Monitoring of murine osteosarcoma by serial alkaline phosphatase determinations.

To establish that alkaline phosphatase (AP) was released by osteosarcoma cells, we measured this enzyme in C3H/HeJ mice with im-implanted osteosarcoma and in in vitro cultures of neoplastic cells subjected to short-term incubation. We found that 10(5) osteosarcoma cells synthesized a significant amount of AP in vitro in 30 minutes at 37 degrees C. A good correlation existed between pulmonary metastatic tumors and the AP values. Serum AP measurements indicated approximate sizes of disseminated and localized tumors, but could not monitor early localized tumors.

Reduction of tumor burden in a murine osteosarcoma following hyperthermia combined with cyclophosphamide.

A radiation- and chemotherapy-resistant murine osteosarcoma was used to investigate the effect of local hyperthermia (42.5 +/-0.1 degrees, 30 min) alone and in combination with cyclophosphamide. The cytotoxicity of cyclophosphamide on murine osteosarcoma was established previously in our laboratory. Local hyperthermia (42.5 +/- 0.1 degree, 30 min) had little or no effect on the 16-day-old (206 X 10(6) osteosarcoma tumor cells/mouse) tumor as shown by the changes in the tumor cell marker, alkaline phosphatase. A 2.5 +/- 3.5% reduction in the number of tumor cells was seen. Large tumors treated at 21 days postimplantation (357 X 10(6) tumor cells) showed a reduction of 24 +/- 14%. The effect of combination treatment with cyclophosphamide and hyperthermia produced greater reduction in the numbers of tumor cells than did either treatment used alone.

Effect of immune response to sheep red blood cells on plasmacytoma MOPC 104 E.

BALB/c mice were given 1 x 10(6) MOPC 104E plasmacytoma cells i.v. to disseminate the neoplasm to various organs. Twenty-five days after implantation and at a time when the neoplastic B-cell clone was in the exponential growth phase, the mice were given i.p. injections of a mixture of antigens containing sheep red blood cells and levan. Each mouse was monitored simultaneously for immunoglobulin M (IgM) anti-dextran myeloma protein produced by the plasmacytoma and anti-sheep red blood cell hemolysin. The increase and decrease of these markers permit assessment of the expansion of the abnormal B-cell clone during the rise and fall of a normal B-cell clone in response to a specific antigen. The model was used to determine (a) the extent of the suppression of myeloma protein, (b) how long inhibition can be maintained, and (c) how soon it occurs after antigen is administered. The results showed that, as the IgM antibody response to sheep red blood cells begins to peak, it exerts a transient suppressive effect on either the MOPC 104E growth or on the cellular release of MOPC 104E IgM. The suppressive effect was noticeable 4 days after antigen administration for only 24 hr. These results indicated that plasmacytoma cells in vivo can recognize signals for either suppression of growth or release of the idiotypic MOPC 104E IgM and were not inconsistent with the view that myeloma may be the result of a defect in B-cell regulation.

MOPC 104E plasmacytoma functional heterogeneity and maturational potential in culture.

MOPC 104E plasmacytoma secretes an immunoglobulin M (IgM) paraprotein which reacts specifically with dextran B-1355. We separated plasmacytoma cells into subfractions by density gradient centrifugation. The majority of cells isolated from the peritoneal washes 3 days following i.p. transplantation of tumor were in active cell cycle and had a density of 1.055 or 1.065 g/ml. Cells isolated from 9-day-old ascites were confined to the heavy-density fraction (1.085 g/ml). The majority of these cells were not in proliferative phase. Functional analysis of the fractions using rosette formation and plaque formation assays indicated that cells in active cell cycle had less surface IgM and secreted less IgM than did cells not in cell cycle. An attempt was made to establish a cultured cell line of MOPC 104E plasmacytoma. The majority of the cells of the continuously cultured cell line were in active cell cycle, had less surface IgM, and secreted less IgM. Cultured cells acquired more cell surface IgM and actively secreted IgM following their secondary colonization in 0.8% methylcellulose. These studies showed that the new line of MOPC 104E retained the properties of the original ascites tumor. An important feature of these studies is that the density of cells within a colony is a stable property and is probably not related to cell cycle.

Maintenance of MOPC 104E myeloma in plateau phase.

MOPC 104E myeloma cells are brought under host regulation after treatment with cyclophosphamide, 1,3-bis(2-chloroethyl)-1-nitrosourea, or cis-dichlorodiammineplatinum(II). The first indication of this phenomenon is the plateau level of immunoglobulin M(lambda) [IgM(lambda)]. The myeloma recurs more frequently in animals with high plateau levels of IgM(lambda) even when remission is maintained for greater than 200 days. The growth rate of the recurring tumor is altered when compared with the original tumor in the same individual. Different drugs and dosages produce stable myeloma of different sizes. Treatment with cyclophosphamide (10 to 200 mg/kg), or 1,3-bis(2-chloroethyl)-1-nitrosourea (25 mg/kg) gives stable myeloma that produces low plateau levels of IgM(lambda). This myeloma does not show late recurrence. Combination of 1,3-bis(2-chloroethyl)-1-nitrosourea, cyclophosphamide, and cis-dichlorodiammineplatinum(II) in low doses or cis-dichlorodiammineplatinum(II) alone gives a stable myeloma clone(s) which produces IgM(lambda) which plateaus at higher levels, and the myeloma clone recurs relatively late in the life of the animal. These results show that treatment does not lead to the elimination of the dominant myeloma clone. Clonal dominance is, however, broken when the proliferative potential is interrupted by drug treatment. The resulting long stable phase supports the view that the proliferation, the expression of the plasma cell maturation sequences, and the secretion of IgM(lambda) are under normal host regulation. Aging presumably causes a loss of regulatory control permitting clonal expansion and recurrence of the myeloma in animals with high plateau levels of the IgM(lambda). The MOPC 104E myeloma model demonstrates for the first time a conversion of the malignant form to the indolent form as seen for humans.

Tumor proliferation and chemotherapy in immunosuppressed mice.

The influences on host immunosuppression by treatment with cyclophosphamide (200 mg/kg), steroid (prednisolone, 12.0 mg/kg for seven doses or 235 mg/kg for one dose), and adult thymectomy on tumor growth were compared. Treatment with cyclophosphamide 24 hr prior to MOPC 104E tumor transplantation produced the greatest facilitation of tumor growth. The role of prednisolone in rendering the MOPC 104E cells more vulnerable to conventional chemotherapy was also investigated. The combination of prednisolone with melphalan added measurably to the cytotoxicity of the treatment and increased the percentage of disease-free survivors. The observed effects of prednisolone might have been due to the increase in the cycling of myeloma cells directly, or the drug may have facilitated growth of the myeloma by blocking host expansion of T-cell immunity. Alterations of the host by adult thymectomy and immunosuppression with cyclophosphamide or prednisolone led to growth facilitation of myeloma. The limited studies reported here point out the usefulness of facilitation of tumor growth to accomplish increased neoplastic cell kill and increased percentage of disease-free survivors.

An experimental approach to relate a tumor-associated enzyme marker to tumor cell numbers.

Alkaline phosphatase was monitored in 17 mice with s.c.-implanted tumors to relate the total circulating alkaline phosphatase to the total number of tumor cells in each mouse. There was a semilogarithmic relationship between the alkaline phosphatase units and the number of tumor cells. A time-independent standard plot of alkaline phosphatase and the number of tumor cells was used to estimate the size of disseminated and localized tumors. In animals treated with cyclophosphamide, the alkaline phosphatase marker was used to monitor the regression and recurrence of the neoplasm in vivo.

cis-dichlorodiammineplatinum(II) chemotherapy in experimental murine myeloma MOPC 104E.

Data are presented indicating marked antineoplastic activity for cis-dichlorodiammineplatinum(II) in MOPC 104E myeloma. One-eighteenth of the dose that produced 100% cures can be combined with noncurative, low doses of cyclophosphamide and 1,3-bis(2-chloroethyl)-1-nitrosourea to produce antineoplastic activity of the same degree as that produced by much higher dose regimens which regularly produce cures. Since, in the past, results of therapeutic trials in plasma cell tumors in humans have paralleled results in this animal model, clinical trials of cis-dichlorodiammineplatinum in multiple myeloma appear warranted.

Conditioning: a new approach to immunotherapy.

It has been demonstrated by Parmiani et al. (Int. J. Cancer, 29: 323-332, 1982) that a significant protective effect can be obtained against the transplanted syngeneic YC8 lymphoma by prior immunization of BALB/c mice with normal allogeneic DBA/2 spleen cells. Using this well established tumor model, we investigated a novel approach, conditioning of specific immunotherapeutic activity. For this purpose, we used the odor of camphor as the conditioning stimulus and allogeneic DBA/2 spleen cells as unconditioning stimulus. We associated the conditioning and unconditioning stimuli two, three, and four times. Following this the conditioned animals were reexposed to the odor of camphor only. In each case, we observed a delay in tumor growth and in some instances the conditioned group performed better than the immunotherapy control group. These results indicate that a limited number of treatments with the antigen is better than the continuous treatment in maintaining the immunity and the homeostasis of the system.

In vitro effect of monoclonal anti-idiotype antibodies (anti-M104E) on MOPC 104E myeloma cells.

We investigated the effect of three monoclonal anti-idiotype antibodies (anti-M104E) on various functions of MOPC 104E myeloma cells in vitro. The antibodies used were N-20-2 [immunoglobulin M (IgM), BALB/c], SJL18-1 [IgM, BALB/c X SJL F1], and CD3-2 [immunoglobulin G1 (IgG1), BALB/c X A/J F1]. The two IgM antibodies were very efficient in blocking surface M104E IgM as shown by rosette inhibition, whereas the IgG1 isotype was not very effective. The reexpression of surface M104E IgM was different from antibody to antibody. The secretion of M104E IgM by MOPC 104E cells was partially blocked by the two IgM antibodies, but the IgG1 antibody had no effect. All three anti-idiotype antibodies inhibited the stem cell renewal activity of MOPC 104E cells assayed by colony formation assay. On the other hand, in suspension culture, the two IgM antibodies inhibited the growth of MOPC 104E cells in the absence of complement or effector cells of antibody-dependent cellular cytotoxicity, but IgG1 antibody had no effect. The starting tumor inoculum size was critical in the observations of the effects seen on both the growth and the colony-forming activity of MOPC 104E cells. The results of this study show the functional differences between various monoclonal anti-idiotype antibodies and also indicate that some anti-idiotype antibodies can inhibit the growth of MOPC 104E myeloma cells directly without any help of complement or effector cells of antibody-dependent cellular cytotoxicity.

Inhibitory effect of L-homoarginine on murine osteosarcoma cell proliferation.

An organ-specific alkaline phosphatase inhibitor, L-homoarginine, at 44.5 mM concentration inhibited [3H]thymidine uptake by C3H/He mouse osteosarcoma (OS) cells, while L-arginine, L-phenylalanine, and glycine had little effect on the uptake. This inhibitory effect of L-homoarginine persisted even after the cells were washed free of the amino acid with fresh media. L-Homoarginine did not affect [3H]thymidine uptake by mouse myeloma MOPC 104E cells. In long-term culture, 22.3 mM L-homoarginine inhibited proliferation of OS cells. L-Arginine at the same concentration inhibited the proliferation to a lesser extent. On the other hand, L-phenylalanine and glycine did not affect in vitro proliferation of OS cells. When the same number of viable OS cells was inoculated s.c. after culturing the 24 hr with 44.5 mM L-homoarginine or L-arginine, the tumor growth in mice given injections of L-homoarginine (but not L-arginine)-treated cells was delayed markedly. Electron microscopic studies indicated that the inhibiting effect on OS cell proliferation was associated with a marked increase in lysosomal granules and a decrease in virus-like structures. Similarly, biochemical assay for acid phosphatase of cell homogenates demonstrated a 2-fold increase of activity in L-homoarginine-treated cells when compared to controls and L-arginine-treated cells. Thus, L-homoarginine inhibits proliferation and alkaline phosphatase activity of mouse OS cells and appears to increase acid phosphatase activity in synthesis of lysosomal granules.

A methodological approach to the prediction of anticancer drug effect in humans.

Tumor cells from animals and humans were treated with drugs under tissue culture conditions. Tumor cells from the sensitive L1210 model were studied first. A dose-response curve was derived between drug exposure and subsequent cytotoxicity in L1210. The concentration of drug and duration of exposure were factors critical to the subsequent development of in vitro cytotoxicity. The in vitro dosage which effected 50% leukemic cell death in L1210 cells correlated with reported in vivo drug levels. Other tumor models and human neoplastic cells were studied at this dosage level. A good correlation was noted in these studies between the in vivo responsiveness and the in vitro chemotherapy results in both animals and humans. It was suggested by these results that it may be possible to predict cancericidal drug activity for individual neoplasms by assaying the tumor cells in vitro for drug sensitivity.

In vitro and in vivo growth control of transformed lymphoid cells expressing plasma membrane galactosyltransferase.

The level of beta 1-4 galactosyltransferase activity was examined in a number of spontaneously, chemically, or virally transformed murine tumor cell lines. Increased levels of enzyme activity were observed for the murine myeloma cell line K181 and in vivo MOPC 104E. The Maloney Sarcoma Virus (MSV) transformed T-cell lymphoma, YC-8, also demonstrated elevated levels of enzyme activity when compared to a second independently MSV transformed T stem-cell lymphoma, LSTRA. Cell surface immunofluorescence was also detected in YC-8 with a monoclonal antibody for galactosyltransferase. The introduction of galactosyltransferase specific substrates, both in vivo and in vitro, led to the retardation of growth in the cell lines K181, MOPC 104E, and YC-8, but not in the cell line LSTRA; this suggests the selective growth control of transformed cells demonstrating elevated levels of galactosyltransferase.

Age related changes in auto-erythrocyte rosettes in the C57BL/6J and NZB/BINJ mice.

The changes in the auto-erythrocyte rosetting thymic and splenic lymphocytes and the induction of autoimmunity was followed with age in C57BL/6J and NZB/BINJ mice. The auto-erythrocyte rosetting cells (auto-RFC) showed shifts in their pattern in both thymus and spleen in C57BL/6J and NZB/BINJ mice. Both strains had approximately the same percentage (approximately 3%) of thymic auto-RFC at 1 month of age. In C57BL/6J mice the rosette population increased to 6.6% by 2 months, declined after 3 months and subsequently increased gradually with age. In contrast, the NZB/BINJ thymic auto-rosettes peaked at 4 months and gradually declined thereafter. Both the NZB/BINJ and C57BL/6J strains were tested for the presence of anti-erythrocyte antibodies by the direct Coombs' agglutination test. The results showed that at 6 and 10 months 50% and 90% of the NZB/BINJ mice were positive for antibodies, respectively, and the thymic and splenic auto-RFC dramatically decreased in numbers. In the C57BL/6J mice during this same period, very low incidence of auto-antibodies was detected by the Coombs' test and auto-RFC increased in numbers.

Age dependent reaction of mouse thymus cells with autologous and syngeneic erythrocytes.

The age dependent events influencing the rosette forming capacity of Balb/c thymus and spleen cells against autologous or syngeneic erythrocytes were examined. A large number of autologous and syngeneic rosette forming cells (RFC) were observed in normal Balb/c mice in vitro. RFC were significantly greater in the thymus than in the spleen. The rosette forming T-cells (T-RFC) have the following characteristics: newborn Balb/c thymus has T-cells which react syngeneic erythrocytes from older donors. The T-RFC showed broad cross-reactivity with erythrocytes from other mouse strains but low reactivity with human or sheep erythrocytes. The auto and syngeneic T-RFC could be enhanced by non-specific serum proteins (FCS or BSA) or EDTA but was effectively inhibited by normal mouse serum. T-RFC resided in the cortisone sensitive population. The data indicate that the development of autologous and syngeneic rosette formation of thymus cells is dependent on the age of the erythrocyte donor. The age dependent change on the erythrocyte surface occurred relatively early in the life of the animal. The results also imply that certain subpopulations of thymus lymphocytes are capable of recognizing possible surface antigenic changes of the erythrocytes.

Effect of thymic hormone treatment on several immune functions of nude mice.

Studies of the effect of short-term, intense treatment with thymic hormone on mitogen response, cytotoxicity to EL-4 lymphoma and natural killer cell (NK) activity was investigated Balb/c nude mice (about 12-16-week-old) were treated 5 times per week for 3 weeks with: Facteur Thymic Serique (FTS) and Thymopentin (TP5, Thymopoietin 32-36) at 1 microgram and 10 ng; TM4 1 ng (an enzyme resistant variant of FTS); Thymosin Fraction V (TF5), 10 and 1 microgram; and 0.1 ml saline, and killed 2 days after the last treatment. The animals were monitored for changes in weight, hematocrit, peripheral blood lymphocyte (PBL) and spleen mitogen response. Additional groups of nude mice were immunized with 1 x 10(7) 5000 R irradiated EL-4 cells 10 days before sacrifice and tested for the presence of cytotoxic T-lymphocytes (CTL). The results show that weight and hematocrit were similar among the groups. Treatment with FTS significantly elevated the number of PBL. Spleen stimulation in mice treated with 1 microgram TP5 was depressed to mitogen concanavalin A (ConA) and lipopolysaccharide (LPS) stimulation. The phytohemagglutinin (PHA) response was not different among the treatment groups. The PBL mitogen response to ConA and LPS was generally increased over saline control in the hormone treated groups but was not statistically significant. The PHA response was only slightly elevated. No CTL was generated in nude mice in any of the groups. However, there was a statistically significant general depression of NK activity in all of the hormone treated animals compared with saline. The results indicate that the basic differentiation defect of the T-cells of nude mice cannot be restored to full functional activity by short-term treatment.

Alloreactivity. I. Effects of age and thymic hormone treatment on cell-mediated immunity in C57B1/6NNia mice.

C57B1/6NNia mice 1, 12, and 24 months old showed loss of cellular-mediated cytotoxicity with aging. Treatment of the three age groups with different thymic hormone preparations effected their cellular mediated cytotoxicity differently. When cytotoxicity of the thymic hormone treated groups was compared to that of the physiological saline treated group, 1-month-old mice treated with serum thymic factor (FTS) at 1 microgram/mouse and 10 ng/mouse had significantly higher activity, and lower to similar activities at 12 and 24 months; TP5 (active fragment of thymopoietin) at 1 microgram and 10 ng caused significantly higher activity in 1-month-old mice, and lower to higher and significantly lower to similar activity at 12 and 24 months, respectively; TM4 (an analogue of TP5) at 1 ng showed significantly depressed activity in 1-month-old mice, and significantly enhanced activity in 12- and 24-month-old mice; thymosin at 10 micrograms and 1 microgram had slightly lower, but not significant, depression at 1 month, similar activities at 12 months and significantly depressed to higher activity at 24 months. Unimmunized control mice showed significant protection in the 12-month-old mice in comparison to 1- and 24-month-old mice. Different hormone preparations showed age- and dose-dependent effects on the ability of spleen cells to kill P815 mastocytoma. Partial restoration of cytotoxicity was observed in 24-month-old mice treated with FTS, TP5 and thymosin fraction V.

The identity of the unconditioned stimulus to the central nervous system is interferon-beta.

The specific mechanism of interaction between the central nervous system and immune system was examined using conditioned augmentation of natural killer (NK) cell activity. This study focused on the role of interferon-beta (IFN-beta) as the unconditioned stimulus (US). IFN-beta was found to be the signal responsible for the bidirectional communication which links the central nervous system with the immune system. This was substantiated by injection of small quantities of IFN-beta directly into the cisterna magna, which activated the effector pathway from the central nervous system to the immune system. More importantly, we found that when the conditioned stimulus (CS) was paired with an injection of IFN-beta into the cisterna magna, the conditioned animals were able to raise their natural killer cell activity in response to subsequent exposure to the conditioned stimulus. These studies show the unconditioned response must be the response of the central nervous system (CNS) to the unconditioned stimulus and not the direct effect of the substance injected into the periphery.

Arecoline a muscarinic cholinergic agent conditions central pathways that modulate natural killer cell activity.

The central nervous system plays an active role in the regulation of the immune system. Modulation of immune activities appears to be in part under the control of the hypothalamic-pituitary-adrenal (HPA) axis. We investigated the effect of a muscarinic cholinergic agonist, arecoline, which stimulates the secretion of corticotropin-releasing hormone (CRF) and adrenocorticotropic hormone (ACTH) on the immune system. In this report we demonstrate that peripherally administered arecoline or ACTH can increase activity of pre-activated NK cells. Second, we show that central administration of arecoline at a dose too low to alter peripheral events is sufficient to induce a significant increase in the activity of pre-activated natural killer (NK) cells. Finally, we demonstrate by using a Pavlovian conditioning paradigm that the pairing of a novel odor (camphor) with administration of arecoline can be used to alter NK cell activity. Subsequent to the conditioning trial, exposure to the odor alone is sufficient to raise NK cell activity. From these observations, we infer that the pathway(s) that are conditioned reside in sites located within the CNS and the conditioned response is evoked in the peripheral compartment (NK cell activity).

Role of arcuate nucleus of the hypothalamus in the acquisition of association memory between the CS and US.

A single trial association protocol was used to demonstrate a conditioned increase in natural killer (NK) cell activity. The signals used were odor of camphor as the conditioned stimulus (CS) and polyinosinic-polycytidylic acid (poly I:C) as the unconditioned stimulus (US). This model has been used to dissect the underlying mechanisms of interaction between the central nervous system (CNS) and the immune system (IS) and vice versa. Here, we demonstrate the potential role played by the arcuate nucleus of the hypothalamus in the acquisition of association memory between the CS and the US. Chemical destruction of the arcuate nucleus with monosodium glutamate (MSG) was used for this purpose. Mice with arcuate nucleus lesion prior to the association protocol did not demonstrate a conditioned increase in NK cell activity. However, the lesion has no effect if produced prior to exposure to the CS at recall. These studies demonstrate the significant role played by the hypothalamus (arcuate nucleus) in a conditioned response.