Gerald Pfister

Senescence-associated changes in respiration and oxidative phosphorylation in primary human fibroblasts

Limitation of lifespan in replicative senescence is related to oxidative stress, which is probably both the cause and consequence of impaired mitochondrial respiratory function. The respiration of senescent human diploid fibroblasts was analysed by high-resolution respirometry. To rule out cell-cycle effects, proliferating and growth-arrested young fibroblasts were used as controls. Uncoupled respiration, as normalized to citrate synthase activity, remained unchanged, reflecting a constant capacity of the respiratory chain. Oligomycin-inhibited respiration, however, was significantly increased in mitochondria of senescent cells, indicating a lower coupling of electron transport with phosphorylation. In contrast, growth-arrested young fibroblasts exhibited a higher coupling state compared with proliferating controls. In intact cells, partial uncoupling may lead to either decreased oxidative ATP production or a compensatory increase in routine respiration. To distinguish between these alternatives, we subtracted oligomycin-inhibited respiration from routine respiration, which allowed us to determine the part of respiratory activity coupled with ATP production. Despite substantial differences in the respiratory control ratio, ranging from 4 to 11 in the different experimental groups, a fixed proportion of respiratory capacity was maintained for coupled oxidative phosphorylation in all the experimental groups. This finding indicates that the senescent cells fully compensate for increased proton leakage by enhanced electron-transport activity in the routine state. These results provide a new insight into age-associated defects in mitochondrial function and compensatory mechanisms in intact cells.

Age‐related loss of naïve T cells and dysregulation of T‐cell/B‐cell interactions in human lymph nodes

In this study we analysed the effects of age on T and B lymphocytes in human lymph nodes by comparing lymphocyte subsets in paraffin sections from lymph node tissue taken from healthy young and elderly people. We demonstrate that the relative number of CD8+ T cells decreases with age but that the relative number of CD4+ T cells does not. There is also a very pronounced age‐dependent loss of CD45RA+ naïve T cells. The number and size of follicles and the relative number of CD20+ B cells are similar in young and elderly donors. For polymerase chain reaction analysis of the T‐cell receptor (TCR) repertoire the TCR‐γ gene rearrangements were used as a marker of clonality. This is a reliable tool to detect not only clonal TCR‐γδ populations but also TCR‐αβ populations. Young donors with clonal T‐cell expansions in their lymph node tissue do, however, have a higher number of CD20+ B cells, a higher relative size of germinal centres compared to the follicle mantles and a higher number of immunoglobulin M‐expressing cells than young donors without evidence of clonal T‐cell expansions. Corresponding changes are not observed in elderly donors with clonal T‐cell expansions in their lymph node tissue. In summary our findings demonstrate characteristic effects of aging on human lymph node tissue, the most striking feature being the depletion of naïve T cells and the apparent dysregulation of T‐cell/B‐cell interactions in old age.

Detection of HSP60 on the membrane surface of stressed human endothelial cells by atomic force and confocal microscopy

The highly conserved and ubiquitous heat shock proteins (HSP) are essential for the cellular homeostasis and efficiently trigger cellular responses to stress conditions. Both microbial and human HSP act as dominant antigens in numerous infectious and autoimmune diseases such as atherosclerosis, inducing a strong immune-inflammatory response. In the present study, the surface localization of HSP60 on stressed and unstressed human umbilical venous endothelial cells (HUVECs) was investigated using sensitive high resolution microscopy methods and flow cytometry. Confocal laser scanning microscopy (CLSM) revealed an increase of HSP60 in the mitochondria and on the surface of heat-stressed living and fixed HUVECs compared to unstressed cells. Atomic force microscopy (AFM), which has developed as sensitive surface-probe technique in biology, confirmed the presence of HSP60 on the membrane of stressed cells at an even higher lateral resolution by detecting specific single molecule binding events between the monoclonal antibody AbII-13 tethered to AFM tips and HSP60 molecules on cells. The interaction force (force required to break a single AbII-13/HSP60 bond) was 59±2 pN, which correlated nicely to the 51±1 pN measured with isolated HSP60 attached to mica surfaces. Overall, we found clear evidence for the occurrence of HSP60 on the surface of stressed HUVECs in a very similar patchy distribution pattern in living and fixed cells. The relevance of our findings with respect to the role of HSP60 in atherogenesis is discussed.

Naive T cells in the elderly: are they still there?

Abstract:  One of the most striking changes in the primary lymphoid organs during human aging is the progressive involution of the thymus. As a consequence, the rate of naïve T cell output dramatically declines with age and the peripheral T cell pool shrinks. These changes lead to increased incidence of severe infections and decreased protective effect of vaccinations in the elderly. Little is, however, known of the composition and function of the residual naïve T cell repertoire in elderly persons. To evaluate the impact of aging on the naïve T cell pool, we investigated the quantity, phenotype, function, composition, and senescence status of CD45RA+CD28+ human T cells—a phenotype generally considered as naïve cells—from both young and old healthy donors. We found a significant decrease in the number of CD45RA+CD28+ T cells in the elderly, whereas the proliferative response of these cells is still unimpaired. In addition to their reduced number, CD45RA+CD28+ T cells from old donors display significantly shorter telomeres and have a restricted TCR repertoire in nearly all 24 Vβ families. These findings let us conclude that naïve T cells cannot be classified with conventional markers in old age.

Disruption of vascular endothelial homeostasis by tobacco smoke: impact on atherosclerosis

The World Health Organization (WHO) predicts that by 2020 tobacco will become the largest single health problem worldwide and will cause an estimated 8.4 million deaths annually (http://www5.who.int/tobacco/). Although the impact of smoking on human health is well defined from the medical point of view, surprisingly little is known about the mechanisms by which tobacco smoke mediates its disastrous effects. Here, we demonstrate that tobacco smoke dramatically changes vascular endothelial cell and tissue morphology, leading to a loss of endothelial barrier function within minutes. Long‐term exposure of endothelial cells to tobacco smoke extracts induces necrosis that may trigger a pro‐inflammatory status of the vessel wall. Pre‐incubation of the extracts without cells for 6 h at 37°C led to a complete loss of activity. Further, the endothelium could be rescued by changing to fresh medium even at times when the extracts had lost their activity. Finally, we show that N‐acetyl cysteine and statins inhibit the adverse tobacco smoke effects.

High level HPV-16 E7 oncoprotein expression correlates with reduced pRb-levels in cervical biopsies

High‐risk human papillomaviruses (HPVs) are major etiological agents of cervical cancer. Despite excellent epidemiological evidence for a direct role of HPV‐16 in cervical carcinogenesis, molecular pathways underlying carcinogenesis in vivo remain obscure. The E7 gene is required for immortalization and maintenance of the transformed phenotype in vitro; however, little is known about its role for tumorigenesis in vivo. The E7 gene codes for an unstable protein the abundance of which in cervical biopsies is unknown. We show here that E7 protein levels strongly increase during cervical carcinogenesis, underlining its fundamental role in cervical cancer. The E7 protein was found predominantly in the nucleus and to a minor extent in the cytoplasm in the cervical cancer cell line Ca Ski in vitro and in invasive cervical carcinoma in situ, suggesting that nuclear resident E7 plays a major role in cervical carcinogenesis in humans. The retinoblastoma protein (pRb) is a major E7‐target in vitro. We show here that pRb expression is initially upregulated in LSIL and disappears in later stages concomitant with increased E7 levels, suggesting that E7‐driven degradation of pRb is involved in cervical tumorigenesis in humans.

CD25-Expressing CD8+ T Cells Are Potent Memory Cells in Old Age

We have recently described an IL-2/IL-4-producing CD8+CD25+ nonregulatory memory T cell population that occurs in a subgroup of healthy elderly persons who characteristically still have a good humoral response after vaccination. The present study addresses this specific T cell subset and investigates its origin, clonal composition, Ag specificity, and replicative history. We demonstrate that CD8+CD25+ memory T cells frequently exhibit a CD4+CD8+ double-positive phenotype. The expression of the CD8 αβ molecule and the occurrence of signal-joint TCR rearrangement excision circles suggest a thymic origin of these cells. They also have longer telomeres than their CD8+CD25− memory counterparts, thus indicating a shorter replicative history. CD8+CD25+ memory T cells display a polyclonal TCR repertoire and respond to IL-2 as well as to a panel of different Ags, whereas the CD8+CD25− memory T cell population has a more restricted TCR diversity, responds to fewer Ags, and does not proliferate in response to stimulation with IL-2. Molecular tracking of specific clones with clonotypic primers reveals that the same clones occur in CD8+CD25+ and CD8+CD25− memory T cell populations, demonstrating a lineage relationship between CD25+ and CD25− memory CD8+ T cells. Our results suggest that CD25-expressing memory T cells represent an early stage in the differentiation of CD8+ cells. Accumulation of these cells in elderly persons appears to be a prerequisite of intact immune responsiveness in the absence of naive T cells in old age.

Pelagic ciliates (Protozoa, Ciliophora) of different brackish and freshwater lakes — a community analysis at the species level

Abstract The pelagic ciliate communities from 58 north German lakes differing in their origin (natural lakes and artificial ponds), morphology (from shallow ponds with a maximum depth of below 0.5 m to relatively deep lakes with a maximum depth of more than 10 m, surface areas from below 10 ha to more than 100 ha), trophic state (from mesotrophic to hypertrophic) and salinity (freshwater lakes and brackish water lakes) are described and compared at species level. Each lake was comprehensively sampled quarterly in the years 1996 and 1997, respectively. Applying a quantitative protargol stain, about 140 ciliate species could be identified and quantified in all investigated lakes. 35 species, mainly members of the Prostomatida and Oligotrichida, were found commonly in all types of lakes at all seasons and dominated the pelagic ciliate communities. 3 species were common in freshwaters, but never occurred in brackish lakes. In the brackish waters a mixture of common freshwater species and marine species was found with 13 species exclusively occurring in brackish waters. Lowest ciliate cell numbers were observed for deep freshwater lakes, highest cell numbers were determined for brackish waters. Highest species richness was found in artificial peat ponds with an average of 24 pelagic ciliate species in spring samples. The range of occurrence for the identified species was wide for most common species. However, the influence of some environmental factors could be enlightened.

IgG Opsonization of HIV Impedes Provirus Formation in and Infection of Dendritic Cells and Subsequent Long-Term Transfer to T Cells

Already at initial phases of infection, HIV is coated with complement fragments. During the chronic phase, when HIV-specific IgGs appear, the virus circulates immune complexed with IgG and complement. Thus, we studied the interaction of dendritic cells (DCs) and DC-T cell cocultures with complement (C)-opsonized and C-IgG-opsonized HIV. HIV infection of monocyte-derived DCs and circulating BDCA-1-positive DCs was significantly reduced upon the presence of virus-specific but non-neutralizing IgGs. DCs exposed to C-Ig-HIV or IgG-opsonized HIV showed an impaired provirus formation and p24 production and a decreased transmission rate to autologous nonstimulated T cells upon migration along a chemokine gradient. This reduced infectivity was also observed in long-term experiments, when T cells were added delayed to DCs exposed to IgG-coated HIV without migration. Similar kinetics were seen when sera from HIV-1-infected individuals before and after seroconversion were used in infection assays. Both C- and C-IgG-opsonized HIV were captured and targeted to a tetraspanin-rich endosome in immature DCs, but differed with respect to MHC class II colocalization. The reduced infection by IgG-opsonized HIV is possibly due to interactions of virus-bound IgG with FcγRIIb expressed on DCs. Therefore, the intracellular fate and transmission of immune-complexed HIV seems to differ depending on time and opsonization pattern.

Techniques in gerontology: Cell lines as standards for telomere length and telomerase activity assessment

The length of telomeres is believed to critically influence cellular aging processes and disease development. In order to reliably monitor telomere length and the corresponding cellular telomerase activity by optimized procedures, either based on flow cytometry or quantitative PCR technique, we here propose three commonly used cell lines, HEK293, K562 and TCL1301 as standards. In this contribution, efficient methods to determine mean telomere length of eukaryotic chromosomal DNA and determination of the corresponding telomeras activity are outlined. In particular, wide-range standard curves for a precise assessment of telomere length of genomic DNA by quantitative PCR technique are presented, measures, which greatly simplify the evaluation of respective functional roles of telomeres when studying biological processes such as disease progression and aging.