Questions of Life and Death

The research started with a definition of the general ethical background to be applied in bioethical discussions, particularly regarding aspects of morality that have to be enforced by the community. Only those moral beliefs that can be accepted by consensus in a free discussion can be enforced. It follows that the basic principle of a well ordered society is the equality (and possible upwards extension) of the basic liberties. Therefore, whenever it is possible to respect the principle of autonomy in matters of bioethics (here including questions of abortion, assisted procreation, genetics, organ transplantation and euthanasia) this must be done. On the other hand, there are some common values that can be defended in a free dialogue, values that are needed in order to guarantee stable cooperation in a political society. These values include the respect for human life, which implies the respect for human life in all its forms. This leads to the conclusion that people who are able to be part of the system of cooperation in a political society have rights which are dominant in moral considerations. In all cases where there is no conflict between these rights and the value of life as manifested in human individuals who are not able participate in social cooperation, considerations of the value of the life of the latter must be an important moral consideration. This implies that embryo experimentation should be permitted in order to develop scientific advances needed to save human life, but embryos must not be used for trivial reasons or for the cosmetic industry.

A proteome signature for intrauterine growth restriction derived from multifactorial analysis of mass spectrometry-based cord blood serum profiling

Intrauterine growth restriction (IUGR) is defined as a condition in which the fetus does not reach its genetically given growth potential, resulting in low birth weight. IUGR is an important cause of perinatal morbidity and mortality, thus contributing substantially to medically indicated preterm birth in order to prevent fetal death. We subjected umbilical cord blood serum samples either belonging to the IUGR group (n = 15) or to the control group (n = 15) to fractionation by affinity chromatography using a bead system with hydrophobic interaction capabilities. So prepared protein mixtures were analyzed by MALDI-TOF mass spectrometric profiling. The six best differentiating ion signals at m/z 8205, m/z 8766, m/z 13 945, m/z 15 129, m/z 15 308, and m/z 16 001 were collectively assigned as IUGR proteome signature. Separation confidence of our IUGR proteome signature reached a sensitivity of 0.87 and a specificity of 0.93. Assignment of ion signals in the mass spectra to specific proteins was substantiated by SDS-PAGE in conjunction with peptide mass fingerprint analysis of cord blood serum proteins. One constituent of this proteome signature, apolipoprotein C-III(0) , a derivative lacking glycosylation, has been found more abundant in the IUGR cord blood serum samples, irrespective of gestational age. Hence, we suggest apolipoprotein C-III(0) as potential key-marker of the here proposed IUGR proteome signature, as it is a very low-density lipoprotein (VLDL) and high-density lipoprotein (HDL) member and as such involved in triglyceride metabolism that itself is discussed as being of importance in IUGR pathogenesis. Our results indicate that subtle alterations in protein glycosylation need to be considered for improving our understanding of the pathomechanisms in IUGR.

Detection and distribution of apoptotic cell death in normal and diseased canine cranial cruciate ligaments

One of the possible initiating factors in canine cranial cruciate ligament (CCL) rupture could be an abnormal pattern of ligament cell death. This study compared apoptotic cell death in sections of ruptured CCLs and normal controls, and examined nitric oxide (NO) production in joint tissues and correlated this to apoptosis. CCLs and cartilage from the lateral femoral condyle were harvested from 10 healthy dogs and 15 dogs with CCL rupture and ligaments were further processed to detect cleaved caspase-3 and to determine supernatant NO production in explant cultures. Apoptotic activity was greater in ruptured ligaments compared to controls. NO in ligaments showed a moderate but significant positive correlation with caspase-positive cells. The results suggest that increased apoptosis has a role in CCL rupture and that apoptosis may be influenced by local NO production.

Cardiac glycosides initiate Apo2L/TRAIL-induced apoptosis in non-small cell lung cancer cells by up-regulation of death receptors 4 and 5

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (Apo2L/TRAIL) belongs to the TNF family known to transduce their death signals via cell membrane receptors. Because it has been shown that Apo2L/TRAIL induces apoptosis in tumor cells without or little toxicity to normal cells, this cytokine became of special interest for cancer research. Unfortunately, cancer cells are often resistant to Apo2L/TRAIL-induced apoptosis; however, this can be at least partially negotiated by parallel treatment with other substances, such as chemotherapeutic agents. Here, we report that cardiac glycosides, which have been used for the treatment of cardiac failure for many years, sensitize lung cancer cells but not normal human peripheral blood mononuclear cells to Apo2L/TRAIL-induced apoptosis. Sensitization to Apo2L/TRAIL mediated by cardiac glycosides was accompanied by up-regulation of death receptors 4 (DR4) and 5 (DR5) on both RNA and protein levels. The use of small interfering RNA revealed that up-regulation of death receptors is essential for the demonstrated augmentation of apoptosis. Blocking of up-regulation of DR4 and DR5 alone significantly reduced cell death after combined treatment with cardiac glycosides and Apo2L/TRAIL. Combined silencing of DR4 and DR5 abrogated the ability of cardiac glycosides and Apo2L/TRAIL to induce apoptosis in an additive manner. To our knowledge, this is the first demonstration that glycosides up-regulate DR4 and DR5, thereby reverting the resistance of lung cancer cells to Apo2/TRAIL-induced apoptosis. Our data suggest that the combination of Apo2L/TRAIL and cardiac glycosides may be a new interesting anticancer treatment strategy.

CD40 ligation protects bronchial epithelium against oxidant-induced caspase-independent cell death

CD40 and its ligand regulate pleiotropic biological responses, including cell proliferation, differentiation, and apoptosis. In many inflammatory lung diseases, tissue damage by environmental or endogenous oxidants plays a major role in disease pathogenesis. As the epithelial barrier is a major target for these oxidants, we postulated that CD40, the expression of which is increased in asthma, plays a role in the regulation of apoptosis of bronchial epithelial cells exposed to oxidants. Using 16HBE 14o- cells exposed to oxidant stress, we found that ligation of CD40 (induced by G28-5 monoclonal antibodies) enhanced cell survival and increased the number of cells in G2/M (interphase between DNA synthesis and mitosis) of the cell cycle. This was associated with NF-kappaB and activator protein-1 activation and increased expression of the inhibitor of apoptosis, c-IAP1. However, oxidant stress-induced apoptosis was found to be caspase- and calpain-independent implicating CD40 ligation as a regulator of caspase-independent cell death. This was confirmed by the demonstration that CD40 ligation prevented mitochondrial release and nuclear translocation of apoptosis inducing factor. In conclusion, we demonstrate a novel role for CD40 as a regulator of epithelial cell survival against oxidant stress. Furthermore, we have identified, for the first time, an endogenous inhibitory pathway of caspase-independent cell death.

Causes of death and determinants of outcome in critically ill patients

INTRODUCTION: Whereas most studies focus on laboratory and clinical research, little is known about the causes of death and risk factors for death in critically ill patients. METHODS: Three thousand seven hundred patients admitted to an adult intensive care unit (ICU) were prospectively evaluated. Study endpoints were to evaluate causes of death and risk factors for death in the ICU, in the hospital after discharge from ICU, and within one year after ICU admission. Causes of death in the ICU were defined according to standard ICU practice, whereas deaths in the hospital and at one year were defined and grouped according to the ICD-10 (International Statistical Classification of Diseases and Related Health Problems) score. Stepwise logistic regression analyses were separately calculated to identify independent risk factors for death during the given time periods. RESULTS: Acute, refractory multiple organ dysfunction syndrome was the most frequent cause of death in the ICU (47%), and central nervous system failure (relative risk [RR] 16.07, 95% confidence interval [CI] 8.3 to 31.4, p < 0.001) and cardiovascular failure (RR 11.83, 95% CI 5.2 to 27.1, p < 0.001) were the two most important risk factors for death in the ICU. Malignant tumour disease and exacerbation of chronic cardiovascular disease were the most frequent causes of death in the hospital (31.3% and 19.4%, respectively) and at one year (33.2% and 16.1%, respectively). CONCLUSION: In this primarily surgical critically ill patient population, acute or chronic multiple organ dysfunction syndrome prevailed over single-organ failure or unexpected cardiac arrest as a cause of death in the ICU. Malignant tumour disease and chronic cardiovascular disease were the most important causes of death after ICU discharge.

Multiple thromboembolic events in fetofetal transfusion syndrome in triplets contributing to the understanding of pathogenesis of hydranencephaly in combination with polymicrogyria

Over the last 180 years, several theories concerning the origin of hydranencephaly have been proposed with an emphasis on infectious, aplastic, and vascular etiologies. In this report, we present a case of triplets with fetofetal transfusion syndrome of which 2 fetuses (1 and 2) developed almost similar hydranencephaly, whereas the third exhibited the features of a fetus papyraceus (3). In the monochorial triamniotic placenta, multiple arteriovenous anastomoses were detected, representing a probable route for the transmission of thrombi originating from fetus 3 causing visceral lesions in fetus 2. Hydranencephaly was histologically characterized by necrosis, macrophage invasion, and endothelial proliferation. In addition, polymicrogyria was seen in fetuses 1 and 2. The combination of multiple visceral thromboembolic events and the death of fetus 3 approximately in the 11th week of gestation suggested a vascular thrombotic pathogenesis of hydranencephaly. Polymicrogyria can be considered as postmigratory laminar necrosis. Our findings contribute to the pathogenetic understanding of combined hydranencephaly and polymicrogyria.

Effects of lamotrigine alone and in combination with MK-801, phenobarbital, or phenytoin on cell death in the neonatal rat brain

The neonatal rat brain is vulnerable to neuronal apoptosis induced by antiepileptic drugs (AEDs), especially when given in combination. This study evaluated lamotrigine alone or in combination with phenobarbital, phenytoin, or the glutamate antagonist (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine hydrogen maleate (MK-801) for a proapoptotic action in the developing rat brain. Cell death was assessed in brain regions (striatum, thalamus, and cortical areas) of rat pups (postnatal day 8) by terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay, 24 h after acute drug treatment. Lamotrigine alone did not increase neuronal apoptosis when given in doses up to 50 mg/kg; a significant increase in cell death occurred after 100 mg/kg. Combination of 20 mg/kg lamotrigine with 0.5 mg/kg MK-801 or 75 mg/kg phenobarbital resulted in a significant increase in TUNEL-positive cells, compared with MK-801 or phenobarbital treatment alone. A similar enhancement of phenytoin-induced cell death occurred after 30 mg/kg lamotrigine. In contrast, 20 mg/kg lamotrigine significantly attenuated phenytoin-induced cell death. Lamotrigine at 10 mg/kg was without effect on apoptosis induced by phenytoin. Although the functional and clinical implications of AED-induced developmental neuronal apoptosis remain to be elucidated, our finding that lamotrigine alone is devoid of this effect makes this drug attractive as monotherapy for the treatment of women during pregnancy, and for preterm or neonatal infants. However, because AEDs are often introduced as add-on medication, careful selection of drug combinations and doses may be required to avoid developmental neurotoxicity when lamotrigine is used in polytherapy.

Discovery of insertion element ISCfe1: a new tool for Campylobacter fetus subspecies differentiation

The species Campylobacter fetus is divided into the subspecies C. fetus subsp. venerealis (CFV) and C. fetus subsp. fetus (CFF). CFV is the causative agent of bovine genital campylobacteriosis, a highly contagious venereal disease that may lead to serious reproductive problems, including sterility and abortion. In contrast, CFF can be isolated from the gastrointestinal tract of a wide range of host species, is associated with abortion in sheep and cattle, and can also be isolated from local and systemic infections in humans. Despite differences in host and niche preferences, microbiological differentiation of the two subspecies of C. fetus is extremely difficult. This study describes the identification of a new insertion element, ISCfe1, which is present exclusively in CFV strains, with highly conserved specific ISCfe1 insertion sites. The results are useful for identification and differentiation of the two C. fetus subspecies and will help in understanding the evolution and pathogenesis of C. fetus.

The death-associated protein kinase 2 is up-regulated during normal myeloid differentiation and enhances neutrophil maturation in myeloid leukemic cells

The death-associated protein kinase 2 (DAPK2) belongs to a family of Ca(2+)/calmodulin-regulated serine/threonine kinases involved in apoptosis. During investigation of candidate genes operative in granulopoiesis, we identified DAPK2 as highly expressed. Subsequent investigations demonstrated particularly high DAPK2 expression in normal granulocytes compared with monocytes/macrophages and CD34(+) progenitor cells. Moreover, significantly increased DAPK2 mRNA levels were seen when cord blood CD34(+) cells were induced to differentiate toward neutrophils in tissue culture. In addition, all-trans retinoic acid (ATRA)-induced neutrophil differentiation of two leukemic cell lines, NB4 and U937, revealed significantly higher DAPK2 mRNA expression paralleled by protein induction. In contrast, during differentiation of CD34(+) and U937 cells toward monocytes/macrophages, DAPK2 mRNA levels remained low. In primary leukemia, low expression of DAPK2 was seen in acute myeloid leukemia samples, whereas chronic myeloid leukemia samples in chronic phase showed intermediate expression levels. Lentiviral vector-mediated expression of DAPK2 in NB4 cells enhanced, whereas small interfering RNA-mediated DAPK2 knockdown reduced ATRA-induced granulocytic differentiation, as evidenced by morphology and neutrophil stage-specific maturation genes, such as CD11b, G-CSF receptor, C/EBPepsilon, and lactoferrin. In summary, our findings implicate a role for DAPK2 in granulocyte maturation.

Active caspase 3 and DNA fragmentation as markers for apoptotic cell death in primary and metastatic liver tumours

AIMS: The induction of tumour cell death by apoptosis is a major goal of cancer therapy and the in situ detection of apoptosis in tumour tissue has become an important diagnostic parameter. Different apoptosis detection methods assess distinct biochemical processes in the dying cell. Thus, their direct comparison is mandatory to evaluate their diagnostic value. The aim of this study was to compare the immunohistochemical detection of active caspase 3 and single-stranded DNA in primary and metastatic liver tumours as markers of apoptotic cell death. METHODS: We studied detection of active caspase 3 and single-stranded DNA in 20 primary hepatocellular carcinomas (HCC) and 20 liver metastases from colorectal carcinomas (CRC) using immunohistochemistry on paraffin sections. RESULTS: Our results reveal that both methods are suitable and sensitive techniques for the in situ detection of apoptosis, however, they also demonstrate that immunohistochemistry for active caspase 3 and single-stranded DNA have differential sensitivities in HCC and CRC. CONCLUSION: The sensitivity of apoptosis detection using immunohistochemistry for active caspase 3 and single-stranded DNA may be tumour cell type dependent.