Production of human-human hybridomas secreting antibody to sheep erythrocytes after in vitro immunization.

This report describes the formation of human hybridomas after in vitro immunization of tonsillar lymphocytes and fusion of the stimulated lymphocytes with a human lymphoblastoid cell line, WI-L279HF2. Lymphocytes were stimulated in vitro with sheep erythrocytes (SRBC) and polyclonal activators (PCA). By varying the duration of in vitro immunization before fusion and varying the PCA concentrations, the optimal conditions for cell fusion, hybrid cell growth, nonspecific immunoglobulin (Ig) secretion, and secretion of antibody to SRBC were determined. Lymphocytes cultured allogeneically at 5 X 10(6) cells per well in 24-well plates with SRBC and PCA (PWM, 1/10,000 dilution or LPS, 5 micrograms/ml) fused with the greatest efficiency. PWM-stimulated cultures fused on day 6 of in vitro stimulation yielded the greatest number of wells containing Ig-secreting hybridomas, whereas LPS-stimulated cultures produced more wells with hybridomas secreting Ig when fused on day 7 of stimulation. In vitro stimulation conducted in tissue culture flasks produced fewer wells with hybrid cell growth and Ig secretion. A fusion ratio of 1:1 (lymphocyte:WI-L2729HF2) yielded the most hybrid formation. Ig levels in wells with hybrid cell growth varied greatly, from less than 10 ng/ml up to 30 micrograms/ml. Ig of IgG, IgA, and IgM isotypes were produced. The addition of lymphocyte conditioned medium to in vitro immunization cultures does not improve the yield of Ig-secreting hybridomas. Hybrid cells secreting antibody to SRBC were cloned and analyzed for HLA specificities and chromosome numbers. Additionally hybridoma cells were formed after in vitro stimulation of peripheral blood lymphocytes (PBL) of normal individuals by fusion of the stimulated lymphocytes with WI-L2-729HF2. Hybridoma formation with PBL was less successful, but hybridoma cells secreting antibody to SRBC after in vitro stimulation were isolated.

Alterations in protein kinase activity following exposure of cultured human lymphocytes to modulated microwave fields.

Cultures of human tonsil lymphocytes were exposed in a Crawford cell to a 450-MHz field (peak envelope intensity 1.0 mW/cm2), sinusoidally amplitude modulated (depth 80%) at frequencies between 3 and 100 Hz for periods up to 60 min. The Crawford cell was housed in a temperature-controlled chamber (35 degrees C) and control cultures were placed in the same chamber. Activity of cAMP-dependent protein kinase relative to controls remained unaltered by fields modulated at 16 or 60 Hz with exposures of 15, 30, and 60 min. By contrast, total non-cAMP-dependent kinase activity fell to less than 50% of unexposed control levels after 15 and 30 min exposures, but, despite continuing field exposure, returned to control or preexposure levels by 45 and 60 min. A smaller reduction (20-25%) also occurred with 60-Hz modulation and was also restricted to exposure durations of 15 and 30 min. CW 450-MHz fields were without effect. Reduced enzyme activity occurred with 16-, 40-, and 60-Hz modulation frequencies, but not with 3-, 6-, 80-, or 100-Hz modulation. The specific identity of this kinase is unknown. This rapid but transient reduction in lymphocyte protein kinase activity restricted to modulation frequencies between 16 and 60 Hz and to less than 30 min exposure is consistent with "windowing" with respect to modulation frequency and exposure duration.

Induction of murine fibrosarcomas by low dose treatment with 3-methylcholanthrene followed by promotion with 12-O-tetradecanoyl-phorbol-13-acetate.

Repeated application of the tumor promoter 12-O-tetradecanoyl phorbol-13-acetate (TPA) to the skin of mice previously treated with an initiating dose of the carcinogen 7,12-dimethylbenz[a] anthracene has been shown to lead to an increased incidence of papilloma. The studies presented here describe a modified murine two-stage carcinogenesis model in which a single subcutaneous administration of the carcinogen 3-methylcholanthrene (3-MC) is followed by multiple applications of TPA administered subcutaneously or intraperitoneally. TPA was observed to act as a promoter under these conditions when given either subcutaneously or intraperitoneally. When a carcinogenic dose of 3-MC was administered (0.5 mg/mouse) followed by regular treatment with TPA (10 micrograms/mouse) the percent of tumor-bearing mice increased and the length of time until tumors developed significantly shortened. At a subcarcinogenic dose of 3-MC (0.025 mg/mouse), repeated treatment with TPA led to tumor development whereas no tumors were observed in mice not treated with TPA. All tumors were found to be fibrosarcomas. Thus, TPA is capable of acting as a systemic promoter of mesenchymally derived tumors.

Suppression of T-lymphocyte cytotoxicity following exposure to sinusoidally amplitude-modulated fields.

Significant inhibition of allogeneic cytotoxicity of the target cell MPC-11 by the murine cytotoxic T-lymphocyte line CTLL-1 was observed when the 4-h cytotoxicity assay was conducted in the presence of a 450-MHz field sinusoidally amplitude-modulated at 60 Hz. Exposure of the effector cells to the field prior to adding them to the target cells in the cytolytic assay resulted in a similar inhibition, suggesting a direct interaction of the field with the cytolytic T lymphocyte. The inhibition was preferentially expressed during the early allogeneic recognition phase. Field-exposed cytolytic cells recovered their full cytolytic capacity in 12.5 h. A differential susceptibility was observed with modulation frequencies from 0 to 100 Hz. Peak suppression occurred at 60 Hz modulation, with progressively smaller effects at 40, 16, and 3 Hz. The unmodulated carrier wave did not affect the cytotoxicity. Effects with 80- and 100-Hz modulation were smaller than at 60 Hz. These results demonstrate an inhibitory but recoverable effect by certain amplitude modulations of weak nonionizing radiation upon the cell-mediated cytolytic immune response.

Separation of functional subpopulations of murine and human lymphoid cells on colloidal silica density gradients. I. Preparation of the Ludox AM gradient material and characterization of separation capacities.

This report describes a unique modification of an isopycnic density gradient system utilizing as a separating menstrum colloidal silica (Ludox AM). The primary advantages of this preparation are: (1) It is chemically defined, allowing extremely reproducible cell separation employing different lots of material; (2) the physical parameters (pH, density, salt concentration) of the final gradient suspension can be manipulated over a wide range of values, allowing for the separation of many different biological materials; (3) it allows separation of very large numbers of lymphoid cells with greater than 95% recovery of applied cells; (4) separated cellular subpopulations can be easily washed free of silica and cellular function is retained. This paper is a report of the preparation and functional characteristics of the gradient material as it relates to the separation of very large numbers of lymphoid cell subpopulations in both mouse and man. Subpopulations of murine and human lymphocytes separated by this gradient material were assayed for IgM synthesis, T-cell mediated cytotoxicity, and lymphokine production.

A potential diagnostic method for food allergy: clinical application and immunogenicity evaluation of an elemental diet.

The use of a simple, hypoallergenic elemental diet would appear well suited for diagnosing food allergy. Vivonex was used in 21 patients (5 to 40 years old) suspected of food allergy or those who had failed to respond to the usual management of inhalant allergy. To study immunogenicity, five New Zealand rabbits were immunized with Vivonex, milk, and egg and were evaluated for the production of precipitin and passive cutaneous anaphylactic antibodies, the latter was evaluated in three Hartley guinea pigs. The clinical study was conducted over a 2- to 3-week period with evaluation of symptom and medication scores, physical examination, and hematological and biochemical measurements made before and after the Vivonex trial, which was a minimum of 1 week. No consistent, significant improvement of allergic manifestations were seen while patients received Vivonex. On the other hand, there were no serious side effects noted either clinically or by laboratory measurements, although four patients discontinued the study because of Vivonex palatibility. Vivonex was not immunogenic by either the precipitin reaction or passive cutaneous anaphylactic response. Although Vivonex did not prove helpful in these severe, refractory allergic individuals, we were encouraged by its safety and acceptance in the outpatient setting. Further studies in young allergic children who are more likely to have clear-cut food sensitivity are being planned.

Enhancement of early human E rosette formation by cholinergic stimuli.

The effects of the cholinergic stimuli carbamylcholine (carbachol) and dibutyrl cyclic guanosine monophosphate (DBCGMP) were determined on both 'early' and 'total' E rosette formation. Ficoll-Hypaque-separated lymphocytes were preincubated with either carbachol or DBCGMP over a 10(-3) M to 10(-13) M dose range. Both agents significantly enhanced 'early', but not 'total' E rosette formation. Peak enhancement above control values occurred at 10(-7) M (72%) and 10(-9) M (69%) for carbachol and 10(-5) M (70%) and 10(-7) M (70%) for DBCGMP. Kinetic studies showed a rapid onset of enhancement (2.5 min) for carbachol, whereas DBCGMP required 15 min for significant enhancement to occur. The muscurinic nature of carbachol enhancement of E rosettes was demonstrated. Atropine at 10(-7) M completely abolished the carbachol effect while showing little inhibition of the DBCGMP effect on rosette formation. These studies indicate that the cholinergic stimuli carbachl and DBCGMP significantly enhance the 'early' E rosette former in man. Human T lymphocytes appear to have functional cholinergic receptors that can be blocked by the muscurinic antagonist atropine. The role of the cyclic nucleotides and their stimulants on the immune system is incompletely understood, but it would appear that they are extremely important in the differentiation and function of the T lymphocyte. E rosette formation may be a useful model in man for studying the effects of the cyclic nucleotides on the human T lymphocyte.

Cellular immune response in vitro: I. A requirement for time-dependent T-lymphocyte cell division of cytotoxic cells in the allogeneic response.

Immunocompetent lymphoid cells cultured in vitro with allogeneic stimulator cells have been shown to produce T-lymphocyte populations which are specifically cytotoxic in vitro to the stimulatory cells whether normal or malignant. Although the culture requirements as well as the allogeneic requirements are known, the events leading to the production of T-lymphocyte cytotoxic cells is poorly understood. This study examines the role of cell division in the production of allogeneic cytotoxic T-cells in vitro. The elimination of cell division during the first 24 hr of allogeneic culture does not affect the cell-mediated cytotoxic immune response in vitro. Cell division is required, however, from 24 hr through 96 hr in culture and not necessary after 96 hr.

Lymphocyte effector molecules: an vitro production method for obtaining liter volumes of supernatants from mitogen-stimulated human lymphocytes.

An in vitro method has been developed utilizing phytohemagglutinin (PHA) activated lymphocytes obtained from human tonsils and adenoids which permit the accumulation of multi-liter quantities of cell-free supernatants containing lymphotoxin and other lymphocyte effector molecules (LEM). An enriched media is employed which contains a large molecular weight, heat stable bovine serum fraction which supports lymphoid cell activation and levels of LEM secretion equal to that of cultures maintained in medium supplemented with whole serum. Elimination of whole serum from the media greatly reduces overall protein concentrations and facilitates concentration and purification studies. Various technical aspects of these cultures have been examined, i.e.: 1) cell concentration, 2) kinetics of LT production over a ten-day period, 3) mitogen dosage, and 4) types of media. Supernatants can be harvested repeatedly from a single culture over the ten day period, thus doubling the yield of LEM collected from a single culture.

Phytohemagglutinin-stimulated immune response. Assay in colorectal carcinoma patients.

Tritiated thymidine incorporation by peripheral human lymphocytes stimulated with phytohemagglutinin (PHA) was investigated in 18 colorectal carcinoma patients (seven with localized tumor and 11 with metastatic tumors). The effect of sera obtained from these patients on lymphocytes from control patients was also studied. The mean mitotic index of PHA-stimulated versus nonstimulated lymphocytes from all tumor patients was 29.2; from patients with localized tumors, 23.0; from patients with metastatic tumors, 26.0; and from controls, 29.4. In the presence of sera from tumor patients, the mitotic index in all tumor lymphocytes was 47.1; from localized tumor patients, 53.7; from metastatic tumor patients, 43.8; and from control lymphocytes, 47.4. No substantial difference in mitotic index was detected in normal compared to tumor patient lymphocytes with or without normal or tumor serum.