Vithal K. Ghanta

NMR study of in vivo RIF-1 tumors. Analysis of perchloric acid extracts and identification of 1H, 31P and 13C resonances

Perchloric acid extracts of radiation-induced fibrosarcoma (RIF-1) tumors grown in mice have been analyzed by multinuclear NMR spectroscopy and by various chromatographic methods. This analysis has permitted the unambiguous assignment of the 31P resonances observed in vivo to specific phosphorus-containing metabolites. The region of the in vivo spectra generally assigned to sugar phosphates has been found in RIF-1 tumors to contain primarily phosphorylethanolamine and phosphorylcholine rather than glycolytic intermediates. Phosphocreatine was observed in extracts of these tumor cells grown in culture as well as in the in vivo spectra, indicating that at least some of the phosphocreatine observed in vivo arises from the tumor itself and not from normal tissues. In the 31P-NMR spectra of the perchloric acid extract, resonances originating from purine and pyrimidine nucleoside di- and triphosphate were resolved. HPLC analyses of the nucleotide pool indicate that adenine derivatives were the most abundant components, but other nucleotides were present in significant amounts. The 1H and 13C resonance assignments of the majority of metabolites present in RIF-1 extracts have also been made. Of particular importance is the ability to observe lactate, the levels of which may provide a noninvasive measure of glycolysis in these cells in both the in vitro states. In addition, the aminosulfonic acid, taurine, was found in high levels in the tumor extracts.

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.

Rat immunoglobulins in serum and secretions: purification of rat IgM, IgA and IgG and their quantitation in serum, colostrum, milk and saliva.

Abstract Rat immunoglobulin A, M, G 2a and G 2b and anti-rat α, μ, and γ were purified by ion exchange and immunoadsorbent chromatography. These purified immunoglobulins and antisera were employed in the quantitation of the principal immunoglobulins in serum and saliva of young rats from birth until 45 days of age and in milk, saliva and serum of adult rats. The level of serum IgG 2a in young rats rapidly increased prior to weaning (days 15–19) and approached the adult level (6·32 mg/ml). This increase in pup serum IgG 2a was apparently due to passive transfer since, (a) an increase in IgG 2a was observed in rat colostrum and milk between day 0 (0·67 mg/ml) and day 19 (1·53 mg/ml) and (b) upon weaning the level dropped and gradually increased to a level of 4·05 mg/ml by day 45. Serum IgM was first detected in 10 days old rats and approached the adult level (·82 mg/ml) this reached the adult level (1·19 mg/ml) by day 30. Adult levels of salivary IgG 2a (0·06 mg/ml protein) was observed in 19 day old rats. However, salivary IGA was not detectable until day 25 and this Ig approached the adult level (0.·04 mg/mg protein) by day 35. No IgM could be detected in rat colostrum, milk or saliva.

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.

XIAP-mediated protection of H460 lung cancer cells against cisplatin.

Molecular mechanism(s) responsible for drug resistance of non-small cell lung cancer (NSCLC) cells to cisplatin was investigated. Results showed that cisplatin (50muM)-induced cell death (apoptosis) was more significant in CH27 and A549 cell lines than in H460. The high protein levels of X-linked inhibitor-of-apoptosis protein (XIAP) observed in H460 cells appeared to play a key role in the regulation of cisplatin resistance of H460 cells. XIAP can bind to and suppress the activities of caspase 3 in H460 cells and lead to apoptosis inhibition of these cells. Blockade of XIAP activity by Embelin (XIAP inhibitor) or siRNA has increased caspase 3 activities and promoted cisplatin-induced cell death of H460 cells. The results indicate a therapeutic value of Embelin and/or XIAP siRNA in the control of cisplatin-resistant NSCLC cells (H460).

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.

Ischemic brain cell-derived conditioned medium protects astrocytes against ischemia through GDNF/ERK/NF-kB signaling pathway.

Conditioned medium (CM) collected from cultures of ischemic microglia, astrocytes, and neurons were protective to astrocytes under the in vitro ischemic condition (deprivation of oxygen, glucose and serum). Molecular and signaling pathway(s) responsible for the CMs protective activity were investigated. Results showed that CMs from the ischemic microglia (MCM), astrocytes (ACM) and neurons (NCM) contained glial cell line-derived neurotrophic factor (GDNF), which protects astrocytes against the in vitro ischemia. Expression of extra cellular signal-regulated kinase (ERK1/2) and nuclear factor-kappa B (NF-kB) by GDNF led to the inhibition of apoptosis of the ischemic astrocytes in a caspase 3-independent manner. However, CMs- and GDNF-mediated protection of the ischemic astrocytes was protein kinase B (Akt) independent. These results provided mechanistic data regarding how GDNF- and CMs-mediated protection of the ischemic astrocytes is taking place. These observations provide information for the use of GDNF and GDNF containing CMs in the control of cerebral ischemia.

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.