Vaccination with microneme protein NcMIC4 increases mortality in mice inoculated with Neospora caninum

NcMIC4 is a Neospora caninum microneme protein that has been isolated and purified on the basis of its unique lactose-binding properties. We have shown that this protein binds to galactosyl residues of lactose; antibodies directed against NcMIC4 inhibit host cell interactions in vitro, thus making it a vaccine candidate. Because of this feature, NcMIC4 was first purified on a larger scale in its native, functionally active form using lactose-agarose affinity chromatography. Second, NcMIC4 was expressed in Escherichia coli as a histidine-tagged recombinant protein (recNcMIC4) and purified through Ni-affinity chromatography. Third, NcMIC4 cDNA was cloned into the mammalian pcDNA3.1 DNA vector and expression was confirmed upon transfection of Vero cells in vitro. For vaccination studies, we employed the murine cerebral infection model based on C57Bl/6 mice, employing experimental groups of 10 mice each. Two groups were injected intraperitoneally with purified native NcMIC4 and recNcMIC4, respectively, employing RIBI adjuvant. The third group was vaccinated intramuscularly with pcDNA-NcMIC4. Control groups included an infection control, an adjuvant control, and a pcDNA3.1 control group. Following 3 injections at 4-wk intervals, mice were challenged by i.p. inoculation of 2 x 10(6) N. caninum tachyzoites (Nc-1 isolate). During the course of parasite challenge (3 wk), mice from the 3 different test groups showed varying degrees of symptoms bearing a semblance to neosporosis, i.e., walking disorder, rounded back, apathy, and paralysis of the hind limbs. Control groups showed no symptoms at all. Most notably, vaccination with pcDNA-MIC4 proved antiprotective, with 60% of mice succumbing to infection within 3 wk, and all mice lacking a measurable anti-NcMIC4 IgG response. NcMIC4 in its native form elicited a substantial humoral IgG1 immune response and a reduction in cerebral parasite load compared to the controls, but 20% of mice succumbed to infection. Vaccination with recNcMIC4 also resulted in 20% of mice dying; however, in this group, cerebral parasite load was similar to the controls, and recNcMIC4 vaccination elicited a mixed IgG1/IgG2 response. In conclusion, vaccines based on NcMIC4, especially pcDNA-NcMIC4, render mice more susceptible to cerebral disease upon challenge with N. caninum tachyzoites.

Pneumococcal meningitis induces apoptosis in recently postmitotic immature neurons in the dentate gyrus of neonatal rats

Bacterial meningitis is associated with high rates of morbidity and mortality, despite advances in antibiotic therapy. Meningitis caused by Streptococcus pneumoniae is associated with a particularly high incidence of neurological sequelae including deficits resulting from damage to the hippocampus. Previous studies have documented that in neonatal rats with experimental pneumococcal meningitis, cells in the subgranular layer of the dentate gyrus undergo apoptosis. The aim of the present study was to define in more detail the nature of the dying cells in the dentate gyrus. Using bromodeoxyuridine labeling at different times before infection combined with immunocytochemistry, we identified the vulnerable cells as those which underwent mitosis 6-10 days before infection. A majority of these cells are of neuronal lineage. Thus, immature neuronal cells several days after the last cell division are preferentially triggered into apoptosis during pneumococcal meningitis. The loss of these cells may contribute to the long-lasting impairment of hippocampal function identified in animal models and in humans after bacterial meningitis.

Clinical diagnosis of posttraumatic stress disorder after myocardial infarction

BACKGROUND: Clinician-rated large-scale studies estimating the prevalence of posttraumatic stress disorder (PTSD) related to myocardial infarction (MI) and identifying predictors of clinical PTSD are currently lacking. HYPOTHESES: We hypothesized that PTSD is prevalent in post-MI patients and that the subjective experience of the MI determines PTSD status. METHODS: We approached 951 post-MI patients with a questionnaire screening for PTSD symptoms related to their MI. Those responding and meeting a cutoff of PTSD symptom levels were invited to participate in a structured clinical interview to diagnose PTSD following Diagnostic and Statistical Manual of Mental Disorders (DSM-IV) criteria. Fear of dying, feelings of helplessness, and severity of pain perceived during the MI were also assessed by visual analog scales. RESULTS: The screening questionnaire was completed by 394 patients, whereby 77 met the cutoff for the interview (8 patients declined the interview). Forty of 394 patients (10.2%) had clinical PTSD (subsyndromal and syndromal forms combined). Younger age (OR 0.95, 95% CI 0.91-0.99), greater fear of dying (OR 2.77, 95% CI 1.28-5.97), and more intense feelings of helplessness (OR 2.97, 95% CI 1.42-6.21) were independent predictors of PTSD status. Perceived pain intensity during MI, sex, type of index MI, left ventricular ejection fraction, number of coronary occlusions, and highest level of total creatinine kinase were not significant predictors. CONCLUSIONS: Clinical PTSD is prevalent in post-MI patients. Demographic and particularly psychological variables related to the subjective experience of the event were stronger predictors of PTSD status than were objective measures of MI severity.

Viable neutrophils release mitochondrial DNA to form neutrophil extracellular traps

Neutrophil extracellular traps (NETs) represent extracellular structures able to bind and kill microorganisms. It is believed that they are generated by neutrophils undergoing cell death, allowing these dying or dead cells to kill microbes. We show that, following priming with granulocyte/macrophage colony-stimulating factor (GM-CSF) and subsequent short-term toll-like receptor 4 (TLR4) or complement factor 5a (C5a) receptor stimulation, viable neutrophils are able to generate NETs. Strikingly, NETs formed by living cells contain mitochondrial, but no nuclear, DNA. Pharmacological or genetic approaches to block reactive oxygen species (ROS) production suggested that NET formation is ROS dependent. Moreover, neutrophil populations stimulated with GM-CSF and C5a showed increased survival compared with resting neutrophils, which did not generate NETs. In conclusion, mitochondrial DNA release by neutrophils and NET formation do not require neutrophil death and do also not limit the lifespan of these cells.

Guidelines for the use and interpretation of assays for monitoring cell death in higher eukaryotes

Cell death is essential for a plethora of physiological processes, and its deregulation characterizes numerous human diseases. Thus, the in-depth investigation of cell death and its mechanisms constitutes a formidable challenge for fundamental and applied biomedical research, and has tremendous implications for the development of novel therapeutic strategies. It is, therefore, of utmost importance to standardize the experimental procedures that identify dying and dead cells in cell cultures and/or in tissues, from model organisms and/or humans, in healthy and/or pathological scenarios. Thus far, dozens of methods have been proposed to quantify cell death-related parameters. However, no guidelines exist regarding their use and interpretation, and nobody has thoroughly annotated the experimental settings for which each of these techniques is most appropriate. Here, we provide a nonexhaustive comparison of methods to detect cell death with apoptotic or nonapoptotic morphologies, their advantages and pitfalls. These guidelines are intended for investigators who study cell death, as well as for reviewers who need to constructively critique scientific reports that deal with cellular demise. Given the difficulties in determining the exact number of cells that have passed the point-of-no-return of the signaling cascades leading to cell death, we emphasize the importance of performing multiple, methodologically unrelated assays to quantify dying and dead cells.

Intracellular Ca(2+) operates a switch between repair and lysis of streptolysin O-perforated cells

Pore-forming (poly)peptides originating from invading pathogens cause plasma membrane damage in target cells, with consequences as diverse as proliferation or cell death. However, the factors that define the outcome remain unknown. We show that in cells maintaining an intracellular Ca(2+) concentration [Ca(2+)](i) below a critical threshold of 10 microM, repair mechanisms seal off 'hot spots' of Ca(2+) entry and shed them in the form of microparticles, leading to [Ca(2+)](i) reduction and cell recovery. Cells that are capable of preventing an elevation of [Ca(2+)](i) above the critical concentration, yet are unable to complete plasma membrane repair, enter a prolonged phase of [Ca(2+)](i) oscillations, accompanied by a continuous shedding of microparticles. When [Ca(2+)](i) exceeds the critical concentration, an irreversible formation of ceramide platforms within the plasma membrane and their internalisation drives the dying cells beyond the 'point of no return'. These findings show that the extent of [Ca(2+)](i) elevation determines the fate of targeted cells and establishes how different Ca(2+)-dependent mechanisms facilitate either cell survival or death.

A MIF haplotype is associated with the outcome of patients with severe sepsis: a case control study

BACKGROUND: Macrophage migration inhibitory factor (MIF) plays an important regulatory role in sepsis. In the promoter region a C/G single nucleotide polymorphism (SNP) at position -173 (rs755622) and a CATT5-8 microsatellite at position -794 are related to modified promoter activity. The purpose of the study was to analyze their association with the incidence and outcome of severe sepsis. METHODS: Genotype distributions and allele frequencies in 169 patients with severe sepsis, 94 healthy blood donors and 183 postoperative patients without signs of infection or inflammation were analyzed by real time PCR and Sequence analysis. All included individuals were Caucasians. RESULTS: Genotype distribution and allele frequencies of severe sepsis patients were comparable to both control groups. However, the genotype and allele frequencies of both polymorphisms were associated significantly with the outcome of severe sepsis. The highest risk of dying from severe sepsis was detectable in patients carrying a haplotype with the alleles -173 C and CATT7 (p = 0.0005, fisher exact test, RR = 1,806, CI: 1.337 to 2.439). CONCLUSION: The haplotype with the combination of the -173 C allele and the -794 CATT7 allele may not serve as a marker for susceptibility to sepsis, but may help identify septic patients at risk of dying.

Solid organ transplantation across the ABO histo-blood group barrier: a case report

The ABO blood group system until recently constituted an insuperable barrier for solid organ transplantation, but cases of heart transplantation in infants and kidney transplantation in adults have been reported, wherein ABO-incompatible grafts have been successful. In 1990, the molecular genetic basis of three major alleles at the ABO locus was elucidated; A and B glycosyltransferases are specified by a variety of functional alleles at this locus. The antibody response to ABH antigens, namely, naturally occurring anti-A/B IgM and IgG isotype agglutinins, are controlled preoperatively by recipient conditioning using plasma exchange, immunoadsorption, and immunosuppressive regimens. We report an O-type patient who accidentally received a B-type cardiac allograft in 1997 who survived for 5 years, dying for an unrelated reason. Over a period of 45 months semiquantitatively we monitored the expression of ABO-type antigens in graft heart vessels using monoclonal antibodies on sections of formalin-fixed, paraffin-embedded biopsies. We observed a progressive change in the antigenic profile of graft endothelial cells from B- to O-type, which was first detected at 1 year posttransplant and most prominent 3 years later, the end of the observation period. No temporal relationship was observed between the transition from B to O expression, the anti-B antibody levels or the immunosuppressive regimen.

ESPEN Guidelines on Parenteral Nutrition: geriatrics

Older subjects are at increased risk of partial or complete loss of independence due to acute and/or chronic disease and often of concomitant protein caloric malnutrition. Nutritional care and support should be an indispensable part of their management. Enteral nutrition is always the first choice for nutrition support. However, when patients cannot meet their nutritional requirements adequately via the enteral route, parenteral nutrition (PN) is indicated. PN is a safe and effective therapeutic procedure and age per se is not a reason to exclude patients from this treatment. The use of PN should always be balanced against a realistic chance of improvement in the general condition of the patient. Lower glucose tolerance, electrolyte and micronutrient deficiencies and lower fluid tolerance should be assumed in older patients treated by PN. Parenteral nutrition can be administered either via peripheral or central veins. Subcutaneous administration is also a possible solution for basic hydration of moderately dehydrated subjects. In the terminal, demented or dying patient the use of PN or hydration should only be given in accordance with other palliative treatments.

Valorization of agrobiodiversity products and strengthening of local identities in the Peruvian Andes: Experiences from the BioAndes Programme

In the Peruvian Andes, a long history of interaction between the local populations and their natural environment has led to extraordinary levels of agrobiodiversity. However, in sharp contrast with this biological wealth, Andean indigenous populations live under most precarious conditions. Moreover, natural resources are undergoing severe degradation processes and local knowledge about biodiversity management is under serious pressure. Against this background, the BioAndes Programme is developing initiatives based on a biocultural approach that aim at fostering biodiversity through the enhancement of cultural processes. On the basis of intercultural dialogue, joint learning and capacity development, and transdisciplinary action-research, indigenous communities, development practitioners, and researchers strive for the creation of innovative ways to contribute to more sustainable economic, socio-cultural, and political valorization of Andean biodiversity. Project activities are diverse and range from the cultivation, transformation, and commercialization of organic Andean fruits in San Marcos, Cajamarca Department, to the recuperation of natural dying techniques for alpaca wool and traditional weaving in Pitumarca, Cusco Department, and the promotion of responsible ecotourism in both regions. Based on the projects’ first two-years of experience, the following lessons learnt will be presented and discussed: 1. The economic valorization and commercialization of local products can be a powerful tool for the revival and innovation of eroded know-how; at the same time it contributes to the strengthening of local identities, in parallel with the empowerment of marginalized groups such as smallholders and women. 2. Such initiatives are only successful when they are embedded within activities that go beyond the focus on local products and seek the valorization of the entire natural and cultural landscape (e.g. through the promotion of agrotourism and local gastronomy, more sustainable management of local resources including the restoration of ecosystems, and the realization of inventories of local agrobiodiversity and the knowledge related to it). 3. The sustainability of these initiatives, which are often externally induced, is conditioned by the ability of local actors to acquire ownership of projects and access to the knowledge required to carry them out, which also means developing the personal and institutional capacities for handling the whole chain from production to commercialization. 4. The confrontation of different economic rationalities and their underlying worldviews that occur when local or indigenous people integrate into the market economy implies the need for a dialogical co-production of knowledge and collective action by local people, experts from NGOs, and political authorities in order to better control the conditions relating to the market economy. The valorization of local agrobiodiversity shows much potential for enhancing natural and cultural diversity in Southern countries, but only when local communities can participate in the shaping of the conditions under which this happens. Such activities should be designed in the mid- to long-term as part of social learning processes that are carefully embedded in the local context. Supporting institutions play a crucial role in these processes, but should see themselves only as facilitators, while ensuring that control and ownership remain with the local actors.

Potentiometric platform for the quantification of cellular potassium efflux

Renewed interest in the measurement of cellular K(+) effluxes has been prompted by the observation that potassium plays an active and important role in numerous key cellular events, in particular cell necrosis and apoptosis. Although necrosis and apoptosis follow different pathways, both induce intracellular potassium effluxes. Here, we report the use of potassium-selective microelectrodes located in a microfluidic platform for cell culture to monitor and quantify such effluxes in real time. Using this platform, we observed and measured the early signs of cell lysis induced by a modification of the extracellular osmolarity. Furthermore, we were able to quantify the number of dying cells by evaluating the extracellular potassium concentration. A comparison between the potentiometric measurement with a fluorescent live-dead assay performed under similar conditions revealed the delay between potassium effluxes and cell necrosis. These results suggest that such platforms may be exploited for applications, such as cytotoxicological screening assays or tumor cell proliferation assays, by using extracellular K(+) as cell death marker.

Incidence and risk factors of homicide-suicide in Swiss households: National Cohort study

BACKGROUND Homicide-suicides are rare but catastrophic events. This study examined the epidemiology of homicide-suicide in Switzerland. METHODS The study identified homicide-suicide events 1991-2008 in persons from the same household in the Swiss National Cohort, which links census and mortality records. The analysis examined the association of the risk of dying in a homicide-suicide event with socio-demographic variables, measured at the individual-level, household composition variables and area-level variables. Proportional hazards regression models were calculated for male perpetrators and female victims. Results are presented as age-adjusted hazard ratios (HR) with 95% confidence intervals (95%CI). RESULTS The study identified 158 deaths from homicide-suicide events, including 85 murder victims (62 women, 4 men, 19 children and adolescents) and 68 male and 5 female perpetrators. The incidence was 3 events per million households and year. Firearms were the most prominent method for both homicides and suicides. The risk of perpetrating homicide-suicide was higher in divorced than in married men (HR 3.64; 95%CI 1.56-8.49), in foreigners without permanent residency compared to Swiss citizens (HR 3.95; 1.52-10.2), higher in men without religious affiliations than in Catholics (HR 2.23; 1.14-4.36) and higher in crowded households (HR 4.85; 1.72-13.6 comparing ≥2 with <1 persons/room). There was no association with education, occupation or nationality, the number of children, the language region or degree of urbanicity. Associations were similar for female victims. CONCLUSIONS This national longitudinal study shows that living conditions associated with psychological stress and lower levels of social support are associated with homicide-suicide events in Switzerland.

International palliative care experts' view on phenomena indicating the last hours and days of life

BACKGROUND Providing the highest quality care for dying patients should be a core clinical proficiency and an integral part of comprehensive management, as fundamental as diagnosis and treatment. The aim of this study was to provide expert consensus on phenomena for identification and prediction of the last hours or days of a patient's life. This study is part of the OPCARE9 project, funded by the European Commission's Seventh Framework Programme. METHOD The phenomena associated with approaching death were generated using Delphi technique. The Delphi process was set up in three cycles to collate a set of useful and relevant phenomena that identify and predict the last hours and days of life. Each cycle included: (1) development of the questionnaire, (2) distribution of the Delphi questionnaire and (3) review and synthesis of findings. RESULTS The first Delphi cycle of 252 participants (health care professionals, volunteers, public) generated 194 different phenomena, perceptions and observations. In the second cycle, these phenomena were checked for their specific ability to diagnose the last hours/days of life. Fifty-eight phenomena achieved more than 80% expert consensus and were grouped into nine categories. In the third cycle, these 58 phenomena were ranked by a group of palliative care experts (78 professionals, including physicians, nurses, psycho-social-spiritual support; response rate 72%, see Table 1) in terms of clinical relevance to the prediction that a person will die within the next few hours/days. Twenty-one phenomena were determined to have "high relevance" by more than 50% of the experts. Based on these findings, the changes in the following categories (each consisting of up to three phenomena) were considered highly relevant to clinicians in identifying and predicting a patient's last hours/days of life: "breathing", "general deterioration", "consciousness/cognition", "skin", "intake of fluid, food, others", "emotional state" and "non-observations/expressed opinions/other". CONCLUSION Experts from different professional backgrounds identified a set of categories describing a structure within which clinical phenomena can be clinically assessed, in order to more accurately predict whether someone will die within the next days or hours. However, these phenomena need further specification for clinical use.

Euthanasia and assisted suicide in selected European countries and US states: systematic literature review

BACKGROUND Legal in some European countries and US states, physician-assisted suicide and voluntary active euthanasia remain under debate in these and other countries. OBJECTIVES The aim of the study was to examine numbers, characteristics, and trends over time for assisted dying in regions where these practices are legal: Belgium, Luxembourg, the Netherlands, Switzerland, Oregon, Washington, and Montana. DESIGN This was a systematic review of journal articles and official reports. Medline and Embase databases were searched for relevant studies, from inception to end of 2012. We searched the websites of the health authorities of all eligible countries and states for reports on physician-assisted suicide or euthanasia and included publications that reported on cases of physician-assisted suicide or euthanasia. We extracted information on the total number of assisted deaths, its proportion in relation to all deaths, and socio-demographic and clinical characteristics of individuals assisted to die. RESULTS A total of 1043 publications were identified; 25 articles and reports were retained, including series of reported cases, physician surveys, and reviews of death certificates. The percentage of physician-assisted deaths among all deaths ranged from 0.1%-0.2% in the US states and Luxembourg to 1.8%-2.9% in the Netherlands. Percentages of cases reported to the authorities increased in most countries over time. The typical person who died with assistance was a well-educated male cancer patient, aged 60-85 years. CONCLUSIONS Despite some common characteristics between countries, we found wide variation in the extent and specific characteristics of those who died an assisted death.

Exploring the periodicity of cardiovascular events in Switzerland: Variation in deaths and hospitalizations across seasons, day of the week and hour of the day

Purpose The purpose of this study is to explore the periodical patterns of events and deaths related to cardiovascular disease (CVD), acute myocardial infarction (AMI) and stroke in Swiss adults (≥18years). Methods Mortality data for period 1969–2007 (N=869,863 CVD events) and hospitalization data for period 1997–2008 (N=959,990 CVD events) were used. The annual, weekly and circadian distribution of CVD-related deaths and events were assessed. Multivariate analysis was conducted using multinomial logistic regression adjusting for age, gender and calendar year and considering deaths from respiratory diseases, accidents or other causes as competitive events. Results CVD deaths and hospitalizations occurred less frequently in the summer months. Similar patterns were found for AMI and stroke. No significant weekly variation for CVD deaths was found. Stratification by age and gender showed subjects aged <65years to present a higher probability of dying on Mondays and Saturday, only for men. This finding was confirmed after multivariate adjustment. Finally, a circadian variation in CVD mortality was observed, with a first peak in the morning (8–12am) and a smaller second peak in the late afternoon (2–6pm). This pattern persisted after multivariate adjustment and was more pronounced for AMI than for stroke. Conclusion There is a periodicity of hospitalizations and deaths related to CVD, AMI and stroke in Switzerland. This pattern changes slightly according to the age and sex of the subjects. Although the underlying mechanisms are not fully identified, preventive measures should take into account these aspects to develop better strategies of prevention and management of CVD.