Association among 1267 A/G HSP70-2, -308 G/A TNF-α polymorphisms and pro-inflammatory plasma mediators in old ZincAge population.

Proinflammatory cytokines and heat shock proteins play relevant roles in the pathogenesis of inflammatory diseases. We investigated whether Hsp70 1267 A/G and TNF-α -308 G/A polymorphisms are associated with proinflammatory mediators, zinc status and laboratory parameters in 1,078 healthy elderly from ZincAge study. Hsp70 1267 A/G genotype and allele distribution were similar among various European countries, while a TNF-α genetic heterogeneity was observed between the Northern and the Southern European populations, with a major frequency of the -308 A variant in France, Germany and Poland. We used linear regression models to test additive, dominant or recessive associations of each SNP with proinflammatory mediators, laboratory parameters, metallothioneins and zinc status. Hsp70 1267 A/G SNP, but not TNF-α -308 G/A SNP, influences TNF-α and IL-6 plasma levels under additive, dominant and recessive models (for TNF-α only). An association between Hsp70 1267 A/G SNP and zinc plasma levels was observed in the dominant model. In particular, G allele carriers showed increased circulating pro-inflammatory cytokines and zinc. Moreover, both these SNPs affect creatinine levels suggesting a possible influence on renal function. In conclusion, Hsp70 1267 A/G SNP is associated with pro-inflammatory cytokine production in healthy elderly and might represent a possible determinant of individual susceptibility to inflammatory diseases.

Zinc, metallothioneins and immunosenescence.

Ageing is an inevitable biological process with gradual and spontaneous biochemical and physiological changes and increased susceptibility to diseases. The nutritional factor, zinc, may remodel these changes with subsequent healthy ageing, because zinc improves the inflammatory/immune response as shown by in vitro and in vivo studies. The intracellular zinc homeostasis is regulated by buffering metallothioneins (MT) and zinc transporters (ZnT and ZIP families) that mediate the intracellular zinc signalling assigning to zinc a role of 'second messenger'. In ageing, the intracellular zinc homeostasis is altered, because high MT are unable to release zinc and some zinc transporters deputed to zinc influx (ZIP family) are defective leading to low intracellular zinc content for the immune efficiency. Physiological zinc supplementation in the elderly improves these functions. However, the choice of old subjects for zinc supplementation has to be performed in relation to the specific genetic background of MT and IL-6, because the latter is involved both in MTmRNA and in intracellular zinc homeostasis. Old subjects carrying GG genotypes (C-carriers) in the IL-6-174G/C locus display high IL-6, low intracellular zinc content, impaired innate immunity and enhanced MT. Old subjects carrying GC and CC genotypes (C+carriers) display satisfactory intracellular zinc content, adequate innate immunity and are more prone to reach longevity. Zinc supplementation in old C-carriers restores natural killer cell cytotoxicity and zinc status. The genetic variations of the IL-6-174G/C locus when associated with those of the MT1A+647A/C locus are useful tools for the choice of old people for zinc supplementation.

Effects of interleukin-6 -174C/G and metallothionein 1A +647A/C single-nucleotide polymorphisms on zinc-regulated gene expression in ageing.

Decreased zinc ion availability in ageing is associated with altered immune response. One of the main regulators of zinc availability is metallothionein. Metallothionein induction is under the control of interleukin-6, a pro-inflammatory cytokine whose production is associated with poor ageing. The production of interleukin-6 is controlled, in part, by variability in the -174 nucleotide position. Under conditions of chronic inflammation, such as in ageing, zinc release by metallothionein is limited and may reduce zinc availability. Understanding the precise nature of the interactions between interleukin-6 and metallothioneins will aid in identifying individuals who are at risk of zinc deficiency. In the current study, we used gene arrays to investigate the effects of in vitro zinc supplementation on gene expression in elderly donors with described interleukin-6 and metallothionein 1a polymorphisms. Ingenuity Pathway Analysis identified several zinc-responsive genetic networks uniquely regulated only in elderly individuals with the pro-inflammatory interleukin-6 polymorphism. These include zinc-dependent decreased transcription of pro-inflammatory cytokines and alterations in metabolic regulatory pathways. The genomic effects of zinc increased in significance in the presence of the metallothionein 1a +647 C/A transition, suggesting that the interleukin-6 and metallothionein 1a genes act in a concerted manner to control zinc-regulated gene expression.

+647 A/C and +1245 MT1A polymorphisms in the susceptibility of diabetes mellitus and cardiovascular complications.

Diabetes mellitus is a chronic disease characterized by an overproduction of reactive oxygen species, which perturbs zinc metabolism and promotes the onset of cardiovascular disease (CVD) in diabetic patients. Metallothioneins (MT) are cysteine-rich metal-binding proteins which, by means of their antioxidant and zinc-buffering properties, might prevent the development of diabetic cardiovascular complications. A recent investigation shows that a polymorphism (+647 A/C) in the human MT-1A gene, affects the intracellular zinc ion release (iZnR) from the proteins and is associated with longevity in Italian population. The aim of the present study is to assess the involvement of +647 A/C and +1245 A/G MT1A polymorphisms with the susceptibility to type 2 diabetes (DM2) and cardiovascular complications. The study included 694 old individuals: 242 old healthy controls, 217 DM2 patients without clinical evidence of CVD (DNC) and 235 diabetic patients with diagnosis of CVD (DCVD). +647 A/C MT1A polymorphism, but not the second SNP, was associated with DM2. C allele carriers were more prevalent in DNC and DCVD patients than in control group (OR=1.37, p=0.034; OR=1.54, p=0.002, respectively). C+ carriers was associated with higher glycemia and glycosylated hemoglobin in DCVD patients, but not in DNC or control subjects. No differences in plasma zinc, but a modulation of MT levels and iZnR in PBMCs were observed in DCVD cohort when related to +647 A/C MT1A polymorphism. In summary, this work provides novel evidence on the association of the +647 A/C MT1A polymorphism with DM2. Moreover, C+ carriers in DCVD patients presented a worse glycemic control, a reduced iZnR and a higher MT levels, suggesting a possible role of MT in diabetic cardiovascular complications.

The -308G/A polymorphism of TNF-alpha influences immunological parameters in old subjects affected by infectious diseases.

Abnormal increments of pro-inflammatory cytokines (IL-6 and TNF-alpha) characterize the outbreak of infectious diseases, which are the major cause of death in the elderly. A counterbalance to the inflammation is exerted by IL-10 with an inhibitory role on TNF-alpha production. As is well known, some cytokine gene polymorphisms influence the cytokine production, playing a role as susceptibility or resistance factors against immune-mediated and infectious disease. Genetic variations in the -308A/G locus for TNF-alpha seems to affect the clinical outcome of some infectious diseases. In fact, the -308A allele is associated with severe septic shock and death. On this basis, we have screened healthy old subjects, nonagenarians and old patients affected by the acute phase of chronic obstructive bronchitis and bronchopneumonia of bacteria origin for the -308G/A locus (PCR-RFLP). Subjects are grouped in A+ (AG, AA genotypes) and A- (GG genotype) and data on IL-6, TNF-alpha, IL-10, NK cell cytotoxicity, zinc and metallothioneins (MTs) gene expression (RT-PCR) were stratified according to different TNF-alpha genotypes. The frequency of the A allele was increased in infected patients in comparison with healthy old controls. No differences existed between A+ and A- young adult, old and nonagenarian controls in tested parameters. Conversely, A+-infected patients displayed elevated IL-6, TNF-alpha and MTmRNA, low IL-10 coupled with impaired NK cell cytotoxicity and lower zinc ion than A- patients. However, the data reported are gender independent. Therefore, the -308A polymorphism at the locus of TNF-alpha may be one of the susceptibility factor for infectious diseases in old persons, particularly considering its association to the increased release of pro-inflammatory cytokines and to the reduction of zinc release and MTs synthesis involved in the control of the inflammatory response. These data strongly suggest that the genetic screening of the -308G/A polymorphism may be a valid tool for identification of subjects needing a more appropriate therapy when affected by acute and/or recurrent infectious diseases.

Different age-related effects of thymectomy in myasthenia gravis: role of thymoma, zinc, thymulin, IL-2 and IL-6.

Different age-related immune pathogenetic mechanisms in myasthenia gravis (MG) have been suggested because of restoration after thymectomy (Tx) of altered zinc, thymulin (TH) and T-cell subsets exclusively in early-onset patients (younger <50 years), not in late-onset patients (older >50 years). In this context interleukin-2 (IL-2), interleukin-6 (IL-6) and thymoma are crucial because both involved in MG pathogenesis and correlated with acetylcholine receptors (AchRs) Ab production. Moreover, IL-2 and IL-6 are zinc-dependent, are altered in aging and related with zinc and TH age-dependent declines. Moreover, zinc is relevant for immune efficiency. In order to confirm these different age-related pathogenetic mechanisms further, the role of thymoma, zinc, TH, IL-2 and IL-6 is studied in MG patients with generalized MG with and without thymoma before and 1 month and 1 year after Tx. The high IL-2, IL-6, zinc, and AChR Ab levels observed before Tx are significantly correlated each other in younger MG patients (<50 years) independently by thymoma and in older MG patients (>50 years) with thymoma. No correlations exist in older MG patients without thymoma. Thymulin is not correlated with other parameters considered to be both in younger and older MG patients independently by the thymoma. Thymectomy restores zinc; immune parameters and AChR Ab are exclusively in the younger group, not in the older one. These findings suggest that IL-2 and IL-6, via zinc, rather than TH, may be involved in different age-related pathogenetic mechanisms mainly in early-onset MG. By contrast, thymoma may be involved in MG etiology in late-onset representing, as such, a useful discriminant tool for MG etiology between early and late-onset MG patients. Because autoimmune phenomena may rise in aging, a parallelism with altered immune functions during aging is discussed.

Zinc, metallothioneins, immune responses, survival and ageing.

Zinc is required as a catalytic, structural (zinc fingers) and regulatory ion. In this capacity, it is involved in many homeostatic mechanisms, including immune responses. Metallothioneins (MTs) may play key roles because of their preferential binding to zinc especially in ageing. MTs protect from oxidative damage during transient stress conditions at young-adult age. This protection no longer exists in ageing and in age-related diseases (cancer and infections) because the stress condition is constant. As such, MTs may constantly deplete zinc from plasma and tissues. This phenomenon causes increased MTs levels on the one hand, but on the other hand induces low zinc ion bioavailability for normal immune responses. This may be particularly relevant for thymic functions and natural killer activity. Therefore MTs which are protective in young-adults may become dangerous in immune responses during ageing. Physiological supplementation of zinc in ageing corrects central and peripheral immune defects, resulting prolonged survival and decreased mortality (50%) from infections and tumours, especially during middle age. Because of increased MT gene expression and protein levels in the liver and atrophic thymus of old mice, MTs are proposed as genetic markers of immunosenescence.

Melatonin administration in tumor-bearing mice (intact and pinealectomized) in relation to stress, zinc, thymulin and IL-2.

Melatonin (MEL) may counteract tumors through a direct oncostatic role. MEL is also an antistress agent with immunoenhancing properties against tumors due to a suppressive role of MEL on corticosterone release. Rotational stress (RS) (spatial disorientation) facilitates metastasis progression in mice. Also, MEL counteracts tumors because of its influence on immune responses via the metabolic zinc pool, which, is reduced in tumors and stress. Zinc is required for normal thymic endocrine activity (i.e. thymulin) and interleukin-2 (IL-2) production. Because in vivo data is still controversial, exogenous MEL treatment (22 days in drinking water) in both intact and pinealectomized (px) mice bearing Lewis lung carcinoma leads to significant decrements of metastasis volume, restoration of the negative crude zinc balance, recovery of thymulin activity and increment of IL-2 exclusively in intact and px tumor bearing mice subjected to RS. Significant inverse correlations are found in both stressed intact and px tumor bearing mice after MEL treatment between zinc and corticosterone (r = 0.78, P < 0.01; r = 0.80, P < 0.01, respectively). Positive correlations between zinc and IL-2 (r = 0.75, P < 0.01; r = 0.73, P < 0.01, respectively) or thymulin (r = 0.75, P < 0.01; r = 0.82, P < 0.01, respectively) are observed in same models of mice. These findings suggest a MEL action to decrease metastasis mediated by a possible interplay between zinc and MEL, via corticosterone, with consequent restoration of thymic efficiency and IL-2 production. MEL as an antistress agent with immunoenhancing properties in cancer deserves further consideration.nuclear factor-kb; POMC, proopiomelanocortin; Px, pinealectomized mice; RIA, radioimmunoassay; RS, rotational stress; SDI, stressed intact mice; SDPx, stressed pinealectomized mice; TNF-alpha, tumor necrosis factor-alpha; ZnFTS, active zinc-bound thymulin; ZnFTS + FTS, total thymulin.

Role of zinc and alpha2 macroglobulin on thymic endocrine activity and on peripheral immune efficiency (natural killer activity and interleukin 2) in cervical carcinoma.

Decreased natural killer (NK) activity as well as interleukin 2 (IL-2) are risk factors for the progression of cervical carcinoma. NK activity and IL-2 may be thymus controlled. Plasma levels of active thymulin, a zinc-dependent thymic hormone (ZnFTS), are reduced in cancer because of the low peripheral zinc bioavailability. Zinc and thymulin are relevant for normal immune functions. Alpha2-macroglobulin is an inhibitor of matrix metalloproteases (MMPs) against invasive tumour proliferation. Because alpha2-macroglobulin has a binding affinity (Kd) for zinc that is higher than does thymulin, it may play a key role in immune efficiency in cancer. Plasma samples of 22 patients (age range 35-60 years) with locally advanced squamous cervical carcinoma and with FIGO stage Ib2-IIb were examined. They showed reduced active thymulin, decreased NK activity and IL-2 production, increased soluble IL-2 receptor (sIL-2R) and augmented alpha2-macroglobulin in the circulation, whereas plasma zinc levels were within the normal range for age. Significant positive correlations were found between zinc or active thymulin and alpha2-macroglobulin (r = 0.75, P < 0.01, r = 0.78, P < 0.01, respectively) in cancer patients. In vitro zinc increases IL-2 production from peripheral blood mononuclear cells (PBMCs) of cancer patients. These data suggest that an increase in alpha2-macroglobulin, which competes with thymulin for zinc binding, may be involved in causing a thymulin deficit with a consequent decrease of IL-2 and NK cytotoxicity. Thus, physiological zinc treatment in cervical carcinoma maybe restores impaired central and peripheral immune efficiency.