C. Franceschi

Extremely low frequency pulsed electromagnetic fields increase cell proliferation in lymphocytes from young and aged subjects.

The effect of the in vitro exposure to extremely low frequency pulsed electromagnetic fields (PEMFs) on the proliferation of human lymphocytes from 24 young and 24 old subjects was studied. The exposure to PEMFs during a 3-days culture period or during the first 24 hours was able to increase phytohaemagglutinin-induced lymphocyte proliferation in both groups. Such effect was greater in lymphocytes from old people which showed a markedly reduced proliferative capability and, after PEMF exposure, reached values of 3H-TdR incorporation similar to those of young subjects. The relevance of these data for the understanding and the reversibility of the proliferative defects in cells from aged subjects and for the assessment of risk related to the environmental exposure to PEMFs has to be considered.

Changes in circulating B cells and immunoglobulin classes and subclasses in a healthy aged population

The study of 87 adults of different ages, including 15 centenarians, selected for their healthy status, showed that profound changes of humoral immunity occur throughout life. In particuIar, a statistically significant age‐reIated increase of the serum level of immunoglobulin cIasses (IgG and IgA but not IgM) and IgG subcIasses (IgGI, 2 and 3, but not IgG4) was detected. A parallel age‐related decrease of circuIating B cells was also observed. The hypothesis of a complex derangement of B cell function and/or compartmentalization with age is put forward, together with the proposal that healthy centenarians (as representative of successful ageing) may be helpful in identifying the physiological age‐reIated modifications of the immune system.

Mitochondria alterations and dramatic tendency to undergo apoptosis in peripheral blood lymphocytes during acute HIV syndrome

Objective:To study alterations of mitochondrial membrane potential (Δψ) and the propensity to undergo apoptosis in peripheral blood lymphocytes (PBL) from subjects with acute HIV syndrome; and to evaluate possible modulations of these phenomena by antioxidants that can be used in therapy, such as N-acetyl-cysteine (NAC), nicotinamide (NAM), or L-acetyl-carnitine (LAC). Methods:Mitochondrial function and the tendency of PBL to undergo spontaneous apoptosis were studied on freshly collected PBL from patients with symptomatic, acute HIV-1 primary infection, which were cultured for different durations in the presence or absence of NAC, NAM or LAC. By a cytofluorimetric method allowing analysis of Δψ in intact cells, we studied the function of these organelles under the different conditions. PBL apoptosis was evaluated by the classic cytofluorimetric method of propidium iodide staining, capable of revealing the typical DNA hypodiploid peak. Results:Significant Δψ alterations and tendency to undergo apoptosis were present in PBL from the subjects we studied. Indeed, when cultured even for a few hours in the absence of any stimulus, a consistent number of cells died. However, the presence of even different levels of NAC, NAM or LAC was able to rescue most of them from apoptosis. Both a fall in Δψ and apoptosis were evident in PBL collected in the earliest phases of the syndrome (before seroconversion), and changed significantly after a few days. A significant correlation was found between spontaneous apoptosis and tumour necrosis factor (TNF)-α or p24 plasma levels, as well as between apoptosis and the percentages of circulating CD4+ or CD8+ T cells. Conclusions:PBL from patients with acute HIV syndrome are characterized by both significant mitochondrial alterations and a dramatic tendency to undergo apoptosis. The use of NAC, NAM or LAC seems to rescue cells through a protective effect on mitochondria, a well-known target for the action of TNF-α and for reactive oxygen species, the production of which is strongly induced by this cytokine. Thus, our data could provide the rationale for the use of such agents in addition to antiviral drugs in primary infection.

Extremely low frequency pulsed electromagnetic fields increase interleukin-2 (IL-2) utilization and IL-2 receptor expression in mitogen-stimulated human lymphocytes from old subjects

The effects of the exposure of mitogen‐stimulated human lymphocytes from aged subjects to low‐frequency pulsed electromagnetic fields (PEMFs) were studied by measuring the production of interleukin‐2 (IL‐2) and the expression of IL‐2 receptor. PEMF‐exposed cultures that presented increased [3H]thymidine incorporation showed lower amounts of IL‐2 in their supernatants, but higher percentages of IL‐2 receptor‐positive cells and of T‐activated lymphocytes. Taken together, these data suggest that PEMFs were able to modulate mitogen‐induced lymphocyte proliferation by provoking an increase in utilization of IL‐2, most likely acting on the expression of its receptor on the plasma membrane.

Pro-Opiomelanocortin-Derived Peptides, Cytokines, and Nitric Oxide in Immune Responses and Stress: An Evolutionary Approach

In vertebrates, including man, the study of stress has contributed substantially to unravelling the complex relationship between immune-neuroendocrine interactions and the systems involved. On the basis of data on the presence and distribution of the main actors (POMC products, cytokines, biogenic amines, and steroid hormones) in different species and taxa from invertebrates to vertebrates, we argue that these responses have been deeply connected and interrelated since the beginning of life. Moreover, the study of nitric oxide suggests that the inflammatory reaction is located precisely between the immune and stress responses, sharing the same fundamental evolutionary roots. The major argument in favor of this hypothesis is that the immune, stress, and inflammation responses appear to be mediated by a common pool of molecules that have been conserved throughout evolution and that from a network of adaptive mechanisms. One cell type, the macrophage, appears to emerge as that most capable of supporting this network critical for survival; it was probably a major target of selective pressure. All these data fit the unitarian hypothesis we propose, by which evolution favors what has been conserved, rather than what has changed, as far as both molecules and functions are concerned.

Age-dependent modifications of Type 1 and Type 2 cytokines within virgin and memory CD4+ T cells in humans

Several alterations in immune function and a concomitant progressive increase in pro-inflammatory status are the major characteristics of ageing process. Cytokines play a key role during ageing acting both in regulatory communication among cells and in effector activity during an immune response. The impact of age on intracellular Type 1 (IFN-gamma and TNF-alpha) and Type 2 (IL-4) cytokines, after stimulation with PMA/ionomycin, was determined in three CD4+ T subsets, i.e. CD95- CD28+ (virgin), CD95+ CD28+ (activated/memory), and CD95+ CD28- (effector/memory) from 47 subjects aged between 21 and 99 years. The percentage of IFN-gamma positive cells significantly decreased in virgin CD4+ subset both in old and nonagenarian subjects, as well as in activated/memory T cells from old in comparison with young subjects. The percentage of TNF-alpha positive cells significantly decreased in activated/memory CD4+ subset from old people. Regarding Type 2 cytokines, IL-4 positive cells significantly increased in activated/memory CD4+ subset from nonagenarians. On the whole our data indicate that: (1) different Type 1 and Type 2 cytokine-positive CD4+ T subsets are differently affected by ageing process; (2) activated/memory T cells appear to be the most affected subset; (3) a shift towards an increased role of Type 2 cytokines and a diminished role of Type 1 cytokines emerges with ageing.

In vivo accumulation of sulfated glycoprotein 2 mRNA in rat thymocytes upon dexamethasone-induced cell death.

Two hours after a single intraperitoneal injection of dexamethasone (20 micrograms/Kg b.w.) into adult male rats, a typical ladder of DNA fragments was detectable upon separation on agarose gels of DNA from thymocytes. This became maximally evident at 4 hours. Accumulation of sulfated glycoprotein-2 (SGP-2) mRNA, whose rate of expression has been associated to the processes of programmed cell death, preceded the appearance of DNA degradation, starting to increase as early as 30 min after steroid injection, and maintained higher than controls until 8 hrs; a different time course was shown by changes in the levels of beta-actin mRNA. In the spleen, under the same conditions, the SGP-2 message also increased at 30 min, prior to DNA fragmentation, but decreased thereafter below the control value.

Spontaneous and mitomycin-C-induced micronuclei in human lymphocytes exposed to extremely low frequency pulsed magnetic fields.

The cytokinesis block micronucleus method, a very sensitive cytogenetic assay, was used to ascertain the possible genotoxic effects of extremely low frequency pulsed magnetic fields in phytohemagglutinin-stimulated human lymphocytes cultures from 16 healthy donors. Four conditions were studied: i) lymphocytes not exposed to the field (control cultures); ii) lymphocytes exposed to the field; iii) lymphocytes treated with mitomycin-C and not exposed to the field; iv) lymphocytes treated with mitomycin-C and exposed to the field. Mitomycin-C-treated cultures were used as control for the micronucleus method, because it is known that mitomycin-C is a potent genotoxic agent, capable of inducing micronuclei. The frequency of micronuclei in field-exposed cultures was similar to the spontaneous frequency observed in control unexposed-cultures. Moreover, the exposure to pulsed magnetic fields did not affect the frequency of micronuclei induced by mitomycin-C, suggesting that, in the experimental conditions used, this kind of field neither affected the integrity of chromosomes nor interfered with the genotoxic activity of mitomycin-C.

50 Hz AC sinusoidal electric fields do not exert genotoxic effects (micronucleus formation) in human lymphocytes

Electromagnetic fields produce a variety of effects in several biological systems, including human peripheral blood lymphocytes. A great concern exists regarding possible carcinogenic effects in subjects exposed environmentally to such fields in the vicinity of power lines and electric domestic appliances. Using human lymphocytes from 33 healthy donors and a sensitive cytogenetic method, the cytokinesis-block micronucleus assay, we have demonstrated that the exposure to 50-Hz AC sinusoidal electric fields over a wide range of intensities (0.5-10 kV/m in air) does not increase the spontaneous frequency of micronuclei. Moreover, these fields did not affect the mitomycin-C-induced micronucleus formation, suggesting that they did not exert any synergistic or antagonistic effect.

DECREASE IN MITOCHONDRIAL ENERGY COUPLING BY THYROID HORMONES : A PHYSIOLOGICAL EFFECT RATHER THAN A PATHOLOGICAL HYPERTHYROIDISM CONSEQUENCE

The effect of the in vivo thyroid status on mitochondrial membrane potential (ΔΨm) in isolated rat hepatocytes was studies by means of a cytofluorimetric technique and the ΔΨm‐specific probe JC‐1. It is shown that the ΔΨm level decreases in the order hypothyroid>euthyroid>hyperthyroid. Polarographic measurement of the hepatocyte respiratory rates revealed an opposite trend of values: the highest respiratory rate in hepatocytes from hyperthyroid animals, the lowest in those from hypothyroid ones. This means that mitochondrial energy coupling is highest in hypothyroid hepatocytes and lowest in hyperthyroid hepatocytes. 6‐Ketocholestanol added to hepatocytes failed to counterbalance the uncoupling effect of thyroid hormones on ΔΨm and respiration rate. Under the same conditions, 6‐ketocholestanol appeared to be effective in recoupling of respiration uncoupled by low concentrations of the artificial protonophore FCCP. The mechanism and possible physiological functions of the thyroid hormone‐induced decrease in mitochondrial energy coupling are discussed.