The NIM revolution.

This paper is dedicated, with great admiration and affection, to a giant of modern science, Ion Baciu. More than a half-century ago, he began to experimentally explore the influence of the central nervous system on the peripheral vascular system including the mysteriously complex actions of phagocytosis and erythropoiesis. To this day, he has never stopped! Other great scientists, in many countries, examining neuro-endocrine-immune interactions (NIM), came and went: Ion Baciu is still at it, all the while writing high-quality textbooks, directing a great research institute, training several generations of young scientists and physicians, publishing many first-rate research reports (in several languages) in diverse scientific journals, working often under very difficult circumstances with inadequate financial resources, and never uttering one word of complaint. This is indeed a most exceptional scientist and an exemplary human being who has set high standards that few will ever equal.

Neuroimmunomodulation: a brief review. Can conditioning of natural killer cell activity reverse cancer and/or aging?

Modern scientific evidence of interactions among the nervous, endocrine, and immune systems dates from the late nineteenth century, although this concept was certainly known to the ancients in Asia, Europe, Africa, and the Americans. Within the past two decades, the mechanisms of these interactions, known today as neuroimmunomodulation (NIM), have been investigated, from the subcellular to the behavioral levels, using the modern tools of receptor and membrane physiology, biochemistry, pharmacology, immunology, chronobiology, and genetics. One of the best demonstrations of neural-immune interactions was classical (Pavlovian) conditioning of immune responses, first reported by Metal'nikov et al. at the Pasteur Institute in the 1920's. Within the past 10 years, my colleagues at the University of Alabama in Birmingham and the Gerontology Research Center in Italy, and I, in experiments with mice, demonstrated (a) a 3- to 39-fold enhancement of natural killer (NK) cell activity by conditioning; (b) reversal of an otherwise fatal cancer (myelo-MOPC-104 E) by perinoculation conditioning ; and (c) that very old animals can be similarly conditioned to increase, many fold, their NK activity in the absence of an antigen. NK cells are among the body's first-line defenses against cancer and viral infections. Research on NIM and its mechanisms is growing exponentially; indeed, it may be the fastest growing field in biomedical sciences. (The International Society for NIM, founded only a few years ago, now has active members in 40 countries.) This revolution in the basic sciences will undoubtedly lead to a corresponding revolution in clinical practice and, most importantly, in the area of preventive medicine.

Age-related regulation of mouse brain cortex adrenoceptors following camphor vapor exposition.

The paper deals with the ability of adrenergic receptors (AR) of mouse brain cortex to be differentially regulated in response to single or multiple expositions to camphor vapor. The regulation of alpha- and beta-adrenoceptors has been studied in young and old Balb/c-nu mice. Results confirm the decrease of total beta-adrenoceptor density previously observed in untreated mice with advancing age; in addition, receptor density decreases in both young and old mice after a single exposition to camphor vapor, followed by an adaptation after multiple stimuli. The beta1, subtype is mainly responsible for density decrease in young animals, while both beta1, and beta2 subtypes contribute to the decrease in old mice. On the contrary, beta2 subpopulation gives the major contribution to the adaptive recovery in both young and old mice. alpha-Adrenoceptors also show an age-related decrease in the control group; after a single exposition they show an increased density with the exception of alpha1-subset in the young group. Repeated expositions lead to a rather general adaptive response towards pre-stimuli conditions. The differential behaviour of receptor subtypes in response to camphor vapor exposition can be related to the differential alterations of receptor characteristics observed during aging and also suggests a possible mechanism through which these alterations may occur.

Immune enhancement by conditioning of senescent mice. Comparison of old and young mice in learning ability and in ability to increase natural killer cell activity and other host defense reactions in response to a conditioned stimulus.

It has been clearly demonstrated that immune responses may be conditioned in a manner similar to that of the classical Pavlovian experiments. Evidence of impaired immune function in aging has raised the question of whether psychological conditioning of an immune response can also be effective in old age. The knowledge that aged mice have decreased spleen cell natural killer (NK) activity and that NK cytotoxicity, at least in young mice, can be psychologically conditioned led us to explore in old mice the possibility of conditioning the response of NK cell activity using the odor of camphor as the conditioned stimulus (CS) and the injection of Poly I:C as the unconditioned stimulus (US). Young and old male mice were divided into five and six groups, respectively. They received the CS and/or the US in association (conditioning) trials (sessions 1-9). Mice were exposed to the camphor odor alone at 72 hours after the final association trial to observe the conditioning phenomenon (session 10). The group conditioned with Poly I:C and camphor and receiving the CS at session 10 showed statistically significant increases in spleen cell NK activity over those of the control groups that did not receive the CS treatment at session 10 (2.6- and 4.0-fold increase in young and old, respectively). Treatment with camphor odor alone had no effect on boosting NK cell activity. These findings demonstrate the possibility of conditioning immune responses in old age, offering a valuable tool for attenuating age-related immune deterioration in various species, including the human. In addition, these results again confirm highly significant immune enhancement by classical conditioning and extend previous findings from female mice to males as well.

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.

Conditioned enhancement of natural killer cell activity, but not interferon, with camphor or saccharin-LiCl conditioned stimulus.

Pavlovian conditioning of the natural killer (NK) cell response has been demonstrated by pairing camphor with polyinosinic:polycytidylic acid (poly I:C) in nine association trials. The NK cell response could be conditioned also by using combined saccharin and lithium chloride (LiCl) as the conditioned stimulus. The camphor and saccharin-LiCl paradigms were tested to determine if the conditioned NK cell activity was the result of conditioning of the interferon response. Interferon levels were measured at 6 hr and NK cell activity at 24 hr after application of the conditioned stimulus. The interferon levels measured in separate experiments were not uniformly elevated in conditioned animals compared with controls.

Old and new strategies in the conditioning of immune responses.

Classical (Pavlovian) conditioning of immune responses was demonstrated by Metal'nikov and his colleagues at the Pasteur Institute in Paris during the 1920s. These experiments, although controversial, were repeated and extended, largely in the Soviet Union, by Dolin, Krylov, Flerov, Luk'yanenko and many others during the 1950s and '60s. Both immunosuppression and immunoenhancement were reported, with many antigens and in several species including man. After a long hiatus, new interest in this subject was revived in the United States, starting with the work of Ader and Cohen on one-trial association learning leading to immunosuppression, and extending again to a new wave of reports from the United States, Canada, Germany and other countries on both conditioned suppression and conditioned enhancement of several host-defense systems, including natural killer cell activity. It already has been demonstrated that conditioning in mice can slow down the growth of tumors and, in some instances, even completely reverse tumor growth. This work is briefly discussed, with emphasis on some of the more recent findings. Applications to human subjects are suggested. The doors are just being opened: the possibilities for new research, for new discoveries, and for new clinical applications are endless.

Neural and environmental influences on neoplasia and conditioning of NK activity.

The effects of physical restraint and poly I:C treatment on the growth of MOPC 104E myeloma and murine osteosarcoma and survival of animals bearing such tumors were investigated. Our studies show that in the Balb/c mice with MOPC 104E myeloma the effects of restraint stress were detrimental and lead to early death of the mice. When the restraint was combined with poly I:C, during the early course of the disease, restraint stress neutralized the beneficial effects of the poly I:C treatment. These studies show that under certain circumstances, restraint stress can negate the effects of therapy. On the other hand, restraint stress produced an opposite effect in C3H/He mice with murine osteosarcoma tumor treated in the same fashion. In mice with osteosarcoma, restraint stress delayed tumor growth and increased the median survival time. When restraint stress was combined with poly I:C treatment, the mean tumor size was smaller and median survival was substantially increased over the control group. Because poly I:C therapy delayed tumor growth and increased survival in both models, efforts to strengthen this response were tested by conditioning. Our studies show the response to poly I:C as measured by elevation of the NK activity could be conditioned with camphor smell. Conditioning of the host to raise immune activity to alter the outcome of disease is a new area yet to be explored and has important clinical significance. These studies, we believe, are important because they explore the connection between mind and body and resistance to disease.

Restraint alters the effects of morphine and heroin on core temperature in the rat.

The importance of restraint in determining the magnitude of alteration in the rat's core temperature (Tc) after the administration of morphine sulphate (M) and heroin hydrochloride (H) was investigated. M, in doses of 5, 15 and 30 mg/kg, or H, in doses of 0.1, 1 and 5 mg/kg was administered IP to either the restrained or free-moving rats as Tc was measured. After the administration of 5 mg/kg of H or 30 mg/kg of M to the restrained rat, a marked hypothermia was observed which reached a maximum mean depth of 3.1 and 4.5 degrees C below the baseline Tc, respectively. Conversely, a mean increase in Tc of 1.5 and 1.9 degrees C occurred following the administration of these same doses of M and H in the unrestrained animal. Furthermore, the hypothermic effect of the highest dose of M was not observed when the third of 3 consecutive injections of M, administered at 48-hr intervals, was administered to the restrained rat. On the other hand, when M was repeatedly administered to the free-moving rat, the hyperthermic response was consistently observed. Pretreatment with naloxone hydrochloride (5 mg/kg IP) effectively blocked the opiate-induced hypothermia in the restrained animal, but a total dose of 10 mg/kg was necessary to completely block the hyperthermic response in the free-moving rat. Although the factor of restraint itself did not alter the rat's Tc, it did dramatically alter the action of M and H on the body temperature of the rat.

Effects of heroin on lever pressing for intracranial self-stimulation, food and water in the rat.

Male rats maintained with continuous access to levers for intracranial self-stimulation (ICSS), water, and food were subjected to five consecutive daily injections of heroin (5 mg/kg). Rates of lever pressing for ICSS were increased in 8 rats, 2--6 hrs after administration of heroin. Rates of lever pressing for water and food were not significantly changes during this period. Naloxone (5 mg/kg) pretreatment attenuated by 82% the facilitative effect of heroin on ICSS. A second group of 8 rats maintained at reduced ICSS rates failed to show an increase in lever pressing for ICSS with heroin. The facilitative effect of heroin described in this study is consistent with previously reported studies describing the effects of morphine on ICSS.