto's thyroiditis, one with idiopathic hypothyroidism, and three with hyperthyroid Graves' disease were selected for the presence of microsomal antibody, but undetectable thyroglobulin antibody. One additional serum sample from a patient with Graves' disease, which was negative for both thyroid antibodies was used. Sera from eight normal subjects were used as controls. Indirect immunofluorescence was applied to FRTL5 cells that were viable or fixed with acetone in order to expose intracellular antigens. When FRTL5 cells were cultured in Coon's medium containing TSH (300 microU/ml), positive indirect immunofluorescence surface staining was observed with sera containing microsomal antibody, but not with control sera or with the Graves' serum negative for microsomal antibody. A rough correlation was found between the intensity of the surface fluorescence and microsomal antibody titer. After acetone treatment, microsomal antibody-positive sera produced typical cytoplasmic staining. When FRTL5 cells were cultured in the absence of TSH, disappearance of the surface fluorescence and reduction of the cytoplasmic microsomal antigen occurred. Readdition of TSH progressively restored both the surface and cytoplasmic microsomal antigen. The present data indicate that the thyroid microsomal antigen is represented on the surface of FRTL5 cells, and its expression is modulated by TSH.     

Islet cell surface antibodies in Type 1 (insulin-dependent) diabetes mellitus: Use of human fetal pancreas cultures as substrate

Summary Sixteen pancreases from 11–24 week old human fetuses were cultured for up to 11 days to investigate islet cell surface antibodies. Hormonal content and presence of cytoplasmic autoantigen were assessed by immunofluorescence with specific antihormone sera and high titre cytoplasmic islet cell antibody positive sera. Viable islet cells cultured on coverslips were tested with 21 islet cell antibody positive sera from Type 1 (insulin-dependent) diabetics, one islet cell antibody positive serum from a non-diabetic and four normal control sera. Surface binding immunoglobulins were detected by indirect immunofluorescence in nine out of 11 newly diagnosed Type 1 diabetics and in two out of ten longstanding diabetics with another coexistent autoimmune endocrinopathy. The four-layer double immunofluorescence technique showed that the surface antibody stained insulin secreting cells, but owing to rarity of A and D cells in the fetal cultures it has not yet been possible to exclude the reactivity of islet cell surface antibodies with glucagon or somatostatin cells.     

Characterization of anticytoplasmic antibodies in patients with systemic autoimmune diseases

We characterized the cytoplasmic staining patterns identified by indirect immunofluorescence (IF) using human epithelial (HEp-2) cells as substrates, and identified autoantigens using enzyme-linked immunosorbent assay (ELISA) and cognate RNA immunoprecipitation techniques in cytoplasmic antibody-positive sera (CA(+)) in patients with systemic autoimmune diseases. Twenty-three sera (3.7%) of 630 patients were found to have a cytoplasmic staining pattern by IF on HEp-2 cells. The fine-pattern IF specificities were as follows: 12 diffuse fine speckled; 7 coarse granular filamentous speckled; 2 diffuse coarse speckled; 1 condensed large speckled; 1 cytoskeletal. No relationship was found between the staining patterns and the diseases. Anti-SS-A antibodies and antimitochondrial (M2) antibodies were detected by ELISA in 6 and 4 sera, respectively, and antismooth muscle antibody was detected by IF in 1 serum. In RNA immunoprecipitation assays, 6, 11, 3, and 1 patients had antibodies that recognized aminoacyltransfer RNA (tRNA) synthetases (including 2 EJ, 2 PL-7, 1 PL-12, and 1 unidentified tRNA-related), SS-A, ribosomes, and SRP, respectively. Moreover, several other autoantigens were detected by Western blotting using human epithelial (HEp-2) cell lysates. This study suggests that autoantibodies against tRNA synthetases, SS-A, ribosomes, mitochondria, and other autoantigens are present in CA(+) sera from patients with a variety of systemic autoimmune diseases.     

Humoral antibodies to the capsid antigen of Epstein-Barr virus in Hodgkin's disease.

By the method of indirect immunofluorescence it has been shown in P3HR-I cells that sera from patients with Hodgkin's disease contain high titers of humoral antibodies to the capsid antigen of Epstein-Barr virus (EBV). Higher titers of antibodies of EBV were found in histological variants of Hodgkin's disease with an unfavorable course. The variant of lymphocyte depletion is accomplished by higher titers of the virus and poorer prognosis than the nodular-sclerotic variant having a course with lower titers of antibodies and better prognosis. At the same time, the level of antibodies does not depend on the results of the therapy applied. In the sera of patients with reticulosarcoma or lymphosarcoma no increase in the level of antibodies to EBV has been discovered in comparison with a group of healthy donors; in acute leukemia a certain tendency to decrease in the level of antibodies to this virus can be observed.     

A cytotoxic monoclonal antibody detecting a novel B cell membrane antigen expressed predominantly on cells bearing surface membrane immunoglobulin

A new human B lymphocyte membrane antigen, CB2, has been detected by a mouse monoclonal IgM antibody. CB2 appears to be predominantly expressed on normal and malignant cells expressing surface membrane immunoglobulin (SmIg). By indirect immunofluorescence, the number of CB2-positive cells in normal peripheral blood correlated well with the number of SmIg-positive cells. Cytotoxicity studies on isolated cell populations showed that CB2 was present on normal B cells isolated from the spleens of 52 donors and on peripheral blood B cells from 8 donors. Monocytes, T cells, granulocytes, platelets, and red cells were CB2 negative. Only malignant cells expressing SmIg were positive. These included B-CLL, B lymphoma, prolymphocytic leukemia, and B lymphoma cell lines Daudi, Raji, and Conception. SmIg-negative leukemia cells, such as common acute lymphoblastic leukemia, acute and chronic myelogenous leukemia, and T cell leukemias, were negative. Blocking studies with human immunoglobulin suggests that the CB2 antigen is not directed against immunoglobulin determinants. Immunoperoxidase studies on normal lymph node sections show that CB2-positive cells are predominantly present in the mantle region of the follicle, whereas B1- positive cells are mainly in the germinal center.     

Human pancreatic B cells in vitro: Microtubule and insulin immunofluorescence

Summary Primary monolayer cultures of human B cells established using collagenase digestion, assume a characteristic epithelial morphology which is ideal for indirect immunofluorescence studies. Using double label indirect immunofluorescence, it was possible to identify an extensive cytoplasmic microtubule complex and numerous insulin antigen positive granules within the human B cells. The microtubules are discrete rod-like structures which terminate at, or near, the plasma membrane and are observed sometimes in close association with insulin antigen positive granules. This culture system is well-suited for studying the role of the microtubules in human B cell function.     

Interactions between human osteoarthritic chondrocytes and synovial fibroblasts in co-culture

OBJECTIVE: To imitate the in vivo joint situation and to allow cell interactions, a co-culture system of human osteoarthritic chondrocytes and synovial fibroblasts from a single joint was established and characterized with or without stimulation by IL-1 beta. METHODS: Culture settings included chondrocytes in alginate alone, synovial fibroblasts in monolayer alone and a co-culture of both. Proteoglycan (PG) synthesis was measured by 35S-incorporation, PG content by a dimethylmethylene blue assay, DNA content by a fluorometric assay, and prostaglandin-E2 and IL-1 beta release by ELISA. RESULTS: In co-culture PG synthesis by chondrocytes was significantly reduced in the presence of IL-1 beta (1 ng/ml) compared to controls. PG content of chondrocyte cultures was reduced for controls and IL-1 beta treated co-cultures. Synovial fibroblasts in co-culture did not show significant change of PG synthesis or content when compared to cells in mono-cell culture. PG release into the medium was relatively high in co-cultures. IL-1 beta significantly decreased the proliferation rate of chondrocytes in co-cultures and slightly increased prostaglandin-E2 release. CONCLUSIONS: Co-culturing of osteoarthritic chondrocytes and synovial fibroblasts from a single human joint allows interactions between both entities and may offer a useful tool to study the effects of mediators or new drugs under more in vivo like conditions compared to mono-cell cultures.