Zinc-bound metallothioneins as potential biological markers of ageing.

Metallothioneins (MTs) (I+II) play pivotal roles in metal-related cell homeostasis because of their high affinity for metals forming clusters. The main functional role of MTs is to sequester and/or dispense zinc participating in zinc homeostasis. Consistent with this role, MT gene expression is transcriptionally induced by a variety of stressing agents to protect cells from reactive oxygen species. In order to accomplish this task, MTs induce the secretion of pro-inflammatory cytokines by immune and brain cells, such as astrocytes, for a prompt response against oxidative stress. These cytokines are in turn involved in new synthesis of MTs in the liver and brain. Such protective mechanism occurs in the young-adult age, when stresses are transient. Stress-like condition is instead constant in the old age, and this causes continuous stealing of intracellular zinc by MTs and consequent low bioavailability of zinc ions for immune, endocrine, and cerebral functions. Therefore, a protective role of zinc-bound MTs (I+II) during ageing can be questioned. Because free zinc ions are required for optimal efficiency of the immune-endocrine-nervous network, zinc-bound MTs (I+II) may play a different role during ageing, switching from a protective to a deleterious one in immune, endocrine, and cerebral activities. Physiological zinc supply, performed cautiously, can correct deficiencies in the immune-neuroendocrine network and can improve cognitive performances during ageing and accelerated ageing. Altogether these data indicate that zinc-bound MTs (I+II) can be considered as novel potential markers of ageing.

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 and immunoresistance to infection in aging: new biological tools.

Infections can cause mortality when the immune system is damaged. The catalytic, structural (in zinc-finger proteins) and regulatory roles of zinc mean that this ion is involved in the maintenance of an effective immune response. Both zinc deficiency and impaired cell-mediated immunity combine during aging to result in increased susceptibility to infection. Dietary supplementation with the recommended daily allowance of zinc for between one and two months decreases the incidence of infection and increases the survival rate following infection in the elderly. This article reviews the biochemical pathways through which zinc might act to increase immunoresistance to infection in the elderly.

Therapeutic application of zinc in human immunodeficiency virus against opportunistic infections.

The relevance of zinc in resistance to infections by virus, fungi and bacteria is recognized because of its pivotal role in the efficiency of the entire immune system, in particular in conferring biological activity to a thymic hormone called thymulin, which has differentiation properties on T-cell lines. In infection with human immunodeficiency virus (HIV), the zinc-bound form of thymulin (active thymulin, ZnFTS) is strongly reduced in stage IV of the disease (Centers for Disease Control and Prevention classification) with concomitant decrements in CD4(+) cell count and zincemia values. The zinc-unbound form of thymulin (inactive thymulin, FTS) is, in contrast, very high. The in vitro addition of zinc to plasma samples induces a recovery of the thymulin active form, suggesting low zinc bioavailability as the cause of impaired thymic functions with consequent CD4(+) depletion. An analysis of risk factors for the incidence of recidivism opportunistic infections shows CD4(+) depletion and zinc deficiency to have significant scores. Supplementation with zinc for 1 mo (45 mg Zn(2+)/d) associated with zidovudine (AZT) therapy in stage IV induces recovery of active zinc-bound thymulin, of zincemia, of CD4(+) cells with concomitant reduction (50%) of recidivism opportunistic infections compared with the AZT-treated group. Complete disappearance of recidivism by Candida aesophagea or Pneumocystis carinii is observed after supplementation with zinc. The relative risk factors (CD4(+) depletion and zinc-deficiency) have lower scores in the HIV-positive zinc-treated group, confirming, as such, the relevance of zinc in opportunistic infections that involve extracellular matrix. Such an assumption is indirectly confirmed with new HAART, where no opportunistic infections occur. Indeed, HIV RNA is inversely correlated with both CD4(+) and zincemia values (r = -0.73, P<0.01) in HAART-treated subjects. Lower scores for the same relative factors for the appearance of opportunistic infections are present in HAART-treated subjects compared with those treated with AZT. These findings, on the one hand, show the poor efficacy of AZT therapy compared with HAART therapy for the progression of HIV, but on the other hand, they suggest that the lack of occurrence of opportunistic infections by HAART may also result from major zinc bioavailability. This further supports the key role played by zinc against opportunistic infections in HIV with a possible independent effect by either HIV or the pathogens involved.

Zinc, infections and immunosenescence.

Infections may cause mortality in old age due to damaged immune responses. As zinc is required as a catalyst, structural (zinc fingers) and regulatory ion, it is involved in many biological functions, including immune responses. Low zinc ion bioavailability and impaired cell-mediated immunity are common in ageing and may be restored by physiological supplementation with zinc for 1-2 months, impacting upon morbidity and survival. This article reviews the role of zinc in immune efficacy during ageing, and also describes the main biochemical pathways involved in the role of zinc in resistance to infections in ageing in order to better understand the possible causes of immunosenescence.

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.

Contribution of zinc to reduce CD4+ risk factor for 'severe' infection relapse in aging: parallelism with HIV.

Aging and HIV have parallelism in immunodeficiency status because of the appearance of infections or relapse leading to death in both conditions. HIV-RNA is predictor for HIV progression correlated with CD4+ depletion. CD4+ and plasma zinc levels (zincaemia) may be predictors for infections relapse in aging because of zinc relevance for normal immune efficiency against infections and for CD4+ growth. Moreover, zincaemia decreases in aging and infection. A total of 67 elderly subjects affected by infections resistant to antibiotic therapy were recruited. A total of 28 HIV+ subjects with HAART therapy were also used. CD4+ depletion (507 mm3) and zincaemia deficiency (76 microg/dl), as compared to CD4+ (700-1100 mm3) and zincaemia (85-100 microg/dl; age 40-75 years) normal ranges, are possible limits (Cox hazard regression) for severe infections relapse, such as chronic obstructive bronchitis and bronchopneumonia by bacteria or Candida complication, in aging. CD4+ and zincaemia values are within the lower limits of normal range in urinary tract infections. Zincaemia and HIV-RNA or CD4+ are inversely correlated (r = 0.57 and r = 0.72, respectively) in HIV+ HAART treated subjects. Consequently there is no appearance of opportunistic infections. Parallelism between aging and HIV may exist because of the resemblance in marked zinc deficiency and CD4+ depletion with high scores in relative risks for severe infections relapse. Supplementing zinc (12 mg Zn++/day) for one month in infected elderly subjects and HAART therapy in HIV+ subjects reduces risk scores in CD4+ and zincaemia deficiencies for infections relapse, suggesting that the zinc beneficial effect may be independent either by HIV-virus or pathogen agents involved. While HAART may reduce the occurrence of opportunistic infections in HIV by means of also major zinc bioavailability, supplementing zinc can be recommended in elderly people as resistance to infections. Since zinc deficiency is correlated with CD4+ depletion, this latter may also be good diagnostic marker to detect 'clear immunodeficiency' in aging, as in HIV condition.

Cisplatin-based polychemotherapy reduces the natural cytotoxicity of peripheral blood mononuclear cells in patients with advanced ovarian carcinoma and their in vitro responsiveness to interleukin-12 incubation.

BACKGROUND: The aim of the current study was to evaluate the in vitro effect of IL-12 on the natural cytotoxicity of peripheral blood mononuclear cells (PBMCs) obtained from patients who underwent adjuvant-based cisplatin polychemotherapy for advanced ovarian carcinoma. The authors also investigated amifostine, a cytoprotective agent that appears to protect against chemotherapy damage to healthy tissues, to determine its effects on natural immune function. METHODS: Twenty-one women with advanced ovarian serous cystoadenocarcinoma who underwent adjuvant cisplatin-based polychemotherapy were included in the study, and 20 normal volunteer women matched for age served as controls. Six of the 21 women who underwent polychemotherapy received 1:3 amifostine pretreatment. Blood samples were obtained immediately before the first cycle of cisplatin-based polychemotherapy and within 24 hours after the completion of polychemotherapy infusion to evaluate the natural cytotoxic activity of PBMCs against the K562 cell line and the in vitro responsiveness of cytotoxic cells to interleukin-12 (IL-12). RESULTS: The in vivo administration of cisplatin-based polychemotherapy significantly reduced the natural killer cytotoxicity of PBMCs toward undetectable levels (2.2+/-3.1 vs. 9.2+/-7.0 lytic units, respectively, after and before cisplatin; P < 0.01), and the in vitro exposure to IL-12 did not increase the cytolytic activity of PBMCs (1.9+/-2.1 lytic units). PBMCs from the 6 patients who received random amifostine pretreatment were shown to have retained natural killer cytotoxicity after in vivo administration of cisplatin polychemotherapy (9.7+/-6.7 vs. 9.6+/-6.0 lytic units, respectively, after and before cisplatin; P = 0.9), and the incubation with IL-12 increased cytotoxic activity (13.4+/-6.9 lytic units) toward the levels observed in PBMCs of controls (14.0+/-4.6 lytic units). CONCLUSIONS: These data suggest that cisplatin-based polychemotherapy reduces the natural cytotoxicity of PBMCs in patients with advanced ovarian carcinoma as well as their in vitro responsiveness to IL-12 incubation. Amifostine demonstrated a protective effect on natural killer cell cytotoxicity and responsiveness to IL-12 in this small cohort of patients.

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.

Presence of links between zinc and melatonin during the circadian cycle in old mice: effects on thymic endocrine activity and on the survival.

Links between zinc and melatonin in old melatonin treated mice with a reconstitution of thymic functions have been recently documented. Concomitant increments of the nocturnal peaks of zinc and melatonin, with a synchronization of their circadian patterns, are achieved in old mice after melatonin treatment. A recovery of the nocturnal peaks of thymulin plasma levels and of the number of thymulin-secreting cells with a synchronization of their circadian patterns are also achieved. The existence of significant positive correlations between melatonin and zinc and between melatonin and thymulin or the number of thymulin-secreting cells supports the presence of links between zinc and melatonin also during the circadian cycle with a beneficial effect on thymic functions. The altered circadian pattern of corticosteron in old mice is normalized by melatonin. The existence of inverse correlations between corticosteron and melatonin, between corticosteron and zinc and between corticosteron and thymulin or the number of thymulin-secreting cells during the whole circadian cycle, suggests the involvement of glucocorticoids pathway in the melatonin thymic reconstitution, via zinc. The presence of an interplay among zinc, melatonin, glucocorticoids and thymulin may be, therefore, supported during the circadian cycle. 'In vitro' experiments from old thymic explants show a direct action of zinc, rather than melatonin, on thymulin production, further suggesting that the action of melatonin on the thymic efficiency is mediated by the zinc bioavailability. The beneficial effect of the links between zinc and melatonin on thymic functions during the circadian cycle, may be extended to a prolonged survival in aging, where, however, zinc may be more involved.

Decrease in peripheral blood polymorphonuclear leukocyte chemotactic index in endometriosis: role of prostaglandin E2 release.

OBJECTIVE: To investigate the effect of disease on peripheral blood polymorphonuclear leukocyte chemotactic index and natural killer cell cytotoxicity and to provide additional information concerning the cell-mediated immune function in endometriosis. METHODS: Chemotactic index of peripheral blood polymorphonuclear leukocytes, natural killer cell activity, and plasma estradiol (E2) and plasma prostaglandin (PG) E2 levels were evaluated in 46 women who underwent laparoscopy or laparotomy for pelvic pain, infertility, and/or benign adnexal masses. RESULTS: The 20 women (43%) with endometriosis showed a decrease in peripheral blood polymorphonuclear leukocyte chemotactic index, related to advanced disease stage (P < .001). A significant inverse correlation was observed between plasma PGE2 levels and chemotactic index in stage III and IV endometriosis (r = -.73, P = .004). Similarly, natural cytotoxicity was decreased significantly with respect to the stage of endometriosis (P = .004) and related inversely to plasma PGE2 levels (r = -.74, P = .003). A direct relationship was observed between PGE2 and plasma E2 levels (r = .59, P = .006). CONCLUSION: Advanced endometriosis is associated with decreased peripheral blood polymorphonuclear leukocyte chemotactic index and natural killer cytotoxicity, which may be related to plasma PGE2 and E2 levels.

Zinc, T-cell pathways, aging: role of metallothioneins.

Zinc is an essential trace element for many biological functions, including immune functions. Indeed zinc is required for the biological activity of a thymic hormone, called thymulin in its zinc-bound form, important for the maturation and differentiation of T-cells. With advancing age zinc, thymic functions and peripheral immune efficiency show a progressive decline. Supplementing zinc in old age restores them. Zinc is also relevant for liver extrathymic T-cell pathway, being preeminent in old age. Since zinc is also required for metallothioneins (MTs) biological functions, binding zinc with high affinity, aim of the present article is to summarize findings from our laboratory regarding the role of zinc on T-cell pathways, investigating also the possible cause of thymic involution and impaired liver extrathymic T-cell pathway in aging. Partial hepatectomy and liver regeneration are good models for this aim because of the likeness with aging for many immune functions, including thymic functions. MTs levels are increased, other than into the liver, also into the thymus during aging and in young hepatectomized (pHx) mice as compared to young sham controls. MTs may be one of the possible causes of reduced thymic efficiency and impaired liver extrathymic T-cell pathway in old age because of their higher zinc binding affinity rather than thymulin with consequent reduction of the free quota of zinc available for normal cell-mediated immunity. Following that, MTs may contribute to thymic involution and impaired peripheral immune efficiency in aging and in young pHx mice with different roles during the whole life of an organism: protective in young-adult age which may became, at least, dangerous for immune responses in aging. In order to limit or avoid this latter MTs possible role in aging, supplementing physiological zinc may be useful to improve immune responses in old age because of no interference of endogenous zinc on already high thymus MTs levels, but with caution for competition phenomena with copper, as documented in old mice and in syndrome of accelerate aging.

Zinc and metallothioneins on cellular immune effectiveness during liver regeneration in young and old mice.

Partial hepatectomy in young mice (pHx) induces thymic atrophy, disregulation of thymocytes subsets and a strong accumulation of zinc in thymic tissue after 1-2 days of liver regeneration. Zinc is relevant for good immune functioning. Restoration of zinc into both the thymus and thymocytes subsets in the late period of liver regeneration is observed in young pHx mice. These findings have suggested a link between the thymus and the liver influencing T-cell functions and involving zinc. This kind of link could be relevant in aging because thymic involution, negative crude zinc balance and crippled immune functions are constant events. The preminence of a liver extrathymic T-cell pathway after pHx or during aging has been suggested. Thus the study of pHx in young and old mice may offer a good model to better understand the role played both by thymic involution and by liver extrathymic T-cell pathway and the role of zinc in these physiological processes during aging. Young pHx mice after 1-2 days of liver regeneration show: reduced thymic endocrine activity, increment of double negative (DN) thymocytes subsets, impairment of peripheral immune efficiency (PHA, NK activity and IL-2) and negative crude zinc balance, which are all restored in the late period of liver regeneration. By contrast the thymic and peripheral immune defects and the negative crude zinc balance, already present in old sham mice, are not modified during liver regeneration in old pHx mice. Circulating leukocytes and lymphocytes are not significantly modified both in young and old pHx mice as compared to respective sham controls. Zinc may also be crucial for extrathymic T-cells pathway, being preminent in aging, rather than in young age, due to its metallothioneins (MT) binding capacity. MT are significantly increased in young pHx and in aging inducing a low zinc-free quota for thymic and peripheral immune efficiency in young pHx mice, and for extrathymic T-cell pathway, in old age. Thus low zinc bioavailability, due to MT, may play a pivotal role, not only for thymocytes but also for liver extrathymic T-cell pathway.

Natural cytotoxicity and GnRH agonist administration in advanced endometriosis: positive modulation on natural killer activity.

OBJECTIVE: To investigate the effects of pharmacologic suppression of ovarian function on the immune system, with respect to the clinical outcome of endometriosis and the possibility of an immunoendocrine combined treatment. METHODS: After informed consent, 25 of 37 patients with revised American Fertility Society stage III and IV endometriosis who underwent postoperative medical treatment were selected and enrolled for this immunoendocrine longitudinal study. Medical treatment consisted of tryptorelinum depot injection, 3.75 mg/month for 24 weeks. Blood samples were collected before the first injection in the early follicular phase, day 2-3 of the cycle, and during medical treatment (every 4 weeks) and follow-up (every 6 months). At the end of the study, we had ten blood samples per patient to evaluate the cytotoxic activity, the number of natural killer cells, and the serum levels of estradiol. Natural killer activity was determined against the K562 cell line by target cell retention of the fluorescent dye carboxyfluorescein diacetate. RESULTS: A positive immunomodulating effect was observed during GnRH agonist administration. In particular, a significant progressive increase in natural killer cell activity was defined within the first 12 weeks of medical treatment; after three injections, we observed the highest values of cytotoxicity, with a median of 7.1 lytic units (range 0.3-14.0; P = .02). Natural cytotoxicity then decreased toward a plateau, which persisted during therapy completation and follow-up, with slight fluctuations. In patients who had recurrence, the values of natural killer cell activity were constantly lower than those in patients with disease-free follow-up, particularly within the first 12 weeks of medical treatment.

The zinc pool is involved in the immune-reconstituting effect of melatonin in pinealectomized mice.

Melatonin (MEL) affects the immune system by direct or indirect mechanisms. An involvement of the zinc pool in the immune-reconstituting effect of MEL in old mice has recently been documented. An altered zinc turnover and impaired immune functions are also evident in pinealectomized (px) mice. The present work investigates further the effect of "physiological" doses of MEL on the zinc pool and on thymic and peripheral immune functions in px mice. Daily injections of MEL (100 micrograms/mouse) for 1 month in px mice restored the crude zinc balance from negative to positive values. Thymic and peripheral immune functions, including plasma levels of interleukin-2, also recovered. The nontoxic effect of MEL on immune functions was observed in sham-operated mice. Because the half-life of MEL is very short (12 min), interruption of MEL treatment in px mice resulted, after 1 month, in a renewed negative crude zinc balance and a regression of immune functions. Both the zinc pool and immunological parameters were restored by 30 further days of MEL treatment. The existence of a significant correlation between zinc and thymic hormone after both cycles of MEL treatment clearly shows an involvement of the zinc pool in the immunoenhancing effects of MEL and thus suggests an inter-relationship between zinc and MEL in px mice. Moreover, the existence of significant positive correlations between zinc or thymulin and interleukin-2 suggests that interleukin-2 may participate in the action of MEL, via zinc, on thymic functions in px MEL-treated mice.

Relationship between 17-beta-estradiol and prolactin in the regulation of natural killer cell activity during progression of endometriosis.

Endometriosis is an estrogen-dependent disease affecting women during their reproductive years. An abnormal immune function and, in particular, a decreased natural killer (NK) cell activity have been found in endometriosis, suggesting a role of the immune system in the pathophysiology of the disease. We have recently evidenced a significant inverse relationship between 17-beta-estradiol plasma levels and NK cytotoxicity in endometriosis patients. In this study we have investigated the combined role of 17-beta-estradiol (E2) and prolactin (PRL) in the regulation of NK cell activity during the progression of endometriosis, by evaluating the correlation among E2, PRL, and other immunomodulating neurohormones on both the cytotoxic activity and the number of NK cells in women at different stages of endometriosis. The early stages (I/II) of endometriosis are characterized by increased plasma levels of either E2 or PRL without significant alterations of NK cell activity in comparison with healthy subjects. The progression to advanced stages (III/IV) of the disease is associated with a further increase of E2 levels, a decrease of PRL plasma concentrations (with an increase of E2/PRL ratio), and an impairment of NK cytotoxicity. The plasma levels of both E2 and PRL and the E2/PRL ratio are significantly correlated with the values of NK cytotoxicity in advanced stages of endometriosis. Either the absolute number or the relative percentage of CD16+ or CD56+ peripheral lymphocytes are not significantly different between patients at either stages I/II or III/IV and healthy controls. Plasma levels of progesterone (P) and luteinizing hormone (LH), are not significantly changed in different stages of endometriosis with respect to healthy controls. The significant decrease of follicle-stimulating hormone (FSH) plasma levels found in either stages I/II or III/IV endometriosis patients is not correlated with the NK cell activity. In conclusion, at advanced stages of endometriosis the impairment of NK cell activity occurs with increased E2, and decreased PRL plasma levels. Additional studies are required to determine whether the E2/PRL ratio represents a possible biochemical marker of endometriosis.

Reversibility of the thymic involution and of age-related peripheral immune dysfunctions by zinc supplementation in old mice.

With advanced ageing the zinc pool undergoes progressive reduction as shown by the low zinc plasma levels and the negative crude zinc balance, both in humans and in rodents. It has been suggested that such zinc deficiency might be involved in many age-related immunological dysfunctions, including thymic failure. The relevance of zinc for good functioning of the entire immune system is, at present, well documented. In particular, zinc is required to confer biological activity to one of the best-known thymic peptides, thymulin, which is responsible for cell-mediated immunity. In deep zinc deficiencies, in humans and other animals, the low thymulin levels are due not to a primary failure of the thymus, but to a reduced peripheral saturation of thymic hormones by zinc ions. In aged mice both a reduced peripheral saturation of the hormone and a decreased production by the thymus were present. Oral zinc supplementation in old mice (22 months old) for 1 month induced a complete recovery of crude zinc balance from negative (-1.82) to positive values (+1.47), similar to those of young animals (+1.67). A full recovery of thymic functions with a regrowth of the organ and a partial restoration of the peripheral immune efficiency, as measured by mitogen responsiveness (PHA and ConA) and natural killer cell (NK) activity, were observed after zinc supplementation. These findings clearly pin-point for relevance of zinc for immune efficiency and suggest that the age-related thymic involution and peripheral immunological dysfunctions are not intrinsic and irreversible events but are largely dependent on the altered zinc pool.

MIB 1 immunostaining in stage I squamous cervical carcinoma: relationship with natural killer cell activity.

The aim of the study was to investigate the prognostic significance of estimating tumor cell proliferation in stage I cervical squamous carcinoma by analyzing MIB 1 immunostaining with respect to the lesion size, lymphatic spread, and clinical outcome. A possible relationship between MIB 1 index and natural killer activity was also discussed. The medical records of 34 patients with stage I squamous cervical carcinoma who had undergone primary radical surgery at the Institute of Gynecologic and Obstetrics, Ancona University, between 1988 and 1993, were recruited from our series of 57 consecutive cases and reviewed. Thirty-one patients were considered eligible for the study and evaluated for age, demographic characteristics, tumor histologic grade, tumor size, lymphatic spread, and adjuvant radiotherapy. The expression of primary tumor proliferation related to Ki67 antigen was immunohistochemically evaluated by monoclonal MIB 1 antibody (Immunotech, Marseille Cedex, France) on microwave oven-processed formalin-fixed paraffin-embedded tissue. The basal natural killer cell activity of peripheral blood lymphocytes was evaluated against K562 cell line and expressed in lytic units for each patient. The MIB 1 immunostaining was significantly related with tumor size (P = 0.001) and lymphatic spread (P = 0.009); in contrast, there was no relationship between grade of histologic differentiation and MIB 1 immunostaining. The Cox proportional hazards analysis showed a significant independent relationship between MIB 1 immunostaining and disease-free survival (P = 0.004). The analysis of natural cytotoxicity defined a significant inverse relationship between peripheral blood lymphocyte's natural killer activity and tumor MIB 1 immunostaining (r = -0.07, with P = 0.03). Our data defined the prognostic significance of tumor cell proliferation immunostaining, an interesting parameter correlated with the disease-free survival in locally advanced cervical carcinoma. The relationship between MIB 1 index and natural killer activity is interesting; natural cytotoxicity seems to be altered in the host with respect to the cervical carcinoma characteristics.

Natural killer activity in stage III and IV endometriosis: impaired cytotoxicity and retained lymphokine responsiveness of natural killer cells.

Our objective was to investigate the role of estrogens in the development and progression of endometriosis, and evaluate the in vitro boosting effect of lymphokines on the activity of natural killer cells from endometriosis patients, with respect to the estradiol concentrations. Natural killer activity of peripheral blood was evaluated in 42 endometriosis patients who underwent laparoscopy for pelvic pain, infertility and benign adnexal masses, and it was correlated with serum estradiol levels. Twenty-five women with moderate and severe disease were re-evaluated for immune and endocrine parameters 4-8 weeks after surgery, before any specific adjuvant medical treatment, and analyzed for in vitro responsiveness of cytotoxic cells to interferon (IFN) alpha 2 beta and interleukin-2 (IL-2) incubation. Patients with moderate and severe endometriosis showed a significant decrease of natural cytotoxicity when compared with patients with mild and minimal disease (p = 0.01). The decrease of immune reactivity was independent of a reduced representation of natural killer cells, and persisted after surgical removal of all macroscopic endometriosis foci. A significant inverse relationship was observed between natural killer activity and serum estradiol levels, which resulted in moderate and severe disease (r = -0.4, p = 0.009) but not in stages I and II. The in vitro responsiveness of cytotoxic cells to lymphokine incubation was preserved; both IFN alpha 2 beta and IL-2 were able to increase the cytotoxicity of natural killer cells significantly from advanced-stage patients (p = 0.014 and p = 0.006 for IFN alpha 2 beta and IL-2 respectively).(ABSTRACT TRUNCATED AT 250 WORDS)

HPV DNA positivity and natural killer cell activity in the clinical outcome of mild cervical dysplasia: integration between virus and immune system.

The objective was to examine the prevalence of human papillomavirus (HPV) DNA infection in mild cervical dysplasia and to evaluate longitudinally the persistence of HPV DNA positivity in an observational study, aiming at identifying the role of peripheral blood lymphocyte natural killer activity in the natural history of dysplastic disease. Twenty-three patients with histologically proven mild cervical dysplasia were selected. The HPV DNA positivity, determined by polymerase chain reaction, and cervical dysplasia were monitored cytologically and colposcopically at the 3rd (time 1), 6th (time 2) and 12th months (time 3), and defined by biopsies for routine histology taken at times 2 and 3. For each patient included in the study, the immune reactivity was evaluated at the time of diagnosis and afterwards, longitudinally during the follow-up. The immune status analysis included T lymphocyte subsets (CD3, CD4, CD8, CD56, CD16 monoclonal antibodies by Beckton Dickinson, Mountain View, Calif., USA) and determinations of natural killer cell activity (against the sensitive cell line K 562). Eighteen out of the 23 women with mild cervical dysplasia (78.3%) were found positive for HPV DNA, with a significantly high representation of HPV DNA type 16 (55.6% of cases). At the end of the study, 12 out of 18 HPV-DNA-positive women became negative (defined by two or more negative tests) for the original HPV DNA type, with 66.7% of spontaneous HPV DNA negativization rate (p = 0.6).(ABSTRACT TRUNCATED AT 250 WORDS)