The aged, the dying, the dead: Medical-legal considerations

Some of the special considerations involved in medical care of the aged and dying, along with the medical-legal problems that may arise, are discussed. Legal cases involving the rights of the dying and their families are reviewed, and precautions given for avoiding difficulties.     

The origin of extracellular DNA during the clearance of dead and dying cells

DNA is a nuclear molecule that has both an intracellular and extracellular role. Inside the cell, it is the essential molecule of heredity while outside the cell it can have immunological activity, both alone and in the context of immune complexes. Furthermore, extracellular DNA has information content that can be mined by genomic techniques. Because of the association of extracellular DNA with clinical conditions marked by cell death, dead and dying cells have been considered the origin of this material. To investigate this process, in vitro and in vivo systems have been used to determine the release of DNA from cells, using Jurkat T cells as a model. Thus, in vitro, apoptotic Jurkat cells release DNA whereas necrotic cells do not. The presence of macrophages in these cultures, however, modifies the release process, causing release from necrotic cells as well. In in vivo experiments in which Jurkat cells are administered to normal mice, both apoptotic and necrotic cells give rise to DNA in the blood in a process that requires macrophages and can be modified by glucocorticoids. In this model, female and male mice differ in the extent of DNA release from the administered Jurkat cells. Together, these results indicate that, while apoptosis and necrosis can lead to a blood DNA response, this process requires macrophages and may be hormonally mediated.     

The origin of extracellular DNA during the clearance of dead and dying cells

DNA is a nuclear molecule that has both an intracellular and extracellular role. Inside the cell, it is the essential molecule of heredity while outside the cell it can have immunological activity, both alone and in the context of immune complexes. Furthermore, extracellular DNA has information content that can be mined by genomic techniques. Because of the association of extracellular DNA with clinical conditions marked by cell death, dead and dying cells have been considered the origin of this material. To investigate this process, in vitro and in vivo systems have been used to determine the release of DNA from cells, using Jurkat T cells as a model. Thus, in vitro, apoptotic Jurkat cells release DNA whereas necrotic cells do not. The presence of macrophages in these cultures, however, modifies the release process, causing release from necrotic cells as well. In in vivo experiments in which Jurkat cells are administered to normal mice, both apoptotic and necrotic cells give rise to DNA in the blood in a process that requires macrophages and can be modified by glucocorticoids. In this model, female and male mice differ in the extent of DNA release from the administered Jurkat cells. Together, these results indicate that, while apoptosis and necrosis can lead to a blood DNA response, this process requires macrophages and may be hormonally mediated.     

The influence on the immunomodulatory effects of dying and dead cells of Annexin V

Apoptotic and necrotic cells expose phosphatidylserine (PS). This membrane modification ensures a swift recognition and uptake by phagocytes of the dying and dead cells. Annexin V (AxV) preferentially binds to anionic phospholipids and thereby, modulates the clearance process. First, we analyzed the influence of AxV on the immunogenicity of apoptotic cells. The addition to apoptotic cells of AxV prior to their injection into mice increased their immunogenicity significantly. Next, we studied the influence of endogenous AxV on the allogeneic reaction against apoptotic and necrotic cells. To preserve heat-labile, short-lived "danger signals," we induced necrosis by mechanical stress. Wild-type mice showed a strong, allogeneic delayed-type hypersensitivity (DTH) reaction. In contrast, AxV-deficient animals showed almost no allogeneic DTH reaction, indicating that endogenous AxV increases the immune response against dead cells. Furthermore, AxV-deficient macrophages had a higher immunosuppressive potential in vitro. Next, we analyzed the influence of AxV on chronic macrophage infection with HIV-1, known to expose PS on its surface. The infectivity in human macrophages of HIV-1 was reduced significantly in the presence of AxV. Finally, we show that AxV also blocked the in vitro uptake by macrophages of primary necrotic cells. Similar to apoptotic cells, necrotic cells generated by heat treatment displayed an anti-inflammatory activity. In contrast, mechanical stress-induced necrotic cells led to a decreased secretion of IL-10, indicating a more inflammatory potential. From the experiments presented above, we conclude that AxV influences the clearance of several PS-exposing particles such as viruses, dying, and dead cells.     

The effect of dexamethasone on the generation of plasma DNA from dead and dying cells

To determine the effects of glucocorticoids on the clearance of apoptotic and necrotic cells, the influence of dexamethasone on plasma levels of DNA was assessed in BALB/c mice receiving Jurkat cells treated with etoposide or ethanol. In untreated mice, administration of 10(8) apoptotic or necrotic Jurkat cells led to the appearance of DNA in the plasma. In mice treated 24 hours previously with dexamethasone, levels of DNA were reduced in a dose-dependent manner, with mice receiving 1 and 2.5 mg showing no appreciable plasma DNA levels. Similar results were obtained with assay of lactate dehydrogenase in mice receiving apoptotic cells. The effects of dexamethasone on anti-Fas treatment were also characterized. While treatment with a monoclonal anti-Fas reagent caused a significant plasma DNA response in untreated mice, mice pretreated with dexamethasone showed much lower levels. Blood levels of caspase 3 and TUNEL staining of liver were also reduced in dexamethasone-treated mice compared to controls receiving anti-Fas antibody. These results indicate that glucocorticoids can affect the clearance of apoptotic and necrotic cells as well as the induction of apoptosis in at least some tissues. These activities may be relevant to the efficacy of glucocorticoids in the treatment of inflammatory disease.     

Apoptosis of macrophages and T cells in tuberculosis associated caseous necrosis

Immunity against mycobacteria is almost exclusively confined to epithelioid cell granulomas, where a long-lasting but labile balance exists between host and bacilli. The relationship between immunity and mycobacteria results in regression, growth, or caseation of granulomas. To prove whether caseation is associated with apoptosis, biopsy specimens of patients with tuberculosis were analysed by electron microscopy and by in situ end-labelling combined with immunofluorescence. Apoptotic cells were not detected in regressive granulomas. Whereas productive granulomas without histologically recognizable caseous necrosis revealed only single apoptotic cells, large numbers of apoptotic CD68+ macrophages and apoptotic CD3+, CD45RO+ T cells were observed within caseous foci. As prime candidates undergoing and/or eliciting apoptosis, vital cells surrounding caseous foci were characterized. Immunohistochemistry showed that the majority of vital CD68+ macrophages surrounding caseous foci are negative for the anti-apoptotic protein bcl2, but positive for the pro-apoptotic protein bax. In situ hybridization combined with immunofluorescence demonstrated that the majority of the adjacent lymphocytes are activated CD3+, CD45RO+ cells expressing the pro-inflammatory cytokine interferon gamma (IFN gamma) and the death ligand FasL. These results suggest that caseation is strongly associated with apoptosis of macrophages and T lymphocytes; that the onset of apoptosis in macrophages may be promoted by the lack of bcl2 and the abundance of bax; and that activation-induced cell death (AICD) may be responsible for the apoptosis of T cells.     

Apoptosis and necrosis: interrelation of the phenomena

Cell death types in the multicellular organism may be divided into two groups: (1) natural cell death--programmed, or apoptotic cell death, which involves the activity of regulatory factors including genetic mechanisms, and (2) occasional (pathologic) death, encompassing numerous types of death that result from the severe cell injuries, caused by damaging factors and which are incompatible with cell life. It is suggested that the term "necrosis" should be applied to describe the process of cell destruction and fragmentation within the living organism, following any type of its death. For the cells, that have died by the mechanism of apoptosis, the period of necrosis is usually short and seems to correspond to the duration of apoptotic body existence. In some cases, specific apoptotic bodies may persist for a longer intervals, performing important functions in the organism (epidermal cornified scales, platelets etc.).     

细胞凋亡、胀亡和坏死--关于细胞死亡的新认识

一、一种不同于凋亡的细胞死亡方式———胀亡细胞死亡和细胞坏死代表的是两个完全不同的概念 ,这一点往往被人们忽视。近 2 0年来 ,人们习惯将凋亡(ａｐｏｐｔｏｓｉｓ)和坏死 (ｎｅｃｒｏｓｉｓ)进行比较 ,把凋亡和坏死看作为两种不同的细胞死亡方式。事实上 ,细胞死亡是生物学上表示细胞不可逆损伤的功能性定义 ,凋亡是细胞死亡方式之一 ,而坏死代表了活体内局部组织、细胞死亡后所发生的终末变化的总和 ,并不管其死亡前过程如何 ,是一形态学诊断。当今很多文献所提及的“坏死性细胞死亡”并非真正的坏死[1] ,实际上是另一种不同于凋亡的…     

The effect of inflammation on the generation of plasma DNA from dead and dying cells in the peritoneum

To assess the effects of inflammation on the generation of circulating DNA from dead and dying cells, plasma DNA levels were determined in BALB/c mice, administered apoptotic or necrotic Jurkat cells following induction of peritonitis by treatment with thioglycollate (TG), peptone (PT), or sodium periodate (NaIO(4)). In mice receiving TG or NaIO(4), plasma DNA levels following intraperitoneal administration of Jurkat cells were significantly reduced compared with controls, whereas they were not affected in mice receiving PT. To determine the basis of these differences, the cellular composition of peritoneal fluids prior to the administration of the dead cells was analyzed. Among agents tested, TG administration led to the largest increase in cells, both neutrophils and monocytes. As shown by flow cytometry, the exudates contained apoptotic neutrophils and macrophages, with the highest levels in the TG-induced exudates. Analysis of DNA and caspase 3 in the fluids also showed differences. TG exudates showed increases in DNA and caspase 3, while NaIO(4)-induced exudates had an increase only in DNA. Fluid from PT-treated mice did not have increases in DNA or caspase 3. Together, these results indicate that prior inflammation can affect the generation of blood DNA from apoptotic or necrotic cells, although this effect may vary depending on the composition of the exudates with respect to cells as well as DNA.     

Apoptosis and necrosis in vaccinia virus-infected HeLa G and BSC-40 cells

In most cells, vaccinia virus (VACV) infection is considered to cause a lytic cell death, an equivalent of necrosis. However, upon infection of the epithelial cell lines HeLa G and BSC-40 with VACV strain Western Reserve (WR), we have previously observed an increased activation of and activity attributable to caspases, a typical sign of apoptosis. In this paper, we have further analyzed the type of cell death in VACV-infected cells HeLa G and BSC-40. In a cell-based flow cytometric assay, we showed a specific activation of caspase-2 and 4 in HeLa G and BSC-40 cells infected with VACV, strain WR, while we did not find any effects of inhibitors of calpain and cathepsin D and E. The actual activity of the two caspases, but also of caspase-3, was then confirmed in lysates of infected HeLa G, but not in BSC-40 cells. Accordingly, poly(ADP)-ribose polymerase (PARP) cleavage was found increased only in infected HeLa G cells. Consequently, we have determined morphological features of apoptosis and/or activity of the executioner caspase-3 in infected HeLa G cells in situ, while only a background apoptosis was observed in infected BSC-40 cells. Finally, vaccination strains Dryvax and Praha were found to induce apoptosis in both HeLa G and BSC-40 cells, as characterized morphologically and by PARP cleavage. These findings may be important for understanding the differences in VACV-host interactions and post-vaccination complications in different individuals.     

Apoptosis, necrosis, and proliferation: possible implications in the etiology of keloids.

Keloids are collagenous lesions acquired as a result of abnormal wound heating. In this study we have assessed the potential role of proliferation, apoptosis, and necrosis in keloids. Samples were immunolabeled for proliferating cell nuclear antigen or DNA strand breaks or stained with acridine orange. Proliferating cells were observed in the basal layer of the epidermis and fibroblasts in the dermis, the numbers of the latter being increased in comparison with normal skin. No proliferating cells were observed in the central region of the keloid. In normal skin, apoptotic cells were restricted to the basal layer of the epidermis. In keloid samples, numerous apoptotic cells were observed in the epidermis and dermis; the number and distribution of positive cells decreased more distal to the keloid lesion. Apoptotic endothelial cells of a small proportion of blood vessels in the dermis were also observed. Evidence of necrosis was also seen in the dermis. These results suggest that, with maturity, progressive cell degeneration primarily by apoptosis results in clearance of certain cellular populations resulting in the typical keloid lesion. However, the persistence of fibroblast proliferation at the dermal/keloid interface propagates the fibrosis.     

Release of DNA from dead and dying lymphocyte and monocyte cell lines in vitro

DNA is a nuclear macromolecule that circulates in the blood where its levels can reflect the activity of inflammatory and malignant diseases. While dead and dying cells have usually been considered the source of blood DNA, the mechanisms for its release during apoptosis and necrosis are not well defined. To elucidate DNA release, an in vitro model system was used, assessing DNA in the media of living, apoptotic or necrotic Jurkat and U937 cells. Apoptosis was induced by etoposide, camptothecin or staurosporine, while necrosis was induced by heating at 56 degrees C. DNA release was measured by fluorometry with the dye PicoGreen while the extent of death was measured by fluorescence-activated cell sorter analysis with propidium iodide and annexin. Apoptotic Jurkat cells released significantly more DNA in the media than untreated cells while necrotic cells did not show significant DNA release. U937 cells showed similar findings. Pretreatment of Jurkat cells with z-VAD-fmk, a caspase inhibitor, reduced both apoptosis and DNA release. By gel electrophoresis, extracellular DNA from apoptotic cells showed laddering with low molecular weight fragments. These studies suggest that extracellular release of DNA is a consequence of apoptosis and may account for some of the DNA in the blood.     

Apoptosis and necrosis induced by cyclic mechanical stretching in alveolar type II cells

Alveolar type II (ATII) cells are exposed to mechanical stretch during breathing and mechanical ventilation. Increased stretch may contribute to lung injury. The influence of three stretching patterns (characterized by frequency [min(-1)] - increase in surface area [%]: S40-13, S60-13, S40-30) on parameters of apoptosis, necrosis, and membrane integrity of rat ATII cells was compared with that in static cultures. The S40-13 stretching pattern simulated normal breathing. The other patterns were chosen to study increased amplitude and frequency. There were no significant differences between the S40-13 group and static cultures. Lactic acid dehydrogenase (LDH) release and early apoptotic cells were significantly increased in S60-13 and S40-30 in comparison with static cultures (LDH: 0.089 +/- 0.014 microg/ml and 0.177 +/- 0.050 microg/ml versus 0.050 +/- 0.011 microg/ml; early apoptosis: 17 +/- 3.5% and 23 +/- 3.1% versus 9.7 +/- 1.4%) at 24 h. Necrosis was significantly increased only in the S40-30 group (13 +/- 2.4% versus 6.1 +/- 0.9% in static culture at 24 h). Captopril as well as L-Arginine prevented apoptosis and reduced apoptotic cells to static culture levels in the S40-30 group, but did not influence necrosis and LDH release. Increased mechanical stretch may contribute to lung injury by induction of apoptosis and necrosis in ATII cells. Apoptosis induced by high-amplitude mechanical stretch is prevented by captopril and L-Arginine.     

Molecular mechanisms of liver injury: Apoptosis or necrosis

Hepatic apoptosis is thought of as a prevalent mechanism in most forms of liver injury. However, the role of hepatic apoptosis is often intermixed with the cellular necrosis. It remains unknown how apoptosis is relevant to the progression of the liver injury. This review summarizes the characteristics of both hepatic apoptosis and necrosis in pathogenesis of liver diseases. Apoptosis and necrosis represent alternative outcomes of different etiology during liver injury. Apoptosis is a main mode of cell death in chronic viral hepatitis, but is intermingled with necrosis in cholestatic livers. Necrosis is the principal type of liver cell killing in acetaminophen-induced hepatotoxicity. Anti-apoptosis as a strategy is beneficial to liver repair response. Therapeutic options of liver disease depend on the understanding toward pathogenic mechanisms of different etiology.     

Clearance of dying cells and autoimmunity

Phagocytic clearance of apoptotic cells is an important physiologic homeostatic mechanism that is associated with non-inflammatory or anti-inflammatory sequalae. Disruption of the process of apoptotic cell clearance may contribute to development of a number of inflammatory and autoimmune diseases. In this review, we summarize the molecular pathways that have been suggested to account for phagocytic clearance of apoptotic cells. We discuss potential mechanisms for regulation of phagocytosis and the implications for development of autoimmunity.     

Apoptosis, oncosis, and necrosis. An overview of cell death.

The historical development of the cell death concept is reviewed, with special attention to the origin of the terms necrosis, coagulation necrosis, autolysis, physiological cell death, programmed cell death, chromatolysis (the first name of apoptosis in 1914), karyorhexis, karyolysis, and cell suicide, of which there are three forms: by lysosomes, by free radicals, and by a genetic mechanism (apoptosis). Some of the typical features of apoptosis are discussed, such as budding (as opposed to blebbing and zeiosis) and the inflammatory response. For cell death not by apoptosis the most satisfactory term is accidental cell death. Necrosis is commonly used but it is not appropriate, because it does not indicate a form of cell death but refers to changes secondary to cell death by any mechanism, including apoptosis. Abundant data are available on one form of accidental cell death, namely ischemic cell death, which can be considered an entity of its own, caused by failure of the ionic pumps of the plasma membrane. Because ischemic cell death (in known models) is accompanied by swelling, the name oncosis is proposed for this condition. The term oncosis (derived from ónkos, meaning swelling) was proposed in 1910 by von Reckling-hausen precisely to mean cell death with swelling. Oncosis leads to necrosis with karyolysis and stands in contrast to apoptosis, which leads to necrosis with karyorhexis and cell shrinkage.     

Clearance of dying cells and autoimmunity

Phagocytic clearance of apoptotic cells is an important physiologic homeostatic mechanism that is associated with non-inflammatory or anti-inflammatory sequalae. Disruption of the process of apoptotic cell clearance may contribute to development of a number of inflammatory and autoimmune diseases. In this review, we summarize the molecular pathways that have been suggested to account for phagocytic clearance of apoptotic cells. We discuss potential mechanisms for regulation of phagocytosis and the implications for development of autoimmunity.     

Apoptosis and necrosis in human ejaculated spermatozoa

BACKGROUND: Features of both apoptosis and necrosis have been reported in ejaculated human spermatozoa. This study examines the relative contribution of these two modes of cell death to the demise of these terminally differentiated cells. METHODS: Sperm fractions were prepared from aliquots of semen samples from young normozoospermic donors by simple washing from seminal plasma, by discontinuous density gradient centrifugation or by swim-up technique. They were subsequently incubated in vitro at 37 degrees C for 24 h. Sperm motility, viability, and the presence of two apoptotic markers, phosphatidylserine externalization (annexin-V binding) and DNA fragmentation (TUNEL), were examined before incubation and again after 4 and 24 h of incubation. RESULTS: The swim-up technique was the most efficient in terms of the recovery of viable, motile and non-apoptotic spermatozoa, followed by density gradient centrifugation and finally simple washing. No changes in the parameters tested were observed after 4 h of incubation, but a significant decrease in sperm motility and viability was detected after 24 h irrespective of the sperm preparation technique employed. However, these changes were not accompanied by any increase in the incidence of spermatozoa showing markers of an active apoptotic process. CONCLUSIONS: Healthy human ejaculated spermatozoa appear incapable of initiating apoptosis, at least under in vitro conditions.     

Introduction: the immune response against dying cells

The innate immune system is charged with the daunting task to discriminate harmless cell death events, as they occur in normal tissue homeostasis, and potentially harmful cell deaths, as they are elicited by infectious microorganisms or as a byproduct of malignant transformation. The distinction between harmful and harmless cell death relies on subtle biochemical differences that precede or accompany cell death and that act on a series of receptors, including pattern recognition receptors that are present on cells of the innate immune system, particularly dendritic cells. The present series of articles provides an up-to-date compendium on the molecular crosstalk between cell death and immune cells.     

Armed response: how dying cells influence T-cell functions

Immune responses during infection, injury, and cancer proceed in the presence of tissue injury and cell death. Consequently, the system must deal with its own dead cells while it determines the appropriate response to the invader. As apoptotic cells are known to induce immune tolerance and necrotic cells can be potent stimulators of immunity, this decision becomes more complex. The key to understanding the immunologic choices made during cell death is to examine the mechanisms of tolerance induction by dying cells and then relate them to the mechanisms of immunity. Ideally, immunogenic cell death should be directed toward tumor cells and infected cells, whereas tolerogenic cell death should be associated with preventing unwanted immune responses to self. In this review, we discuss how the decision is made by focusing on the biochemical process of cell death and how its key components can influence both tolerance and immunity.