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The Natural Killer cell maturation from CD34(+) hematopoietic cell precursors is a complex process that requires the synergistic effect of different cytokines and growth factors. Although there have been a number of important advances in our understanding of the Natural Killer differentiation, the developmental step leading to mature Natural Killer cells is still poorly defined. We evaluated the effect of two zinc concentrations (10 and 20microM) on the kinetic of development of CD34(+) cell progenitors towards Natural Killer cells. CD34(+) cells were purified from peripheral blood and cultured in medium supplemented with interleukin-15, interleukin-7, Flt 3 ligand, and stem cell factor. CD34(+) cells underwent proliferation and progressively lost CD34 antigen and acquired a CD56(+) phenotype. These CD56(+) cells exerted cytotoxic activity and expressed the CD94 inhibitory receptor. The supplementation with zinc greatly increased both the number of cells in culture and the absolute number of CD56(+) cells. Zinc induced higher levels of cytotoxic activity and a higher number of perforin-producing and of CD94-bearing CD56(+) cells in comparison with zinc unsupplemented cultures in early stages of Natural Killer cell development. The zinc-induced changes in CD34-derived CD56(+) cells were associated with an increased expression of GATA-3, a zinc-finger transcription factor providing for maturation and activity of T and Natural Killer cells. The increase was related to a higher CD56(+) cell number (10microM zinc), or to an increased GATA-3 mRNA transcription in CD56(+) cells (20microM zinc). Our data demonstrate that zinc influences the proliferation and differentiation of CD34(+) progenitors.

Influenza is an important cause of morbidity and mortality in the elderly and influenza vaccination has shown a decreased efficacy in aged people. Both gammadelta- and alphabeta-T cell responses, which are believed to play an important role in controlling influenza infection, are impaired during aging. The aim of this study was to evaluate the ability of influenza vaccine to induce cellular immune responses mediated by gammadelta- and alphabeta-T cells in healthy elderly subjects. After influenza vaccination, an increased proportion of Vgamma9Vdelta2 T cells having reduced proliferative capacity and increased perforin production, and then a differentiated effector/memory phenotype, was present. The peripheral number and the cytokine production of gammadelta T cells were not changed. A significant decrease of CD4 and CD8 naïve T cells and a corresponding increase of CD4 and CD8 memory T cells were found. The in vitro stimulation of PBMCs from elderly subjects with influenza antigens increased their proliferative capacity and the production of both IFNgamma and IL-4. The vaccine was clinically effective, since in the outbreak period, only one influenza case was noted. The results reported in this study demonstrate the activation of both gammadelta- and alphabeta-T cell responses in healthy elderly after influenza vaccination.

Studies of DNA vaccination against HER2/neu showed the effectiveness of immunization protocols in models of transplantable or spontaneous tumors; scarce information, however, has been provided to identify the procedure of DNA administration that more effectively contributes to the activation of immune system against spontaneously arising HER2/neu-positive tumors. We compared the effectiveness of different procedures of DNA vaccine delivery (intradermic injection (ID), gene gun (GG) delivery and intramuscular injection (IM) alone or with electroporation) in a murine transgenic model of mammary carcinoma overexpressing HER2/neu. We highlighted the role of DNA delivery system in the success of DNA vaccination showing that, among the analysed methods, intramuscular injection of the vaccine, particularly when associated to electroporation, elicits a better protection against HER2/neu spontaneous tumor development inducing antibody and cell-mediated immune responsiveness against HER2/neu and a Th1 polarization of the immune response.

We performed in vitro studies to elucidate the bactericidal activity of the antibiotics in an adherent-cell biofilm model. Efficacy studies were performed in a staphylococcal central venous catheter (CVC) infection rat model. Silastic catheters were implanted into the superior cava. Via the CVC the rats were challenged with 1.0 x 10(6) CFU of a live Staphylococcus aureus strain. Twenty-four hours later, the antibiotic-lock technique was started. All animals were randomized to receive daily isotonic sodium chloride solution, quinupristin-dalfopristin (Q/D), linezolid, vancomycin, or ciprofloxacin at the minimal bactericidal concentration (MBC) and at 1,024 microg/ml in a volume of 0.1 ml that filled the CVC. The main outcome measures were MICs and MBCs for both planktonic and adherent cells, quantitative culture of the catheters and surrounding venous tissues, and quantitative peripheral blood cultures. The killing activities of all antibiotics against the adherent bacteria were at least fourfold lower than those against freely growing cells, with the exception of Q/D, which showed comparable activities against both adherent and planktonic organisms. Overall, Q/D at 1,024 microg/ml produced the greatest reduction in the number of cells recovered from the catheters, while at the same concentration, Q/D and vancomycin demonstrated higher activities than ciprofloxacin or linezolid in reducing the number of organisms recovered from the blood cultures. This study points out that treatment outcome of device-related infections cannot be predicted by the results of a standard susceptibility test such as the MIC. Our findings suggest that the clinically used antibiotics cannot eradicate the CVC infection through the antibiotic-lock technique, even at a concentration of 1,024 microg/ml.

Studies in mammals have led to the suggestion that hyperglycemia and hyperinsulinemia are important factors both in aging and in the development of cancer. Insulin/insulin-like growth factor 1 (IGF-1) signaling molecules that have been linked to longevity include DAF-2 and InR and their homologues in mammals, and inactivation of the corresponding genes is followed by increased life span in nematodes, fruit flies and mice. It is possible that the life-prolonging effects of calorie restriction are due to decreasing IGF-1 levels. A search of pharmacological modulators of insulin/IGF-1 signaling pathway (which mimetic effects of life span extending mutations or calorie restriction) could be a perspective direction in regulation of longevity. The chronic treatment of female transgenic HER-2/neu mice with metformin (100 mg/kg in drinking water) slightly decreased the food consumption but failed in reducing the body weight or temperature, slowed down the age-related rise in blood glucose and triglycerides level, as well as the age-related switch-off of estrous function, prolonged the mean life span by 8% (p < 0.05), the mean life span of last 10% survivors by 13.1%, and the maximum life span by 1 month in comparison with control mice. The demographic aging rate represented by the estimate of respective Gompertz's parameter was decreased 2.26 times. The metformin-treatment significantly decreased the incidence and size of mammary adenocarcinomas in mice and increased the mean latency of the tumors.

Resveratrol (Res) has been reported to possess cancer chemopreventive activity on the basis of its in vitro effects on tumor cells and in vivo experimental models of rodents transplanted with parental tumors or treated with carcinogens. We investigated the effects of Res on the development of mammary tumors appearing spontaneously in HER-2/neu transgenic mice at an early age. The mechanisms involved in the Res antitumor effect were evaluated by studying the immune effectiveness, tumor apoptosis and expression of mRNA and protein for HER-2/neu in tumoral mammary glands from Res-treated mice and in tumor cell lines. Res supplementation delayed the development of spontaneous mammary tumors (p < 0.001), reduced the mean number and size of mammary tumors (p < 0.0001) and diminished the number of lung metastases in HER-2/neu transgenic mice. The effects of Res were associated with downregulation of HER-2/neu gene expression and increased apoptosis both in tumoral mammary glands and in murine (N202) and human (SKBr3) tumor cell lines. Neither the basal, the IL-2-induced NK activity nor the lymphocyte number and proliferation was modified in Res-supplemented compared to control mice. Our results demonstrate that Res supplementation delays the development and reduces the metastasizing capacity of spontaneous mammary tumors in HER-2/neu transgenic mice. The antitumor effect of Res might be related to the downregulation of HER-2/neu expression and the induction of apoptosis in tumor cells., ((c) 2005 Wiley-Liss, Inc.)

BACKGROUND: In a previous study we demonstrated the existence of numerical and functional alterations of gammadelta T cells in healthy elderly. Recently, we analysed the involvement of gammadelta T lymphocytes in malignant melanoma, describing a lower frequency of circulating gammadelta T cells, an altered pattern of cytokine production, and an impaired in vitro expansion of these cells in primary cutaneous melanoma patients. METHODS: In this study we investigated the existence of numerical and functional alterations of circulating gammadelta T cells in young/adult and old melanoma patients, comparing the data obtained with age-matched healthy subjects. RESULTS: We demonstrated that the number of circulating gammadelta+ T cells was significantly and similarly reduced in young/adult and old melanoma patients and in old healthy subjects in comparison with young healthy donors. The decrease was due to a reduction of Vdelta2 T cells whereas the number of Vdelta1 T cells was not affected. A higher percentage of gammadelta+ T cells producing TNF-alpha was found in old healthy donors, whereas a reduced number of TNF-alpha-producing gammadelta+ T cells was present in melanoma patients independently by age. No significant difference was observed in IFN-gamma production. After a 10-day in vitro culture, both the percentage and the expansion index of gammadelta T cells, and in particular of Vdelta2 subset, were significantly and similarly reduced both in young/adult and old melanoma patients, and in healthy aged people, in comparison with young/adult healthy subjects. CONCLUSIONS: Our study demonstrates that the numerical and functional impairment of gammadelta T cells found in melanoma patients is not correlated with age and that it has characteristics very similar to the alterations of gammadelta T cells found in old healthy subjects. We suggest that a similar impairment of gammadelta T cell population may be related to the increased susceptibility to tumors present in the elderly as well as in the pathogenesis of malignant melanoma.

Over the last few years there has been a growing interest in geriatric oncology, mainly because of the evidence that advanced age is the greatest risk factor for the development of cancer and that, since the elderly population is rapidly expanding, so too will the number of cancer patients. This forecast necessitates the development of new and more specific strategies for the prevention and cure of cancer in the elderly and as a result an ever-increasing need for oncologists, geriatricians and researchers to work closely together. The increased incidence of cancer in elderly people has been related to the age-associated changes occurring in the immune system, the so-called immunosenescence. This phenomenon is best characterised by a remodelling of the immune system, which appears early on and progresses throughout a person's life and mainly involves a decrease in cellular functions. This review aims to provide a rationale for the development of specific immunotherapeutic and immunopreventive regimens for the elderly. We also include a discussion on the influence that immunosenescence has on the growth of tumours and the effectiveness of immunogene therapy and cancer vaccination following a brief analysis of the age-related alterations of the cell populations involved in antitumour immunity.

The increased age-associated incidence of infectious and cancer diseases has been related to the alteration of immune functioning found in the elderly (immunosenescence). The reduction of naive T cells, which determine an impaired ability to mount immune responses to new antigens, represents a hallmark of the aging process. The aim of this study was to evaluate the susceptibility to apoptosis of purified naive and memory CD4(+) T cells from peripheral blood of healthy people ranging in age from 20 to 98 years. Two mechanisms of T cell elimination by apoptosis have been evaluated: cytokine deprivation and activation-induced cell death. After Interleukin-2 deprivation, the percentage of naive and memory CD4(+) apoptotic cells significantly increased with donor age concomitantly with a reduction of Bcl-2 expression and an increase of intracellular content of reactive oxygen species. After phytohemagglutinin addition, the percentage of apoptotic cells, the expression of CD95, and the intracellular reactive oxygen species, were not significantly correlated with age both in naive and memory CD4(+) T cells. Our data demonstrate the existence of functional alterations of naive and memory T cell populations during ageing. These alterations are mainly related to the mechanism of the apoptotic event rather than to the type of cell population involved (naive or memory). The alterations of naive and memory T cells may have implications in the age-related susceptibility to diseases.

The aim of this study was to evaluate the peripheral representation and the clonogenic capacity of CD34(+) progenitor cells from 130 healthy subjects (80 females and 50 males) ranging in age from 16 to 100 years. We demonstrated that the absolute number of circulating CD34(+) cells progressively and significantly decreased with advancing age, with a 2-fold reduction in subjects aged more than 80 years. The number of granulocyte-macrophagic (CFU-GM), erytroid (BFU-E), and mixed (CFU-GEMM) colonies which developed from the number of CD34(+) purified cells per ml, progressively and significantly decreased with advancing age. The reduction of both CD34(+) cell number and clonogenic capacity during aging was statistically significant in males but not in females. When evaluated on a per cell bases, a significant age-related decrease in the number of CFU-GM colonies was observed in female but not in male subjects. Our study demonstrates the influence of gender on age-related alterations of the number and clonogenic capacity of CD34(+) cells in the peripheral blood. This evidence deserves particular consideration for the future planning of stem cell therapy in age-associated debilitating diseases.

We recently demonstrated that numerical and functional alterations of gammadelta T cells are present in healthy elderly. Here we observed that the decreased absolute number of Vgamma9Vdelta2 T cells present in old subjects in comparison with young/adult and middle aged donors is due to the reduction of naive and central memory Vgamma9Vdelta2 T cells bearing CD27 and CCR7 antigens. The proportion of effector/memory Vgamma9Vdelta2 T cells lacking CD27 or CCR7 markers was significantly increased in the peripheral blood of old subjects in comparison with younger donors. Moreover, the percentage of CD69+ gammadelta T cells was significantly increased in old subjects in comparison with younger donors after overnight activation, confirming that more effector cells are available in aged people. A functional analysis in young/adult and middle aged donors revealed that effector/memory CD27- Vgamma9Vdelta2 T cells are increased after 10-days of in vitro colture in the presence of isopentenylpyrophosphate (IPP) and IL-2. In contrast, the IPP+IL-2 mediated differentiation and expansion of CD27- effector/memory cells was absent in old subjects, confirming a lack of naive and central memory cells responding to IL-2. Accordingly, the expansion index of effector/memory CD27- Vgamma9Vdelta2 T cells was negatively correlated with the donor age. Finally, terminally differentiated Vgamma9Vdelta2 T cells measured as perforin content after 10-day in vitro expansion showed no age-related difference. These data demonstrated a shift of the circulating gammadelta T cell population towards CD27- and CCR7- effector T cells in the elderly with the reduction of immature CD27+ and CCR7+ T cell precursors.

Objectives: The purpose of this study was to determine whether psychological well being as well as metabolic, neuroendocrine and immune functions were different in non spousal primary caregivers of disabled elderly than in controls., Setting and Design: We randomly recruited 38 primary family carers of over 65 year old recipients of health home care services and 37 controls stratified according to sex and age., Method: Data were collected on psychological wellbeing (including anxiety, depression and self-perceived quality of life), on neuroendocrine and immune conditions (haemanalysis and metabolic signs, plasma ACTH, cortisol, prolactin, intra-lymphocyte content of beta-endorphins, NK cell activity and number), as well as on the incidence and severity of influenza disease during previous winter., Results: Caregivers showed greater anxiety, although mean scores did not reach pathological levels. Neither depression nor satisfaction on quality of life did differ significantly, nor differences in haemanalisis and metabolic signs were found, apart from leukocyte and lymphocyte number, which was significantly lower in carers. Plasma levels of ACTH, cortisol and prolactin, the intra-lymphocyte content of beta-endorphins as well as the NK cell number and cytotoxicity did not show significant differences. Incidence and severity of influenza episodes was also similar, whereas the duration of influenza disease showed to be significantly longer., Conclusions: Non spousal caregivers of disabled elderly suffer from only slight alterations of psychological, endocrine and immune parameters, and do not respond very differently to influenza disease. This does not support therefore any generic privilege for them in the allocation of public support or respite services.

We studied the peripheral representation, in vitro expansion, cytokine production, and cytotoxicity of gamma delta T lymphocytes from 23 patients with cutaneous primary melanoma and 28 healthy subjects. We demonstrated that the absolute number and the percentage of circulating gamma delta + T cells were significantly reduced in melanoma patients in comparison with healthy subjects. The decrease was due to a reduction of V delta 2 T cells, whereas the number of V delta 1 T cells was not affected. As a consequence, the V delta 2/V delta 1 ratio was inverted in melanoma patients. A lower percentage of gamma delta + T cells producing tumor necrosis factor-alpha or interferon-gamma was found in melanoma patients. After a 10 d in vitro culture, both the percentage and the expansion index of gamma delta T cells, and in particular of V delta 2 subset, were significantly reduced in melanoma patients in comparison with healthy subjects. The cytotoxicity of sorted gamma delta T cells against tumor cell lines and the percentage of gamma delta T cells producing perforins were preserved in melanoma patients. The numerical and functional impairment of gamma delta T cells could contribute to the inadequate immune response found in melanoma patients and offers the potentiality for the planning of new approaches of immune therapy of malignant melanoma.

We evaluated the effectiveness of vaccination with a HER-2/neu DNA plasmid to induce protective immunity against HER-2/neu overexpressing syngeneic TUBO tumour cells in old ages. Young and old Balb/c mice received three immunizations with a pCMVneuNT DNA plasmid and, successively, were challenged with TUBO cells. Young mice were completely protected whereas less than 60% protection was observed in old mice. Anti-p185(neu) antibodies were found in the sera from both young and old immunized mice, even if antibody production was significantly higher in young in comparison with old mice. Similarly, higher anti-p185(neu) lymphocyte proliferation was induced in young than in old mice. No anti-p185(neu) cytotoxicity was found in lymphocytes from old animals. We conclude that anticancer DNA vaccination has a lower effectiveness in old than in young ages.

Female FVB/N HER-2/neu transgenic mice from the age of 2 months were subcutaneously injected with saline, the peptide Epitalon(R) (Ala-Glu-Asp-Gly) or with the peptide Vilon(R) (Lys-Glu) in a single dose of 1 microg/mouse for 5 consecutive days every month. Epitalon treatment reduced the cumulative number and the maximum size of tumors (p < 0.05). Furthermore, the number of mice bearing 1 mammary tumor was increased, whereas the number of mice bearing 2 or more mammary tumors was reduced in Epitalon-treated in comparison to saline-treated animals (p < 0.05). The size but not the number of lung metastases was reduced in Epitalon-treated compared to saline-treated mice (p < 0.05). The treatment with Vilon produced significant negative effects when compared to the control group, with an increased incidence of mammary cancer development (p < 0.05), a shorter mean latent period of tumors (p < 0.05) and an increased cumulative number of tumors (p < 0.05). A 3.7-fold reduction in the expression of HER-2/neu mRNA was found in mammary tumors from HER-2/neu transgenic mice treated with Epitalon compared to control animals. The expression of mRNA for HER-2/neu was also partially reduced in Vilon-treated mice, but it remained significantly higher in Vilon- than in Epitalon-treated animals (1.9-fold increase). The data demonstrate the inhibitory effect of Epitalon in the development of spontaneous mammary tumors in HER-2/neu mice, suggesting that a downregulation of HER-2/neu gene expression in mammary adenocarcinoma may be responsible, at least in part, for the antitumor effect of the peptide., (Copyright 2002 Wiley-Liss, Inc.)

In the present paper, we investigated whether the phenotype, the antigen-presenting capacity, and the migration of antigen-presenting cells (APCs), are affected by the aging process. APCs were obtained incubating peritoneal monocyte-macrophage cells with granulocyte-macrophage colony-stimulating factor (GM-CSF) (immature APCs) or GM-CSF and IFNgamma (mature APCs). Phenotypically, after 8 days incubation, APCs cultures were composed of CD11c and Mac-3 cells, with a similar representation, both in young and old mice. The absolute number and the expression of MHC I and II, CD80, and CD86 both on immature and mature APCs were not significantly different in young and old mice. APCs from old mice induced similar lymphocyte proliferative responses but lower lymphocyte cytotoxicity and a reduced number of CD8(+) T cells producing IFNgamma in comparison with APCs from young animals. Lymphocyte responses were antigen-specific, since TS/A pulsed APCs induced lymphocyte cytotoxicity against TS/A but not against syngeneic TUBO tumor cells. The low expression of the mRNA for the migratory CCR7 chemokine receptor present in immature APCs from old mice was greatly increased in mature APCs up to the levels found in APCs from young animals. The in vivo migration of APCs was higher in old than in young mice. These results demonstrate that some alterations in APCs function are present in aging, suggesting that an increased migratory capacity of old APCs may be required to balance their reduced antigen presentation to cytotoxic lymphocytes.

The aim of this study was to evaluate the peripheral representation, in vitro expansion, cytokine production, and cytotoxicity of gammadelta T lymphocytes from 104 healthy subjects ranging in age from 19 to 103 years. We demonstrated that the absolute number of circulating gammadelta(+) T cells was reduced significantly in old people and centenarians in comparison with young subjects as a consequence of the age-related decreased lymphocyte number. The decrease was a result of an age-dependent reduction of Vdelta2 T cells, whereas the absolute number of Vdelta1 T cells was unaffected by age. As a consequence, the Vdelta2/Vdelta1 ratio was inverted in old subjects and centenarians. A higher percentage of gammadelta(+) T cells producing tumor necrosis factor alpha was found in old donors and centenarians, whereas no age-related difference was observed in interferon -gamma production. After a 10-day in vitro expansion, a twofold lower expansion index of gammadelta T cells, and particularly of a Vdelta2, but not of a Vdelta1 subset, was found in old people and centenarians in comparison with young subjects. The cytotoxicity of sorted gammadelta T cells was preserved in old people and centenarians. The alteration of gammadelta T cells could contribute to the age-related derangement of T cell-mediated, adoptive responses and may represent a new characteristic of immunosenescence.

We evaluated the kinetics of transgene expression and humoral and cellular immune responses against viral antigens and the product of the reporter gene LacZ in young (4 months) and old (20 months) Wistar rats. Animals received the intramuscular injection of a recombinant E1-deleted human type 5 adenovirus encoding beta-gal (Ad-LacZ) on days 0 and 30. The transgene expression evaluated on day 2 after infection revealed a significantly higher beta-gal activity in young than in old animals (1.9-fold increase, p<0.05). beta-gal expression decreased on day 6, and on day 15 transgene activity was undetectable in muscles from both groups. Ad-LacZ inoculation was repeated on day 30 in both animal groups. However, after the second adenovirus administration, no increase in beta-gal activity was observed. Humoral and cellular immune responses, evaluated after the first and second Ad-LacZ injection, developed with similar kinetics in young and old rats. In particular, the antigen specific antibodies were able to kill adenovirus-infected tumor cells in both complement-dependent cytotoxicity (CDC) and antibody-mediated cell-dependent cytotoxicity (ADCC) assays. Lymphocyte proliferation in response to the in vitro stimulation with specific antigens was significantly lower in old than in young animals whereas no difference was found in cytotoxic T lymphocyte activity against adenovirus-infected tumor cells. Our results demonstrate that repeated immunization with AdCMV.LacZ induces minor age-related differences in immune response which precludes gene expression both in young and old animals.

Female transgenic FVB mice transfected with the mammary erbB-2/neu oncogene were injected 0.1 ml 0.9% solution of sodium chloride (control), 1 meg Vilon peptide (Lys-Glu) or Epitalon peptide (Ala-Glu-Asp-Glu), s.c., 5 days in succession once a month, beginning from the age of 2 months. The characteristics of mammary tumor induction in the control and experimental groups did not differ until the age of 9 months. Later on, Epitalon-treated mice revealed distinct inhibition of carcinogenesis. One tumor per animal was detected in 7% (control), 4% (Vilon) and 16% (Epitalon) (p < 0.05). Two or more tumors per animal were in 75%, 95% and 56%, respectively (p < 0.05). Largest diameter of mammary adenocarcinoma in the Epitalon group was smaller than in controls by 33% (p < 0.05). Although the number of mice with metastases to the lung in all three groups was practically identical, their incidence in the Vilon group was 2.6 times higher than in Epitalon-treated animals (p < 0.05). Largest diameter of metastasis in the Epitalon group was the smallest, too. Our data point to inhibition of mammary carcinogenesis by Epitalon in transgenic erbB-2/neu mice.

Objectives and Design: The effect and the mechanism of light regimen and melatonin on the development of mammary tumors in HER2/neu transgenic mice were investigated. Female HER-2/neu mice starting from the age of 2 months were kept under standard light/dark regimen (LD) or constant light illumination (LL) and a part of each group was given melatonin (20 mg/l) during the night time., Results: The exposure to LL failed to change the incidence of spontaneous mammary adenocarcinoma development, the size of mammary tumors, as well as the incidence and size of lung metastases. However, the number of tumors per mouse was significantly increased in the LL group as compared to the LD group. The number of mice bearing 4 and more tumors was higher in the LL group than in the LD group, whereas the number of mice bearing 1 to 3 tumors was lower in the LL group in comparison with the LD group. Melatonin decreased the incidence and size of mammary adenocarcinomas, and the incidence of lung metastases in the LD group but not in the LL group. The mean number of tumors per mouse was not changed by melatonin treatment in both light regimens. The number of mice bearing 4 and more tumors was reduced by melatonin more significantly in the LL group than in LD group. Melatonin treatment resulted in a 2.5-fold reduction in the expression of HER-2/neu mRNA in mammary tumors from HER-2 /neu transgenic mice., Conclusion: The data demonstrate the influence of the LD light regiment and melatonin treatment in the development of spontaneous mammary tumors in HER-2/neu mice suggesting a melatonin-dependent modulation of HER-2/neu gene expression in mammary adenocarcinoma.

Anticancer immunotherapy with cytokines is often limited by the occurrence of severe toxicity, particularly in older age groups, which are characterized by a reduced tolerance to antineoplastic therapies. We, and others, have recently demonstrated the efficacy of pulsing procedures with IL-2 as a new therapeutic strategy to induce antitumor cytotoxic cells. The aim of this paper was to evaluate the effect of IL-12 on NK cell activity in young and old mice and to investigate the possibility of inducing NK cytotoxicity and perforin and granzyme B gene expression through a brief exposure of spleen lymphocytes from young and old mice to IL-12. Pulsed lymphocytes were compared with non-pulsed cells cultured continuously in IL-12. IL-12 was able to boost both endogenous and IL-2-induced NK cell activity in young and old mice; the levels of cytotoxicity were lower in old than in young animals although the relative increase of IL-12 plus IL-2 versus IL-2 alone was greater for old mice. Comparable levels of NK cell activity were obtained in pulsed (5 min-1 hour) and non-pulsed lymphocytes from both young and old mice after one or three days of culture. The efficacy of the pulsing procedure was evident in both endogenous and IL-2-induced NK cytotoxicity. The mRNA encoding perforin and granzyme B were markedly and similarly enhanced in both IL-12-pulsed and non-pulsed lymphocytes in comparison with control cells. The results demonstrate the effectiveness of IL-12 pulsing in inducing antitumor cytotoxic cells, suggesting the possibility of using IL-12 pulsing, alone or in combination with IL-2, in the immunotherapy of both young and old subjects.

Apoptosis represents the main mechanism involved in the intrathymic cell selection. The involution and atrophy of the thymic gland during aging has been associated with an altered representation of thymocyte subsets and particularly of CD4+CD8+ double-positive (DP) thymocytes, i.e., the cell population mainly involved in thymocyte selection. The aim of the present study was to evaluate the responsiveness of thymocytes from old mice to factors regulating the apoptotic cell death, such as antibodies to CD3/T cell receptor complex, zinc, and dexamethasone (DEX). Balb/c mice were used at the ages of 2 months (young), 21-22 months (old), and 24-26 months (very old). Thymocytes from these mice were incubated overnight with anti-CD3 monoclonal antibody (8 microg/ml), Zn2+ (150 microM), or DEX (10[-7] M) and then analysed for number of apoptotic nuclei, cell cycle phase, and phenotype by flow cytometry. A significant decrease of both the total number and the proportion of DP thymocytes was present in old and very old mice in comparison with young animals. Antibodies to CD3 antigen induced thymocyte apoptosis in both young and old mice. The stimulation of the CD3/TCR complex was more effective in giving apoptosis in very-old than in old and young mice. An impairment of the inhibiting effect of zinc on apoptosis induced by either serum deprivation or DEX was found in old and very old mice, whereas zinc was less effective in inhibiting CD3-induced apoptosis only in very old animals. Reduced DEX-induced apoptosis was also present in old age; this effect was more evident in very old than in old mice. Thymocyte apoptosis in old mice required protein synthesis being blocked with cycloheximide. Apoptosis was exerted on thymocytes in a specific cell cycle phase, i.e., on G0/G1 phase cells. Anti-CD3 antibodies, Zn2+, or DEX regulated apoptosis by modulating the proportion of DP thymocytes. The results demonstrate an altered in vitro responsiveness of thymocytes from old and very old mice to factors regulating apoptosis and suggest further investigations to determine if this altered responsiveness is associated with increased apoptosis of thymocyte populations occurring with increasing age.

The toxicity of high-dose interleukin 2 (IL-2) treatments limits its use in tumour therapies, particularly in older age groups, characterized by a reduced tolerance to antineoplastic therapies. Here, we evaluated the possibility to induce cytotoxic lymphokine-activated killer (LAK) cells through a brief exposure (1-h pulse) of peripheral blood mononuclear cells (PBMC) from elderly cancer patients to high concentrations of IL-2. The cytotoxic activity, phenotype, apoptosis, and cell cycle phase of IL-2 pulsed PBMC were determined and compared with those of non-pulsed PBMC cultured continuously in IL-2. Significant levels of LAK cytotoxicity were obtained in pulsed PBMC from all patients examined. The mean values of lytic activity on day 6 of culture were lower, even if not significantly, in pulsed than non-pulsed cultures. The pulsed cells were phenotypically similar to non-pulsed lymphocytes with regards to the expression of CD3, CD4, CD8, CD16, and CD56 antigens. The induction of activation markers, like CD25 and CD122 IL-2 receptors and CD71 transferrin receptor, was also comparable in pulsed and non-pulsed cultures. When a lower cytotoxicity was found in pulsed cultures, a lower number of CD54+ (ICAM-1) cells was also found. LAK cell cytotoxicity and number of CD54 cells were significantly correlated. No difference was found between pulsed and non-pulsed cultures in their cell cycle phase or in the percentage of apoptotic cells. Autologous plasma did not inhibit the differentiation of pulsed PBMC into LAK cells. The IL-2 pulse of PBMC from healthy young donors resulted in the induction of LAK cytotoxicity as observed in elderly cancer patients. The results demonstrate the effectiveness of IL-2 pulse to generate cytotoxic LAK cells in elderly cancer patients suggesting the potential application of pulsing procedures to treatment of older age groups.

Thymic regrowth and reactivation of thymic endocrine activity may be achieved even in old animals by different endocrinological or nutritional manipulations. In particular: a) intrathymic transplant of pineal gland or treatment with melatonin; b) implantation of a growth hormone secreting tumor cell line or treatment with exogenous growth hormone; c) castration or treatment with exogenous LH-RH; d) treatment with exogenous thyroxine or triiodothyronine, and e) nutritional interventions such as arginine or zinc supplementation. These data strongly support the idea that thymic involution is a phenomenon secondary to age-related alterations in neuroendocrine-thymus interactions and that it is the disruption of such interactions in old age which is responsible for most of age-associated dysfunctions. With regard to the mechanisms involved in hormone-induced thymic reconstitution, it is, at present, difficult to draw any definitive conclusion. The effect of GH, thyroid hormones and LH-RH may be due to the presence on thymic epithelial cells, supposed to produce thymic peptides, of the specific hormone receptors. Melatonin or pineal derived factors may as well act through specific receptors but experimental demonstration is still lacking. The role of zinc, whose turnover is usually reduced in old age, is of quite wide-range: from the reactivation of zinc-dependent enzymes, required for both cell proliferation and apoptosis, to the reactivation of thymulin, a zinc-dependent thymic hormone. The role of zinc may be even more crucial. According to recent preliminary data obtained both in animal and in man, it appears that the above reported endocrinological manipulations, capable of restoring thymic activity in old age, may act also by normalizing the altered zinc pool.

In this study we evaluated the effect of long-term melatonin (MEL) treatment on the cytotoxic activity and number of natural killer (NK) cells and the proliferative response of spleen lymphocytes to phytohemagglutinin (PHA) or interleukin-2 (IL-2) in old mice. Seventeen-eighteen month-old Balb/c mice were supplemented with MEL (40-50 microg/day/mouse) and sacrificed after eight months. The MEL supplementation was unable to recover the low levels of both endogenous and IL-2-induced NK cell activity found in old untreated mice. Also the NK cell number was unaffected by MEL treatment. The spleen lymphocyte proliferative response to both PHA and IL-2 was not different in old MEL-treated compared to old untreated mice. These results indicate that long-term MEL supplementation does not recover the age-related deterioration of NK cell activity and lymphocyte proliferative capacity.

The passive transfer of antibodies and vaccination procedures against p185, the erbB2/neu oncoprotein, are approaches being explored for treatment of human breast cancer. We now report the possibility of using the erbB2/neu gene as an immunogen. This study demonstrates that intramuscular or intradermal injections of rat neuNT full-length DNA into mice generate anti-p185 autoantibodies. Anti-p185 polyclonals were also shown to bind the homologous human receptor ErbB2 and to stain specimens of breast adenocarcinoma from both neu-transgenic mice and humans. Further, in vitro assays demonstrated that anti-p185 IgG (probably dependent on CD4+ Th1) were able to inhibit human SKBR3 tumour cell growth and to mediate their lysis by natural killer cells. The continuous presence of circulating neu autoantibodies in mice did not cause any discernible toxic effects on normal tissues expressing low levels of self-antigen, even after 1 year. The experiments reported here raise the possibility that boosting anti-ErbB2 immunity by DNA vaccination will not induce harmful autoimmunity in humans.

In a previous work we demonstrated that chronic in vivo antalgic therapy of cancer patients with morphine reduced the endogenous cytotoxic activity of natural killer (NK) cells, while increasing the development of lymphokine activated killer (LAK) cell cytotoxicity. In order to investigate the mechanisms by which morphine affects NK and LAK cell function further, we evaluated the modulation exerted by short- or long-term morphine administration on either NK/LAK cell cytotoxicities or plasma levels of prolactin (PRL) and other immunomodulating neurohormones. An intravenous morphine injection (10 mg) significantly increased the plasma levels of PRL, reduced the cytotoxic activity of NK cells, and increased the development of LAK cell activity 30 min after drug injection in neoplastic patients. The administration of bromocriptine before the injection of morphine prevented both PRL augmentation and the increase in LAK cell activation, although it did not prevent the inhibition of NK cytotoxicity. The chronic oral administration of morphine (90 +/- 30 mg/day for 1 month) also resulted in higher PRL levels; the NK and LAK cell activities were, respectively, lower than or higher than those found in neoplastic patients untreated with morphine. The plasma levels of thyrotropin (TSH), adrenocorticotropic hormone (ACTH) and cortisol were not significantly modified in either short- or long-term experiments. The absolute number and the percentages of lymphocyte populations, as well as the percentage of IL-2 receptors, were not modified after short-term morphine administration whereas little changes of T lymphocyte populations and NK cell number were observed after oral treatment with morphine. In vitro morphine did not affect the development of LAK cell activity. In conclusion, our findings indicate that morphine reduces NK cytotoxicity and increases the development of LAK cell cytotoxicity after short- and long-term administration. The effect of morphine on LAK cell activation but not on NK cell reduction is related to the modulation of PRL levels determined by the opioid drug.

We have evaluated the effect of chronic melatonin (MEL) treatment or pineal grafting (PG) in old mice on the apoptosis of both thymocytes and spleen lymphocytes under conditions of either serum deprivation or glucocorticoid or zinc administration. The apoptosis was correlated with the modulation of thymus and adrenal weight and corticosterone and zinc plasma levels induced by MEL treatment or PG in old mice. Balb/c mice (17-18 months old) were given supplements of MEL (40-50 micrograms/day/mouse) or grafted with a young pineal gland and then sacrificed after 8 months. Both the MEL treatment and PG partially prevented thymic involution in very old mice. Both treatments protected the thymic and spleen lymphocytes in old mice from the apoptosis induced by serum deprivation and recovered the reduced thymocyte sensitivity to the apoptosis induced by dexamethasone (DEX), present in old untreated animals, to the values found in young mice. DEX caused a bigger loss of G D /G 1 phase cells in MEL treated mice than in old untreated mice. The protective action of MEL treatment or PG on serum deprivation induced apoptosis was correlated with increased thymus weight, reduced adrenal weight and corticosterone levels and increased zinc plasma levels. The greater DEX-induced apoptosis found in MEL treated and PG mice was correlated with reduced adrenal weight and function. In vitro MEL did not affect thymocyte apoptosis in young or old mice. These results suggest that MEL treatment or PG prevent age-related thymus involution through regulation of thymocyte apoptosis which, in turn, occurs through modulation of the pituitary-adrenal axis and zinc turnover determined by the pineal hormone.

Various chemoantiblastic agents cause DNA damage followed by apoptotic cell death through the activation of the p53 suppressor gene. The aim of our study was to evaluate the relationship between p53 protein expression, apoptosis of autologous tumor cells, and clinical response to neoadjuvant chemotherapy in patients with cervical carcinoma. Our study included 14 women with stage II squamous cervical carcinoma who had been admitted to the Institute of Gynecology and Obstetrics, Ancona University, between January 1990 and December 1995. The patients received neoadjuvant combination chemotherapy, consisting of three cycles of cisplatin (80 mg/m2) and bleomycin (30 mg/m2). After chemotherapy, radical surgery was performed. Bioptic specimens were obtained from cervical tumors before and after chemotherapy, and processed for DNA staining and apoptosis, and immunohistochemical staining with a monoclonal antibody against p53. Ten patients (71.4%) showed a clinical response (2 complete, and 8 partial), while of the remaining 4 cases (28.6%) 3 had no change and 1 showed progression after neoadjuvant combination chemotherapy. A significant relationship was observed between the overexpression of p53 and sensitivity to chemotherapy; responder patients showed a higher frequency of p53 positive cells than non-responders (p = .05). A significant direct relationship was observed between p53 protein immunostaining and apoptosis of tumor cells both before (p = .02) and after (p = .01) chemotherapy. Our study seems to define the relationship between p53 expression and sensitivity to cisplatin based chemotherapy in locally advanced cervical carcinoma, supporting the notion that the cytotoxic action of cisplatin can activate p53 mediated apoptosis. However, the limited number of patients in our series does not permit judgement on the clinical implications of the expression of p53 in patients undergoing neoadjuvant combination chemotherapy for locally advanced cervical carcinoma.

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.

Our previous studies have shown that prolactin (PRL), a pituitary and lymphocyte hormone and a ligand of the cytokine/hemopoietin receptors (R) superfamily, acts synergistically with interleukin (IL)-2 on the development of lymphokine activated killer (LAK) cells and enhances the effects of GM-CSF and IL-3 on myeloid progenitors' proliferation and differentiation. More recently, we have demonstrated that GM-CSF and IL-3 increase the sensitivity of acute myeloid leukemic (AML) cells to LAK activity. Together, these findings have prompted us to study the role of PRL on the target arm of the LAK response. We show here that CD33+ blasts from AML patients express membrane PRL-R and that the PRL/PRL-R interaction is followed by increased susceptibility to natural killer (NK) (p < 0.02) and LAK (p < 0.001) cells. As predicted from the dimerization model of PRL-R and in agreement with previous reports, the response of AML blasts to PRL was bell-shaped with a trend peak at 25 ng/ml. Although enhanced lysis occurred at the target recognition level, it was not accompanied by changes in the MHC class I, cellular adhesion molecules, or myeloid differentiation antigens. Cell cycle recruitment and lysis increased concurrently in three cases studied, suggesting a modulatory action of PRL on the expression of putative cycle-related NK/LAK-target structures. Together, these data strengthen the role of PRL in the LAK response.

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.

Zinc is a crucial nutritional component required for the normal development and maintenance of immune functions. It has been reported that zinc is a potent inhibitor of DNA fragmentation, the specific marker of apoptosis. The effect of zinc on apoptotic cell death has been previously studied in a narrow range of high zinc concentrations, and the role of physiological zinc doses has not yet been elucidated. In this paper we evaluate the effect of in vitro Zn2+ administration at concentrations higher than, corresponding to, and lower than the physiological concentration, in thymocytes from young mice. We demonstrate that Zn2+ has an opposite effect on apoptosis, inhibiting or increasing it depending on the Zn2+ concentration used. High Zn2+ concentrations (from 600 to 75 microM) inhibit both serum-free medium and DEX-induced thymocyte apoptosis. Low Zn2+ concentrations (from 15 to 7.5 microM) induce apoptosis or increase serum-free medium-induced apoptosis. The effect of low Zn2+ concentrations on DEX-induced apoptosis is dependent on the length of incubation, since Zn2+ has an additive effect with DEX in inducing DNA fragmentation at 8 h of culture, whereas it blocks DEX-induced apoptosis after 20 h incubation. Both DEX and 15 microM Zn(2+)-induced DNA fragmentation require protein synthesis, being blocked through cycloheximide. The inhibiting and inducing effects of Zn2+ on apoptosis are exerted on G0/G1 phase thymocytes. The inhibiting effect of Zn2+ on apoptosis is related to an increase in the number of CD4+CD8+ thymocytes. Concentrations of Zn2+ inducing apoptosis sometimes cause a decrease of CD4+CD8+ cells with a corresponding increase of CD4+CD8-thymocytes. These data show that in vitro Zn2+ has a dose-dependent opposite effect on apoptosis, suggesting that Zn2+ not only acts as an inhibitor but also plays a more complex role in physiological intrathymic cell selection.

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)

An altered cellular immune response as a secondary phenomenon has been suggested to be probably involved in the bronchopulmonary infections by Pseudomonas aeruginosa in cystic fibrosis (CF). The difficulty to eradicate with modern anti-pseudomonal antibiotics the bronchopulmonary infections has led us to further investigate the possible existence of other cellular immune defects and their cause. Alterations in zinc turnover are present in CF. Zinc is relevant for good immune functioning. In particular, zinc is required to confer biological activity to thymulin (ZnFTS), a biochemically defined thymic hormone with a modulating action on cell-mediated immunity. The zinc-unbound form (FTS) is inactive and it can be unmasked by in vitro zinc addition to the plasma samples revealing the total amount of circulating thymulin (active + inactive). Marginal zinc deficiencies may prevent peripheral biological activation of active thymulin. Total zinc-saturable thymulin fractions in CF are similar to those observed in normal subjects, whereas the active quota is strongly reduced associated with concomitant high plasma levels of inactive thymulin compared to the values of healthy children (P < 0.01). A strict correlation exists between zinc and thymic hormone-saturable fraction (r = 0.87, P < 0.01) in CF. These findings suggest that the defect is not due to a thymic failure but to a reduced peripheral saturation of thymulin by zinc ions. This defect might depend on augmented plasma concentration of alpha 2-macroglobulin, which has a higher binding affinity for zinc than thymulin. T cell subsets are normal in CF. Reduced NK cell number and activity are present. Also, plasma IL-2 levels are reduced. The existence of positive correlations between zinc and IL-2 (r = 0.79, P < 0.01) and between zinc or active thymulin and NK activity (r = 0.70, P < 0.01 and r = 0.88, P < 0.01, respectively) suggest a close link among zinc failure, impaired IL-2 activity, low thymulin level, and reduced NK activity in CF patients with both normal and growth retardation. Although the role of NK cells is unknown in CF, a zinc supplementation, in order to induce a complete saturation of thymulin molecules, to correct some cellular immune defects and to improve the growth, may be suggested.

The kinetics of the development of lymphokine-activated killer (LAK) cell activity, the surface phenotype, and the expression of p55 and p75 interleukin 2 receptors (IL-2R) on IL-2-activated peripheral lymphocytes have been investigated in young and old healthy humans selected according to the Senieur Protocol criteria. No difference is present between young and old healthy subjects in terms of LAK cell activity development. The proliferative capacity of lymphocytes incubated with IL-2 shows similar kinetics in young and old subjects. The mean percentages of CD56+ and CDl6+ cells reach higher levels in old than in young donors. The proportion of CD56+/CD25+ cells in culture is significantly higher in old than in young individuals. A progressive decrease of CD4+ population occurs during LAK cell development, both in young and old subjects. The proportion of CD4+ T cells in culture is lower in old than in young individuals. No age-related difference is present in the mean percentage of cells positive for p55 or p75 IL-2R at any culture time. Thus, our findings show similar kinetics of development of LAK cell activity in young and old subjects, indicating that aging per se does not represent an exclusion criterion of cancer patients from clinical trials of adoptive immunotherapy. A higher proportion of CD56+ and CDl6+ cells seems to be required in old subjects to obtain the same levels of LAK cell activity present in young age.

A good body of experimental and clinical results has supported the existence of numerous reciprocal interactions among the nervous, endocrine and immune systems. Increasing evidence has been accumulated in the last years on the interaction between pituitary-thyroid hormones and the immune system on the basis of either the existence of receptors for thyreotropic and thyroid hormones on lymphocytes or the frequent immune alteration in physiological and pathological fluctuations of thyroid hormones. The data were obtained either in animals with experimentally induced hyper- or hypothyroidism or in humans with various hyperthyroid or hypothyroid situations. Conversely, immune-derived products such as lymphokines and monokines have been shown able to influence the pituitary-thyroid axis modulating either the thyroid hormone levels or the hormone/cytokine production by thyrocytes. The present paper aims at summarizing the data available on the existence of thyroid-immune interactions, and at analyzing the possible integration between pituitary-thyroid hormones and immune factors in favoring the development and maintenance of both thymic and peripheral immune efficiency. The relevance of pituitary-thyroid-immune interactions is discussed for its implication in the ageing process.

It is well known that immune efficiency is frequently deteriorated in elderly people. The age-diminished antibody response to T-cell dependent antigens, such as influenza virus antigens, may explain the low protection offered by influenza vaccination in the elderly population. To investigate the possibility of increasing the antibody response to influenza virus vaccinations, we have conducted a nursing home-based study on the efficacy of IL-2. Seventy-five institutionalized elderly subjects (82 +/- 8 years) were enrolled in the study in the course of winter season 1991-1992. Thirty-nine subjects were treated with three subcutaneous daily injections of interleukin-2 (IL-2, 1 x 10(6) I.U./day) before vaccination and their antibody response was compared to that of 36 aged people receiving the vaccine only. An increased antibody response against influenza virus was present in vaccine plus IL-2 treated subjects (P < 0.001) but not in subjects treated with vaccine only. The number of protected subjects 45 days after vaccination was increased only in the IL-2-treated group (P = 0.045). The low-dose of IL-2 administered and the short-term treatment allowed a good tolerance to the IL-2 injection. In conclusion, the low-dose IL-2 treatment represents an effective means of inducing antibody response to influenza virus antigens in elderly subjects without appreciable toxicity.

It has been clearly demonstrated that immune responses may be conditioned in a manner similar to that of the classical Pavlovian experiments. Evidence of impaired immune function in aging has raised the question of whether psychological conditioning of an immune response can also be effective in old age. The knowledge that aged mice have decreased spleen cell natural killer (NK) activity and that NK cytotoxicity, at least in young mice, can be psychologically conditioned led us to explore in old mice the possibility of conditioning the response of NK cell activity using the odor of camphor as the conditioned stimulus (CS) and the injection of Poly I:C as the unconditioned stimulus (US). Young and old male mice were divided into five and six groups, respectively. They received the CS and/or the US in association (conditioning) trials (sessions 1-9). Mice were exposed to the camphor odor alone at 72 hours after the final association trial to observe the conditioning phenomenon (session 10). The group conditioned with Poly I:C and camphor and receiving the CS at session 10 showed statistically significant increases in spleen cell NK activity over those of the control groups that did not receive the CS treatment at session 10 (2.6- and 4.0-fold increase in young and old, respectively). Treatment with camphor odor alone had no effect on boosting NK cell activity. These findings demonstrate the possibility of conditioning immune responses in old age, offering a valuable tool for attenuating age-related immune deterioration in various species, including the human. In addition, these results again confirm highly significant immune enhancement by classical conditioning and extend previous findings from female mice to males as well.

Peripheral blood natural killer (NK) cell activity against K562 tumor line was monitored in 16 patients with locally advanced squamous cervical carcinoma (II and III FIGO stages) during neoadjuvant polychemotherapy (cisplatin at 80 mg/m2 and bleomycin at 30 mg/m2). There was a significant progressive decrease in NK activity during the three cycles of antiblastic treatment (P = 0.008) without significant depletion of NK cell (CD56 and CD16 monoclonal antibody positive cell percentage and absolute number). A significant relationship was shown between basal NK activity levels and response to polychemotherapy; in fact, nonresponder patients had a significantly lower mean value of NK activity before polychemotherapy than responders (P = 0.044). In conclusion, NK activity declines as a result of antiblastic therapy; although polychemotherapy reduces tumor spread, its antineoplastic action may be affected by this decreased immune reactivity.

Objective: To evaluate the correlation between natural killer cell activity and serum estradiol (E2) levels in patients with different stages of endometriosis., Methods: Natural killer cell activity of peripheral blood lymphocytes and serum E2 levels were evaluated in 73 women who underwent laparoscopy for pelvic pain, infertility, and benign adnexal masses., Results: The 33 patients (45%) with endometriosis showed a significant decrease in natural killer cell activity in relationship to an increase in disease stage (correlation coefficient r = -0.83, P < .001). A significant inverse relationship was observed between cytotoxicity and serum E2 levels (correlation coefficient r = -0.89, P < .001)., Conclusion: The relationship between natural killer cell activity and serum E2 levels suggests that an immunoendocrine interaction plays an important role in the progression of endometriosis.

This paper describes the comparison between a fluorimetric NK assay based on the target cell retention of fluorescent dye carboxyfluorescein diacetate (cFDA) and standard 51Cr release assay. The results provide several suggestions to improve the cytotoxic assay based on the use of the fluorogenic substrate showing that the measurements of cFDA retained by target cells represent a method of evaluating cytotoxicity completely comparable to the 51Cr release assay.

In the present study we investigated the effect of thyrotropin (TSH) on both the proliferative capacity and the natural killer (NK) cell activity of murine spleen lymphocytes. It was found that TSH at various concentrations significantly increased the proliferative response of mouse lymphocytes to both concanavalin A (Con A) and phytohemagglutinin (PHA). This increase was particularly evident when suboptimal concentrations of mitogens were used (40-50% increase). The administration to cell cultures of TSH alone could not induce a significant stimulation of proliferative capacity. In order to provide a better knowledge about the mechanism by which TSH improved the mitogen-induced lymphocyte proliferation, the effect of the pituitary hormone on lymphocytes directly stimulated with recombinant interleukin-2 (RIL-2), was examined. It was observed that there was a great increase in IL-2-induced lymphocyte proliferation by TSH. The improvement in proliferative capacity of lymphocytes was particularly evident by using suboptimal rIL-2 concentrations (25-30% increase). The studies carried out on the cytotoxic activity of NK cells showed that TSH was able to significantly increase the IL-2-induced NK cell activity without modifying the basal levels of cytotoxicity. The results support the immunoregulatory role of TSH and contribute towards understanding the mechanisms of interaction between neuroendocrine and immune systems.

The cytotoxic activity of natural killer (NK) cells under both basal and interferon (IFN)-induced conditions, as well as the proliferative response of lymphocytes to ConA in old rats treated in vivo with AL-721 have been analyzed. The low levels of basal NK cell activity present in spleen of old rats were completely recovered to the levels of NK cytotoxicity obtained in young rats in animals treated in vivo with AL-721. Similar results were obtained for in vitro IFN-boosting of NK cells, in so far as the reduced IFN-responsiveness of NK cells from old untreated rats was restored to the levels of IFN-induced young NK activity in animals treated in vivo with AL-721. The evaluation of the proliferative response of lymphocytes to ConA revealed that old rats treated in vivo with AL-721 showed an increased proliferative capacity in comparison to old untreated animals even if the mitogenic response was not recovered up to the levels observed in young rats.

The cytotoxic activity of Natural Killer (NK) and Lymphokine Activated Killer (LAK) cells in neoplastic patients with or without antalgic treatment was studied. NK cell activity was found reduced in untreated neoplastic patients when compared to healthy subjects. The atalgic treatment with morphine (orally or intrathecally administered) was able to significantly reduce the mean values of NK cell activity found in cancer patients. In three patients the cytotoxicity of NK cells significantly decreased during transfer from oral to intrathecal administration of morphine. In contrast to the NK cell function, the development of LAK cell activity significantly increased in neoplastic patients when compared to healthy controls. Further increments were obtained during treatment with morphine. The oral treatment with morphine was able to determine a higher induction of LAK cells than the intrathecal administration of the drug. Besides providing new knowledge on the effect of morphine on immune system our findings suggest that, in order to include neoplastic patients in clinical trials of adoptive immunotherapy with LAK cells and interleukin-2 (IL-2), the antalgic therapy with oral administration of morphine may represent a better solution than the intrathecal administration of the drug.