Raymond N. Hiramoto

31P NMR spectroscopy of in vivo tumors

Abstract A probe, suitable for any wide-bore NMR spectrometer, was constructed for monitoring high-resolution spectra of in vivo subcutaneously implanted tumors in mice. Preliminary studies of a variety of murine tumors (MOPC 104E myeloma, Dunn osteosarcoma, colon-26, ovarian M5, and mammary adenocarcinoma as well as human colon, mammary, and lung tumors in athymic mice) indicate that the 31P NMR spectrum is a sensitive monitor of progressive metabolic changes that occur during untreated tumor growth and an early indicator of tumor response to chemotherapy, hyperthermia, and X radiation. Response to each of these therapeutic modalities is accompanied by distinctly different spectral changes.

Influence of conditioned natural immunity on tumor growth.

We studied the effect of classical (Pavlovian) conditioning of the natural killer cell response on survival of tumor-bearing mice. Mice were given repeated injections of poly I:C every three days paired with exposure to the odor of camphor for 4 hours. First, we investigated the possible therapeutic effect of repeated exposure to the odor of camphor on the growth of MOPC 104E murine myeloma. The results indicate that camphor alone had no therapeutic effect when the mice were exposed to the odor of camphor after tumor transplantation. We then investigated the effect of repeated exposure to camphor prior to tumor transplantation and subsequent repeated exposure to camphor following tumor transplantation. Again, we observed no therapeutic benefit. In a third experiment, we examined the effect of the conditioned poly I:C response on the growth of the murine myeloma. Animals in the conditioned group had an increase in median survival (day 43, as compared to days 34, 38, 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. During the course of the study conditioned mice received no additional treatment other than being reexposed to camphor every third day.

Expression of the conditioned NK cell activity is β-endorphin dependent

We are interested in identifying the pathways which are responsible for triggering the conditioned enhancement of natural killer (NK) cell activity. Earlier studies have suggested that central opioid(s) are involved in eliciting the expression of the conditioned NK cell activity. The purpose of this study was to identify the central opioid peptides that allow the central nervous system (CNS) to communicate with the immune system. Mediators that activate the efferent pathway of communication between the CNS and immune system was examined by injection of the mediator via the cisterna magna (CM). Conditioning was used as a tool to show that the bi-directional communication between the CNS and the immune system does take place. We found that beta-endorphin but not dynorphin could stimulate NK cell activity, when beta-endorphin or dynorphin was injected into the CM. In addition, when anti-beta-endorphin or anti-dynorphin antibody was injected into the conditioned animals via CM the conditioned response was blocked by anti-beta-endorphin but not by anti-dynorphin antibody. These observations suggest that beta-endorphin appears to be one of the signals that is induced in the brain at the CS recall step of the conditioned response to trigger the elevation of NK cell activity.

Naltrexone blocks the expression of the conditioned elevation of natural killer cell activity in BALBc mice

An elevation of natural killer (NK) cell activity was conditioned by the association of a camphor odor conditioning stimulus (CS) with an injection of 20 micrograms polyinosinic:poly-cytidylic acid (poly I:C), the unconditioned stimulus (US). Poly I:C elicits the production and secretion of interferon (IFN), which induces an increase in NK cell activity. Reexposure to the CS occurred on Days 3 and 5 after the association trial on Day 0. Immediately following the CS exposure on Day 5, 1 microgram poly I:C was administered to all animals. This procedure resulted in an increased NK cell activity in the conditioned (CND), but not the nonconditioned (NC), mice. In this study we have shown that the expression of the conditioned response was blocked by an injection of naltrexone (NTX) at 10 mg/kg ip when given immediately prior to the two test CS odor exposures. Peripheral treatment (ip) with a quaternary form of naltrexone (QNTX), which is a less potent opiate antagonist, at the same dose and at the same time relative to the CS odor reexposure did not block the conditioned response. The formation of the conditioned association did not appear to be disrupted by NTX at the 10 mg/kg dose when given immediately prior to the trial odor exposure on Day 0. No modulation of NK cell activity was observed in any of the control groups treated with naltrexone or the quaternary analog. Because of the inability of the QNTX to block the conditioned response, we hypothesize that the opiate receptors involved in the conditioned response and blocked by NTX were within the central nervous system (CNS). Whether this response is peripherally or centrally mediated, we have shown that opiate receptors represent part of the mechanism which mediates the conditioned augmentation of NK cell activity.

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.

The central effect of methionine-enkephalin on NK cell activity.

The central effect of opioid peptide on natural killer (NK) cell activity in BALB/c mice was investigated. Injection of methionine-enkephalin (Met-Enk), 0.02 microgram/mouse or 1 microgram/kg, directly into the cisterna magna (CM) of the brain, resulted in a significant enhancement of NK cell activity. This enhancement was blocked by opiate antagonists, naltrexone and quaternary naltrexone. The same dose of Met-Enk had no effect on NK cell activity when given to the mouse intraperitoneally or intravenously. Moreover, des-tyrosine-methionine-enkephalin injected into the CM at 1 microgram/kg, had no effect on NK cell activity. The results indicate that activation of an opioid-mediated pathway in the central nervous system is capable of activating the pathways that stimulate the NK cell response in the periphery.

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.

A Simple, Single, Trial-Learning Paradigm for Conditioned Increase in Natural Killer Cell Activity

Abstract A change in natural killer (NK) cell activity can be conditioned with one trial learning when conditioned stimulus (CS) precedes the unconditioned stimulus (US). To avoid the problems associated with two reexposures in our earlier studies, we have developed a reliable and simple conditioning protocol utilizing the one trial learning and one reexposure to the odor CS. The conditioned change in NK cell activity was significantly different (P < 0.05) from the control groups of mice. The paradigm is short and simple in that the conditioned change could be demonstrated within 3 days. We have also compared the effects of temporal association of CS and US on conditioned increase in NK cell activity. Forward conditioning (CS preceded the US) demonstrated a conditioned change, but the backward conditioning protocol did not. The paradigm provides a reliable approach to the study of mechanisms of the phenomenon of odor-NK conditioning.

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.

Cytokine and hormone profiles in mice subjected to handling combined with rectal temperature measurement stress and handling only stress

Stress is known to either up or down regulate immunity. In this study, mice were subjected to handling combined with rectal temperature measurement (RTM) stress or handling only stress. We investigated whether there were any significant differences in the effect of handling combined with RTM and handling only on NK cell activity, serum cytokine (IL-1beta, IL-6, and TNF-alpha) and ACTH and beta-endorphin levels, and splenic cytokine (IL-1beta, IL-6, TNF-alpha, IFN-alpha, and IFN-beta) levels. Circulating cytokines and hormones and splenic cytokine mRNA levels were measured in individual mice. NK cell activity was significantly increased in both stress groups when compared to the control group. Handling combined with RTM produced significantly increased serum levels of IL-1beta, IL-6, and beta-endorphin. Serum IL-1beta, ACTH, and beta-endorphin were elevated significantly in the handling only group. Splenic TNFalpha mRNA in both of the stress groups and IL-6 mRNA in handling only group decreased significantly. Our observations are supported by existing literature demonstrating that various stressors have differential effects on immune functions and the neuroendocrine hormones and cytokines, which regulate them.