Programmed Cell Death in the Developing Nervous System

Virtually all cell populations in the vertebrate nervous system undergo massive "naturally-occurring" or "programmed" cell death (PCD) early in development. Initially neurons and glia are overproduced followed by the demise of approximately one-half of the original cell population. In this review we highlight current hypotheses regarding how large-scale PCD contributes to the construction of the developing nervous system. More germane to the theme of this symposium, we emphasize that the survival of cells during PCD depends critically on their ability to access "trophic" molecular signals derived primarily from interactions with other cells. Here we review the cell-cell interactions and molecular mechanisms that control neuronal and glial cell survival during PCD, and how the inability of such signals to suppress PCD may contribute to cell death in some diseases such as spinal muscular atrophy. Finally, by using neurotrophic factors (e.g. CNTF, GDNF) and genes that control the cell death cascade (e.g. Bcl-2) as examples, we underscore the importance of studying the mechanisms that control neuronal and glial cell survival during normal development as a means of identifying molecules that prevent pathology-induced cell death. Ultimately this line of investigation could reveal effective strategies for arresting neuronal and glial cell death induced by injury, disease, and/or aging in humans.     

Senescent Keratinocytes Die by Autophagic Programmed Cell Death

Normal cells reach senescence after a specific time and number of divisions, leading ultimately to cell death. Although escape from this fate may be a requisite step in neoplastic transformation, the mechanisms governing senescent cell death have not been well investigated. We show here, using normal human epidermal keratinocytes, that no apoptotic markers appear with senescence. In contrast, the expression of several proteins involved in the regulation of macroautophagy, notably Beclin-1 and Bcl-2, was found to change with senescence. The corpses occurring at the senescence growth plateau displayed a large central area delimited by the cytokeratin network that contained a huge quantity of autophagic vacuoles, the damaged nucleus, and most mitochondria. 3-methyladenine, an inhibitor of autophagosome formation, but not the caspase inhibitor zVAD, prevented senescent cell death. We conclude that senescent cells do not die by apoptosis, but as a result of high macroautophagic activity that targets the primary vital cell components.Senescence is described as a tumor suppressor mechanism that limits proliferation of altered cells.¹ It occurs in vivo with advancing age, as well as in culture because of both telomere erosion and increasing oxidative damage.² Despite these alterations, senescent cells are not dead cells: they maintain a metabolic activity, different from that of young cells.³ However, they remain in this state only for a while, and finally die. Although escape from this fate could be a requisite step in neoplastic transformation, how cells die once they have become senescent has not been much investigated, and the results remain controversial.Cell death can occur accidentally, in response to an insult, or as the result of a genetic program, activated for example during development or in response to a specific signal. Death following senescence fits with the definition of programmed cell death, because (i) it is time-scheduled and (ii) senescent cells express a genetic program different from that of young cells.^{4,5,6,7,8,9,10,11} Two main types of programmed cell death have been described: apoptosis (type I), and autophagic programmed cell death (type II). Apoptosis involves characteristic morphological and biochemical changes, including cytoplasmic and nuclear condensation, blebbing, chromatin condensation, oligonucleosomal DNA degradation, and final cell fragmentation into apoptotic bodies. Apoptosis is attributed mainly to caspase activation.¹² Autophagic programmed cell death is accompanied by an increase in macroautophagic activity and is believed to occur through the ensuing degradation of many vital cell components.^13,14 The macroautophagic process starts with sequestration of a damaged cell component by a double membrane whose origin is controversial.^15,16,17 The autophagosome resulting from closure of this membrane then fuses with lysosomes to form an autophagolysosome, inside which the sequestered material is degraded by hydrolytic enzymes at acidic pH. The formation and migration of all these vesicles are orchestrated by about 30 Atg genes^17,18 and depend on the integrity of the cytoskeleton, which is in contrast degraded during apoptosis.¹⁹ Caspase activation is not required²⁰ and may even inhibit the autophagic pathway.²¹ Necrosis, although originally described as the process of accidental cell death, might be partly programmed. It is defined as a type of cell death lacking the characteristics of apoptosis and autophagy, but both its cellular manifestations and its molecular pathways are poorly described. It is generally recognized, however, that necrosis involves early plasma membrane lysis, organelle swelling and lysis, and some vacuolization.^22,23The purpose of this study was therefore to establish whether senescent cells die by apoptosis, autophagic programmed cell death, necrosis, or some other undescribed or mixed type of cell death. Throughout this text, we use the expression senescent-cell death to refer to the mechanism of death following senescence.     

Programmed Cell Death (Apoptosis) in Cord Blood Lymphocytes

Cord blood lymphocytes are functionally immature and have deficient immune responses. In order to determine whether the process of programmed cell death is distinct between cord blood and peripheral blood lymphocytes, we analyzed the expression of fas and bax (apoptosis promoting genes) and bcl-2 and bcl-x _L (apoptosis inhibiting genes) at protein or mRNA levels using flow cytometry and quantitative PCR methods, respectively. The susceptibility of T cell subsets from cord blood and adult peripheral blood to undergo apoptosis following restimulation with anti-CD3 or anti-Fas monoclonal antibodies was also studied. We observed that cord blood T cell subsets expressed lower levels of Fas and Bcl-2, a low bcl-2/bax ratio, and higher bcl-x _L compared to peripheral blood. Additionally, upon primary stimulation with anti-CD3, cord blood T cell subsets were more resistant to apoptosis compared to peripheral blood. In contrast, rechallenge of previously stimulated lymphocytes with anti-CD3 monoclonal antibody triggered apoptosis in a larger proportion of T cells from cord blood as compared to peripheral blood, whereas the number of T cells undergoing anti-Fas-induced programmed cell death were lower in cord blood compared to peripheral blood.     

Fibroblast Programmed Cell Death Ligand 1 Promotes Osteoclastogenesis in Odontogenic Keratocysts

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DNA梯度化分析软件系统在细胞凋亡定量检测中的应用

目的:检验我们设计的DNA梯度化分析软件在细胞凋亡定量检测中的应用价值 方法:用Camptothesin(CAM)诱导U937细胞株凋亡,通过该系统计算DNA琼脂糖凝胶电泳后DNA的梯度值,并与凋亡细胞胞浆寡聚核小体的Elisa检测值进行相关性分析.结果:随着CAM浓度的增加,DNA琼脂糖凝胶电泳后梯度化程度越明显,测出的梯度值也越大,而且与胞浆寡聚核小体的Elisa检测值呈正相关(r=0.989,p<0.01)结论:利用DNA梯度化分析系统可对琼脂糖凝胶电泳后的凋亡细胞DNA片段,进行定量分析,提供简便直接的方法.     

Polymorphisms of Genes for Programmed Cell Death 1 Ligands in Patients with Rheumatoid Arthritis

To investigate the role of ligands for programmed cell death 1 (PD-L) in the pathogenesis of rheumatoid arthritis (RA), 129 patients with RA and 125 unrelated healthy controls were enrolled in this study. The PD-L1 and PD-L2 polymorphisms were determined by the method of polymerase chain reaction (PCR)/direct sequencing or PCR/reaction fragment length polymorphisms. The genotype distributions of PD-L1 6777 C/G were not significantly different between the patients with RA and healthy controls. There was also no significant difference in the allele frequencies of PD-L1 6777 C/G polymorphisms between the patients with RA and controls. Similar findings could also be found in the phenotypes and alleles frequencies of PD-L2 47103 C/T and 47139 T/C polymorphisms between the patients with RA and controls. The patients with PD-L1 6777 G had higher prevalence of rheumatoid nodule in comparison with those without PD-L1 6777 G (p = 0.005, OR = 4.0, 95% CI = 1.5–10.9). In contrast, the PD-L2 47103 C/T and 47139 T/C polymorphisms were not related to the occurrence of rheumatoid nodule. This study demonstrated that the PD-L1 and PD-L2 polymorphisms were not associated with susceptibility to RA in Taiwan. PD-L1 6777 G was associated with the prevalence of rheumatoid nodule. Shu-Chen Wang and Chia-Hui Lin contributed equally to this work.     

T Lymphocytes Bearing the γδ T Cell Receptor are Susceptible to Steroid-Induced Programmed Cell Death

The mechanisms by which glucocorticoids suppress immune responses have not yet been clearly defined. In steroid-sensitive pathological conditions, an increase in γδ T cells can occur in certain untreated systemic autoimmune disorders and seems to be a persistent feature in most cases of systemic lupus erythematosus (SLE). Our previously published data demonstrated that immunosuppressive therapy normalized this expanded SLE T cell subset in parallel with clinical remission of the symptoms. To establish how corticosteroid treatment determines the disappearance of peripheral blood γδ T lymphocytes, circulating αβ and γδ T lymphocytes from seven SLE subjects with active disease and seven healthy individuals were cultured in the presence or absence of 10_-7M Dexamethasone (DEX). Cell suspensions were then analysed for DNA fragmentation, characteristic of apoptotic cell death, by a new cytofluorimetric method. Conventional agarose-gel electrophoresis on the same T cell populations was carried out for comparison. Regular follow-ups for 6 months revealed in vivo steroid treatment determined a dramatic fall in SLE blood γδ T cells, and in vitro experiments seem to indicate that DEX-triggered apoptotic signals are confined to the double negative (CD4_- CD8_-) γδ T cell subpopulation which disappears after in vivo immunosuppressive therapy. Clinical and pathological remission of some autoimmune diseases is often obtained by corticosteroids. Our results offer new insights on the mechanisms through these hormones exert their potent inhibitory activities on immune system cells postulated to play a role in the generation of autoimmune responses.     

Programmed Cell Death of Peripheral Myeloid Precursor Cells in Down Patients: Effect of Zinc Therapy

Hemopoietic stem cell differentiation represents the primary rule of self-renewal, proliferation, and specialization modulated by several mechanisms, including growth factors, cell interactions, and bioavailability of various ions, especially Ca²⁺ and Zn²⁺. Apoptotic death, during normal cell turnover, has been widely studied and is recognized as an important pathway for clonal deletion in the hemopoietic system. Multi-parametric analyses have shown that subjects with Down syndrome show low levels of plasmic zinc associated with the presence of immature myeloid cells in the peripheral blood. This arrangement is repaired by in vivo zinc therapy. This study presents morphological and biochemical analyses to show that ZnSO₄ therapy induces the disappearance of peripheral myeloid precursor cells by a programmed cell death mechanism. The programmed zinc-therapy-induced cell death presumably provides a simple way to regulate the myeloid differentiation selecting appropriate cells.     

Programmed Cell Death of Peripheral Myeloid Precursor Cells in Down Patients: Effect of Zinc Therapy

Hemopoietic stem cell differentiation represents the primary rule of self-renewal, proliferation, and specialization modulated by several mechanisms, including growth factors, cell interactions, and bioavailability of various ions, especially Ca²⁺ and Zn²⁺. Apoptotic death, during normal cell turnover, has been widely studied and is recognized as an important pathway for clonal deletion in the hemopoietic system. Multi-parametric analyses have shown that subjects with Down syndrome show low levels of plasmic zinc associated with the presence of immature myeloid cells in the peripheral blood. This arrangement is repaired by in vivo zinc therapy. This study presents morphological and biochemical analyses to show that ZnSO₄ therapy induces the disappearance of peripheral myeloid precursor cells by a programmed cell death mechanism. The programmed zinc-therapy-induced cell death presumably provides a simple way to regulate the myeloid differentiation selecting appropriate cells.     

In Vivo Activation of T-Cell Induction into the Primed Phenotype and Programmed Cell Death by Staphylococcal Enterotoxin B

The authors demonstrate that SEB immunization activates Vβ8⁺ T cells and induces the acquisition of the primed phenotype as defined previously by low MEL-14 and high Pgp-1 expression. SEB-activated spleen CD4⁺ and CD8Vβ8⁺ T cells have different population dynamics and regulate the expression of MEL-14 and Pgp-1 differentially, suggesting that the SEB-MHC class II complex preferentially activates CD4Vβ8⁺ T cells. Interestingly, at day 3 after SEB immunization, Vβ8⁺ T cells expressing low, but not high, levels of MEL-14 undergo apoptosis, indicating that T-cell activation is a prerequisite for triggering programmed cell death. These results might help to trace antigen-reactive cells to the activated or primed pool, as well as to identify those cells which will undergo programmed cell death.     

Streptolysin S Promotes Programmed Cell Death and Enhances Inflammatory Signaling in Epithelial Keratinocytes during Group A Streptococcus Infection

Streptococcus pyogenes, or group A Streptococcus (GAS), is a pathogen that causes a multitude of human diseases from pharyngitis to severe infections such as toxic shock syndrome and necrotizing fasciitis. One of the primary virulence factors produced by GAS is the peptide toxin streptolysin S (SLS). In addition to its well-recognized role as a cytolysin, recent evidence has indicated that SLS may influence host cell signaling pathways at sublytic concentrations during infection. We employed an antibody array-based approach to comprehensively identify global host cell changes in human epithelial keratinocytes in response to the SLS toxin. We identified key SLS-dependent host responses, including the initiation of specific programmed cell death and inflammatory cascades with concomitant downregulation of Akt-mediated cytoprotection. Significant signaling responses identified by our array analysis were confirmed using biochemical and protein identification methods. To further demonstrate that the observed SLS-dependent host signaling changes were mediated primarily by the secreted toxin, we designed a Transwell infection system in which direct bacterial attachment to host cells was prevented, while secreted factors were allowed access to host cells. The results using this approach were consistent with our direct infection studies and reveal that SLS is a bacterial toxin that does not require bacterial attachment to host cells for activity. In light of these findings, we propose that the production of SLS by GAS during skin infection promotes invasive outcomes by triggering programmed cell death and inflammatory cascades in host cells to breach the keratinocyte barrier for dissemination into deeper tissues.     

Francisella tularensis Schu S4 O-Antigen and Capsule Biosynthesis Gene Mutants Induce Early Cell Death in Human Macrophages

Francisella tularensis is capable of rampant intracellular growth and causes a potentially fatal disease in humans. Whereas many mutational studies have been performed with avirulent strains of Francisella, relatively little has been done with strains that cause human disease. We generated a near-saturating transposon library in the virulent strain Schu S4, which was subjected to high-throughput screening by transposon site hybridization through primary human macrophages, negatively selecting 202 genes. Of special note were genes in a locus of the Francisella chromosome, FTT1236, FTT1237, and FTT1238. Mutants with mutations in these genes demonstrated significant sensitivity to complement-mediated lysis compared with wild-type Schu S4 and exhibited marked defects in O-antigen and capsular polysaccharide biosynthesis. In the absence of complement, these mutants were phagocytosed more efficiently by macrophages than wild-type Schu S4 and were capable of phagosomal escape but exhibited reduced intracellular growth. Microscopic and quantitative analyses of macrophages infected with mutant bacteria revealed that these macrophages exhibited signs of cell death much earlier than those infected with Schu S4. These data suggest that FTT1236, FTT1237, and FTT1238 are important for polysaccharide biosynthesis and that the Francisella O antigen, capsule, or both are important for avoiding the early induction of macrophage death and the destruction of the replicative niche.Much of the recent interest in Francisella tularensis, the etiological agent of tularemia, is due to concern about its potential use as an agent of bioterrorism coupled with an incomplete understanding of the molecular basis of its pathogenicity. F. tularensis is highly pathogenic by the pneumonic route, causing disease in humans with an inoculum as small as 10 organisms, and infection by this route carries a mortality rate of 30 to 60% if untreated (43, 67). Due to its extreme virulence and ease of aerosol dissemination, several nations have weaponized F. tularensis and the U.S. Centers for Disease Control and Prevention have classified this organism as a category A select agent (20). F. tularensis has a remarkably broad host range: it is capable of infecting over 250 known species from across the entire phylogenetic tree, including amoebae, insects, small mammals (such as rodents and lagomorphs), and primates (51). Tularemia is primarily a zoonosis, and humans are thought to be accidental hosts (23). The majority of human infections, the pneumonic infections reported on Martha''s Vineyard in 2000 (21) being an notable exception, are cutaneous, lead to ulceroglandular disease, and ensue following exposure to infected animals or animal products (47). The ability of F. tularensis to infect such a wide range of eukaryotes suggests that this organism either co-opts cellular mechanisms common to all hosts, has the requisite virulence genes to adapt to many different intraorganismal environments, or both.Despite the infectivity of Francisella for disparate hosts, relatively little is known about its virulence genetics. F. tularensis invades and replicates within many cell types: phagocytes, such as primary macrophages (human monocyte-derived macrophages [MDMs] and mouse bone marrow-derived macrophages) and macrophage-like cell lines (J774A.1 and THP-1), as well as in nonphagocytic cells such as bronchial airway epithelial cells, hepatocytes, human umbilical vein endothelial cells, and epithelium-derived tissue culture cell lines (i.e., HEp-2, A549, HBE, and HepG2) (17, 24, 31, 40, 61). Macrophages are known to bind and phagocytose F. tularensis using at least three receptors: complement receptor 3 (CR3), which binds to complement 3b (C3b) protein deposited upon the bacterium when exposed to fresh serum (14) (60); mannose receptor (MR) (60); and scavenger receptor A (53). Once internalized, F. tularensis organisms are able to alter intracellular trafficking of their phagosomes and prevent fusion with the lysosome, acquiring late endosomal markers such as lamp-1 transiently and altogether avoiding endosomes containing cathepsin D and S (15). Shortly thereafter, F. tularensis escapes from the phagosome and grows in the macrophage cytosol. The majority of genes known to be important for phagosomal escape and intracellular growth lie within the duplicated Francisella pathogenicity island (FPI), an ∼30-kb region of the chromosome carrying the igl and pdp operons (46). Mutants with mutations in many of the genes in the FPI, such as iglABCD, vgrG, and iglI, as well as pdpA and pdpD, are defective for intracellular growth in both primary and tissue culture cells in vitro, and these genes may encode a novel type VI secretion system (4). Mutational analysis implicates genes such as mglA, sspA, fevR, fslA, pmrA, and migR in regulation of FPI genes (5-8, 10, 38). However, knowledge of other virulence genes residing outside the FPI that play important roles in intracellular growth is limited.We undertook a high-throughput approach to identify genes important for the growth of virulent F. tularensis Schu S4 in primary human macrophages. F. tularensis has two major biovars: type B (F. tularensis subsp. holarctica), which is found throughout the Northern hemisphere, and type A (F. tularensis subsp. tularensis), which is found exclusively within North America and typically causes a more severe disease in humans than type B isolates. These biovars are further subdivided into clades that exhibit differences in virulence phenotypes (45). A significant research effort to date has focused on the virulence properties of the non-human pathogens Francisella novicida and the F. tularensis live vaccine strain (LVS), an attenuated F. tularensis subsp. holarctica variant generated in the Soviet Union in the 1950s. Although both of these strains are avirulent in healthy humans, they can grow in some human cells and cell lines in vitro and are virulent in the mouse model of infection, properties that make them attractive models for studying the pathogenesis of tularemia. Several random transposon mutant libraries generated in F. novicida and F. tularensis LVS (41, 50, 55, 66, 68, 70) have been screened using both in vitro tissue culture models and mice. In addition, Qin and Mann generated an ∼700-member Tn5 (EZ-TN) mutant library in the type A strain F. tularensis Schu S4 and screened each clone through the hepatocyte-like cell line HepG2 to detect mutants defective in intracellular growth. Among the genes identified in this screen is FTT1236 (55).Lipopolysaccharide (LPS) is a major component in the outer membranes of Gram-negative organisms, and O antigens (O Ags) are known virulence factors of many pathogenic bacteria that function in part to protect against damage by serum complement and antimicrobial peptides as well as mask bacterial surface antigens (16). LPS is composed of a lipid A moiety that secures it to the Gram-negative outer membrane on which a core polysaccharide and O Ag are assembled. Whereas the LPS of most bacterial species is recognized as a pathogen-associated molecular pattern (PAMP) through MD-2/TLR4 and is a potent activating signal for the innate immune system, the LPS of F. tularensis is nearly inert (27). Of note, the lipid A of F. tularensis has an atypical structure, does not bind to LPS-binding protein (LBP), and is therefore not recognized by TLR2 or TLR4 (3, 28, 52). In addition, the F. tularensis O Ag is structurally distinct from that of F. novicida (43). Although our understanding of the genetics of O-Ag biosynthesis in F. tularensis is incomplete, it is known that wbt operon mutants of F. tularensis LVS do not express an O Ag. These strains bind C3b more avidly and are more sensitive to bile salts and to complement-mediated lysis than wild-type strains (13, 39). These mutants are also defective for intracellular growth in J774A.1 cells and exhibit reduced virulence and dissemination in mice (41, 56, 62, 69). Of note, an LVS FTL0706 (designated FTT1238 in F. tularensis Schu S4) mutant lacks an O Ag and exhibits decreased replication within, and is cytotoxic to, J774A.1 cells (41). A corresponding Schu S4 FTT1238 mutant also lacks an O Ag, but its virulence properties have not yet been described.In many pathogenic bacteria, capsular polysaccharides also contribute to serum resistance yet, unlike O Ags, diminish phagocytosis (16). Francisella has long been thought to have an extracellular structure that resembles that of a capsular polysaccharide. Observed by Hood in 1976 (30), the nature of this capsule has remained elusive. Acridine orange treatment was used to produce an undefined LVS mutant that appears to lack an extracellular polysaccharide structure (58). This strain was designated Cap⁻ (alternately termed “rough” in more recent work) and is serum sensitive (13, 62). Furthermore, expression of the capsular polysaccharide was observed to be increased by repeated passage of LVS on Chamberlain''s defined medium, which in turn increased virulence in mice (12). Recently, our group has identified a capsular polysaccharide using a new monoclonal antibody that recognizes this structure as distinct from LPS O Ag. This capsule has immunological properties distinct from those of purified LPS and is also a potential vaccine (2).Advances in transposon delivery systems in our lab and others allowed us to generate a near-saturating random transposon mutant library in strain Schu S4 that is capable of high-throughput screening using the transposon site hybridization (TraSH) system previously used by Weiss et al. for F. novicida (70). Here we describe the utilization of this genetic system to identify and characterize new virulence genes important for F. tularensis Schu S4 entry and growth in human MDMs. Among the genes we identified is a locus required for LPS O-Ag and capsular polysaccharide biogenesis and for serum resistance and intracellular growth within MDMs. We further show that defects in intracellular growth are due to their induction of premature macrophage death, which deprives mutant organisms of their replicative niche.     

Normal Location Of Thumb/Big Toe may be Related to Programmed Cell Death in the Preaxial Area of Embryonic Limb

The forelimbs and hindlimbs of newborn Polydactyly Nagoya (Pdn) mice were examined to analyze the roles of programmed cell death (PCD) in the preaxial region of the limb. Special attention was paid to the relationship between the PCD in the preaxial area and the location and shape of the first digit (thumb/big toe). Although a large, bifurcated or duplicated thumb/big toe appeared in Pdn/+ mice, digit I (thumb/big toe) in Pdn/+ mice, as in +/+ ones, was located more ventro‐proximally than the other four digits. On the other hand, abnormal preaxial digits of the fore/hindlimb in Pdn/Pdn mice lay distally and were aligned at the radial/tibial end of a serial curved plane formed by digits II–V; that is, a thumb and big toe of normal shape and location were not detectable in any preaxial digits of Pdn/Pdn mice. In the limb development of Pdn mouse embryos on Day 11‐12, PCD did not occur in the preaxial mesoderm of fore/hindlimb only in one‐fourths of all embryos obtained by Pdn/+ x Pdn/+ mating. In addition to digital rays II‐V, extra preaxial digital rays appeared in the prominent preaxial expansion of fore/hindlimbs in these embryos on early Day 12. These abnormal limb configurations in embryos were closely similar to those in Pdn/Pdn newborn mice. The present findings suggest that PCD in the preaxial region not only prevents the formation of extra digits but also determines the location of the thumb/big toe for the normal limb morphogenesis. Anat Rec, 2011. © 2011 Wiley‐Liss, Inc.     

程序性死亡因子1及其配体在脑胶质瘤中的研究进展

脑胶质瘤一直是中枢神经系统肿瘤研究的重点及难点,高级别胶质瘤因其浸润生长快,位置特殊,综合治疗后效果并不理想。肿瘤免疫治疗通过维持肿瘤与免疫系统的平衡,恢复机体抗肿瘤免疫应答,从而限制肿瘤的发生、发展。目前免疫治疗已运用与多种实体瘤中,并取得了令人欣喜的结果,也为脑胶质瘤的治疗提供了新的思路。阻断PD-1/PD-L1是目前肿瘤免疫治疗的热点。本文就PD-1/PD-L1在脑胶质瘤中的研究及治疗进展进行综述。