Anti-tumor action of tumor necrosis factor against Bomirski Ab melanoma in hamsters.

INTRODUCTION: Tumor necrosis factor (TNF) is a cytokine able to exert anti-tumor activity in various models and modes of applications. However, the exact mechanism mediating the in vivo anti-tumor effect of TNF has not yet been clarified. MATERIALS AND METHODS: The effects of intratumoral injection of rat TNF into hamsters bearing Bomirski Ab amelanotic melanoma, a fast growing tumor of high metastatic potential, were tested. Subcutaneous injections of the anti-angiogenic compound TNP-470 allowed analysis of its influence on the effects of TNF administration. RESULTS: TNF application resulted in a significant inhibition of tumor growth and changes in metastasis pattern. Accelerated hemorrhagic necrosis was also observed, indicating the effect of the cytokine on tumor vessels. Moreover, the synergistic anti-tumor effect of TNF and anti-angiogenic agent TNP-470 suggested a cooperative activity of both substances on tumor vasculature. Microscopically, the effect of TNF injections was expressed by an increase in the amount of tumor cells with nuclear pyknosis and karryorrhexis. In vitro assays indicated a direct cytotoxic effect of TNF against Ab melanoma cells, most probably as an outcome of apoptosis. Intratumoral application of TNF also caused some modulation of cytokine response in melanoma-bearing hamsters as evidenced by increased levels of IL-6 in blood serum. CONCLUSIONS: This study established Bomirski Ab melanoma as a useful model for complex analysis of the anti-tumor activity of TNF.

CD4+CD25+ T regulatory cells inhibit cytotoxic activity of CTL and NK cells in humans-impact of immunosenescence.

We hypothesized that advanced age and medical conditions had an impact on the accumulation of CD4+CD25+ T regulatory cells (Treg), which in turn could deteriorate cytotoxic activity of CD8+ T and NK cells. Volunteers were divided according to the Senieur Protocol into healthy young and elderly and non-healthy young and elderly subjects. The numbers of Treg cells in peripheral blood, their influence on CD8+ T and NK cells and production of IL2 as well as apoptosis intensity of Treg cells were measured. The number of Treg cells was higher in both elderly groups than in respective young ones. Compared to healthy subjects, those with medical conditions were revealed to have higher numbers of Treg cells. In addition, the highest accumulation of Treg cells in non-healthy elderly could be a result of their resistance to undergo apoptosis. The frequency of Treg cells correlated inversely with the activity of autologous cytotoxic cells in PBMC and production of IL2 by autologous CD4+CD25- Th cells. Thus, these parameters were the most highly decreased in non-healthy subjects, notably in the elderly. However, these parameters improved in the cultures of pure sorted cells. The only subset capable of decreasing them to the levels noted in PBMC when added back was Treg cells, which proved the link between the number of Treg cells, cytotoxic activity and production of IL2. Concluding, we found that Treg accumulated as a result of ageing and/or medical conditions were capable of decreasing cytotoxic activity of CD8+ T and NK cells and production of IL2.

Long-term therapy with recombinant human erythropoietin increases CD8+ T-cell apoptosis in haemodialysis patients.

BACKGROUND: We intended to assess the intensity of apoptosis in the CD4+ and CD8+ T-lymphocytes of haemodialysis (HD) patients on recombinant human erythropoietin (rHuEpo). METHODS: The expression of Fas, tumour necrosis factor-alpha receptors (TNFRI and TNFRII) and the CD28 molecule on lymphocytes was evaluated in 15 HD patients before and during treatment with rHuEpo. In cultures of peripheral blood mononuclear cells (PBMCs) stimulated with rHuEpo, phytohaemagglutinin and camptothecin, our measures of apoptosis were the percentages of cells with subdiploid DNA content and of annexin V-stained cells. Results, Therapy with rHuEpo did not affect CD4+ T cells but decreased the percentage of CD8+ T cells in peripheral blood. The intensity of apoptosis in both CD4+ and CD8+ T cells at baseline was lower in HD patients than in healthy volunteers, and increased in those treated with rHuEpo. In vitro, rHuEpo did not induce apoptosis in PBMCs. The percentage of CD8+Fas+ T cells was constant, while that of CD8+TNFRI+ cells declined during follow-up. There was an increase in the percentage of CD28+ T cells, mainly in the CD8+ compartment, as early as 1 month after the introduction of rHuEpo. CONCLUSIONS: Treatment with rHupo caused a decline of CD8+ T cells in HD patients, which most probably was mediated via the TNFRI-related apoptotic pathway and was independent of Fas expression. Apoptosis in vitro was not directly influenced by rHuEpo, suggesting that the process in vivo was only initiated by rHuEpo supplementation.

CD4+CD25+ T regulatory cells inhibit cytotoxic activity of T CD8+ and NK lymphocytes in the direct cell-to-cell interaction.

There are reports suggesting an influence of CD4(+)CD25(+) T regulatory cells (Treg) on cytotoxic lymphocytes. The aim of the study was to evaluate such an influence. Cytotoxic activity was examined in the cultures of peripheral blood mononuclear cells (PBMC) as well as in the cultures of separate T CD8(+) or NK cells mixed with Treg and other subpopulations of PBMC. We found that the production of IFNgamma, perforin and cytotoxic activity of T CD8(+) or NK cells were decreased in the presence of Treg, however, the percentage of conjugates formed by cytotoxic cells with target cells during cytotoxic reaction was decreased only in the cultures of T CD8(+) cells. Inhibition of the cytotoxic reactions in the presence of Treg cells was found to be associated with the generation of conglomerates formed by CD4(+)CD25(+) and the cytotoxic cells, as observed under the fluorescence microscope. Treg produced IL10 when mixed with the cytotoxic lymphocytes, however, an addition of anti-IL10 mAb into the cultures did not affect the results. It is concluded that Treg were able to inhibit both T CD8+ and NK lymphocyte cytotoxic activities in a direct cell-to-cell interaction. Treg decreased the number of T CD8+ cells attached to the target cells, while the mechanism underlying a decrease in NK cytotoxicity remained unclear.

The effect of angiogenesis inhibitor TNP-470 on the blood vessels of the lungs, kidneys and livers of treated hamsters.

The growth of solid tumours and their metastases is dependent on the development of new blood vessels (angiogenesis). Therefore angiogenesis inhibitors are potential antitumour drugs. In our previous studies it was found that the angiogenesis inhibitor TNP-470 given to transplantable melanoma-bearing hamsters can decrease the rate of the tumour growth, although the survival time of the animals treated was not significantly affected. It was found finally that TNP-470 given in the vicinity of the growing tumour can cause complete remission of the melanoma in hamsters treated in this way. To check what side-effects could be evoked by such treatment, an examination of the morphology of the blood vessels of the lungs, kidneys and livers of the treated animals was carried out. It was found that the angiogenesis inhibitor applied did not cause any changes which could be observed by light and electron microscopes in the structure of the examined blood vessels of the treated animals.

Differences in kinetics of donor lymphoid cells in response to G-CSF administration may affect the incidence and severity of acute GvHD in respective HLA-identical sibling recipients.

Granulocyte-colony stimulating factor (G-CSF), in addition to myeloid and stem cells, mobilizes a large number of lymphoid cells. We examined which lymphoid populations were mobilized in 21 consecutive donors of peripheral blood stem cells (PBSC) and whether the differences in mobilization could affect the incidence of acute and chronic GvHD in respective HLA-identical recipients. G-CSF administration induced significant increases of donor B (CD3-CD19+) lymphocytes and slight increases of T (CD3+) and cytotoxic (CD16+CD56+) NK cells. The number of extrathymic cells (CD3+ cells with NK markers, or CD7+) remained unchanged except for an increase of CD3+CD57+CD8+ cells. Donors of patients without subsequent grade II-IV acute GvHD compared to donors of patients who developed significant acute GvHD were found to have in peripheral blood stable numbers of CD3+CD4+ cells producing IL2, with a concomitant increased number of CD3+CD4low+CD25+ T regulatory cells and decreased NK-mediated cytotoxicity, together with a higher number of suppressive extrathymic CD57+CD3+ cells in the blood and G-PBMC grafts. Increasing numbers of activated T and NK cells in the blood were associated with the development of chronic GvHD. We suggest that differences in steady-state levels and kinetics of G-CSF induced mobilization of donor lymphoid cells may in addition to other well-known factors affect the incidence of GvHD in HLA-identical recipients. However, owing to the small number of donor-recipient pairs studied, our results must be verified in a larger group of patients.

Immune consequences of the spontaneous pro-inflammatory status in depressed elderly patients.

INTRODUCTION: The aim of the study was to describe the interrelationship between senescence, depression, and immunity. METHODS: We assessed 10 elderly patients with depression and 10 age- and sex-matched controls: before, at one and at six month intervals after the anti-influenza vaccination. Levels of TNFalpha, IL6, ACTH, and cortisol, titres of anti-hemagglutinins and anti-neuraminidases, lymphocytes secreting IFNgamma, IL2, IL4, and IL10, cytotoxicity of NK and CD3+ CD8+ IFNgamma+ cells, anti-CMV antibodies, and CD28- CD57+ lymphocytes known to be associated with the CMV carrier status were evaluated. RESULTS: Higher levels of anti-CMV, higher percentage of the CD28- CD57+ cells, and elevated levels of TNFalpha, IL6, and cortisol concomitant with decreased levels of ACTH and insufficient production of IL10 (which increased the IFNgamma+ /IL10+ ratio) were found in the patients suffering from depression, in comparison to healthy controls. The subjects with depression revealed a low NK cytotoxicity, while a level of CD3+ CD8+ IFNgamma+ cells was comparable between the groups. Although the levels of anti-hemagglutinins and anti-neuraminidases were low in the depressed patients, they reached the protective titres. The majority of these differences disappeared when CMV titres were entered into the analyses as a covariate. DISCUSSION: The results suggest that the elderly depressed patients were characterised by increased exposure to CMV in the past, which could have resulted in a pro-inflammatory profile demonstrated as elevated levels of TNFalpha, IL6 and deficiency of suppressive IL10+ cells. These changes negatively affect humoral and innate response in the depressed patients.

Association between cytomegalovirus infection, enhanced proinflammatory response and low level of anti-hemagglutinins during the anti-influenza vaccination--an impact of immunosenescence.

We assessed association between prior cytomegalovirus (CMV) infection, proinflammatory status and effectiveness of the anti-influenza vaccination. We examined 154 individuals during the epidemic season dividing them according to the age, response to the vaccine and the Senieur Protocol (SP). The anti-hemagglutinins (HI), tumour necrosis factor alpha (TNFalpha), interleukin (IL) 1beta, IL6, IL10, ACTH/cortisol axis, anti-CMV antibodies and CD28+CD57- lymphocytes were assessed. Non-responders of both ages we characterised by higher levels of anti-CMV IgG and higher percentages of CD57+CD28- lymphocytes (known to be associated with CMV carrier status) together with increased concentrations of TNFalpha and IL6 and decreased levels of cortisol. The anti-influenza vaccine induced increase in TNFalpha and IL10 in the all non-responders, while cortisol increased only in the young. Concluding, CMV carrier status eliciting elevated proinflammatory potential could contribute to unresponsiveness to the anti-influenza vaccine.

Ex vivo apoptotic potential of peripheral blood mononuclear cells of the elderly human subject.

Studies of immunosenescence have led to a detailed knowledge of immune system dysfunctions in the ageing human being. Apoptosis seems to be one of the process regulating an immune response after the antigenic stimulation. We examined whether commonly used methods of assessing apoptosis in the elderly human subject produce comparable results to young subjects. PBMC of young and elderly volunteers were isolated from the venous blood and cultured for 6 or 24 h with antigens of anti-influenza vaccine or PMA. The intensity of apoptosis was measured using the annexinV test, flow cytometric evaluation of DNA content (sub-G1 peak in DNA histograms), 'ladder' by DNA gel electrophoresis, and fluorescence microscope. Apoptosis in 6 h-lasting cultures of the elderly was more intense in annexinV test, while it was decreased assessing subG1 peak. Additionally, in the aged group, those changes were associated with cell cycle arrest. Our results suggest that the apoptosis after the stimulation with the vaccine antigens seems to be some kind of activation-induced cell death (AICD). Different patterns of apoptosis after stimulation may be associated with the cell cycle arrest of the PBMC in the elderly.

Lower percentage of CD8(high+)CD152(+) but not CD8(high+)CD28(+) T lymphocytes in the elderly may be reverted by interleukin 2 in vitro.

An expression of the surface co-stimulatory molecules-the CD152 and the CD28 has been compared between young and old individuals on the CD8(high+) lymphocytes. Sixty five elderly healthy (65-96 years old) and 31 young (19-40 years old) volunteers were examined. An expression of CD152 and CD28 surface antigens was analyzed by flow cytometry ex vivo and on whole blood cell cultures lymphocytes stimulated with interleukin 2 (IL2). The elderly population was characterized by a lower percentage of the CD8(high+) lymphocytes than the young population. The percentages of CD28(+) lymphocytes as well as those of CD8(high+)CD28(+) subpopulation were lower in the old group compared to the young group. The surface expression of CD152 antigen was similar to that of CD28 with a lower percentage of the CD152(+) lymphocytes and CD8(high+)CD152(+) cells in the old group. Stimulation of lymphocytes in vitro with IL2 resulted in an increase of the CD8(high+)CD152(+) cells in the elderly, while it had no effect on lymphocytes of the young group. Our results indicate that lymphocytes of the elderly population are characterized by a lower expression of the surface CD28 and CD152 molecules. An age-related decrease of an expression of the co-stimulatory molecules CD28 and CD152 on the surface of lymphocytes, found in our study, may be compatible with a hypothesis of a 'remodelling' of immune response in the healthy elderly.