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.

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.

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.

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.

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.

Augmentation of natural immunity and regulation of tumor growth by conditioning.

We have reported the effect of classical (Pavlovian) conditioning of natural immunity on survival of tumor-bearing mice. In the first study, we have observed that mice conditioned, transplanted with tumor, and re-exposed to conditioned stimulus (camphor odor) had an increase in median survival (day 43, as compared to days 34, 38, and 37 of various control groups). Two of these conditioned mice lived more than 120 days and showed early tumor growth, but were free of disease at day 97. We report the observations of a repeat study. Two groups of conditioned mice were used for these studies. One group was re-exposed to the conditioning stimulus following transplantation with tumor (CND) and the second group was not re-exposed to odor of camphor (CNDo). Statistically significant delay in growth of MOPC 104E in the CND group was observed when compared with the CNDo group. The survival data supports the observations of tumor IgM values. In an independent study, we investigated the possible mechanisms of MOPC 104E regulation in vitro. Plastic adherent spleen cells (macrophage cells) from mice primed in vivo with MOPC 104E tumor cells suppressed tumor IgM production by MOPC cells by 98% and also reduced colony formation by MOPC cells. The possible mechanism(s) of regulation of tumor growth in conditioned mice might be mediated by plastic adherent activated macrophages.

Regulation of natural immunity (NK activity) by conditioning.

A paradigm is described for the conditioning of the NK response. Mice were exposed to a classical conditioning procedure in which saccharin and lithium chloride (LiCl) served as the conditioned stimulus (CS) paired with an injection of either poly I:C or cytoxan, the unconditioned stimulus (US). Poly I:C was used as the US for enhancement and cytoxan served as the US for suppression. Subsequent exposure to the CS either enhanced or suppressed the NK activity in the conditioned animals. Our studies showed that only one association was needed for the learning to take place, training to consumption of water was not necessary, and the conditioned (enhanced) response could be recalled as soon as 7 days after pairing of the US and CS.

Survey of thymic hormone effects on physical and immunological parameters in C57BL/6NNia mice of different ages.

Immunosenescence occurs with aging, which is seen in decline in response to mitogens PHA, ConA, decline in cell-mediated immunity, increase in anemia, and increase in autoimmune antibodies to erythrocytes and DNA. These studies compared FTS, TP5, TM4, and TF5 in C57BL/6NNia mice. Mice aged 4, 26, 52, 78 and 104 wk were treated with various hormones 5x/wk for 3 wk and monitored for hormonal effects on weight; hematocrit; peripheral blood, spleen, and thymic cell numbers; spleen and peripheral blood cell mitogen responses to PHA, ConA, LPS; IgM hemolysin autoantibody; and cell-mediated cytotoxicity to P815 allogenic cells. Hormone treatments altered mitogen responses, enhanced IgM hemolysin autoantibody production, and modulated cell-mediated immune responses. The effects were not consistent for every hormone. There was a tendency for enhancement in younger mice and suppression in older animals. Treatment with FTS showed the greatest changes in either enhancing or suppressing the different parameters measured. The hormonal effects appeared to be age specific in that certain activities were altered for certain age groups but not in others. Hormone treatment did not restore any immune parameters in old mice to the level of young animals. In general, the different hormones did not consistently produce the same effects in C57BL/6NNia mice of different age groups. Even though all animals received from National Institutes on Aging (NIA) animal models program were held under strictly controlled conditions, intrinsic variations between cohorts of different ages are difficult to control. Cohorts of aging animals tested at different times might be intrinsically different. This inherent variability in the cohorts could affect the range of activity, specificity and reproducibility of hormone effects in vivo. Most importantly, it should be emphasized that cross-sectional data identifies age differences rather than age changes. There is no assurance that age changes in any individual or in all subpopulations follow this pattern. In our studies only healthy animals were used. Old, sick, or tumor-bearing animals were culled out prior to being sent to us. Therefore, the 78- and 104-wk-old mice represent selected healthy cohorts. The age changes that take place can be answered only from repeated measurements made in the same individual over time.

Conditioning the elevation of body temperature, a host defensive reflex response.

We hypothesize that a number of host defense responses such as natural killer (NK) cell activity, cytotoxic lymphocyte (CTL) activity, antibody production, and elevated body temperature (TR) might be conditionable. We have designated such specifically learned response to be a defensive reflex response. Here we describe a simple single trial association paradigm for conditioning the TR response in BALB/c mice. Animals are conditioned on day 0 by exposing them to the odor of camphor for 1 hr, followed by injection of the pyrogen poly I:C 20 microgram ip. Control groups are injected with either poly I:C or saline and not exposed to the camphor odor. Reexposure of all groups to the conditioned stimulus (CS) on day 2 or 3 cause elevation of body temperature in the conditioned group mice but not in the nonconditioned or saline control groups. Since we have conditioned the natural killer cell response with the same paradigm, these results suggest that multiple defensive responses might be conditionable simultaneously and they might have important survival value for the species.

Identification of specific pathways of communication between the CNS and NK cell system.

The specific signals and pathways utilized by the natural killer (NK) cell system and the central nervous system (CNS) that results in the conditioned response (CR) is not clearly understood. Single trial conditioning of the NK cell activity provides us with a model to probe the mechanisms of communication between two major systems (Immune and CNS) which are involved in the health and disease of the individual. The studies show that the IFN-beta molecules possess the properties attributed to the unconditioned stimulus (US). IFN-beta can penetrate the CNS and evoke the elevation of NK cell activity in the spleen. This unconditioned response (UR) can be linked to a specific conditioned stimulus (CS). Specific odors such as camphor provide a neural pathway for the CS to associate with the US. Evidence is presented that in conditioning there are two locations where memory develops. The CS/US association is made centrally and its memory is stored at a central location, but the memory for the specificity of the odor is presumably stored in the olfactory bulbs. The CS recalls the CR by triggering the olfactory neural pathway which, in turn, signals the hypothalamic-pituitary axis to release mediators that modulate the activity of NK cells in the spleen. These results imply that through conditioning one has direct input into the regulatory hypothalamus that controls the internal environment of the organism and the health and disease of the individual. Consequently, it is not inconceivable that through this approach we might be able to alter the course of a disease process.

Lipopolysaccharide and IL-1 alpha activate CNS pathways as measured by NK cell activity.

The effect of lipopolysaccharide (LPS) on murine unstimulated and prestimulated natural killer (NK) cells and its ability to serve as an unconditioned stimulus was investigated. LPS injection induced a statistically significant increase in NK cell activity when compared with saline-treated control groups. To demonstrate the existence of communication between the peripheral immune system and the central nervous system (CNS), we used a single-trial conditioning paradigm in which camphor served as the conditioned stimulus (CS) and LPS as the unconditioned stimulus (US). Once a CS/US association is made, exposure of animals to the CS alone results in the conditioned response (i.e., increase in NK cell activity). Using 50 micrograms of LPS as the US produced a low but significant increase in NK cell activity when compared to control groups. However, 10 micrograms of LPS did not show a significant increase in NK cell activity. We also observed that interleukin-1 alpha (IL-1 alpha) injected intracisternally can serve as a US to condition a central neuroendocrine pathway. Because the dose of IL-1 alpha employed was too small to raise NK cell activity in the spleen, the NK cells themselves were formally not subjected to conditioning. These observations suggest that LPS and IL-1 alpha conditions the brain and that NK cell activity can be used as an indicator system to detect neuroendocrine signals arising from the activated pathway(s).

Effect of reserpine on retention of the conditioned NK cell response.

The effect of reserpine and 6-hydroxydopamine on the learned conditioned natural killer (NK) cell response was investigated in mice. Reserpine given at 2.5 mg/kg, 24 hr prior to reexposure to camphor-conditioned stimulus on days 6 and 8 blocked the recall of conditioned NK cell response to a significant extent. In other words, the NK cell activity of conditioned mice, treated with reserpine and reexposed to the conditioned stimulus, was similar to the nonconditioned (NC) group. A conditioned increase in NK cell response was still evident in mice treated with 6-OHDA.

Indomethacin and sodium carbonate effects on conditioned fever and NK cell activity.

The augmentation of natural killer (NK) cell activity and elevation of body temperature (fever) can both be conditioned using camphor odor as the conditioned stimulus (CS) and poly I:C as the unconditioned stimulus (US). While both responses can be conditioned in parallel fashion as shown previously, our results indicate the conditioned learning of these responses may not follow along a common path. We found that injection of a 1% solution of sodium carbonate was able to consistently block the CS/US learning of the NK cell response but did not block conditioning of the fever response. In contrary fashion, mice treated with indomethacin (which inhibits prostaglandin-induced fever) dissolved in the sodium carbonate solution did not learn in consistent fashion the fever response. However, indomethacin-treated animals were able to recall the NK cell response. These results support the view that although the same mediator, IFN-beta, is responsible for the conditioned learning of the NK cell and fever responses both the learning and recall of the responses are initiated along separate pathways.

Conditioning of the secondary cytotoxic T-lymphocyte response to YC8 tumor.

Studies from our laboratory demonstrated that conditioned resistance to the syngeneic YC8 lymphoma was established by multiple conditioned stimulus (CS)/unconditioned stimulus (US) associations. The conditioned stimulus used was exposure to the odor of camphor for 1 h and the unconditioned stimulus was an injection of DBA/2 spleen cell alloantigen that shares minor histocompatibility determinants with the YC8 lymphoma. To demonstrate a cellular basis for immune resistance to the YC8 tumor, BALB/c mice primed with DBA/2 spleen cell alloantigen were conditioned using a single trial CS/US association paradigm. Conditioned animals showed a measurable conditioned elevation of the cytotoxic T-lymphocyte (CTL) response to the YC8 tumor. Control groups in which the CS and US were not given in the proper sequence were unable to mount a conditioned response. These studies show that a secondary CTL response can be upregulated by the central nervous system (CNS).

Conditioning of the allogeneic cytotoxic lymphocyte response.

Allogeneic cytotoxic T-lymphocyte (CTL) response can be obtained following immunization of BALB/c mice with C57BL/6 spleen cells. We investigated the possibility of behaviorally conditioning this response by associating the C57BL/6 spleen cell immunization [unconditioned stimulus (US)] with camphor odor [conditioned stimulus (CS)]. We reported the possible mechanisms involved in the conditioning of natural killer cell activity. Similar approaches were used to investigate the mechanisms that participate in the conditioned CTL activity. The first mechanism of investigation utilized opioid receptor blockers naltrexone and quaternary naltrexone. Naltrexone, which blocks both the central and peripheral opioid receptors, blocked the recall of the conditioned response, whereas quaternary naltrexone, which does not penetrate the blood-brain barrier, was unable to block the conditioned response, demonstrating that centrally located opioid receptors play a role in the recall of the conditioned response. The studies are of interest because they indicate that resistance or susceptibility to various diseases such as cancer, autoimmunity, and infectious diseases might be influenced by the regulatory network of the CNS.

Stenotrophomonas maltophilia infection in hematopoietic SCT recipients: high mortality due to pulmonary hemorrhage.

To clarify the clinical features and outcome of Stenotrophomonas maltophilia infection among hematopoietic SCT (HCT) recipients, we retrospectively reviewed the records of 1085 consecutive HCT recipients and identified 42 episodes in 31 HCT recipients with S. maltophilia infection. We compared these recipients with 30 non-HCT patients with S. maltophilia infection. The mortality rate in HCT recipients was significantly higher than that in non-HCT patients (relative risk 5.7, P=0.04), and we identified seven patients with pulmonary hemorrhage due to S. maltophilia, exclusively in the HCT cohort. Six of these latter seven patients died within 1 day from the onset of hemorrhage and the isolate was identified after death in most cases; one patient, who received empiric therapy for S. maltophilia and granulocyte transfusion, survived for more than 2 weeks. The patients with pulmonary hemorrhage had a more severe and longer duration of neutropenia, persistent fever despite of the use of broad-spectrum antibiotics, complication by pneumonia and higher C-reactive protein levels than those without pulmonary hemorrhage. In conclusion, S. maltophilia was associated with fulminant and fatal pulmonary hemorrhage in HCT recipients. Empiric therapy with antibiotics before the onset of pulmonary hemorrhage may be effective in HCT recipients who carry the conditions identified.

The direction of the conditioned natural killer cell response can be re-directed with indomethacin and/or handling.

We have used the pairing of camphor odor conditioned stimulus (CS) and injection of poly I:C unconditioned stimulus (US) in a short 3 day single trial conditioning paradigm. Conditioning was done by exposing mice to the CS/US combination on day 0 and reexposing the conditioned animals to the CS on day 2. This results in a conditioned augmentation of the natural killer (NK) cell response. Indomethacin treatment and/or handling stress induced by simply measuring rectal temperature was found to dramatically alter the direction of the conditioned NK cell response. Conditioning of indomethacin treated mice produced a conditioned suppression of the NK cell response mimicking a conditioned tolerance response. If handling stress was superimposed on day 2 the conditioned suppression response was replaced by a conditioned augmentation of the NK cell response. Even with one trial conditioning, drugs and handling stress can serve as additional cues to alter the direction of the conditioned response. The studies also show that the conditioning of the fever response is independent of conditioning of the NK cell response.