Age-related changes of apoptotic cell death in human lymphocytes

Apoptosis seems to be involved in immunosenescence associated with aging. Moreover, in lymphocytes (PBL) of patients with Alzheimer's disease, an increased susceptibility to the apoptotic pathway has been described possibly due to impaired protection of oxidative stress. Accordingly, it seemed to be of particular interest to investigate the contribution of normal aging to the susceptibility from human lymphocytes to programmed cell death. We could show that PBL from elderly individuals (>60 years) accumulate apoptosing cells to a significant higher extent in spontaneous and activation-induced cell death compared to younger controls (<35 years). Treatment with the oxidative stressor 2-deoxy-D-ribose or with agonistic-CD95-antibody pronounced this effect even more implicating a higher sensitivity to reactive oxygen species and a higher functional CD95 expression, respectively. In addition, expression of the activation markers HLA-DR and CD95 was significantly increased in CD3+-cells of aged subjects, while expression of CD25 did not seem to be affected by age. Expression of Bcl-2 was increased in aging and correlated with the number of apoptotic cells

Fc-Engineered Therapeutic Antibodies: Recent Advances and Future Directions.

Monoclonal therapeutic antibodies have revolutionized the treatment of cancer and other diseases. Fc engineering aims to enhance the effector functions or half-life of therapeutic antibodies by modifying their Fc regions. Recent advances in the Fc engineering of modern therapeutic antibodies can be considered the next generation of antibody therapy. Various strategies are employed, including altering glycosylation patterns via glycoengineering and introducing mutations to the Fc region, thereby enhancing Fc receptor or complement interactions. Further, Fc engineering strategies enable the generation of bispecific IgG-based heterodimeric antibodies. As Fc engineering techniques continue to evolve, an expanding portfolio of Fc-engineered antibodies is advancing through clinical development, with several already approved for medical use. Despite the plethora of Fc-based mutations that have been analyzed in in vitro and in vivo models, we focus here in this review on the relevant Fc engineering strategies of approved therapeutic antibodies to finetune effector functions, to modify half-life and to stabilize asymmetric bispecific IgGs

In vivo manipulation of interleukin-2 expression by a retroviral tetracycline (tet)-regulated system

We have used the tetracycline (tet)-regulated system as described previously to evaluate the applicability of controlled gene expression in cancer gene therapy. As a model gene, we used the human interleukin-2 (IL-2) gene, which has been placed under the transcriptional control of the tetO/promoter. Human melanoma cells were transduced by two modified retroviral tet vectors containing the transactivator regulatory unit and the IL-2 gene driven by the tetO/promoter, respectively. In the absence of tet, IL-2 expression in the target cells was stable over several months. IL-2 production was in the range of 40 U/106 cells/24 hours. A fine tuning of IL-2 expression could be achieved by culturing the transduced cells with increasing doses of tet, whereby a concentration of 500 ng/mL tet in the culture medium abrogated IL-2 expression. Most importantly for clinical application, IL-2 expression by the transduced melanoma cells could also be regulated in vivo. When nu/nu mice were inoculated with the transduced tumor cells, they failed to develop tumors. Instead, the inhibition of IL-2 expression in the transduced tumor cells by oral administration of tet led to subcutaneous tumor growth; this growth rate was comparable with the growth rate of subcutaneously inoculated untransduced parental cells. The finding demonstrates the applicability of the tet-regulated system in cancer gene therapy

Mechanismen des apoptotischen Zelltodes an Lymphozyten im Alterungsprozeß und in der Alzheimer Demenz

Aging and age-related diseases are becoming more and more important for our society and our health care system. Alzheimer's disease (AD) is a disorder that destroys some parts of the brain and is characterized by global cognitive decline including a progressive irreversible loss of memory, orientation, and reasoning. “Healthy aging”, therefore, is one of the major aims for modern medicine. Apoptosis, or programmed cell death, plays an important role for example in fetal development, as well as for learning processes. T-lymphocytes usually undergo apoptosis in order to terminate an acute inflammation. The aim of this thesis was to explore the changes in the apoptotic mechanism of peripheral lymphocytes from Alzheimer’s disease (AD) patients in contrast to physiological aging. The experiments were conducted with lymphocytes of healthy volunteers of different ages, AD patients and young and aged mice. Moreover, transgenic mice carrying familiar AD-related mutations were examined. The aging study of peripheral cells of ‘healthy’-aged volunteers revealed an age-related increase of basal apoptosis. In addition, spontaneous apoptosis as well as apoptosis induced by oxidative stress (ROS) or by Fas engagement were enhanced in aging. A closer look at the subcellular basis of the lymphocytes (e.g. B-, NK-, CD4+-, and CD8+-T cells) determined that all lymphocyte subsets were affected by aging. Therefore, it could be concluded that the regulation of apoptosis is generally impaired in lymphocytes of aged persons. The increased susceptibility to oxidative stress supports the ‘Free radical theory of aging’ that claims the radicals to be the cause for the aging-process. In mice an increase of basal, spontaneous and ROS-induced apoptosis was detected in T cells from the spleen, as well. An oral treatment over two weeks with the Ginkgo biloba extract EGb761 showed a clear reduction of ROS-induced apoptosis in the treated group. Interestingly, basal and spontaneous apoptosis, e.g. physiological apoptosis, were not effected by the plant extract. This is an important benefit for therapy since physiological apoptosis has a great relevance in the elimination of cancer-cells for example. In conclusion, the antidementive drug EGb761 reduces specifically ROS-induced apoptosis that a plays an important role in aging as shown in this thesis. Based on the data found in healthy aging, lymphocytes from AD patients were assessed for apoptosis. The cells show enhanced levels of basal, spontaneous, and Fas-induced apoptosis. In subsequent experiments it was demonstrated that mainly the T cells were responsible for the findings. However, the NK-cells provided an important impact as well. In concordance with AD-affected neurons, peripheral lymphocytes of AD patients show clear signs of apoptotic cell death. In addition, basal apoptosis of T cells and the CD4/CD8-ratio showed a correlation with the severity of the dementia. Therefore, it could be speculated that apoptosis is due to activation-induced cell death (AICD) that occurs in acute and chronic activation of adaptive immunity. In AD there is a chronic neuroinflammation in the CNS triggering degeneration of neural tissue. In order to explore this, the experimental model of lymphocyte’s activation was established in healthy aging first. The study included the detection of various events of lymphocyte’s activation on the basis of the T cell subsets (CD4+ and CD8+). The inducibility to mitogenic stimulation clearly decreased in both subsets in aging. In contrast, T lymphocytes from AD patients showed an enhanced activation subsequent to mitogenic stimulation compared with age-matched nondemented persons. Only proliferation of CD8+ T cells was clearly reduced in AD. This data could be clues that an increased generation of memory T cells due to chronic neuroinflammation might be evident in AD. Memory T lymphocytes show increased inducibility upon mitogenic activation. Interestingly, CD8+ memory T cells display decreased prolifertive capacity. Due to activation, cells die by apoptosis later on. It could be concluded that AD patients display an increased amount of memory T cells compared to controls. The data implicate that there could be a cross talk between inflammatory within the brain and inflammatory cells of the periphery. This is an interesting point since the brain used to be assumed as immune-privileged zone. According to the experiment, the information of the diseased brain is transferred to white blood cells. The connection of those two compartments might raise the opportunity to observe and probably to influence easily not-accessible regions like the brain. Transgenic mice carrying mutations in familiar AD-relevant genes (Amyloid-Precursor-Protein, Presenilin-1, respectively) displayed enhanced levels of apoptotic T cells from the spleen, as well. It seems that those mutated proteins influence the regulation of apoptosis. Probably, they are involved in the increased cell death of T- and NK-cells, as well. Animals overexpressing Presenilin-1 showed reduced levels of apoptotic cell death. It was demonstrated with molecuar biology tools that Presenilin-1, processed during apoptosis, has an anti-apoptotic effect.Altern und damit verbundene Alters-bedingte Erkrankungen stellen ein enormes Problem in der westlichen industrialisierten Welt dar. Apoptose, der programmierte Zelltod, spielt bei vielen pathologischen, aber auch bei physiologischen Prozessen eine wichtige Rolle, so zum Beispiel in der Embryonalentwicklung sowie bei Lernprozessen. Lymphozyten beispielsweise, die während einer Immunreaktion stark proliferieren, werden anschließend apoptotisch eliminiert. Ziel der vorliegenden Arbeit war die Erfassung von Veränderungen des apoptotischen Verhaltens an peripheren Lymphozyten in der Alzheimer Demenz und zum Vergleich im physiologischen Alterungsprozess. Die Untersuchungen wurden mit Lymphozyten von gesunden Probanden unterschiedlichen Alters, Alzheimer-Patienten und Mäusen verschiedener Altersgruppen durchgeführt. Ausserdem wurden transgene Mäuse untersucht, die mutierte Gene tragen, die für die familiäre Form der Alzheimer Demenz verantwortlich sind. Der Alt-Jung Vergleich von peripheren Zellen von “gesund”-gealterten Menschen zeigte einen Alters-bedingten Anstieg der basalen Apoptoserate. Ausserdem waren die spontane, die durch oxidativen Stress (ROS)- sowie Fas-induzierte Apoptose deutlich mit zunehmenden Lebensalter erhöht. Eine genauere Untersuchung der einzelnen Zellsubtypen zeigte, dass dieser Anstieg bei allen Lymphozyten (B-, NK-, CD4+- und CD8+- T-Lymphozyten) zu messen ist. Man kann daher vermuten, dass die Regulation der Apoptose im Alter allgemein in peripheren Lymphozyten verändert ist. Ausserdem legte sich eine verstärkte Vulnerabilität gegenüber oxidativen Stress dar, eine weiterer Befund zur Stützung der “Free radical theory of aging”, die den Radikalen eine ursächliche Bedeutung im Alterungsprozess beilegt. In der Jung-Alt-Studie am Mausmodell zeigte sich sowohl an peripheren Blutlymphozyten als auch an T-Lymphozyten aus der Milz, ein deutlicher Anstieg der basalen, spontanen und ROS-induzierten Apoptose. Eine zweiwöchige perorale Behandlung mit dem Ginkgo biloba-Extrakt EGb761 zeigte eine deutliche Reduktion der ROS-induzierten Apoptose, aber keinerlei Beeinflussung der physiologischen basalen und spontanen Apoptose. Dies hat einen besonderen Vorteil für die Therapie, da Apoptose physiologisch eine wichtige Rolle zur Elimination bestimmter Zellen, wie beispielsweise Tumorzellen, hat. Das Antidementivum EGb761 hat daher nur spezifisch Einfluss auf den oxidativen Stress-induzierten Zelltod, der, wie hier gezeigt werden konnte, im Alterungsprozess eine grosse Rolle spielt. Basierend auf den Befunden zur Apoptose im physiologischen Alterungsprozess, wurden Untersuchungen mit peripheren Blutzellen von Alzheimer-Patienten durchgeführt. Die Zellen der Patienten zeigten eine Erhöhung der basalen, spontanen und Fas-induzierten Apoptose, die bei Untersuchungen der Subtypen (B-, T- und NK-Lymphozyten) auf die T-Lymphozyten zurück geführt werden konnte, die NK-Zellen aber auch deutlich betroffen waren. Wie die Neuronen im ZNS, so zeigen auch die peripheren Lymphozyten eindeutige Veränderungen, die die Apoptose kennzeichnen. Ausserdem erwies sowohl die basale Apoptose der T-Zellen, als auch das Verhältnis der CD4- zu CD8-positiven T-Zellen eine Abhängigkeit zum Schweregrad der Erkrankung. Dies lies die Vermutung aufkommen, dass es sich bei diesem verstärkten Zelltod um den Aktivierungs-induzierten Zelltod (AICD) handeln könnte, der bei einer akuten aber auch chronischen Aktivierung der adaptiven Immunität auftritt. In der Alzheimer Demenz kommt es zu einer jahrelangen chronischen Neuroinflammation, die mit starker Degeneration einher geht. Um diesen Aspekt näher zu beleuchten, war es notwendig, die Lymphozyten in vitro zu stimmulieren. Untersuchungen, zur Aktivierbarkeit der T-Lymphozyten wurden zunächst wieder im Alterungsprozess etabliert. Hierbei konnte deutlich in einer Studie, die mehrere Zeitpunkte sowie die genauere Betrachtung der T-Zellsubpopulationen (CD4+ und CD8+) mit einschloss, gezeigt werden, in wieweit das physiologische Altern die Immunfunktion beeinflusst. So konnte insgesamt eine verminderte Aktivierbarkeit beider T-Zellsubsets von alten Probanden festgestellt werden. Im Kontrast dazu zeigten sowohl CD4+ als auch CD8+ T-Zellen von Alzheimer-Patienten eine verstärkte Aktivierbarkeit verglichen mit nicht-dementen Altersgemittelten Kontrollpersonen. Nur bei der Proliferation nach mitogenen Stimulieren zeigten CD8+ T-Lymphozyten eine geringeres Ansprechen. Diese Befunde könnten Hinweise darauf geben, dass bei dieser Demenzerkrankung verstärkt Gedächtnis-T-Zellen, wahrscheinlich bedingt durch die chronische Inflammation im ZNS, generiert werden. Gedächtnis-TLymphozyten zeigen eine verstärkte Aktivierbarkeit nach mitogener Stimulation. Interessanterweise, zeigen CD8+ Gedächtniszellen eine geringere Proliferation. Aufgrund der Aktivierung sterben die Zellen anschliessend apoptotisch ab. Daher liegt die Vermutung nahe, dass bei Alzheimer-Patienten eine erhöhte Anzahl an Gedächtnis-T-Zellen im vorliegt. Diese Befunde stützen die These, daß der im Zentralnervensystem stattfindende Zelltod über die aktivierten Glia auch in das periphere Immunsystem überstrahlt. Über die Verknüpfung dieser beiden Kompartimente des Abwehrsystems ist es möglich, Veränderungen in einer nicht zugänglichen Region wie dem Gehirn zu beobachten und möglicherweise auch zu beeinflussen. Transgene Mäuse, die Mutationen (im Amyloid-Precursor-Protein- bzw. im Presenilin-1 Gen) tragen, die für die familiäre Form der Alzheimer-Demenz verantwortlich sind, zeigen auch eine erhöhte Apoptoserate an T-Lymphozyten aus der Milz, was darauf hindeutet, dass diese Genprodukte einen signifikanten Einfluss auf die Regulation der Apoptose haben. Möglicherweise sind diese Mechanismen auch am erhöhten Zelltod der T- und NK-Zellen beteiligt. Bei Tieren, die das Wildtyp Presenilin-1 Protein überexpremieren, konnte, mittels molekularbiologischer Methoden, gezeigt werden, dass dieses während der Apoptoseinduktion prozessiert wird und einen antiapoptotischen Effekt ausübt

Age-related increase of oxidative stress-induced apoptosis in mice prevention by Ginkgo biloba extract (EGb761)

Enhanced apoptosis and elevated levels of reactive oxygen species (ROS) play a major role in aging. In addition, several neurodegenerative diseases are associated with increased oxidative stress and apoptosis in neuronal tissue. Antioxidative treatment has neuro-protective effects. The aim of the present study was to evaluate changes of susceptibility to apoptotic cell death by oxidative stress in aging and its inhibition by the antioxidant Ginkgo biloba extract EGb761. We investigated basal and ROS-induced levels of apoptotic lymphocytes derived from the spleen in young (3 months) and old (24 months) mice. ROS were induced by 2-deoxy-D-ribose (dRib) that depletes the intracellular pool of reduced glutathione. Lymphocytes from aged mice accumulate apoptotic cells to a significantly higher extent under basal conditions compared to cells from young mice. Treatment with dRib enhanced this difference, implicating a higher sensitivity to ROS in aging. Apoptosis can be reduced in vitro by treatment with EGb761. In addition, mice were treated daily with 100mg/kg EGb761 per os over a period of two weeks. ROS-induced apoptosis was significantly reduced in the EGb761 group. Interestingly, this effect seemed to be more pronounced in old mice

Age-related impairment of human T lymphocytes' activation: specific differences between CD4+ and CD8+ subsets

The relevance of physiological immune aging is of great interest with respect to determining disorders with pathologic immune function in aging individuals. In recent years, the relevance of changes in peripheral lymphocytes in age-associated neurologic diseases has become more evident. Due to the lack of immunological studies, covering more than one event after mitogenic activation, we envisaged a new concept in the present study, aiming to investigate several events, starting from T cell receptor (TCR) ligation up to T cell proliferation. In addition, we addressed the question whether changes are present in the subsets (CD4, CD8) with aging. Phosphorylation of tyrosine residues declines with increasing age in CD4+ cells. Fewer levels of CD69 positive cells after 4 h mitogenic activation, altered expression of cytokines (IL2, IFN-gamma and TNF-alpha; 22 h) and lower proliferation (72 h) were determined in aging. Moreover, it could be shown that CD8+ lymphocytes react more effectively to mitogenic stimulation with reference to CD69 expression and proliferation in both age groups (60 years old). These data indicate that T cell activation, mediated by TCR engagement, is significantly impaired in aging and both subsets are affected. However, bypassing the TCR does not fully restore T cell function, indicating that there are more mechanisms involved than impaired signal transduction through TCR only. The results will be discussed in relation to their relevance in neurodegenerative and psychiatric disorders

Reduced antioxidant enzyme activity in brains of mice transgenic for human presenilin-1 with single or multiple mutations

Alzheimer's disease-related mutations in the presenilin-1 gene (PS1) are leading to an elevated production of neurotoxic beta-amyloid 1-42 and may additionally enhance oxidative stress. Here, we provide in vivo evidence indicating that brains of transgenic mice expressing different human Alzheimer-linked PS1 mutations exhibit a reduced activity of two antioxidant enzymes. For this purpose, mice transgenic for human PS1 and for single and multiple PS1 mutations were generated. Mice with multiple PS1 mutations showed a significantly decreased activity of the antioxidant enzymes Cu/Zn superoxide dismutase and glutathione reductase already at an age of 3-4 months. As expected, this effect was less pronounced for the mice with a single PS1 mutation. By contrast, animals bearing normal human PS1 showed significantly elevated enzyme activities relative to non-transgenic littermate controls

Effects of EGb 761® Ginkgo biloba extract on mitochondrial function and oxidative stress

As major sources of reactive oxygen species (ROS), mitochondrial structures are exposed to high concentrations of ROS and may therefore be particularly susceptible to oxidative damage. Mitochondrial damage could play a pivotal role in the cell death decision. A decrease in mitochondrial energy charge and redox state, loss of transmembrane potential (depolarization), mitochondrial respiratory chain impairment, and release of substances such as calcium and cytochrome c all contribute to apoptosis. These mitochondrial abnormalities may constitute a part of the spectrum of chronic oxidative stress in Alzheimer's disease. Accumulation of amyloid beta (Abeta) in form of senile plaques is also thought to play a central role in the pathogenesis of Alzheimer's disease mediated by oxidative stress. In addition, increasing evidence shows that Abeta generates free radicals in vitro, which mediate the toxicity of this peptide. In our study, PC12 cells were used to examine the protective features of EGb 761(definition see editorial) on mitochondria stressed with hydrogen peroxide and antimycin, an inhibitor of complex III. In addition, we investigated the efficacy of EGb 761 in Abeta-induced MTT reduction in PC12 cells. Moreover, we examined the effects of EGb 761 on ROS levels and ROS-induced apoptosis in lymphocytes from aged mice after in vivo administration. Here, we will report that EGb 761 was able to protect mitochondria from the attack of hydrogen peroxide, antimycin and Abeta. Furthermore, EGb 761 reduced ROS levels and ROS-induced apoptosis in lymphocytes from aged mice treated orally with EGb 761 for 2 weeks. Our data further emphasize neuroprotective properties of EGb 761, such as protection against Abeta-toxicity, and antiapoptotic properties, which are probably due to its preventive effects on mitochondria

Nose-to-Brain delivery of insulin for Alzheimer’s disease

The transport of small molecules, peptides and proteins via the olfactory epithelium and along olfactory and trigeminal nerve pathways from the nasal cavity to the brain is very well known and clinically established for central nervous system (CNS) active drugs like oxytocin, sumatriptan or insulin. Insulin is a clinically well-established biopharmaceutical with a validated function in cognition. Central supply with insulin via intranasal administration improves cognition in animal models and in human, making insulin a so-called cognitive enhancer. Furthermore, dysregulation of insulin is implicated in the pathogenesis of Alzheimer’s disease, which is associated with lower levels of insulin in the cerebrospinal fluid and is involved in amyloid-beta (Ab) regulation. Clinical trials with intranasal insulin implicate positive effects on learning and memory, but a massive lack of pharmacokinetic and efficacy data hamper a pharmacokinetic – pharmcodynamic relation and a possible clinical development as cognition enhancer. A lack of such data also prevents resolving the mechanisms involved in directing insulin to the central or to the peripheral compartment. Here we discuss the basic mechanism of Nose-to-Brain delivery, evidences for intranasal insulin as cognition enhancer, medical devices for intranasal delivery and safety aspects

Alzheimer's disease-like alterations in peripheral cells from presenilin-1 transgenic mice

Many cases of early-onset inherited Alzheimer's disease (AD) are caused by mutations in the presenilin-1 (PS1) gene. Expression of PS1 mutations in cell culture systems and in primary neurons from transgenic mice increases their vulnerability to cell death. Interestingly, enhanced vulnerability to cell death has also been demonstrated for peripheral lymphocytes from AD patients. We now report that lymphocytes from PS1 mutant transgenic mice show a similar hypersensitivity to cell death as do peripheral cells from AD patients and several cell culture systems expressing PS1 mutations. The cell death-enhancing action of mutant PS1 was associated with increased production of reactive oxygen species and altered calcium regulation, but not with changes of mitochondrial cytochrome c. Our study further emphasizes the pathogenic role of mutant PS1 and may provide the fundamental basis for new efforts to close the gap between studies using neuronal cell lines transfected with mutant PS1, neurons from transgenic animals, and peripheral cells from AD patients. Copyright 2001 Academic Press