[Principles of biological aging]. / Grundlagen der biologischen Alterung.

The aging process is the substrate on which aging-associated diseases develop; therefore, the scientific discipline of gerontology aims at understanding this biological aging process, which refers to the progressive increase in the risk of death caused by a loss of body functions. Studies in simple model organisms demonstrate that pharmacological substances, genetic interventions and dietary restriction can slow down the process of aging. The cell culture model of cellular senescence gives researchers the opportunity to conduct studies in a system more closely related to the human organism; therefore, cells from different human tissues are cultured in vitro until they stop proliferating. This permanent growth arrest is called cellular senescence. Recent studies have demonstrated that senescent cells also accumulate in many tissues in vivo and contribute to age-related pathologies.

How immunosuppressive therapy affects T cells from kidney transplanted patients of different age: the role of latent cytomegalovirus infection.

The average age of patients receiving renal transplantation is increasing as programmes have been established which support the donation of organs from elderly donors to older recipients. Little is known about the effect of immunosuppressive therapy on the immune system of older patients. In this study, T cell function and the composition of the T cell repertoire were analysed in immunosuppressed renal transplant recipients of different age and cytomegalovirus (CMV) status in comparison to age- and CMV-matched controls. Independent of age and CMV status, the production of interleukin (IL)-2 and interferon (IFN)-γ by T cells was decreased in the patient groups and autologous serum from patients was capable of inhibiting the proliferation of CD3⁺ T cells. CXCR5 expression on T cells was increased in patients versus controls reflecting reduced endogenous IL-2 signalling under immunosuppressive therapy. In CMV-seronegative patients kidney transplantation and immunosuppressive therapy did not induce changes in the CD8⁺ T cell pool, but there was a moderate increase in CD4⁺CD28⁻ effector T cells when compared to age-matched controls. In contrast, latent CMV infection triggered a shift from early to late differentiated CD4⁺ and CD8⁺ T cells in patients and controls. This shift was most pronounced in elderly transplant patients under immunosuppressive therapy. In conclusion, our results demonstrate that immunosuppressive therapy following kidney transplantation is effective in patients older than 65 years. Latent CMV infection, however, accelerates age-related changes in the T cell repertoire in elderly people under immunosuppressive therapy. These patients should therefore be monitored with special care.

Self-rated health in individuals with and without disease is associated with multiple biomarkers representing multiple biological domains.

Self-rated health (SRH) is one of the most frequently used indicators in health and social research. Its robust association with mortality in very different populations implies that it is a comprehensive measure of health status and may even reflect the condition of the human organism beyond clinical diagnoses. Yet the biological basis of SRH is poorly understood. We used data from three independent European population samples (N approx. 15,000) to investigate the associations of SRH with 150 biomolecules in blood or urine (biomarkers). Altogether 57 biomarkers representing different organ systems were associated with SRH. In almost half of the cases the association was independent of disease and physical functioning. Biomarkers weakened but did not remove the association between SRH and mortality. We propose three potential pathways through which biomarkers may be incorporated into an individual's subjective health assessment, including (1) their role in clinical diseases; (2) their association with health-related lifestyles; and (3) their potential to stimulate physical sensations through interoceptive mechanisms. Our findings indicate that SRH has a solid biological basis and it is a valid but non-specific indicator of the biological condition of the human organism.

Retrobulbar T cells from patients with Graves' ophthalmopathy are CD8+ and specifically recognize autologous fibroblasts.

Graves' ophthalmopathy is an autoimmune condition characterized by T cell infiltration of the retrobulbar tissue. Phenotypic and functional analysis of these infiltrating cells may provide insight into the pathogenesis of the disease. IL-2-responsive cells were therefore grown out of the retrobulbar tissue from two patients with severe Graves' ophthalmopathy undergoing orbital decompression surgery, and six T cell lines were established and characterized. They consisted predominantly of CD8 + CD45RO+ cells and secreted IL-4, IFN-gamma, and IL-10 upon activation. When screened for their antigen reactivity, all lines proliferated in response to stimulation with autologous retrobulbar fibroblasts in an HLA class I-restricted manner, but did not recognize autologous peripheral blood mononuclear cells, crude eye muscle extract, allogeneic cells, or purified protein derivate of Mycobacterium tuberculosis. In contrast, PBMC from the same patients responded readily to purified protein derivate of Mycobacterium tuberculosis and allogeneic PBMC, but did not recognize autologous fibroblasts. Interestingly, only one of the six retrobulbar T cell lines displayed cytotoxicity towards its specific target cell population. These results suggest that the retrobulbar fibroblasts are a major T cell target in Graves' ophthalmopathy. Pronounced cytokine production in the absence of target cell cytotoxicity may explain fibroblast proliferation, glycosaminoglycan secretion, and secondary eye muscle enlargement in this condition.

Pathogenetic relevance of HLA class II expressing thyroid follicular cells in nontoxic Goiter and in Graves' disease.

HLA class II expressing thyroid follicular cells are found not only in classical thyroid autoimmune diseases, such as Graves' disease, but also in presumably nonautoimmune thyroid disorders such as nontoxic goiter. In this study the immunostimulatory function of the HLA class II expressing thyroid follicular cells derived from patients with nontoxic goiter and with Graves' disease was compared by assessing their capacity to stimulate allogeneic and autologous peripheral blood mononuclear cells, as well as cultured intrathyriodal T lymphocytes. Proliferation of allogeneic peripheral blood mononuclear cells was stimulated by thyroid follicular cells from both nontoxic goiter and Graves' disease thyroids, thus demonstrating that thyroid follicular cells from both disorders are capable of presenting alloantigens. In contrast the proliferation of autologous peripheral blood mononuclear cells was more efficiently stimulated by thyroid follicular cells from Graves' disease than from nontoxic goiter. Cultured intrathyroidal T lymphocytes proliferated specifically in response to autologous HLA class II+ thyroid follicular cells in Graves' disease, but not in nontoxic goiter. The responses were dose dependent and HLA class II restricted. Thyroid autoantigen presentation by HLA class II expressing thyroid follicular cells thus only occurs in Graves' disease, suggesting that HLA class II expression on thyroid follicular cells is an essential feature, but by itself not sufficient for the induction of autoimmunity. Additional factors, the possible nature of which is discussed must also be involved.

Transforming growth factor beta regulates thyroid growth. Role in the pathogenesis of nontoxic goiter.

The production and growth regulatory activity of transforming growth factor beta were studied in human thyroid tissue. As estimated by its mRNA expression in fresh tissue samples, transforming growth factor beta was produced in normal and in diseased thyroid glands. Transforming growth factor beta mRNA was mainly produced by thyroid follicular cells and in lesser quantities by thyroid infiltrating mononuclear cells. The concentrations of transforming growth factor beta mRNA were lower in iodine-deficient nontoxic goiter than in Graves' disease and normal thyroid tissue. Transforming growth factor beta protein secretion by cultured thyroid follicular cells was also low in nontoxic goiter, but could be increased by addition of sodium iodide (10 microM) to the culture medium. Recombinant transforming growth factor beta did not affect basal tritiated thymidine incorporation in cultured thyroid follicular cells, but inhibited, at a concentration of 10 ng/ml, the growth stimulatory influence of insulin-like growth factor I, epidermal growth factor, transforming growth factor alpha, TSH, and partly that of normal human serum on cultured thyroid follicular cells. This inhibition was greater in Graves' disease than in nontoxic goiter. These results suggest that transforming growth factor beta may act as an autocrine growth inhibitor on thyroid follicular cells. Decreased transforming growth factor beta production and decreased responsiveness to transforming growth factor beta may be cofactors in the pathogenesis of iodine-deficient nontoxic goiter.

Release and vasoactive actions of catecholamines during inhibition of prostaglandin synthesis in normal man.

To assess the effect of prostaglandin inhibition upon the vasoactive actions of endogenous and exogenous catecholamines in healthy man, indomethacin (150 mg/day for 3 days) was administered to six healthy men in the sodium-repleted state. Pretreatment with indomethacin did not interfere with the response of blood pressure and pulse rate to orthostasis (10 minutes), a cold pressor test (2 minutes), and the intravenous (i.v.) administration of norepinephrine (NE) (50, 100, and 200 ng kg-1 min-1). Basal plasma concentrations of epinephrine (E) and NE as well as the concentrations of E during orthostasis and cold pressor test remained uninfluenced by pretreatment with indomethacin. While the release of NE during orthostasis appeared to be suppressed in the indomethacin-treated state, it was unchanged during the cold pressor test. These results indicate that inhibition of endogenous prostaglandin synthesis may suppress the release of NE, but does not have a major impact on the vasoactive actions of endogenous and exogenous catecholamines in normal men.

Do heat shock proteins play a role in Graves' disease? Heat shock protein-specific T-cells from Graves' disease thyroids do not recognize thyroid epithelial cells.

Thyroid-derived T-cells from patients with Graves' disease were analyzed for their reactivity to recombinant heat shock proteins (hsp) and autologous thyroid epithelial cells (TEC). Five of six uncloned T-cell lines responded to stimulation with recombinant mycobacterial 71-kilodalton (kDa) hsp and cross-reacted with the corresponding amoebial and human proteins. Only one line reacted with recombinant 65-kDa hsp. Thyroid-derived T-cell lines also showed a proliferative response to TEC, which could be increased in four of the lines, when hsp expression was induced in thyroid cells by heat stress before the initiation of coculture. Clonal specificity analysis of thyroid-derived T-cell clones, however, demonstrated that distinct T-cells were responsible for the recognition of recombinant hsp and TEC. None of the clones responsive to recombinant hsp recognized TEC, whereas TEC-responsive clones did not react with recombinant hsp. Interestingly, the response of the majority of TEC-reactive clones could be dramatically increased when heat-shocked TEC were used as stimulator cells. These results suggest that T-cells specific for hsp of the 70- or 60-kDa families do not recognize TEC in the autoimmune thyroid gland. Heat shock-inducible proteins may, however, still play a role in the autoimmune process by facilitating the presentation of thyroid-specific autoantigen(s) to autoreactive T-cells.

The production of an amyloidogenic metabolite of the Alzheimer amyloid beta precursor protein (APP) in thyroid cells is stimulated by interleukin 1 beta, but inhibited by interferon gamma.

Thyroid epithelial cells have been shown to have a high APP expression and to produce large amounts of its metabolic derivatives, namely secreted APPs and a potentially amyloidogenic 41-kDa C-terminal fragment. It was the aim of the present study to analyze how APP production and metabolism were regulated in human thyroid cells. The effects of three cytokines, interferon gamma (IFN gamma), interleukin 1beta (IL-1 beta) and transforming growth factor (TGF) beta, were investigated. Cell extracts and supernatants were studied by immunoblotting using specific N- and C-terminal APP antibodies. Quantification was performed by densitometric scanning. We demonstrate that IFN gamma has a strong suppressive effect on the production and metabolism of APP. From a concentration of 30 U/ml upwards it reduces the cellular APP content, decreases the amounts of secreted APPs and inhibits the generation of the 41-kDa amyloidogenic APP fragment. In contrast, IL-1 beta has a stimulatory influence on the generation of the amyloidogenic 41-kDa APP metabolite, but does not affect the cellular holoprotein or APPs. TGFbeta has no significant effect on APP. Our results demonstrate that cytokines can regulate APP production and metabolism in thyroid cells. IFN gamma is the first naturally occurring agent described to inhibit the generation of amyloidogenic APP fragments. It may be of relevance in preventing amyloid deposition during inflammatory processes in the thyroid gland, but may exert a similar protective effect in other non-neuronal and neuronal tissues.

Thyroid epithelial cells produce large amounts of the Alzheimer beta-amyloid precursor protein (APP) and generate potentially amyloidogenic APP fragments.

The Alzheimer beta-amyloid precursor protein (APP) is a transmembrane glycoprotein from which the amyloid beta-protein is proteolytically derived. The latter is a hydrophobic peptide that can aggregate and forms the core of the senile plaques found in the brains of patients suffering from Alzheimer's disease (AD). In view of the known association between familial AD and thyroid autoimmune disease, the expression pattern and cellular processing of APP in human thyroid cells were investigated. Cultured thyroid epithelial cells and homogenized thyroid tissue from normal and pathological thyroid samples were analyzed by immunoblotting using specific N- and C-terminal APP antibodies as well as by reverse transcription-polymerase chain reaction in which two sets of oligonucleotide primers were used. The results of these studies demonstrated that APP isoforms 770 and 751 were expressed in fresh thyroid extracts as well as in cultured thyroid epithelial cells, with APP 770 being the predominant form. Compared to other types of cells, such as lymphocytes and fibroblasts, thyroid epithelial cells produced larger amounts of APP. Most of the mature protein was cleaved within the amyloid beta region, as a result of which a large N-terminal APP fragment was released into the culture medium, whereas a C-terminal nonamyloidogenic fragment of 14 kilodaltons (kDa) was retained within the cell. Interestingly, thyroid epithelial cells also contained larger C-terminal APP fragments of 21, 35, and 41 kDa. From the sizes of these fragments it could be deduced that they contained the entire amyloid beta sequence and were thus potentially amyloidogenic. The 41-kDa fragment was unique to thyroid cells. These fragments may be released into the circulation after thyroid cell damage. Increased/altered thyroid APP expression in familiar AD may induce alterations in thyroid epithelial cells and cell damage, and thus explain the frequent occurrence of thyroid autoimmunity in this disease.

The impact of euglycemia and hyperglycemia on stimulated pituitary hormone release in insulin-dependent diabetics.

To study the influence of different blood glucose (BG) concentrations on the release of pituitary hormones, the effect of the simultaneous iv administration of LRH (200 micrograms), TRH (400 micrograms), and arginine (30 g/30 min) upon the serum concentrations of LH, FSH, TSH, PRL, and GH was determined in six male insulin-dependent diabetics. BG concentration was clamped by feedback control and an automated glucose-controlled insulin infusion system at euglycemic (BG 4-5 mmol/liter) or hyperglycemic (BG, 14-18 mmol/liter) levels. Increments in serum concentrations of LH, FSH, TSH, and PRL were similar in the euglycemic and hyperglycemic steady states, whereas the GH response to arginine was suppressed during the hyperglycemic clamp (P less than 0.01). Omission of exogenous insulin during hyperglycemia did not modify the observed hormonal responses. Thus, the release of LH, FSH, TSH, and PRL in response to adequate acute stimuli at the pituitary level is not modulated by hyperglycemia in insulin-dependent diabetes, while arginine-induced GH release is suppressed. Since the effect of arginine on GH is most likely mediated by an action on the hypothalamus, the data suggest that elevated glucose concentrations may exert their modulatory influence on GH secretion at the hypothalamic rather than at the pituitary level.

Immunological features of nonimmunogenic hyperthyroidism.

Blood lymphocyte subpopulations (Leu 4+ cells = pan-T cells, Leu 3a+ cells = helper/inducer cells, and Leu 2a+ cells = suppressor/cytotoxic cells), thyroid-stimulating immunoglobulins, microsomal antibodies and antibodies against thyroglobulin were determined in 10 patients with hyperthyroidism due to single autonomously functioning thyroid nodules (ATN), 11 patients with hyperthyroidism due to Graves' disease (GD) and in 20 normal subjects. Thyroidectomy was performed in 8 of the patients with ATN and in 6 of those with GD after 3 weeks of antithyroid drug treatment with methimazole. Lymphocytic infiltration of thyroid tissue, the amount of the various lymphocyte subsets (Leu 4+, Leu 3a+, and Leu 2a+ T cells as well as B+ B cells) in the thyroid gland, as well as the expression of the histocompatibility antigen HLA-DR on thyrocytes and intrathyroidal lymphocytes were examined. Blood Leu 4+ cells were reduced due to a lack of Leu 2a+ cells in patients with ATN and GD when compared to normal subjects. Thyroid-stimulating immunoglobulins were detected in all patients with ATN and GD, but in none of the normal subjects. Lymphocytic infiltration of thyroid tissue was present in patients with ATN and GD. The various lymphocyte subsets in the thyroid gland did not differ between the two patient groups. DR expression on thyrocytes was seen in 6 of the patients operated for ATN and in 5 of those who underwent surgery for GD. Infiltration with DR+-T lymphocytes was found in all thyroid glands investigated. Thus immunological findings usually classified as proof for the autoimmune origin of GD exist also in patients with ATN. An overlap in the pathogenetic background of both diseases seems possible.

APP peptides stimulate lymphocyte proliferation in normals, but not in patients with Alzheimer's disease.

We hypothesized that metabolic products of the Alzheimer beta amyloid precursor protein (APP) might be targets for cells of the immune system. To test this hypothesis, peripheral blood lymphocytes from young and old healthy blood donors and patients with Alzheimer's disease were analysed for their responsiveness upon stimulation with amyloid beta protein as well as with four other synthetic peptides corresponding to parts of the APP sequence. Stimulation of resting blood lymphocytes from young and old healthy blood donors resulted in IL-2 receptor expression and proliferation in both age groups. In contrast, lymphocytes from the majority of patients with Alzheimer's disease did not proliferate, when stimulated with APP peptides, while their proliferative response to anti-CD3 was unimpaired. This lack of proliferative responsiveness to APP peptides was not due to apoptosis, but could reflect T cell anergy, as it was accompanied by unimpaired IL-2 receptor expression. The results suggest that autoreactive lymphocytes with specificity for metabolic products of APP occur in healthy individuals. These cells may be of relevance for the elimination of potentially amyloidogenic substances. This mechanism could be impaired in patients with Alzheimer's disease.

The amyloid beta peptide abeta (25-35) induces apoptosis independent of p53.

Apoptosis of neuronal cells apparently plays a role in Alzheimer's disease (AD). The amyloid beta (Abeta) peptide derived from beta-amyloid precursor protein is found in AD brain in vivo and can induce apoptosis in vitro. While p53 accumulates in cells of AD brain, it is not known if p53 plays an active role in Abeta-induced apoptosis. We show here that inactivation of p53 in two experimental cell lines, either by expression of the papillomavirus E6 protein or by a shift to restrictive temperature, does not affect apoptosis induction by Abeta (25-35), indicating that Abeta induces apoptosis in a p53-independent manner.

Dendritic cells in old age--neglected by gerontology?

Dendritic cells (DC) are known as the most efficient antigen-presenting cell type to activate T cells and are key modulators of the immune response. Due to their central role in immunology, DC have been considered as a useful tool for immunotherapy. They may also compensate failing T cell reactivity in aged persons. Despite numerous recent advances in the molecular and cell biology of DC, only very few groups have addressed the topic of DC and aging. It is the aim of the present contribution to give a short overview on what is known on DC in old age.

Peripheral blood dendritic cells reinduce proliferation in in vitro aged T cell populations.

Dendritic cells (DC) are professional antigen presenting cells which are essential for the initiation of an immune response. Recently we demonstrated that DC, which had been propagated from the peripheral blood of healthy elderly people, were morphologically and functionally intact. It was the aim of the present study to analyze how DC from young and old healthy individuals could affect T cell responsiveness to antigen in an in vitro senescence model. Tetanus toxoid (TT)-specific T cell lines were derived from 3 young (< 30 years) and 3 old (> 65 years) individuals and were kept in long term culture. T cell proliferation in response to stimulation with antigen presented by either autologous peripheral blood mononuclear cells (PBMC) or DC was assessed at three different time points, once soon after the initiation of the cultures and twice after 20 to 30 population doublings at a stage when growth, was slow and programmed cell death imminent. Antigen presentation by DC enhanced T cell proliferation at each time point and reinduced proliferation in in vitro aged T cell populations which had stopped dividing. Terminal apoptosis was thus prevented. DC from old individuals were as effective as cells from young donors. Our results demonstrate that DC stimulate the clonal expansion and postpone the clonal elimination of antigen-specific T cell populations. As a consequence they may increase immunoreactivity, prolong immunological memory and be of particular importance for the maintenance of the T cell repertoire in old age.

Interactions of the Alzheimer beta amyloid fragment (25-35) with peripheral blood dendritic cells.

We have previously demonstrated that soluble amyloid beta protein (A beta) induces IL-2 receptor expression and proliferation in peripheral T cells from young and old healthy individuals, but not from patients with Alzheimer's disease (AD). It seemed of interest to examine how the immune system would react upon stimulation with A beta in its aggregated form. It was the aim of this study to define interactions between the spontaneously aggregating A beta (25-35) and antigen-presenting cells. Human dendritic cells (DC), propagated from the peripheral blood of young healthy individuals, were incubated with A beta (25-35) and its effects on DC survival, cytokine release, and surface marker expression were monitored. The question whether DC could present amyloid to T cells was also addressed. We demonstrated that A beta (25-35) does not induce DC apoptosis or necrosis. This was shown by electron microscopy as well as by nuclear staining with propidium iodide. Some peptide aggregates were found in intracellular vacuoles of DC. This process did not increase production of TNF alpha and did not change the surface expression of CD18, CD11a or CD11b. A decreased surface expression of MHC class II molecules was, however, noted. DC pulsed with A beta aggregates were unable to stimulate T cells in an autologous coculture system. The results demonstrate that amyloid may escape immune recognition by its failure to activate antigen-presenting cells and by inhibiting MHC class II surface expression.

Changes in the expression of CD31 and CXCR3 in CD4+ naïve T cells in elderly persons.

So far, very few studies exist on the naïve T cell population of elderly persons. Only recently an increase in the percentage of long lived CD4(+)CD31(-) naïve T cells has been claimed to occur with aging. We, therefore, characterised CD31(+) and CD31(-) CD45RA(+) CD4(+) T cells in young and healthy elderly persons. The production of IL-2 and IFN-gamma by the different subpopulations was studied following stimulation with PMA and Ionomycin. The expression of CD28, CD11a, CD62L, CXCR3 and CCR7 was also analysed. The results of this study demonstrate a pronounced increase in the percentage of CD31(-) CD45RA(+) T cells within the CD4 subpopulation of elderly persons. Both, CD31(-) and CD31(+) CD45RA(+) cells expressed CD28, CD62L, were CD11a (dim) and produced IL-2 but no IFN-gamma. This phenotype confirms that they were naïve T cells. IL-2 production by naïve T cells was not impaired in elderly persons. Interestingly, CD31(+) as well as CD31(-) naïve T cells contained a subpopulation of CXCR3(+) cells in elderly individuals, but not in young ones. In spite of expressing this chemokine receptor that enables the cells to migrate into inflammatory tissues, they were still CCR7(+) and CD62L(+). We speculate that due to previous contact with local environmental factors, this subset of naïve T cells acquires a different chemokine receptor phenotype, resulting in an altered migratory capacity in old age. Aberrant contact with antigen and effector cell differentiation in unorthodox locations may be the consequence. This could also affect Th1/Th2 polarisation, which is known to be impaired in elderly persons.