Conditions for forming ascitic tumors in hybridoma cultivation in vivo

The conditions of the formation of ascitic cells in BALB/c mice injected with hybridoma cells were studied. All the hybridomas under study, producing monoclonal antibodies to viral antigens, induced the formation of ascitic tumors when introduced into the abdominal cavity of BALB/c mice pretreated with sensitizing agents. In the mice pretreated with pristane hybridoma cells took at a rate of 43-80% and in the mice pretreated with Freund's complete adjuvant, 31-70%. Angara oil and perfume oil, as well as Bayol F, were less effective. The time of the formation of ascites was inversely proportional to the dose of the injected cells, while the volume of ascitic fluid depended rather on the type of hybridoma and not on the dose of the injected cells. The study showed that the use of physiological saline or culture medium without serum for washing the abdominal cavity of mice after withdrawing ascites permitted the additional collection of 2.6-13.7 million hybrid cells, as well as a considerable amount of immunoglobulins.     

Pristane induced effects on chromatin of rat lymphoid cells

The effects of pristane on the conformation of chromatin in cells isolated from the lymphoid tissues of pristane-treated Copenhagen rats were examined by flow cytometry, thermal denaturation, sensitivity to enzymatic digestion, and histone protein analyses. Decreases were observed in the fluorescent intensities of propidium iodide (PI) stained nuclei isolated from lymphoid cells of pristane-treated rats when compared with normal rat lymphoid nuclei. Studies to address the possible basis for the pristane-induced changes in the DNA staining characteristics of lymphocytes demonstrated that 1) there were no decreases in the amount of DNA present in the nuclei, 2) nuclei isolated from pristane treated rats were less sensitive to thermal denaturation, as well as DNase I enzymatic digestion, and 3) there were apparent increases in the expression of the H1 histone proteins. Collectively, these results suggest that pristane elicits a conformational change in the chromatin which may be mediated by altered expression of nuclear-associated histone proteins.     

Monoclonal antibody production in murine ascites. II. Production characteristics

OBJECTIVE: To characterize monoclonal antibody production parameters of five hybridoma cell lines in murine ascites for correlation with clinicopathologic changes in mice. METHODS: Five hybridoma cell lines were grown in groups of 20 mice. Fourteen days prior to inoculation with 10(6) hybridoma cells, mice were primed with 0.5 ml of pristane given intraperitoneally. Ascites fluid was collected a maximum of three times by abdominal paracentesis; volume was measured and antibody concentration was determined by ELISA for each sample. RESULTS: Trends differed among cell lines when comparing ascites volumes and antibody concentrations over time from the first to the third tap. Antibody production was greatest at tap 1 for Groups 2B11 and 2C6D9; tap 2 for Group 3C9; and tap 3 for Groups RMK and 3D6. Total antibody production ranged from 422.90 to 996.64 mg; total ascites fluid volume ranged from 74.2 to 115.7 ml; and mean antibody concentration for taps 1, 2, and 3 ranged from 2.50 to 15.03 mg/ml among cell lines. CONCLUSION: Production characteristics were significantly different among hybridoma cell lines. Determination of production characteristics of hybridomas and correlation with clinicopathologic changes in mice may be valuable in making recommendations for managing mice with ascites.     

IL-2 plasmid therapy of murine ovarian carcinoma inhibits the growth of tumor ascites and alters its cytokine profile

We have evaluated whether i.p. murine ovarian tumors could be treated with an IL-2 plasmid DNA complexed with the cationic lipid, (+/-)-N-(2-hydroxyethyl)-N,N-dimethyl-2, 3-bis(tetradecyloxy)-1-propanaminium bromide/dioleoylphosphatidylethanolamine (DMRIE/DOPE). Reporter gene studies were initially conducted in which mice bearing i.p. murine ovarian teratocarcinoma (MOT) were injected i.p. with reporter gene plasmid DNA (pDNA):DMRIE/DOPE. Histochemical analyses revealed that transfection occurred primarily in the tumor cells of the ascites, with only a minority of other ascitic cells or surrounding tissues transfected. IL-2 levels in the MOT ascites were determined after i. p. injection of either IL-2 pDNA:DMRIE/DOPE or recombinant IL-2 protein. IL-2 was detected in tumor ascites for up to 10 days after a single i.p. injection of IL-2 pDNA:DMRIE/DOPE, but was undetectable 24 h after a single i.p. injection of IL-2 protein. In an antitumor efficacy study, MOT tumor-bearing mice injected i.p. with IL-2 pDNA:DMRIE/DOPE on days 5, 8, and 11 after tumor cell implant had a significant inhibition of tumor ascites (p = 0.001) as well as a significant increase in survival (p = 0.008). A cytokine profile of the MOT tumor ascites revealed that mice treated with IL-2 pDNA:DMRIE/DOPE had an IL-2-specific increase in the levels of IFN-gamma and GM-CSF. Taken together, these findings indicate that i. p. treatment of ovarian tumors with IL-2 pDNA:DMRIE/DOPE can lead to an increase in local IL-2 levels, a change in the cytokine profile of the tumor ascites, and a significant antitumor effect.     

Characterization of mouse-human hybridoma as a useful fusion partner for the establishment of mouse-human-human hybridoma secreting anti-tetanus toxoid human monoclonal antibody of IgM or IgG class

We succeeded in establishing a mouse-human (M-H) heterohybridoma clone which provides parental cells useful for human monoclonal antibody (hMoAb) production. Electron micrographs show that the M-H hybridoma cells retain characteristics of murine origin with regard to chromatin patterns, small granules, granular endoplasmic reticulum and Golgi apparatus. The human DNA incorporated into the M-H hybridoma is estimated to be about 1% of the total human chromosomal DNA. Mouse-human-human (M-H-H) hybridomas obtained by hybridization of the M-H hybridoma cells with Epstein-Barr virus(EBV)-transfected human B cells secrete immunoglobulin (Ig) in amounts comparable to those of murine hybridomas. Also the M-H-H hybridomas grow in nude mice and are capable of producing ascites containing large quantities of Ig. The Ig class switching takes place in the M-H-H hybridomas at a much higher frequency than in the original EBV transformant and the M-H hybridoma. Cells secreting specific monoclonal antibody of different Ig classes could be separated and concentrated by the use of fluorescence activated cell sorter (FACS).     

Cytofluorometric and cytochemical comparisons of normal and abnormal human cells from the female genital tract.

Acridine orange staining of exfoliated cells from epithelial tissues facilitates discrimination between normal and abnormal cells: abnormal cells develop highly elevated nuclear fluorescence. Comparisons of acridine orange (AO) staining with propidium iodide (PI) or Feulgen staining have shown that: (a) PI staining also provides highly elevated nuclear fluorescence from abnormal cells; (b) the distributions of nuclear fluorescence following AO or PI staining were usually not significantly different as judged by the Kolmogorov-Smirnov test; (c) fluorescence emission spectra from AO and PI stained cells are consistent with the hypothesis that both fluorochromes bind to DNA within cell nuclei; (d) DNAse treatment of AO stained normal cells eliminates the nuclear fluorescence peak from slit-scan contours; RNAse treatment has no effect on nuclear fluorescence; (e) the distribution of abnormal cell nuclear fluorescence after AO staining is usually, but not always, significantly different from the distribution of abnormal cell nuclear absorbance after Feulgen staining, with relative nuclear fluorescence being greater than relative nuclear absorbance. The hypothesis currently most consistent with these results is that elevated Feulgen DNA content can account for only part of the discrimination provided by AO staining, and that the chromatin within abnormal cells is altered so as to increase accessibility of DNA to intercalating dyes.     

Differential expression of fibronectin in a rat histiocytoma: possible role of fibronectin in tumor cell adhesion

The specific role of fibronectin in tumor cells has been investigated using the transplantable histiocytic tumor line AK-5 [A. Khar (1986) J. Natl. Cancer Inst. 76, 871]. These cells, capable of growth as both ascites and solid tumors in rats, can be separated into four subpopulations by buoyant density centrifugation on Percoll. These subpopulations are shown to contain different amounts of fibronectin on the cell surface when studied by immunofluorescent staining followed by cytofluorometric analysis. Cells which contain high amounts of fibronectin can grow both as ascites and as solid tumors while those cells which contain low amounts of fibronectin cannot grow as solid tumors but can grow as ascitic tumors. Pretreatment of high-fibronectin-containing cells with anti-fibronectin antibody abolished their capacity to grow as solid tumors; however, the cells retained their capacity to grow as ascitic tumors. These results have been discussed with reference to the specific role of fibronectin in tumor-forming cells.     

Moloney murine leukemia virus-induced myeloid tumors in adult BALB/c mice: requirement of c-myb activation but lack of v-abl involvement.

BALB/c mice treated with pristane and Abelson virus have been used as an animal model system for the rapid induction of plasmacytomas. Myelomonocytic tumors with helper Moloney murine leukemia virus clonally inserted into the c-myb locus were observed in about 10% of pristane-primed BALB/c mice infected with Abelson virus. However, v-abl was absent in almost all of those tumors. Since Moloney virus is thought to induce mostly T-cell lymphomas, we have carried out studies to investigate this alteration of disease specificity and to determine whether v-abl played an obligatory role in the development of these tumors. We found that, whereas lymphomas developed late (greater than 3 months) in both pristane-primed and unprinted control mice, the myelomonocytic tumors arose at a high frequency, within 3 months, but only in pristane-treated mice. Clonal Moloney virus insertion was again found in each of the seven myelomonocytic tumors examined. Northern blot analyses and S1 mapping studies revealed the presence of virally promoted chimeric mRNAs that lack the three 5'-most myb coding exons. Hence it appears that the requirement for the v-abl gene product in tumor induction is not obligatory. Our results also indicate that tumor-specific alteration at the 5' end of the myb gene plays an important role in the development of these tumors.     

Isolation and characterization of a monoclonal nonspecific suppressor factor (MNSF) produced by a T cell hybridoma

By fusing Con A-activated BALB/c mice spleen cells with AKR thymoma BW5147 cells, we prepared a hybridoma producing a monoclonal nonspecific suppressor factor (MNSF). This factor inhibits a generation of LPS-induced immunoglobulin-secreting cells. We used ELISA for the bioassay of MNSF activity. With this method, a stable E17 hybridoma clone was selected, and its product in culture medium was isolated and characterized. MNSF fractionated on Sephadex G-100 in saline buffer shows a form with multiple m.w., but fractionated in 0.4 M pyridine-acetic buffer, it is limited to two species of approximately 24Kd and 16Kd. The MNSF was purified by hydroxyapatite chromatography, with marked effectiveness. MNSF activity was found exclusively in the 0.35 M sodium phosphate elution, and the content was further fractionated on subsequent gel filtration in the high ionic strength buffer described above. The purified factor exhibited two forms, of 24Kd and 16Kd, and showed peaks of pI 5.3 and 5.7, respectively, on isoelectric focusing. The MNSF preparation described here is stable at 56 degrees C and unaffected by 2-mercaptoethanol, but is unstable at pH 2.0 and is sensitive to tryptic proteolysis. We injected the hybridoma cells into the peritoneal cavity of pristane-primed F1 (AKR/J X BALB/c) mice, and a large amount of pure MNSF was obtained from the ascites, the characteristics of which were similar to those in the culture supernatant. Thus, the MNSF obtained from the E17 hybridoma consists of functionally identical but physicochemically different discrete proteins. This simple method of purification can serve as a probe for further characterization of MNSF and its application in in vivo experiments.     

Decrease of DNA per cell during development of the lens in chickens.

Developmental changes in the amount and conformation of DNA in chicken lens were studied. For this, DNA in situ in lens fiber cell nuclei of chickens was examined by microfluorometry with Hoechst 33258 (Hoe) fluorochrome. On 1 M NaCl-aided Hoe staining, by which the amount of DNA can be determined accurately, the fluorescence intensity of lens fiber cells was found to decrease with no change in that of the lens epithelial cells during development. On the contrary, on normal NaCl-free Hoe staining the fluorescence intensity of the lens cells was found to increase gradually during development. These results suggest that during development the amount of DNA in lens fiber cells decreases in association with some change in its conformation.     

Comparison of the action of pristan and Freund's incomplete adjuvant on the development of ascites in mice--the recipients of hybridoma cells

Production of immunodiagnostic preparations based on monoclonal antibodies requires large amounts of such antibodies. Most frequently preparative quantities of monoclonal antibodies are provided by ascitic fluid from hybridoma-carrying mice. Prior to intraperitoneal administration of hybridoma cells mice are subjected to stimulation with pristan, a mineral oil component. The authors showed that the Freund's incomplete adjuvant (FIA) may be as well used for this purpose. The effect of pristan and the FIA on ascites development in mice with hybridomas producing monoclonal antibodies to the Ia-like antigens of man was studied. The stimulants were administered in amounts of 0.5 ml per a mouse. It was shown that irrespective of the stimulant injection time and the number of the administered hybridoma cells the amount of the ascitic fluid formed in female mice BALB/c stimulated with the FIA was 1.5-3 times higher as that in the animals stimulated with Pristane, the antibody titer in the ascitic fluid being unchanged.     

Effect of 5-azacytidine on DNA methylation in Ehrlich's ascites tumor cells

5-Azacytidine inhibited in vivo DNA methylation in Ehrlich's ascites tumor cells depending upon the dose at which 5-azacytidine did not inhibit DNA synthesis significantly. This drug did not inhibit DNA methylation in vitro. The DNA methylase activity in ascitic cells decreased with the increasing dose of 5-azacytidine. Hypomethylated DNA was obtained from the 5-azacytidine treated ascitic cells.     

Thymus-dependent in vivo suppression of IgE synthesis in a murine IgE-secreting hybridoma

In previous studies we demonstrated that BALB/c mice bearing ascitic tumors of the IgE-secreting hybridoma B53 (epsilon, kappa, anti-dinitrophenyl) developed large numbers of Lyt-1-2+ Fc epsilon R(+) T lymphocytes (T cells with membrane Fc receptors) in response to the elevated serum IgE concentration. The development of Fc epsilon R(+) T lymphocytes was followed by a progressive decrease in the levels of serum IgE in spite of continued proliferation of the hybridoma cells. This sequence of events suggested that the IgE-secreting hybridoma triggered a suppressive immunoregulatory circuit of the host that inhibited IgE expression by the hybridoma cells. The present studies were undertaken to investigate the basis for the subsequent decline in serum IgE levels in mice with B53 tumors and to identify host factors that might be involved in this process. We observed that ascitic B53 cells recovered at increasing time points from BALB/c mice exhibited a selective decline in steady state levels and rates of synthesis of epsilon-heavy chain protein and mRNA. The expression of kappa-light chain protein and mRNA appeared relatively unchanged. The decrease in epsilon-heavy chain gene expression did not occur when B53 tumors were passaged in nu+/nu+ mice or in BALB/c mice depleted of Lyt-2+ cells (suppressor/cytotoxic cell lineage), but did occur in nu+/nu+ mice reconstituted with neonatal BALB/c thymus and in BALB/c mice depleted of L3T4+ cells (helper/inducer cell lineage). That Fc epsilon R(+) T lymphocytes were directly involved in the inhibition of IgE expression was supported by the earlier and more pronounced inhibition of B53 IgE in mice infused with Fc epsilon R(+) T lymphocytes. We conclude from these findings that: 1) the decline in serum IgE levels that occurs toward the end of each generation of in vivo passage of the B53 hybridoma is due to decreased production of IgE by the hybridoma cells, 2) the decreased production of IgE is due to a selective loss of epsilon mRNA expression, 3) the decrease production of IgE by B53 cells is dependent on the presence of Lyt-2+ cells, and 4) Fc epsilon R(+) T lymphocytes participate in the mechanism by which IgE production is suppressed.     

Decrease of DNA per cell during development of the lens in chickens

Summary Developmental changes in the amount and conformation of DNA in chicken lens were studied. For this, DNA in situ in lens fiber cell nuclei of chickens was examined by microfluorometry with Hoechst 33258 (Hoe) fluorochrome. On 1 M NaCl-aided Hoe staining, by which the amount of DNA can be determined accurately, the fluorescence intensity of lens fiber cells was found to decrease with no change in that of the lens epithelial cells during development. On the contrary, on normal NaCl-free Hoe staining the fluorescence intensity of the lens cells was found to increase gradually during development. These results suggest that during development the amount of DNA in lens fiber cells decreases in association with some change in its conformation.     

Observations on the replication of ectromelia virus in mouse-derived cell lines: implications for epidemiology of mousepox

Ectromelia virus was shown to replicate in vitro in all lymphoma cell lines and in a small proportion of hybridoma lines tested. It was demonstrated that certain hybridoma cell lines, which were passed in ectromelia virus-infected mice, yielded ectromelia virus infectivity on explantation into tissue culture. This finding further substantiated the belief that ascitic fluid and hybridoma cell lines exposed to virus during mouse-passage could be important in the epidemiology of mousepox.     

Production of monoclonal antibodies to antigens of specific mouse T-suppressors

F1(MSU X WAG) rats were immunized with anti B6 BALB/c specific suppressor T cells (SSTC), purified by absorption/elution technique, with the following fusion of splenocytes to NS-I myeloma cell line. Hybrids were screened for their ability to affect SSTC, cytotoxic T lymphocytes (CTL) and producers of macrophage migration inhibition factor (MIF-producers) all triggered by in vivo priming with allogeneic cells. Two hybridoma cell lines--C1 and C4 inactivated SSTC by approximately 50%, leaving CTL and MIF-producers intact. C4 were also active in vivo, if injected as ascitic fluid from nu/nu mice, though to a lesser extent than in vitro.     

Monoclonal antibody production in murine ascites. I. Clinical and pathologic features

BACKGROUND AND PURPOSE: Murine ascites production has been associated with appreciable morbidity and mortality, thus raising animal-welfare concerns. To address these concerns, the clinicopathologic changes associated with in vivo production of monoclonal antibodies in mice were characterized, and results were compared among cell lines. METHODS: Five hybridoma cell lines were grown in groups of 20 mice. Fourteen days prior to inoculation with 10(6) hybridoma cells, mice were primed with 0.5 ml of pristane given intraperitoneally; 12 mice were sham treated (controls). Ascites fluid was collected a maximum of three times by abdominal paracentesis. Clinical observations and pre- and postabdominal tap body weights were recorded. Necropsies were performed on all mice. RESULTS: For all groups combined, overall survival to tap 1 was 98%, to tap 2 was 96%, and to tap 3 was 79%; survival among groups ranged from 90 to 100% for tap 1, 85 to 100% for tap 2, and 35 to 100% for tap 3. Disseminated intra-abdominal seeding with irregular soft tissue and/or solid tumor masses was observed at necropsy. CONCLUSIONS: Significant clinicopathologic changes were associated with monoclonal antibody production in mice, and differences between various hybridoma cell lines were apparent.     

A stable propidium iodide staining procedure for flow cytometry

A propidium iodide (PI) staining procedure is described in which 50 micrograms/ml PI in 10(-2) M Tris, pH 7.0, with 5 mM MgCl2 is used to stain murine erythroleukemia cells (MELC) grown in suspension culture as well as single cell suspensions derived from rat kidney adenocarcinoma and human prostatic carcinoma. Specificity of staining of nuclear DNA is achieved by enzymatic removal of RNA using RNAse in the staining solution. Virtually identical histograms, with the same G1 peak height and closely similar coefficients of variation (CVs), are obtained using a wide range of RNAse concentrations on replicate samples of MELC if the incubation times are sufficiently prolonged when employing the lower enzyme concentrations. For 1 mg/ml RNAse on logarithmically growing MELC, 30 min incubation at 37 degrees C is needed to obtain a maximum G1 peak height and optimal CV and there is no significant change in the histogram if the incubation is prolonged to 4 hr. For every 4-fold decrease in RNAse concentration, the incubation time at 37 degrees C must be doubled to obtain the same maximal G1 peak height and optimal CV. Unfixed cell preparations, whether derived from suspension or monolayer cultures or from solid tumors, are stable for 2 or more weeks if stored at 4 degrees C between flow cytometric analyses and histograms are usually only minimally altered if the stained cell samples are stored for 1-2 months at 4 degrees C. Sample decay is associated with bacterial contamination. If sterile preparative techniques are used initially, subsequent contamination of the stained preparations may be minimized by adding sodium azide to the stained samples at 0.1% without influencing fluorescence intensity. Glycerine may be added to 10% and the samples slowly frozen for storage without altering DNA histogram shapes. The simplicity of sample preparation and the stability of the resulting stained cell samples makes this procedure suitable for repetitive comparative sampling of tissue and cell populations over prolonged time spans.     

Passage of hybridomas in male BALB/c mice

For cultivating hybridomas in the ascitic form there are usually used female mice BALB/c and not male ones. Efficiency of production of monoclonal antibodies with cultivation of the hybridomas in male and female mice BALB/c was studied comparatively. The animals were stimulated to form ascite by administration of the incomplete Freund's adjuvant or 3 per cent peptone with petrolatum oil. Some parameters of the ascite formation were studied: viability of the hybridoma cells, ascitic fluid formation period and volume, hybridoma cell concentration and titers of monoclonal antibodies in the ascitic fluid. In regard to all the parameters studied the male animals were not inferior to the female ones and in case of one of the hybridomas even surpassed them twofold by the volume of the ascitic fluid formed. This is evident of possible using male mice for mass cultivation of hybridoma cells with a purpose of obtaining preparative amounts of monoclonal antibodies in production of immunodiagnostic agents on their basis.     

In vivo and in vitro regulation of IgE production in murine hybridomas

Normal BALB/c mice injected i.p. with the IgE-secreting hybridomas B53 (epsilon, kappa anti-DNP), SE1.3 (epsilon, kappa, anti-arsonate) or A3B1 (epsilon, kappa, anti-TNP) were monitored for serum IgE concentrations and frequencies of splenic T lymphocytes with surface membrane receptors for the Fc portion of IgE (Fc epsilon R+ T lymphocytes). Mice with B53 or SE1.3 hybridomas initially developed high concentrations of IgE and CD8+ Fc epsilon R+ T lymphocytes, followed by a progressive decline in both serum IgE and expression of cytoplasmic epsilon-chains in the hybridoma cells. Serum IgE concentrations in mice with A3B1 hybridomas progressively increased without development of Fc epsilon R+ T lymphocytes nor a subsequent decline in IgE or change in cytoplasmic epsilon-chain expression in the A3B1 cells. An in vitro system in which the IgE-secreting hybridoma cells were cocultured with spleen cells harvested from mice with established B53 tumors was used to investigate the mechanisms involved in the inhibition of IgE production by the hybridoma cells. The results of these studies indicate that: 1) the induction/upregulation of Fc epsilon R on CD8+ T lymphocytes in vivo requires factors in addition to high serum IgE concentrations; 2) in addition to CD8+ Fc epsilon R+ T lymphocytes and monocytes, another, as yet unidentified, splenic cell component appears to contribute to the process by which epsilon-chain expression in IgE-secreting hybridoma cells is suppressed, and 3) a hybridoma (A3B1) that fails to induce CD8+, Fc epsilon R+ T lymphocytes in vivo and is not inhibited in IgE expression in vivo, nonetheless is inhibited in IgE expression in vitro when cocultured with spleen cells from mice with B53 tumors.