Chemical Biology, Molecular Mechanism and Clinical Perspective of ��-Secretase Modulators in Alzheimer��s Disease

Comprehensive evidence supports that oligomerization and accumulation of amyloidogenic Aβ42 peptides in brain is crucial in the pathogenesis of both familial and sporadic forms of Alzheimer''s disease. Imaging studies indicate that the buildup of Aβ begins many years before the onset of clinical symptoms, and that subsequent neurodegeneration and cognitive decline may proceed independently of Aβ. This implies the necessity for early intervention in cognitively normal individuals with therapeutic strategies that prioritize safety. The aspartyl protease γ-secretase catalyses the last step in the cellular generation of Aβ42 peptides, and is a principal target for anti-amyloidogenic intervention strategies. Due to the essential role of γ-secretase in the NOTCH signaling pathway, overt mechanism-based toxicity has been observed with the first generation of γ-secretase inhibitors, and safety of this approach has been questioned. However, two new classes of small molecules, γ-secretase modulators (GSMs) and NOTCH-sparing γ-secretase inhibitors, have revitalized γ-secretase as a drug target in AD. GSMs are small molecules that cause a product shift from Aβ42 towards shorter and less toxic Ab peptides. Importantly, GSMs spare other physiologically important substrates of the γ-secretase complex like NOTCH. Recently, GSMs with nanomolar potency and favorable in vivo properties have been described. In this review, we summarize the knowledge about the unusual proteolytic activity of γ-secretase, and the chemical biology, molecular mechanisms and clinical perspective of compounds that target the γ-secretase complex, with a particular focus on GSMs.     

Long-lived antitumor CD8+ lymphocytes for adoptive therapy generated using an artificial antigen-presenting cell.

PURPOSE: Antitumor lymphocytes can be generated ex vivo unencumbered by immunoregulation found in vivo. Adoptive transfer of these cells is a promising therapeutic modality that could establish long-term antitumor immunity. However, the widespread use of adoptive therapy has been hampered by the difficulty of consistently generating potent antitumor lymphocytes in a timely manner for every patient. To overcome this, we sought to establish a clinical grade culture system that can reproducibly generate antigen-specific cytotoxic T lymphocytes (CTL). EXPERIMENTAL DESIGN: We created an off-the-shelf, standardized, and renewable artificial antigen-presenting cell (aAPC) line that coexpresses HLA class I, CD54, CD58, CD80, and the dendritic cell maturation marker CD83. We tested the ability of aAPC to generate tumor antigen-specific CTL under optimal culture conditions. The number, phenotype, effector function, and in vitro longevity of generated CTL were determined. RESULTS: Stimulation of CD8(+) T cells with peptide-pulsed aAPC generated large numbers of functional CTL that recognized a variety of tumor antigens. These CTLs, which possess a phenotype consistent with in vivo persistence, survived ex vivo for prolonged periods of time. Clinical grade aAPC(33), produced under current Good Manufacturing Practices guidelines, generated sufficient numbers of CTL within a short period of time. These CTL specifically lysed a variety of melanoma tumor lines naturally expressing a target melanoma antigen. Furthermore, antitumor CTL were easily generated in all melanoma patients examined. CONCLUSIONS: With clinical grade aAPC(33) in hand, we are now poised for clinical translation of ex vivo generated antitumor CTL for adoptive cell transfer.     

Genetic animal models for retinal degeneration

Inherited retinal degenerations are a common cause of blindness in Western countries. A mechanism for most retinal degenerations is still unknown; hence, a suitable treatment for most of these diseases has yet to be found. Before one can rationally design a treatment, it is necessary to understand the pathway from a gene mutation to the phenotype in patients. Animal models are crucial to understand this process and to develop a treatment. Some naturally occurring animal models are known. However, over the past few years, transgenic engineering has allowed the generation of a rapidly growing number of animal models. In this review, we give an overview of the broad variety of genetic animal models for retinal degeneration.     

Induction of autoimmune diabetes through insulin (but not GAD65) DNA vaccination in nonobese diabetic and in RIP-B7.1 mice

Insulin has been used to modify T-cell autoimmunity in experimental models of type 1 diabetes. In a large clinical trial, the effect of insulin to prevent type 1 diabetes is currently investigated. We here show that insulin can adversely trigger autoimmune diabetes in two mouse models of type 1 diabetes, using intramuscular DNA vaccination for antigen administration. In female nonobese diabetic (NOD) mice, diabetes development was enhanced after preproinsulin (ppIns) DNA treatment, and natural diabetes resistance in male NOD mice was diminished by ppIns DNA vaccination. In contrast, GAD65 DNA conferred partial diabetes protection, and empty DNA plasmid was without effect. In RIP-B7.1 C57BL/6 mice (expressing the T-cell costimulatory molecule B7.1 in pancreatic beta-cells), autoimmune diabetes occurred in 70% of animals after ppIns vaccination, whereas diabetes did not develop spontaneously in RIP-B7.1 mice or after GAD65 or control DNA treatment. Diabetes was characterized by diffuse CD4(+)CD8(+) T-cell infiltration of pancreatic islets and severe insulin deficiency, and ppIns, proinsulin, and insulin DNA were equally effective for disease induction. Our work provides a new model of experimental autoimmune diabetes suitable to study mechanisms and outcomes of insulin-specific T-cell reactivity. In antigen-based prevention of type 1 diabetes, diabetes acceleration should be considered as a potential adverse result.     

Detection of MDM2 alterations in cultured human hepatocytes treated with 17beta-estradiol or 17alpha-ethinylestradiol

BACKGROUND: Preneoplastic and neoplastic lesions of the liver are suspected to arise as a result of estrogen treatment. Here we present the first report on the modulational effects of the steroids 17beta-estradiol (E2) and 17alpha-ethinylestradiol (EE2) on oncogene MDM2 in human hepatocytes. MATERIALS AND METHODS: Collagen-embedded cultures of hepatocytes stimulated with different E2/EE2 concentrations were analyzed by immunocytochemistry, RT-PCR and sequencing for MDM2 protein/mRNA expression, MDM2 mRNA splicing and MDM2 gene mutation. RESULTS: The hepatocytes responded to stimulation with steroid E2/EE2 concentrations from 1-100 nmol/l with the overexpression of MDM2 protein while non-stimulated cells were negative. Stimulation with 1 nmol/l E2 and 10-100 nmol/l EE2 induced MDM2 splicing variants. Hepatocytes treated with 100 nmol/l E2 contained full-length MDM2 mRNA carrying a new type of MDM2 gene mutation. Unstimulated hepatocytes revealed neither mRNA splicing nor alteration of the MDM2 genes. CONCLUSION: The data show that steroid hormones are involved in the induction of MDM2 alterations in benign human hepatocytes. We speculate that some of the alterations may influence MDM2 function, thus possibly favouring genesis of liver changes.     

Wind-dispersed pollen mediates postglacial gene flow among refugia

A long-term genetic legacy of refugial isolation has been postulated and was demonstrated for maternal refugial lineages for numerous plant and animal species. The lineages were assumed to have remained separated from each other for several glacial periods. The conifer Abies alba Miller, silver fir, is an excellent model to test whether pollen-mediated gene flow may eliminate the genetic imprints of Pleistocene refugial isolation. Two DNA markers with contrasting modes of inheritance were applied to 100 populations covering the entire range of silver fir in Europe. The markers exhibited each two highly conserved alleles based on an insertion/deletion of 80 bp in the fourth intron of the mitochondrial nad5 gene and on a synonymous substitution in the chloroplast psbC gene. The geographical distribution of the maternally inherited mitochondrial variation supported the existence of at least two refugia with two recolonizing maternal lineages remaining largely separated throughout the range. The cline of the nad5 allele frequencies was much steeper than the one of the two psbC alleles. The psbC cline was as wide as the whole range of the species. Our results provide striking evidence that even a species with very long generation times and heavy pollen grains was able to establish a highly efficient pollen-mediated gene flow between refugia. Therefore we postulate that an exchange of genetic information between refugia by range-wide paternal introgression is possible in wind-pollinated plant species.     

Thrombogenicity of various endovascular stent types: an in vitro evaluation

PURPOSE: The aim of this study was to evaluate the thrombogenicity of different peripheral stent types in a standardized in vitro model with fresh human whole blood. MATERIALS AND METHODS: Different stents (N = 77; n = 7 of each of 11 types) were implanted in polyvinyl chloride tubing loops and filled with donor blood samples. After 120 minutes of blood circulation, the thrombin-antithrombin III complex (TAT) levels, beta-thromboglobulin (beta-TG) levels, and platelet counts were assessed. RESULTS: After 2 hours, significant differences were seen. TAT values (+/- SD) with the investigated stents were 31 micro g/mL +/- 20 (control, no stent), 328 micro g/mL +/- 206 (Saxx stent, peripheral medium CrNi31 L), 651 micro g/mL +/- 760 (Palmaz Corinthian Stent, 316 L stainless steel, electropolished), 1,609 micro g/mL +/- 1,264 (Palmaz Corinthian Stent, 316 L stainless steel, not electropolished), 810 micro g/mL +/- 578 (Palmaz Schatz long medium stent), 569 micro g/mL +/- 347 (Smart Nitinol stent), 1,037 micro g/mL +/- 577 (Megalink peripheral stent), 543 micro g/mL +/- 487 (peripheral stent, electropolished), 1,674 micro g/mL +/- 2,057 (peripheral stent, not electropolished), 3,128 micro g/mL +/- 1,812 (SelfX Nitinol stent, polished), 5,897 micro g/mL +/- 2,380 (SelfX Nitinol stent, unpolished), and 1,458 micro g/mL +/- 887 (bridge stent). The platelet count (x1,000/ micro L +/- SD) was 218 +/- 35 (control, no stent), 188 +/- 22 (Saxx stent), 187 +/- 20 (Palmaz Corinthian stent, electropolished), 135 +/- 37 (Palmaz Corinthian stent, not electropolished), 170 +/- 24 (Palmaz Schatz stent), 180 +/- 36 (Smart Nitinol stent), 159 +/- 26 (Megalink peripheral stent), 173 +/- 17 (peripheral stent, electropolished), 133 +/- 51 (peripheral stent, not electropolished), 123 +/- 37 (SelfX Nitinol stent, polished), 52 +/- 27 (SelfX Nitinol stent, unpolished), and 130 +/- 31 (bridge stent). CONCLUSION: This standardized study showed a wide range of platelet activation after stent implantation. Electropolishing clearly reduced the thrombogenicity of the stents.