Inhibition of cell growth in vitro by adenosine 3',5'-monophosphate.

Adenosine 3',5'-monophosphate, at a concentration of 40 micrograms per milliliter, inhibits the growth of HeLa and strain L cells in culture. The inhibition becomes progressively greater during the incubation of the cells. Adenosine 5'-monophosphate and adenosine, metabolites of adenosine 3',5'-monophosphate, do not affect the growth of either cell culture. This suggests that 3',5'-monophosphate enters the cell without alteration. Dibutyryl-adenosine 3',5'-monophosphate, reported to have a greater activity than adenosine 3'5'-monophosphate on several tissues, inhibited the growth of the cells much less.

Stimulation of malignant skin cells by antibody to normal skin cells of mice.

The ability of antibodies developed against normal skin cells to stimulate skin cells transformed by 7,12-dimethylbenz[a]anthracene (DMBA) was investigated. Primary cultures of normal skin, containing both fibroblasts and epithelial cells, were established from epidermis of the back skin of adult strain A/J mice. Malignant skin cells were obtained by treating a subculture of normal cells with DMBA. Transformation was demonstrated by increased growth rate, growth in soft agar, and production of tumors in strain A/J mice. Antisera developed in New Zealand White rabbits against the normal cells were cytotoxic to both normal and malignant cells in the presence of complement of 1:320 dilution. However, greater dilutions of the antisera (1:500-1:1,000) in the absence of complement produced growth enhancement of the malignant but not of the normal cells. The growth-enhancing properties were present in the gamma-globulin fraction of the antisera that contained IgG, IgM, and IgA antibodies. Immunofluorescence studies indicated that antibodies from the sera bound to the membranes of both normal and malignant cells. These data indicate that antibodies to normal cells are stimulatory to DMBA-transformed cells and confirm previous data obtained with spontaneously transformed cells.

Susceptibility to lipid peroxidation and accumulation of fluorescent products with age is greater in T-cells than B-cells.

The age-related loss of immune function, which is primarily due to loss of T-lymphocyte function, is also associated with accumulation in spleen lymphocytes of autofluorescent products indicative of peroxidation damage. In this study, we examined T-cell membranes for age-related changes which could be related to lipid peroxidation. Using fluorescence spectroscopy of CHCl3:CH3OH membrane extracts, we observed that old T-cells have a 2-fold greater accumulation of fluorescent products than old B-cells and that young T-cells, when exposed to free radicals in an in vitro system, accumulate significantly more fluorescent products over time than young B-cells. We used fluorescence polarization to show that young T-cell membranes are more fluid than young B-cell membranes. However, T-cell membrane fluidity decreases with age, whereas B-cell membrane fluidity does not change; in old mice, T-cell membranes are significantly less fluid than old B-cell membranes. Using two-dimensional electrophoresis, we showed that membrane extracts of old T-cells contain many more proteins than extracts of young T-cells. Our results indicate that age-related changes occur in T-cell membranes which could be due to their increased susceptibility to lipid peroxidation and these changes may contribute to the age-related decline in immune function.

Restoration of impaired immune functions in aging animals. IV. Action of 2-mercaptoethanol in enhancing age-reduced immune responsiveness.

The enhancing effect of 2-mercaptoethanol (2-ME) on the immune responses of young and old unseparated spleen cells and purified populations of T-cells, B-cells and macrophages was studied. While 2-ME enhances both the normal responses of young cells and the age-reduced responses of old cells, the sulfhydryl compound has a relatively greater enhancing effect on old spleen cells in the humoral response to sheep red blood cells and the blastogenic response to concanavalin A (Con A). The greater enhancing effect in the humoral response appears to be due to 2-ME overcoming a suppressive effect of old T-cells which develops with increasing age. The greater enhancing effect in Con A stimulation appears to be an effect on the Con A responsive sub-population of T-cells and not on the synergistic effect of the presence of B-cells.

Adenosine deaminase and purine nucleoside phosphorylase activity in spleen cells of aged mice.

The activities of two enzymes of purine metabolism, adenosine deaminase (ADA) and purine nucleoside phosphorylase (PNP), were determined in spleen lymphocytes from mice of various ages. We found that in the older animals, which have depressed responses to concanavalin A and phytohemagglutinin, there is a decrease in the activity of PNP but normal activity of ADA. The decline of PNP activity was seen at 7.5 months of age and appears to be concurrent with a decline in T-cell function. These results suggest that a decrease in PNP activity may be a contributing factor in the immunodeficient state of the aged.

Effect of dietary 2-mercaptoethanol on the life span, immune system, tumor incidence and lipid peroxidation damage in spleen lymphocytes of aging BC3F1 mice.

The age-related decline in immune function, which is thought to be responsible for the increased incidence with age of certain diseases, including cancer, has been attributed primarily to a loss of T-lymphocyte function. As free radical reactions may contribute to cellular deterioration and loss of cell function with age, we investigated the effect of adding an immunopotentiating antioxidant, 2-mercaptoethanol (2-ME), to the diet of BC3F1 mice in a longitudinal study. For the study, young mice were divided into two groups, one of which received the 2-ME-supplemented diet. Approximately every 3 months for 2.5 years, mice from each group were sacrificed and the spleen lymphocytes assessed for immune function (proliferative response to concanavalin A, phytohemagglutinin, and lipopolysaccharide and the humoral response to sheep red blood cells). The accumulation of fluorescent products indicative of free radical damage was measured in the spleen lymphocytes and the cytochrome P-450 content and activity assessed in the liver. The effect of the 2-ME-supplemented diet on the mean and maximum life span and tumor incidence was also determined. The results showed that the animals fed the 2-ME diet had an increased mean and maximum life span and a postponed onset and decreased incidence of tumors. In general the T-cell-dependent immune responses were higher in the 2-ME-fed mice compared to the controls when the animals were young. No difference was observed between the two groups during mid-life. The responses declined in both groups during the latter half of the life span, but the responses of the 2-ME-fed animals declined to a lesser extent. The accumulation of fluorescent products of lipid peroxidation damage was also delayed in the lymphocytes of the 2-ME-fed mice. Cytochrome P-450 content and activity in the liver was not different in the two groups. The results suggest that the antioxidant activity of 2-ME delayed the accumulation of free radical damage in spleen lymphocytes, which resulted in a delay in the decline of immune function and was associated with the decreased tumor incidence and increased life span.

Neurotrophins and their receptors in nerve injury and repair.

Cytokines are a heterogenous group of polypeptide mediators that have been associated with activation of numerous functions, including the immune system and inflammatory responses. The cytokine families include, but are not limited to, interleukins (IL-I alpha, IL-I beta, ILIra and IL-2-IL-15), chemokines (IL-8/ NAP-I, NAP-2, MIP-I alpha and beta, MCAF/MCP-1, MGSA and RANTES), tumor necrosis factors (TNF-alpha and TNF-beta), interferons (INF-alpha, beta and gamma), colony stimulating factors (G-CSF, M-CSF, GM-CSF, IL-3 and some of the other ILs), growth factors (EGF, FGF, PDGF, TGF alpha, TGF beta and ECGF), neuropoietins (LIF, CNTF, OM and IL-6), and neurotrophins (BDNF, NGF, NT-3-NT-6 and GDNF). The neurotrophins represent a family of survival and differentiation factors that exert profound effects in the central and peripheral nervous system (PNS). The neurotrophins are currently under investigation as therapeutic agents for the treatment of neurodegenerative disorders and nerve injury either individually or in combination with other trophic factors such as ciliary neurotrophic factor (CNTF) or fibroblast growth factor (FGF). Responsiveness of neurons to a given neurotrophin is governed by the expression of two classes of cell surface receptor. For nerve growth factor (NGF), these are p75NTR (p75) and p140trk (referred to as trk or trkA), which binds both BDNF and neurotrophin (NT)-4/5, and trkC receptor, which binds only NT-3. After binding ligand, the neurotrophin-receptor complex is internalized and retrogradely transported in the axon to the soma. Both receptors undergo ligand-induced dimerization, which activates multiple signal transduction pathways. These include the ras-dependent pathway utilized by trk to mediate neurotrophin effects such as survival and differentiation. Indeed, cellular diversity in the nervous system evolves from the concerted processes of cell proliferation, differentiation, migration, survival, and synapse formation. Neural adhesion and extracellular matrix molecules have been shown to play crucial roles in axonal migration, guidance, and growth cone targeting. Proinflammatory cytokines, released by activated macrophages and monocytes during infection, can act on neural targets that control thermogenesis, behavior, and mood. In addition to induction of fever, cytokines induce other biological functions associated with the acute phase response, including hypophagia and sleep. Cytokine production has been detected within the central nervous system as a result of brain injury, following stab wound to the brain, during viral and bacterial infections (AIDS and meningitis), and in neurodegenerative processes (multiple sclerosis and Alzheimer's disease). Novel cytokine therapies, such as anticytokine antibodies or specific receptor antagonists acting on the cytokine network may provide an optimistic feature for treatment of multiple sclerosis and other diseases in which cytokines have been implicated.

Immunological aspects associated with suppression of hormone levels in rats infected with plerocercoids of Spirometra mansonoides (Cestoda).

The effect of the plerocercoid larvae of Spirometra mansonoides on hormone levels and the immune system of intact and hypophysectomized (Hx) rats was studied. Intact rats exhibited a decrease in serum growth hormone (GH) and thyroxine (T4) concentrations and a suppression of the humoral immune response 2 wk postinfection. At 4 and 9 wk postinfection, hormone concentrations and the humoral immune response returned to normal levels in the rats. When rats were infected with plerocercoids and then received daily injections for 2 wk of either GH or T4, the immune response returned to normal in the T4-injected, but not the GH-injected animals. Plerocercoid infection had no effect on the humoral immune response of Hx rats. The results of the present study suggest that PGF may act via an endocrine pathway to suppress the host's immune response enabling the parasite's survival in this host.

Immunomodulatory action of levamisole--II. Enhancement of concanavalin A response by levamisole is associated with an oxidation degradation product of levamisole formed during lymphocyte culture.

Previously we determined that levamisole (LMS), when stored for a period of time, breaks down to three degradation products at neutral and alkaline pH. At low concentrations (10(-6) M), Product 1 inhibits the lymphocyte response to concanavalin A (Con A). Product 2 enhances the response and Product 3 has no effect. At higher concentrations (10(-5) M) all three products inhibit the response. To determine if these products are formed in culture media under culture conditions (e.g. in RPMI-1640 bicarbonate buffered medium, 37 degrees C, pH 7.0-7.5, during a 72 h culture period), we added freshly prepared LMS solutions to culture media with and without lymphocytes present and maintained the pH at 7.0, 7.25 or 7.5 by varying the amount of CO2 present. Periodically over a 72 h period, aliquots of the media were removed and analyzed for the presence of LMS and the three degradation products. Within 4 h, two of the degradation product began to form in culture media with or without lymphocytes present. Product No. 1, 3-(2-mercaptoethyl)-5-phenylimidazolidine-2-one or dl-2-oxy-3-(2-mercaptoethyl)-5-phenylimidazolidine (OMPI), which inhibits the lymphocyte response to concanavalin A (Con A) at concentrations above 0.4 micrograms/ml, was formed at pH 7.0, 7.25 and 7.5, but the compound did not reach inhibitory concentrations in the lymphocyte cultures during the 72 h culture period. Product No. 2, 6-phenyl-2,3-dihydroimidazo (2,1-b) thiazole, which enhances the Con A response between concentrations of 0.5 and 10 micrograms/ml, was detected at concentrations between 2.5 and 3.5 micrograms/ml at pH 7.25 and 7.5. Product 2 was not detected in cultures at pH 7.0 and subsequently when we cultured lymphocytes with freshly prepared LMS and maintained the pH at 7.0, no significant enhancement of the Con A response was observed.(ABSTRACT TRUNCATED AT 250 WORDS)

Immunomodulatory action of levamisole--I. Structural analysis and immunomodulating activity of levamisole degradation products.

In our laboratory we observed that solutions of levamisole (LMS) stored at 4 degrees C consistently enhanced the lymphocyte proliferation response to concanavalin A (Con A) more than freshly prepared solutions did. To determine if the increased immunopotentiation observed with the stored solutions of LMS was due to products formed from LMS, we assessed the stability of LMS when stored at 4 or 37 degrees C at pH 6, 7, 7.5 and 8. Analysis of the various solutions by high pressure liquid chromatography demonstrated that LMS decomposes during storage in neutral and alkaline conditions to form three products. The formation of the products was accelerated by increasing the temperature from 4 to 37 degrees C. The three degradation products were purified by preparative high pressure liquid chromatography and their structures determined by mass spectrometry, infrared spectrometry and homo- and heteronuclear two dimensional nuclear magnetic resonance spectroscopy. The degradation products, denoted as No. 1, No. 2 and No. 3, based on their high pressure liquid chromatography retention times, were identified as: No. 1, 3-(2-mercaptoethyl)-5-phenylimidazolidine-2-one; No. 2, 6-phenyl-2,3-dihydroimidazo (2,1-b) thiazole and No. 3, bis [3-(2-oxo-5-phenylimidazolidin-1-yl) ethyl] disulfide. Product 2 significantly enhanced murine lymphocyte proliferation responses to concanavalin A (Con A) at concentrations between 0.5 and 10.0 micrograms/ml (whereas the optimum concentration of LMS is 10-100 fold higher (50-100 micrograms/ml)). Products 1, 2 and 3 significantly inhibited the lymphocyte proliferative response at concentrations greater than 2.2, 10.0 and 10.0 micrograms/ml, respectively. These studies indicate that under relatively mild conditions, including physiological conditions, LMS may decompose to products which inhibit or enhance lymphocyte responses to Con A.

Immunodeficiency and autoimmunity in aging.

In general, both cell-mediated and humoral immune activities decline with advancing age and, associated with this decline, is an increase in the incidence of certain types of autoimmune disease, cancer and infection. Not only may the relationship be causal, but the former may be a significant factor involved in the expression of the latter. Discussion is focused primarily on the relative contribution of extrinsic and intrinsic factors responsible for the immunodeficiency of aging individuals.

Suppressed proliferation of lymphatic tissue in diabetic and adrenalectomized-diabetic rats.

One week following induction of diabetes in rats by alloxan administration, the thymus and spleen showed marked involution and a highly significant depression of in vivo incorporation of [3H]deoxycytidine into the DNA of these tissues. Adrenalectomy of diabetic rats reduced the decline of lymphatic tissue weight, but uptake of [3H]deoxycytidine into thymic and spleen DNA remained suppressed. These results indicate that in alloxan-induced diabetes, the observed depressed lymphatic tissue weight and cellular proliferation are not simply due to the elevated plasma level of corticosteroids, but are either a result of the insulin deficiency per se or due to other metabolic alterations associated with the diabetic condition.

The binding of FITC-insulin to ANAE-positive cells in rat thymus.

To determine if thymic macrophages have insulin receptors, alternate sections of rat thymus were stained with FITC-insulin and examined for nonspecific esterase (ANAE) activity. Cells showing a diffuse ANAE staining pattern also bound FITC-insulin. These cells were concentrated in the cortico-medullary border and increased in number following administration of cortisol. Thymic macrophages may be insulin-dependent and therefore could be malfunctional in diabetes.

Reduced humoral immune activity in long-lived old mice: an approach to elucidating its mechanisms.

Spleen cells from young (3-5 months) and old (22-27 months) mice were assessed in cultures, both in vivo and in vitro, for their anti-sheep RBC response separately and in mixtures. Pooled young spleens, pooled old spleens, and individual old spleens were analysed. The response of pure young spleen cells was always higher than that of pure old spleen cells (approximately 30 times). The responses of mixtures were either less than (i.e. reduced; frequency approximately 65%), comparable to (i.e. additive; frequency approximately 10%), or greater than (i.e. elevated; frequency approximately 30%) the sum of the responses given by equivalent numbers of pure young and pure old spleen cells. The reduced response was observed in mixtures containing cells from histologically normal old spleens, old spleens with tumours and old spleens with atrophic follicles. Additive and elevated responses were observed only in mixutres containing cells from histologically normal old spleens. The reduced response is explicable in terms of excessive numbers of suppressor cells in old spleens that can prevent young immunocompetent cells from responding maximally to the test antigen. The additive response can be accounted for by a reduction in number of immunocompetent cells in old spleens and/or a decrease in their functional efficiency. The elevated response can be explained by a reduction in number of at least one type of immunocompetent cell in old spleens that exists in excess in young spleens. These results indicate that there are several types of cellular changes responsible for the decrease in humoral immune activity in old mice. Pooling of old spleens, as was commonly done in the past, should therefore be discouraged. Not only might it selectively favour the expression of old spleens with an excess of supressor cells but it conceivably could result in an elevated response.

Regulation of carbohydrate metabolism in mouse liver. Effect of glucagon on gluconeogenic/glycolytic flux in isolated perfused livers.

1. Glucagon stimulated gluconeogenesis from both [U-14C]lactate and [14C]xylitol in isolated perfused mouse liver. 2. Addition of cyclic AMP also stimulated gluconeogenesis from [U-14C]lactate. 3. Glucagon caused a rapid (2.5 min) 12-fold increase in hepatic cyclic AMP but not cyclic GMP concentration. 4. Glucagon caused a rapid and stable decrease in hepatic fructose 1,6-diphosphatase activity measured in vitro. 5. The results are interpreted to indicate that glucagon stimulates hepatic gluconeogenesis in mice via cyclic AMP by two different mechanisms: (a) increased substrate uptake (i.e. utilization) and (b) increased gluconeogenic efficiency (i.e. inhibition of alternate substrate fates).