A cytokinetic model for heterogeneous mammalian cell populations. III. Tritiated thymidine studies: Correlations among multiple kinetic parameters in human tumors

A computer model for the simulation of radioautographic studies in growing mammalian cell populations was used to study multiple tritiated thymidine (³HTdR) associated parameters in model populations whose properties are comparable to those found in human tumors. Several different DNA synthesis rate patterns are distinguishable, designated type I, intermediate, and type II. Correlations among percent labelled mitosis (PLM) curves, interphase cell labelling patterns, and continuous ³HTdR labelling studies suggest a type I pattern in human breast cancer, an intermediate pattern in human melanoma, and a type II pattern in human adult leukemia. Detailed simulation studies were carried out in human adult acute leukemia and human melanoma. It was possible to fit all available kinetic data in leukemia and melanoma, both with respect to low threshold data, and with respect to radioautographic labelling intensity, provided that simulated experimental conditions and simulated radioautographic conditions corresponded to those actually employed. Kinetic differences between leukemia and melanoma were demonstrated which are in keeping with the natural histories and clinical drug response behavior of these two malignancies.     

Is it possible to modify serotonin receptor sensitivity?

It is possible to induce sub- and super-sensitivity of β-adrenergic receptors by long-term treatment with drugs acting on catecholamine systems. In contrast, analogous treatment with drugs acting on serotoninergic systems does not modify serotonin receptor sensitivity as measured by serotonin binding. Firstly, chronic 1-5HTP, the precursor amino acid, increases serotonin turnover but does not decrease serotonin binding. Secondly, chronic clomipramine, a predominantly serotonin uptake inhibitor also has no effect on serotonin binding. Thirdly, chronic metergoline, a selective serotonin antagonist in the cortex, does not induce supersensitivity.This apparent intractability of the serotonin receptor to changes after long-term treatment indicates a different post-synaptic regulatory mechanism than that found in catecholaminergic neurones. Furthermore, modification of serotonin receptor sensitivity is probably not relevant to the mode of action of antidepressant drugs. Rather, the response of pineal melatonin stimulation found after chronic clomipramine in these experiments implicates induction of β-receptor subsensitivity.     

Cerebrospinal fluid flow and iodide131 transport in the spinal subarachnoid space

Free Na¹³¹ I and ¹³¹I-Albumin were injected in the cisterna magna of rhesus monkeys. The dynamics of descent into the spinal subarachnoid space and transport out of the cerebrospinal fluid were determined by gamma scintigraphy. ¹³¹I-Albumin moved slowly caudally, reaching the sacral CSF in three hours. Free Na¹³¹I was rapidly absorbed locally and did not descend. When its transport out of cerebrospinal fluid was inhibited by the addition of unlabeled isotonic Na I, ¹³¹I descended slowly at a rate parallel to that of tagged albumin. Injection of Na¹³¹I in hypertonic solutions caused immediate descent. Two minute periods of tumbling activity caused rapid movement of Na¹³¹I and ¹³¹I-Albumin into the lumbar spinal fluid. Na¹³¹I dynamics may serve as a model for other molecules actively transported out of cerebrospinal fluid, such as 5-hydroxy-indoleacetic acid; descent into caudal spinal fluid may depend on the degree of efflux from cerebrospinal fluid and on the animal's activity.     

Abnormal locomotion in rats after bilateral intrastriatal injection of kainic acid.

The locomotion changes, after bilateral injection of kainic acid into the striata, are characterized by increased swing time and decreased stance time. This may be analogous to the locomotion pattern in patients with Huntington's Disease.     

Assay of proteolytic enzymes by the fluorescence polarization technique.

Neuraminidase substrates of high specific activity (>300 μCi/μmol) were prepared by reduction of sialyllactose with NaB³H₄, followed by separation of the 2 → 3 and 2 → 6 isomers of [³H]sialyllactitol by paper chromatography. Hydrolysis of sialyllactitol by neuraminidase was monitored by measuring the radioactivity in the neutral reaction product, which was separated from the charged substrate by passage over a small anion exchange column. The assay was applied to the neuraminidase activity of cultured human skin fibroblasts. The K_m was found to be 1.1 mm for both substrates; the pH optimum, 4.0; the 2 → 3 isomer was hydrolyzed twice as fast as the 2 → 6. In several genetic disorders associated with neuraminidase deficiency, the activity toward both isomers was reduced almost completely (mucolipidoses I and II; Goldberg syndrome), or only partially (mucolipidosis III; adult myoclonus syndrome); however, the relative activity towards the two isomers remained approximately the same in all cases.     

Lack of B-cell participation in acute lymphocyte choriomeningitis disease of the central nervous system.

The pattern of acute lymphocytic choriomeningitis (LCM) virus-induced central nervous system (CNS) disease was studied in BALB/c mice rendered deficient in B cells and incapable of producing a humoral immune response. The CNS disease in mice treated from birth with goat anti-mouse μ-chain globulin (μ-suppressed mice) was clinically and histopathologically indistinguishable from that of controls. This result demonstrates that neither B cells nor antibody participate in the pathogenesis of acute LCM of the CNS. These observations support the view that the host cell-mediated response to viral antigens is solely responsible for acute CNS disease induced by the intracerebral inoculation of LCM virus.     

The influence upon mitogenic and cellular immunologic reactive systems in vitro by poly(I:C) and BCG murine interferons induced in vivo.

We have previously reported that lymphocytes from W/Fu rats immunized with syngeneic (C58NT)D tumor cells were cytotoxic against these cells in a 4-hr ⁵¹Cr release assay. We have investigated the feasibility of cryopreserving lymphocytes and target cells and have selected freezing conditions which provide good yields of viable cells and functional activity. Lymphocytes from different animals had a recovery of 60–80% viability which resulted in a corresponding 55–75% recovery of cytotoxic activity. Repeated testing of lymphocyte cytotoxicity from a pool of frozen spleen cells against either fresh or frozen (C58NT)D cells gave reproducible cytotoxicity. In addition, recovery of high levels of lymphocyte function was also demonstrated when cryopreserved cells were employed in long-term cytotoxic assays, i.e., ³H-proline and ¹²⁵IUdR release assays, in the lymphoproliferative response to mitogens (PHA and Con A)³ or tumor cells (MLTI) as measured by ³H-thymidine incorporation, and in the in vitro generation of secondary cytotoxicity.By employing these cryoprotective techniques it is possible to have: 1) a population of lymphoid cells with known functional activity and 2) a pool of target cells with known susceptibility to lysis and antigenic content. Furthermore, the use of frozen cells as internal standards in each test also permits the analysis of assay variation as well as the study of variation in various cell types.     

Inhibition of rat mixed lymphocyte cultures by suppressor macrophages.

Normal rat spleens contain suppressor cells which can inhibit proliferative and cytotoxic responses of lymphocytes to alloantigens in vitro. The suppressor cells are adherent, phagocytic, resistant to treatment with ATS and C, radioresistant, resistant to treatment with mitomycin C, apparently absent from the thymus, and found in very high concentrations in peritoneal exudates. These characteristics indicate that the suppressor cell is a macrophages and not a T cell. When suppressor cells were removed from spleen cell suspensions, strong in vitro proliferative and cytotoxic responses to alloantigens could consistently be observed.     

Natural cytotoxic autoantibody against thymocytes in spontaneously hypertensive rats

A strain of SHR rats, which spontaneously develops hypertension and periarteritis nodosa, had a decreasing number of rosette-forming T cells in their thymuses and a progressive decline in cellular immune functions by aging. They were found to produce natural thymocytotoxic autoantibody (NTA) detected by a complement-dependent cytotoxicity test. This autoantibody occurred from 1 month of age and throughout life; the incidence was more than 60% of SHR rats at any age. Thymocytes from all six rat strains tested showed similarly high sensitivity to NTA but none of the strains tested produced NTA except the SHR strain. Rosette-forming thymocytes of WKA rats, which were separated by Ficoll gradient, showed much higher cytotoxic sensitivity to NTA than did whole thymocytes and nonrosetting thymocytes. The cytotoxicity of NTA was weak or negative for spleen cells, lymph node cells, bone marrow cells, and blood lymphocytes of WKA rats. However, the cytotoxic activity of NTA was completely absorbed with the thymus, spleen, lymph node cells, and brain homogenates and was partially absorbed with bone marrow cells, but not with liver and kidney homogenates. NTA in SHR rats was an IgM-globulin as determined by sensitivity to 2-ME treatment and by Sephadex G-200 column chromatography. These results suggest that NTA is responsible for the selective suppression of T-cell functions in SHR rats.     

Ability of dialysates containing transfer factor to induce lymphocyte activating factor by human mononuclear cells.

Dialysates of human leukocyte lysates containing transfer factor (TF_d) stimulated human mononuclear cells (MNL) to produce lymphocyte activating factor (LAF). Both unfractionated and adherent MNL cultures were stimulated by TF_d to produce a factor which was mitogenic for murine thymocytes and had the biochemical characteristics of LAF as determined by Bio-Gel P-100, DEAE cellulose, and hydroxylapatite chromatography. Fractionation of TF_d on Sephadex G-25 showed that the specific transfer factor activity of converting in vivo skin tests was present in the major uv-absorbing peak, whereas the substance(s) that induced LAF activity was present in a number of the other fractions. Therefore, the capacity of TF_d to induce monocytes to produce LAF is not a measure of classical transfer factor activity. However, this effect of TF_d may instead participate in the nonspecific immunoenhancing effects of TF_d.     

In vitro inhibition of lymphoproliferative responses to tumor associated antigens and of lymphoma cell proliferation by rat splenic macrophages.

Spleens from W/Fu rats bearing a syngeneic progressively growing (C58NT)D tumor contain cells which can inhibit lymphoproliferative responses in a mixed lymphocyte-tumor interaction designed to demonstrate suppressor activity. Spleens from rats having rejected (C58NT)D tumors also contained suppressor cells but to a lesser degree. The growth inhibition assay, which measures inhibition of proliferation of tumor cells, was evaluated as a simple assay system to screen for suppressor cell activity. The effector cells in both assays had the same characteristics, indicating a predominant role of macrophages. Normal rat spleens were found to contain growth inhibition activity which led to the demonstration of suppressor cell activity in spleens of normal animals. Removal of suppressor cells from the spleens of immunne rats results in consistently higher lymphoproliferative responses to tumor associated antigens on the tumor cells.     

Inhibition of in vitro lymphoproliferative responses by in vivo passaged rat 13762 mammary adenocarcinoma cells. I. Characteristics of inhibition and evidence for an infectious agent.

Small numbers of X-irradiated 13762 cells added as third-party cells to mitogen response assays or mixed lymphocyte cultures caused a significant reduction in viability of the cocultivated lymphocytes, and completely inhibited the expected lymphoproliferative responses. Results showed that the factor(s) responsible for the inhibitory effect was preserved after ultrasonic disruption of the tumor cells, could be sedimented by ultracentrifugation, and was sensitive to treatment with ultraviolet light. Further, cytopathic effects could be serially propagated using cell-free supernatants obtained from sonicated 13762 tumor cells. The results suggest that the 13762 adenocarcinoma line, as carried in vivo in this laboratory, harbors an infectious particle which can affect the proliferative responses of lymphocytes in vitro.     

Studies of the T-lymphocytes resident in the spleens of thymectomized, irradiated, bone marrow-reconstituted (TXB) mice.

The T-lymphocytes resident in the spleens of thymectomized, lethally irradiated mice that had been reconstituted with syngeneic bone marrow (TXB) were characterized. Both recently reconstituted N-TXB, (approximately 3 weeks after bone marrow injection) and aged (>6 months after reconstitution) A-TXB animals were studied. The T-lymphocytes from spleens of recently reconstituted N-TXB donors did not respond to PHA but did react significantly to Concanavalin A (Con A). The lack of PHA sensitivity was not due to dilution of reactive cells by other cell types. Removal of adherent cells, likewise, did not restore N-TXB spleen cell PHA responsiveness. N-TXB splenic T-cells were cortisone resistant. N-TXB spleen cells by themselves did not cause a graft vs host response. However, N-TXB spleen cells amplified the graft vs host response of normal lymph node cells but not N-TXB lymph node cells. Addition of cyclic GMP enhanced [³H]thymidine uptake of N-TXB spleen cells caused by Con A. N-TXB spleen cells were exclusively spleen seeking. The Con A reactive cell within N-TXB spleens was demonstrated to be of donor origin. Fetal liver as well as syngeneic bone marrow contained cells capable of reconstituting the Con A response. Spleen cells from aged. (>6 months) A-TXB were found to be PHA sensitive. Competitive inhibition assays measuring θ expression in A-TXB spleen cells indicate a significant increase in the θ positive lymphocyte population occurred with time. The data indicate that considerable reconstitution of θ positive cells had occurred in A-TXB donors. The results also suggest that the T-lymphocyte population of the TXB spleen may be a unique subpopulation of T-lymphocytes that resides exclusively in spleen and bone marrow.     

Cell-mediated immunity in experimental filariasis: lymphocyte reactivity to filarial stage-specific antigens and to B- and T-cell mitogens during acute and chronic infection.

To study immunological responses in chronic filarial infections, a model utilizing inbred Lewis rats infected with Brugia pahangi was developed. Microfilaria were found in the bloodstream of over 90% of the rats by 16 weeks of infection. Using in vitro lymphocyte blastogenesis, cell-mediated immune responses of blood, splenic, and mesenteric node lymphocytes were followed during 1.5 years of infection. Lymphocyte responses to antigen prepared from infective stage filarial larvae were detectable in the early weeks of infection, whereas responses to microfilarial antigen only developed late as microfilaremia waned. Lymphocyte responses to antigen from adult filaria vacillated during the infection. With the mitogens, phytohemagglutinin, pokeweed mitogen, and bacterial lipopolysaccharide, periods of B and T-cell hyporesponsiveness were demonstrable. Between 16 and 36 weeks of infection node lymphocytes from many rats were unresponsive to all mitogens and antigens. The model of B. pahangi in inbred rats offers advantages for immunological studies of filarial infections.     

Immunological reactivity of the lung: VII. Effect of corticosteroids and cyclophosphamide on the Fc receptor function of alveolar macrophages

The effects of various in vitro and in vivo regimens of either corticosteroid or cyclophosphamide administration on guinea pig alveolar macrophages were studied. Corticosteroid- and cyclophosphamide-induced immunosuppression was assessed by the effect of drug administration on the capacity of alveolar macrophages to attach to and/or ingest antibody-coated sheep red blood cells (SRBC). In vitro hydrocortisone (up to 20 μg/ml) had no effect on either the binding or ingestion of antibody-coated SRBC. Two separate regimens of in vivo corticosteroids were given: a single dose of iv hydrocortisone (100 mg/kg), which is a short-acting soluble preparation, and sc doses of cortisone acetate (100 mg/kg for 7 days), which is a depot preparation resulting in sustained levels of plasma cortisol of the magnitude of that found for a brief period of time following iv injection of hydrocortisone. Both regimens resulted in similar degrees of peripheral blood lymphocytopenia and monocytopenia 4 and 24 hr, respectively, following injection. The regimen of hydrocortisone has previously been reported to have no effect on alveolar macrophage cytotoxic effector function in antibody-dependent cellular cytotoxicity (ADCC), whereas the cortisone acetate regimen markedly suppressed ADCC. In the present study, hydrocortisone had no effect on either the binding or ingestion of antibody-coated SRBC by alveolar macrophages. In contrast, cortisone acetate caused a marked decrease in both the binding and ingestion of antibody-coated SRBC. This suppressive effect was maximal at suboptimal concentrations of antibody on the SRBC and could be overcome by increasing the concentrations of anti-SRBC antibody. Alveolar macrophages from animals treated with daily cyclophosphamide (a regimen which suppresses ADCC) were capable of binding and ingesting antibody-coated SRBC normally. Thus, prolonged exposure to corticosteroids in vivo causes an alteration in membrane Fc receptor function of alveolar macrophages, which can explain this impaired ability to kill target cells. Since cyclophosphamide therapy did not interfere with the binding and ingestion of antibody-coated target cells, it is concluded that the impairment in killing of target cells by alveolar macrophages is not directly related to an alteration of Fc receptor function but to a defect in the actual killing process.     

Activation of the alternative complement pathway by lymphoblastoid cell lines derived from patients with Burkitt's lymphoma and infectious mononucleosis.

Cultured human lymphoblastoid cell lines derived from patients with Burkitt's lymphoma or infectious mononucleosis were shown to activate the alternative pathway of complement fixation. This reaction does not require any conventional antibody directed against the cells. Although the reaction showed an absolute dependence on the presence of factor B it was relatively independent of the presence of factor D or of properdin. To this extent activation of the alternative pathway by lymphoblastoid cells resembles that produced by “C3-nephritic factor.” Rat and mouse complement were activated in a manner similar to human complement, but guinea pig complement was inactive. Chicken complement, unlike any of the mammalian complements tested, was able to bring about lysis of the lymphoblastoid cell lines by the alternative pathway.     

Author index for volume 31

Kilham rat virus (KRV) was isolated from the lymphocyte cytopathic 13762 summary adenocarcinoma tumor line described in part I of this report, as well as three other in vivo passaged rat tumors maintained in the same animal room. It could not, however, be isolated from the noncytopathic (CS8NT)D tissue culture line. Tests done with CsCl₂-purified KRV preparations showed that the virus could replicate in rat lymphocytes and could profoundly depress lymphocyte viability and lymphoproliferative responses. Heterologous anti-KRV antiserum could reverse the inhibitory effects of the purified virus preparation and the inhibitory effects of ultrasonically disrupted KRV-infected tumor cells, but could only partially reverse the inhibitory properties of X-irradiated whole 13762 tumor cells. The results suggest that KRV could account for some, if not all, of the inhibitory properties of the 13762 tumor line.     

Secretion of proteoglycans by chondrocytes. Influence of colchicine, cytochalasin B, and beta-D-xyloside.

Chondrocytes obtained from epiphyseal cartilage of fetal guinea pigs or ear cartilage of young rabbits were cultured in monolayer. The influence of colchicine, cytochalasin B, and p-nitrophenyl-β-d-xylopyranoside on secretion of proteoglycans was investigated. Radioactive sulfate was used as a precursor. As observed previously in other systems, β-d-xylosides initiated the synthesis of free chondroitin sulfate chains, competing with the endogenous proteoglycan core protein acceptor. The molecular weights of the chondroitin sulfate chains synthesized both on the xyloside and on the core-protein acceptor in maximally stimulated cells were similar and significantly lower than in proteoglycans synthesized in the absence of xyloside. The size of the chondroitin sulfate chains synthesized on the xyloside was inversely related to the concentration of this compound. This finding suggests that the chain length is dependent on the ratio between available acceptor and chain-lengthening enzymes or precursors. Cytochalasin B, a microfilament-modifying agent, inhibited proteoglycan synthesis, without any effect on secretion. Cells treated with cytochalasin B could be stimulated with β-d-xyloside to synthesize free chondroitin sulfate chains to the same relative degree as cells with intact microfilaments. Colchicine, an antimicrotubular agent, partially inhibited synthesis and secretion of proteoglycan. However, cells treated with colchicine could be stimulated with β-d-xyloside to synthesize and secrete free chondroitin sulfate chains to about the same relative degree as cells with intact microtubules. The data suggest that microtubules may have a facilitatory rather than an obligatory role in the secretion of proteoglycans and that at least part of the effect of colchicine is located at or after the site of glycosaminoglycan synthesis.     

Macrophages as effector cells of protective immunity in murine schistosomiasis: IV. Coincident induction of macrophage activation for extracellular killing of schistosomula and tumor cells

Peritoneal macrophages from Schistosoma mansoni-infected mice are activated both for nonspecific tumor cytotoxicity and for killing of skin-stage schistosomula in vitro. In the current study, mechanisms for induction of macrophage tumoricidal and schistosomulacidal activity have been compared. Examination of macrophages activated in vivo by BCG infection or C. parvum treatment, or in vitro by exposure to lymphokine prepared from antigen-stimulated BCG-immune spleen cells, showed that these effector functions were closely linked. Indeed, fractionation of lymphokine-rich supernatant fluids by Sephadex G-100 gel filtraction showed that activities responsible for induction of schistomula killing by inflammatory macrophages and for induction of tumoricidal activity cochromatographed as a single peak in the 50,000 MW region. Thus, development of macrophage-mediated cytotoxicity against these two extracellular (tumor cell or helminth) targets was coincident in several cell populations activated in vivo or in vitro. However, activation for tumoricidal and schistosomulacidal capacity appeared to be quantitatively dissociated in macrophages from mice with chronic schistosomiasis; those cells demonstrated low, yet significant, levels of larval killing ($\text{1}\text{3}$ to $\text{1}\text{5}$ those of BCG or lymphokine-activated cells) but maximal levels of tumor cell cytotoxicity. Furthermore, cytotoxicity by peritoneal cells from S. mansoni-infected mice was not increased in vitro by exposure to lymphokine. Identification of this functional alteration in S. mansoni-activated cells may help to clarify the role of macrophages in the partial immunity against challenge infection which is demonstrated by mice with chronic primary S. mansoni infection.