Zinc status and age-related changes in peripheral blood leukocyte subpopulations in healthy men and women aged 55-70 y: the ZENITH study.

OBJECTIVE: To determine zinc status and age-related changes in the immune function of healthy late-middle-aged men and women (aged 55-70 y). DESIGN: Observational study. SETTING: Population of Northern Ireland. SUBJECTS: Apparently healthy, free-living individuals (45 men, 48 women) aged 55-70 y. INTERVENTION: Zinc status markers were analysed by flame atomic absorption spectrometry and commercially available kits. Immune function was assessed by flow cytometry. RESULTS: Serum and erythrocyte zinc concentrations were 13.0 (s.d. 1.40) micromol/l and 222 (s.d. 48.2) micromol/l, respectively. Serum alkaline phosphatase (ALP) concentrations were 76.8 (s.d. 16.1) U/l; women showed significantly higher concentrations of ALP (P = 0.011). Women demonstrated (1) a significant inverse correlation in naive T lymphocytes, specifically naive T-helper lymphocytes (% expression, r = -0.364, P = 0.007 and absolute count, r = -0.275, P = 0.036) with age and (2) a significant positive correlation between late activation of T lymphocytes (% expression, r = 0.299, P = 0.019 and absolute count, r = 0.260, P = 0.039) with advancing age. Men demonstrated a significant positive correlation in the % expression of (CD3-/CD16+/CD56+) natural killer (NK) cells with age (r = 0.316, P = 0.017). CONCLUSIONS: Between the ages of 55 and 70 y, healthy individuals experience significant alterations in immune function; however, such changes appear largely sex specific. Given the reported importance of adequate zinc status in maintaining optimal immune function, further studies are required to explore the effect of enhanced zinc status on emerging immune deficiencies in cell-mediated immunity in healthy 55-70 y olds.

Inter-relationships between DNA damage, ascorbic acid and glycaemic control in Type 2 diabetes mellitus.

AIMS: The onset of complications in Type 2 diabetes mellitus (DM) patients cannot be predicted in individuals. Evidence suggests a link between complications and hyperglycaemia, oxidative stress and antioxidants, but causality is unclear. This study investigated baseline (entry) fasting plasma ascorbic acid, lymphocytic DNA damage and glycaemic control in Type 2 DM as part of a long-term study, the aim of which is to explore a biomarker profiling approach to identify and improve outcome in high-risk subjects. METHODS: A cross-sectional study, in which DNA damage, glycated haemoglobin (HbA(1c)), fasting plasma glucose (FPG) and ascorbic acid (AA) were measured on fasting blood samples collected from 427 Type 2 DM subjects. RESULTS: DNA damage was significantly (P < 0.0001) and directly correlated to both FPG (r = 0.540) and HbA(1c) (r = 0.282), and was significantly (P < 0.0001), independently and inversely correlated to plasma AA (r = -0.449). In those subjects with both poor glycaemic control and low AA (< 48 microm, the overall mean value for the study group), DNA damage was significantly (P < 0.005) higher compared with those subjects with a similar degree of hyperglycaemia but with AA above the mean. CONCLUSIONS: The novel finding of a significant inverse relationship between plasma AA and DNA damage in Type 2 DM indicates that poorly controlled diabetic subjects might benefit from increased dietary vitamin C. The data also have important implications for biomarker profiling to identify those subjects who might benefit most from intensive therapy. Longer-term follow-up is underway.

Insulin-like growth factor-I (IGF-I) and its association with lymphocyte homeostasis in the ageing cat.

Ageing affects feline lymphocyte homeostasis in a similar pattern to that observed in other long-lived mammalian species, contributing to increased levels of morbidity and mortality in the ageing cat. Insulin-like growth factor-I (IGF-I) is now recognised as an important endocrine regulator of immunity and has been shown to decline with age in humans and rodent species. Analysis of plasma IGF-I in adult and senior cats confirmed that the older cats had significantly lower circulating levels of IGF-I. In order to determine whether an association existed between lymphocyte subpopulations and IGF-I levels in the cat, each parameter was measured and subjected to regression analysis. A highly significant association was found in vivo between plasma IGF-I and CD4(+) T-cell values in the senior group, but no such association was observed in the adult group. In order that this relationship could be examined further, in vitro studies were undertaken to investigate the effects of physiologically relevant concentrations of recombinant human IGF-I (rhIGF-l) on peripheral blood lymphocyte (PBL) cultures from adult and senior cats. While rhlGF-I induced low-level thymidine incorporation in the lymphocytes isolated from the senior group, it did not enhance the proliferative response to T-cell mitogens, Con A and PHA in either group, nor did it rescue cells from oxidatively induced apoptosis. Furthermore, the proliferative response of PBL from seniors did not attain the magnitude of that from the adults at any concentration of rhIGF-l. We propose that the observed association is not a direct effect of IGF-I on PBL, but may be mediated through an effect of IGF-I on the thymus.

Vitamins C and E: acute interactive effects on biomarkers of antioxidant defence and oxidative stress.

Oxidative stress is implicated in the aetiology of many diseases; however, most supplementation trials with antioxidant micronutrients have not shown expected beneficial effects. This randomized, double-blinded, placebo-controlled study evaluated acute effects (at 90, 180min and 24h [fasting] post-ingestion) of single doses of Vitamins C (500mg) and E (400IU), alone and in combination, on biomarkers of plasma antioxidant status, lipid peroxidation and lymphocyte DNA damage in 12 healthy, consenting volunteers. Plasma ascorbic acid increased significantly (P < 0.01) within 2h of ingestion of Vitamin C, and alpha-tocopherol was significantly (P < 0.01) higher at 24h post-ingestion Vitamin E. The pattern of response was not significantly different whether Vitamin C (or Vitamin E) was taken alone or in combination, indicating no augmentation of response to one by co-ingestion of the other vitamin. No significant changes were seen in plasma FRAP in the group overall (although increases (P < 0.05) were seen at 90 and 180min post-ingestion in women after Vitamin C ingestion) or in MDA across treatments, and no evidence of increased DNA damage, or of DNA protection, was seen at any time point after Vitamin C and/or E ingestion. In conclusion, the data from this first controlled study of acute effects of single doses of Vitamin C and/or E show no evidence of either a protective or deleterious effect on DNA damage, resistance of DNA to oxidant challenge, or lipid peroxidation. No evidence of a synergistic or cooperative interaction between Vitamins C and E was seen, but further study is needed to determine possible interactive effects in a staggered supplementation cycle, and study of subjects under increased oxidative stress or with marginal antioxidant status would be useful. It would be of interest also to study the effects of these vitamins ingested with, or in, whole food, to determine if they are directly protective at doses above the minimum required to prevent deficiency, if combinations with other food components are needed for effective protection, or if Vitamins C and E are largely surrogate biomarkers of a 'healthy' diet, but are not the key protective agents.

Age-related differences in parameters of feline immune status.

In order to assess age-related differences in feline immune status, 101 domestic short haired cats were assigned to two groups, adult (2-5 years, n=50) and senior (10-14 years, n=51). Analyses of leucocyte populations, lymphocyte subsets, complement activity, serum immunoglobulins and acute-phase proteins were undertaken and revealed significant differences between the two groups. The senior group had significantly lower WBC, lymphocyte and eosinophil counts than the adult group. Neutrophil, monocyte and basophil counts did not differ between the groups. Flow cytometry analysis, in combination with differential WBC data, revealed that the absolute values (cells/l) of T-cells, B-cells and natural killer (NK) cells were significantly lower in the older animals. While serum immunoglobulins IgA and IgM were higher in the senior group when compared with the adult group, no significant differences were observed in complement activity or in serum acute-phase proteins. Our findings suggest that age-related changes to parameters of immune status in the feline model are likely to follow a similar pattern to those observed in other long-lived mammalian species.

Vitamin C decreases intracellular calcium level in human lymphoid cells.

Human lymphocytes have low levels of many antioxidant enzymes however they are know to concentrate vitamin C. Cell injury, including oxidative stress effects, is associated with calcium influx so the influence of vitamin C on the maintenance of calcium levels in leukocytes was studied. Incubation of Molt-3 human lymphoblastoid cells with physiologically relevant concentrations of vitamin C and the calcium ionophore A23187 reversed the calcium influx and increased nuclear protein level associated with the ionophore alone. It is concluded that intracellular vitamin C can inhibit calcium influx into leukocytes so helping to minimise cell damage.

Expression of oestrogen and progesterone receptors by mast cells alone, but not lymphocytes, macrophages or other immune cells in human upper airways.

BACKGROUND: Nasal polyposis often coexists with asthma in airway inflammatory conditions characterised by the infiltration of a range of immune cells. A potentially important role for ovarian hormones has been implicated in airway inflammation but the cellular target for such action is not known. METHODS: Expression of oestrogen receptors (ER) and progesterone receptors (PR) was examined using immunohistochemistry in formalin fixed nasal polyp tissues from 47 subjects. The cells positive for ER or PR were confirmed by spatial location, dual immunolabelling, and histochemical staining. RESULTS: Consistent with the known features of nasal polyps, CD4+ (T helper/inducer), CD8+ (cytotoxic/suppressor), CD68+ (macrophages), mast cells, eosinophils and neutrophils were all clearly detected by their relevant monoclonal antibodies or appropriate histochemical staining, but only mast cells tested positive for ER/PR labelling with their polyclonal and monoclonal antibodies. The frequencies for expression were 61.7% for ER positive and 59.6% for PR positive cells. The expression of ER/PR was independent of patient sex and age but was highly correlated with the numbers of mast cells (r = 0.973, p<0.001 for ER; r = 0.955, p<0.001 for PR). Fewer than 5% of mast cells were found to be negative for ER/PR expression. CONCLUSIONS: Mast cells alone, but not lymphocytes, macrophages, or other immune cells, express ER/PR in human upper airways. Numerous ER/PR positive mast cells exist in nasal polyps, indicating that this may be a major route for the involvement of sex hormones in airway inflammation when exposed to the higher and varying concentration of oestrogen and progesterone characteristic of females.

The effect of vitamin C or vitamin E supplementation on basal and H2O2-induced DNA damage in human lymphocytes.

There is a wealth of epidemiological information on antioxidants and their possible prevention of disease progression but very little of the research on antioxidants has involved intervention studies. In this study, the potential protective effect of vitamin C or E supplementation in vivo against endogenous and H2O2-induced DNA damage levels in lymphocytes was assessed. The supplementation involved fourteen healthy male and female non-smokers mean age 25-53 (SD 1.82) years, who were asked to supplement an otherwise unchanged diet with 1000 mg vitamin C daily for 42 d or 800 mg vitamin E daily for 42 d. DNA damage in H2O2-treated peripheral blood lymphocytes (PBL) and untreated PBL before and after supplementation, and during a 6-week washout period was assessed using an ELISA. At each sampling time-point, the red cell concentrate activities of superoxide dismutase, catalase and glutathione peroxidase were also determined. Supplementation with vitamin C or vitamin E decreased significantly H2O2-induced DNA damage in PBL, but had no effect on endogenous levels of DNA damage. The activities of the antioxidant enzymes superoxide dismutase and glutathione peroxidase were suppressed during the supplementation period. These supplementation regimens may be used to limit the possible adverse effects of reactive oxygen species (including those produced during the course of an immune response) on lymphocytes in vivo, and so help to maintain their functional capacity.

Radioadaptation in Indian muntjac fibroblast cells induced by low intensity laser irradiation.

Earlier reports have indicated that an adaptive, protective response to ionizing radiation is inducible by pre-treatment with low intensity laser irradiation (LILI). We have investigated the potential of LILI to induce an adaptive response against the damaging effects of ionizing radiation in Indian muntjac fibroblasts. LILI at 660, but not 820 nm, at 11.5 and 23.0 J/cm2, induced an apparent adaptive response in the form of a reduction in the frequency of radiation-induced chromosome aberrations, but not in cell survival. There was also a trend towards a reduction in the level of single-stranded and double-stranded DNA breaks induced by ionizing radiation when cells were preconditioned with LILI. However, this did not contribute to the reduced chromosome aberration frequency. Further analysis revealed that the reduced aberration frequency was caused by a laser-induced extension of G2 delay. The adaptive response was therefore the result of cell cycle modulation by LILI, at a wavelength where there is no known DNA damaging effect to induce the checkpoint mechanisms that are normally responsible for altering cell cycle progression.

Cell-cycle delay is induced in cells of a U937 promonocytic cell line by low-intensity light irradiation at 660 nm.

Visible-light irradiation (VLI) at 660 nm and 11.5 J/cm2 inhibits proliferation of cells of the U937 promonocytic cell line, as monitored by autoradiographical analysis. The S-phase cell population is reduced at 6 h post-radiation treatment. Flow cytometric analysis confirms this, and also shows that light irradiation of cells induces a statistically significant increase in G2/M cells at 6 h post-radiation treatment. It has been postulated that VLI at 660 nm can alter cell-cycle progression by affecting intracellular concentrations of ions, in particular pH and calcium. However, no significant effects of light irradiation on these intracellular ions have been observed. These effects of VLI are not a consequence of radiation-induced DNA strand breaks, therefore events other than direct DNA damage are involved. These findings demonstrate a direct photobiological effect of VLI at 660 nm on the cell cycle, and indicate a previously unsuspected mechanism for the induction of cell-cycle delay that is neither a result of changes in the concentration of intracellular ions nor initiated by DNA strand breaks.

Macrophages and apoptotic cells in human colorectal tumours.

The numerical relationship between tumour associated macrophages (TAM) and apoptotic cells in 12 human colorectal tumours was evaluated. TAM were labelled immunohistochemically and apoptotic cells were visualized by counterstaining with haematoxylin and eosin (H&E). The stereological techniques, Cavalieri's estimator of volume and the Disector were used to estimate both tumour volume and numerical density of both cell types. The occurrence of TAM per unit volume of tissue increased with increasing tumour volume to a maximum in a tumour of 110.5 cm3, after which numbers declined. Levels of apoptosis also increased with tumour volume though more erratically than levels of TAM and declined for tumour volumes greater than 80 cm3. This is the first report of an attempt to assess the relationship between apoptotic cells and TAM in human tumours.

The effects of copper deficiency on human lymphoid and myeloid cells: an in vitro model.

Cu has long been known to influence immune responses. An in vitro model system was established in which human myeloid (HL-60), B-lymphoid (Raji) and T-lymphoid (Molt-3) cell lines could be grown in culture media of varying Cu levels. Initially Cu was removed from the medium by dialysis of fetal calf serum against a metal-ion chelator, minor depletion of other trace metals being obviated by repletion with appropriate metal salts. The growth rate of HL-60 was significantly (P < 0.05) inhibited by 72 h Cu depletion. Molt-3 cells required a longer period, up to 144 h, in Cu-depleted medium before growth was impaired. Raji-cell growth was not affected. These results confirmed clinical observations that T-cell functions were more sensitive to Cu deprivation than B cells. Analysis of intracellular metal levels in Molt-3 cells showed that Cu levels had been significantly lowered (P < 0.05) although Ca2+ levels were raised. Intracellular activity of the antioxidant enzyme superoxide dismutase (EC 1.15.1.1) was significantly impaired (P < 0.05) in Molt-3 cells grown in Cu-depleted medium. Activity of the mitochondrial enzyme cytochrome c oxidase (EC 1.9.3.1) was also significantly impaired (P < 0.05) by Cu depletion. Each of these findings indicates an increase in the potential for cellular damage by reduced antioxidant activity, impairment of normal mitochondrial activity and excessive Ca2+ influx. A major consequence of the type of damage occurring under these circumstances is membrane disruption. This was confirmed by scanning electron microscopy of Molt-3 cells grown under varying Cu levels.

Reactive oxygen species inducible by low-intensity laser irradiation alter DNA synthesis in the haemopoietic cell line U937.

BACKGROUND AND OBJECTIVE: The purpose of this study was to evaluate the possible role of reactive oxygen species (ROS) in mediating previously recorded alterations in DNA synthesis, inducible by low-intensity laser irradiation (LILI), in the haemopoietic cell line U937. STUDY DESIGN/MATERIALS AND METHODS: The ability of LILI (660 nm, 12 mW, 5 kHz) to induce ROS from U937 cells was assessed spectrophotometrically at energy densities (E.D.) from 1.0 to 11.5 J/cm2. In order to assess whether laser-induced ROS could alter cellular proliferation DNA synthesis was measured post-irradiation, by the incorporation of tritiated thymidine (3H-TdR) into the cells in both the presence and absence of the antioxidant catalase (CAT). RESULTS: Detectable ROS were produced post-irradiation only from the differentiated form of the cell line. Analysis by Student's t-test for unrelated groups showed a significant difference, at E.D.s 2.9 and 8.6 J/cm2, in the extent of DNA synthesis occurring in cells irradiated in the presence of CAT or in its absence. CONCLUSION: These findings demonstrate that laser-inducible ROS can mediate laser's effects on this cell line.

Oxidant-induced stress response in lymphoid cells.

The stress response to reactive oxygen species is an important defence system which can reduce their potential to induce biomolecule damage. In this investigation the effect of exposing Molt-3 lymphoblastoid cells or peripheral blood lymphocytes to a non-toxic dose of hydrogen peroxide (10 microM) was studied. Cellular response to a subsequent high dose of hydrogen peroxide (100-200 microM) was assessed by measurement of growth, viability, proliferation and DNA damage (lymphocytes only) and intracellular activities of the enzymes, superoxide dismutase, glutathione peroxidase and catalase (Molt-3 only). The results indicate that pretreatment of lymphocytes with 10 microM hydrogen peroxide can elicit a response which is protective against DNA damage normally inducible in these cells by subsequent exposure to toxic doses of hydrogen peroxide. It appears from the results with Molt-3 cells that altered activities of glutathione peroxidase may contribute to this enhanced resistance to hydrogen peroxide.

Diet and immune function.

Adequate human nutrition is essential to maintain all normal physiological functions including defence of the self. Controversy exists about the precise constituents of diets optimal for all stages in the human life-span. Dietary composition may also need to be altered under such stressful conditions as infection or recovery from major surgery. Understanding how specific nutrients can alter immune responses may add to the quality of human lives by minimising the impact of disease morbidity and mortality. Dietary modification also offers hope of new therapeutic regimens for human diseases.

DNA damage in human T-lymphoblastoid cell line Molt-3 induced by reactive oxygen species.

In the course of studying the effect of reactive oxygen species such as superoxide anion (O2-), hydrogen peroxide (H2O2) on oxidant-sensitive human T-lymphoblastoid cell line Molt-3, O2- has been generated by the interaction of xanthine with xanthine oxidase (XOD). To confirm that H2O2 is a key intermediate for inducing DNA single-strand breaks in Molt-3 cells, studies have been carried out with pure H2O2. In the presence of xanthine + XOD or H2O2 the amount of DNA single-strand breaks has been found to increase as a function of the O2- and H2O2 concentration. Data from studies with antioxidants such as superoxide dismutase and catalase supports a mechanism of DNA damage dependent on the presence of H2O2 in Molt-3 cells. Molt-3 cells are CD4+ and sensitive to reactive oxygen stress and therefore, could be an ideal cell line for determining the relationship between oxidative stress and various diseases such as acquired immunodeficiency syndrome (AIDS).