End-of-Life Decisions: A Cross-National Study of Treatment Preference Discussions and Surrogate Decision-Maker Appointments

Background

Making treatment decisions in anticipation of possible future incapacity is an important part of patient participation in end-of-life decision-making. This study estimates and compares the prevalence of GP-patient end-of-life treatment discussions and patients’ appointment of surrogate decision-makers in Italy, Spain, Belgium and the Netherlands and examines associated factors.

Methods

A cross-sectional, retrospective survey was conducted with representative GP networks in four countries. GPs recorded the health and care characteristics in the last three months of life of 4,396 patients who died non-suddenly. Prevalences were estimated and logistic regressions were used to examine between country differences and country-specific associated patient and care factors.

Results

GP-patient discussion of treatment preferences occurred for 10%, 7%, 25% and 47% of Italian, Spanish, Belgian and of Dutch patients respectively. Furthermore, 6%, 5%, 16% and 29% of Italian, Spanish, Belgian and Dutch patients had a surrogate decision-maker. Despite some country-specific differences, previous GP-patient discussion of primary diagnosis, more frequent GP contact, GP provision of palliative care, the importance of palliative care as a treatment aim and place of death were positively associated with preference discussions or surrogate appointments. A diagnosis of dementia was negatively associated with preference discussions and surrogate appointments.

Conclusions

The study revealed a higher prevalence of treatment preference discussions and surrogate appointments in the two northern compared to the two southern European countries. Factors associated with preference discussions and surrogate appointments suggest that delaying diagnosis discussions impedes anticipatory planning, whereas early preference discussions, particularly for dementia patients, and the provision of palliative care encourage participation.     

<Emphasis Type="Italic">pspA</Emphasis> overexpression in <Emphasis Type="Italic">Streptomyces lividans</Emphasis> improves both Sec- and Tat-dependent protein secretion

Streptomyces is an interesting host for the secretory production of recombinant proteins because of its innate capacity to secrete proteins at high level in the culture medium. In this report, we evaluated the importance of the phage-shock protein A (PspA) homologue on the protein secretion yield in Streptomyces lividans. The PspA protein is supposed to play a role in the maintenance of the proton motive force (PMF). As the PMF is an energy source for both Sec- and Tat-dependent secretion, we evaluated the influence of the PspA protein on both pathways by modulating the pspA expression. Results indicated that pspA overexpression can improve the Tat-dependent protein secretion as illustrated for the Tat-dependent xylanase C and enhanced green fluorescent protein (EGFP). The effect on Sec-dependent secretion was less pronounced and appeared to be protein dependent as evidenced by the increase in subtilisin inhibitor (Sti-1) secretion but the lack of increase in human tumour necrosis factor (hTNFα) secretion in a pspA-overexpressing strain.     

Protein secretion biotechnology in Gram-positive bacteria with special emphasis on Streptomyces lividans

Proteins secreted by Gram-positive bacteria are released into the culture medium with the obvious benefit that they usually retain their native conformation. This property makes these host cells potentially interesting for the production of recombinant proteins, as one can take full profit of established protocols for the purification of active proteins. Several state-of-the-art strategies to increase the yield of the secreted proteins will be discussed, using Streptomyces lividans as an example and compared with approaches used in some other host cells. It will be shown that approaches such as increasing expression and translation levels, choice of secretion pathway and modulation of proteins thereof, avoiding stress responses by changing expression levels of specific (stress) proteins, can be helpful to boost production yield. In addition, the potential of multi-omics approaches as a tool to understand the genetic background and metabolic fluxes in the host cell and to seek for new targets for strain and protein secretion improvement is discussed. It will be shown that S. lividans, along with other Gram-positive host cells, certainly plays a role as a production host for recombinant proteins in an economically viable way. This article is part of a Special Issue entitled: Protein trafficking and secretion in bacteria. Guest Editors: Anastassios Economou and Ross Dalbey.     

Conditional deletion of Smad1 and Smad5 in somatic cells of male and female gonads leads to metastatic tumor development in mice

The transforming growth factor β (TGFβ) family has critical roles in the regulation of fertility. In addition, the pathogenesis of some human cancers is attributed to misregulation of TGFβ function and SMAD2 or SMAD4 mutations. There are limited mouse models for the BMP signaling SMADs (BR-SMADs) 1, 5, and 8 because of embryonic lethality and suspected genetic redundancy. Using tissue-specific ablation in mice, we deleted the BR-SMADs from somatic cells of ovaries and testes. Single conditional knockouts for Smad1 or Smad5 or mice homozygous null for Smad8 are viable and fertile. Female double Smad1 Smad5 and triple Smad1 Smad5 Smad8 conditional knockout mice become infertile and develop metastatic granulosa cell tumors. Male double Smad1 Smad5 conditional knockout mice are fertile but demonstrate metastatic testicular tumor development. Microarray analysis indicated significant alterations in expression of genes related to the TGFβ pathway, as well as genes involved in infertility and extracellular matrix production. These data strongly implicate the BR-SMADs as part of a critical developmental pathway in ovaries and testis that, when disrupted, leads to malignant transformation.     

Secretory production of biologically active rat interleukin-2 by Clostridium acetobutylicum DSM792 as a tool for anti-tumor treatment

A bacterium, isolated from contaminated soils around a chemical factory and named strain DSP3 was capable of biodegrading both chlorpyrifos and 3,5,6-trichloro-2-pyridinol. Based on the results of phenotypic features, phylogenetic similarity of 16S rRNA gene sequences, DNA G+C content, and DNA homology between strain DSP3 and reference strains, strain DSP3 was identified as Alcaligenes faecalis. Chlorpyrifos was utilized as the sole source of carbon and phosphorus by strain DSP3. We examined the role of strain DSP3 in the degradation of chlorpyrifos and 3,5,6-trichloro-2-pyridinol under different culture conditions. Parathion and diazinon could also be degraded by strain DSP3 when provided as the sole sources of carbon and phosphorus. An addition of strain DSP3 (10(8)cells g(-1)) to soil with chlorpyrifos (100 mg kg(-1)) resulted in a higher degradation rate than the one obtained from non-inoculated soils. Different degradation rates of chlorpyrifos in six types of treated soils suggested that soils used for cabbage growing in combination with inoculation of strain DSP3 showed enhanced microbial degradation of chlorpyrifos.     

Receptor-independent Role of Urokinase-Type Plasminogen Activator in Pericellular Plasmin and Matrix Metalloproteinase Proteolysis during Vascular Wound Healing in Mice

It has been proposed that the urokinase receptor (u-PAR) is essential for the various biological roles of urokinase-type plasminogen activator (u-PA) in vivo, and that smooth muscle cells require u-PA for migration during arterial neointima formation. The present study was undertaken to evaluate the role of u-PAR during this process in mice with targeted disruption of the u-PAR gene (u-PAR^−/−). Surprisingly, u-PAR deficiency did not affect arterial neointima formation, neointimal cell accumulation, or migration of smooth muscle cells. Indeed, topographic analysis of arterial wound healing after electric injury revealed that u-PAR^−/− smooth muscle cells, originating from the uninjured borders, migrated over a similar distance and at a similar rate into the necrotic center of the wound as wild-type (u-PAR^+/+) smooth muscle cells. In addition, u-PAR deficiency did not impair migration of wounded cultured smooth muscle cells in vitro. There were no genotypic differences in reendothelialization of the vascular wound. The minimal role of u-PAR in smooth muscle cell migration was not because of absent expression, since wild-type smooth muscle cells expressed u-PAR mRNA and functional receptor in vitro and in vivo. Pericellular plasmin proteolysis, evaluated by degradation of ¹²⁵I-labeled fibrin and activation of zymogen matrix metalloproteinases, was similar for u-PAR^−/− and u-PAR^+/+ cells. Immunoelectron microscopy of injured arteries in vivo revealed that u-PA was bound on the cell surface of u-PAR^+/+ cells, whereas it was present in the pericellular space around u-PAR^−/− cells. Taken together, these results suggest that binding of u-PA to u-PAR is not required to provide sufficient pericellular u-PA–mediated plasmin proteolysis to allow cellular migration into a vascular wound.     

Human herpesvirus 6 infection arrests cord blood mononuclear cells in G(2) phase of the cell cycle

We here report that after infection with human herpesvirus 6A, human cord blood mononuclear cells accumulate in G₂/M phase of the cell cycle. Experiments with foscarnet or ultraviolet (UV)-irradiated virus stocks pointed at an (immediate-)early, newly formed viral protein to be responsible for the arrest. At the molecular level, p53, cyclin B₁, cyclin A and tyrosine¹⁵-phosphorylated cdk1 accumulated after HHV-6A infection, indicating an arrest in G₂. However, no change was observed in the levels of downstream effectors of p53 in establishing a G₂ arrest, i.e. p21 and 14-3-3σ. We thus conclude that the HHV-6A-induced G₂ arrest occurs independently of p53 accumulation.     

Receptor binding studies disclose a novel class of high-affinity inhibitors of the Escherichia coli FimH adhesin

Mannose-binding type 1 pili are important virulence factors for the establishment of Escherichia coli urinary tract infections (UTIs). These infections are initiated by adhesion of uropathogenic E. coli to uroplakin receptors in the uroepithelium via the FimH adhesin located at the tips of type 1 pili. Blocking of bacterial adhesion is able to prevent infection. Here, we provide for the first time binding data of the molecular events underlying type 1 fimbrial adherence, by crystallographic analyses of the FimH receptor binding domains from a uropathogenic and a K-12 strain, and affinity measurements with mannose, common mono- and disaccharides, and a series of alkyl and aryl mannosides. Our results illustrate that the lectin domain of the FimH adhesin is a stable and functional entity and that an exogenous butyl alpha-D-mannoside, bound in the crystal structures, exhibits a significantly better affinity for FimH (Kd = 0.15 microM) than mannose (Kd = 2.3 microM). Exploration of the binding affinities of alpha- d-mannosides with longer alkyl tails revealed affinities up to 5 nM. Aryl mannosides and fructose can also bind with high affinities to the FimH lectin domain, with a 100-fold improvement and 15-fold reduction in affinity, respectively, compared with mannose. Taken together, these relative FimH affinities correlate exceptionally well with the relative concentrations of the same glycans needed for the inhibition of adherence of type 1 piliated E. coli. We foresee that our findings will spark new ideas and initiatives for the development of UTI vaccines and anti-adhesive drugs to prevent anticipated and recurrent UTIs.