No Evidence of Progressive Proinflammatory Cytokine Storm in Brain-dead Organ Donors-A Time-course Analysis Using Clinical Samples.

BACKGROUND: Solid organ transplantation is a cost-effective treatment for end-stage organ failure. Organ donation after brain death is an important source of transplanted organs. Data are limited on the effects of brain injury or donor management on grafts. The consensus view has been that brain death creates a progressively proinflammatory environment. We aimed to investigate time-course changes across a range of cytokines in a donation after brain death cohort of donors who died of intracranial hemorrhage without any other systemic source of inflammation. METHODS: A donor cohort was defined using the UK Quality in Organ Donation biobank. Serum levels of proteins involved in proinflammatory and brain injury pathways (tumor necrosis factor-alpha, interleukin-6, complement C5a, neuron-specific enolase, and glial fibrillary acidic protein) were measured from admission to organ recovery. Moving median analysis was used to combine donor trajectories and delineate a time-course. RESULTS: A cohort of 27 donors with brain death duration between 10 and 30 h was created, with 24 donors contributing to the time-course analysis. We observed no increase in tumor necrosis factor-alpha or interleukin-6 throughout the donor management period. Neuronal injury marker and complement C5a remain high from admission to organ recovery, whereas glial fibrillary acidic protein rises around the confirmation of brain death. CONCLUSIONS: We found no evidence of a progressive rise of proinflammatory mediators with prolonged duration of brain death, questioning the hypothesis of a progressively proinflammatory environment. Furthermore, the proposed approach allows us to study chronological changes and identify biomarkers or target pathways when logistical or ethical considerations limit sample availability.

Socio-Economic Vulnerability to Climate Change in Rural Areas in the Context of Green Energy Development-A Study of the Great Masurian Lakes Mesoregion.

Green energy production has become a common and recognized method of electricity generation. Giving up reliance on non-renewable energy sources is an important trend in the economies of many countries. The paper presents an analysis of the impact of indicators like increased green energy production on the level of vulnerability to climate change. The model of the Climate Change Vulnerability Index (VCC) recommended by the Intergovernmental Panel on Climate Change (considering three aspects: exposure, vulnerability, and adaptive capacity of the studied spatial unit/society) was applied. Sensitivity analysis, spatial heterogeneity, and temporal dynamics of indicators characterizing changes in electricity consumption, renewable energy production, greenhouse gas emissions, and variability of financial losses due to extreme weather events and their number were implemented. Several findings arose. First, the vulnerability to climate change (the level of the VCC index), does not decrease after the implementation of a single action, like the development of green energy production. The level of index of vulnerability to climate change (VCC1) from the reference year (2017) relative to VCC2 (2021) has changed slightly, despite the development of RES. The variation does not exceed a 1% reduction in the value of the VCC1 index. Second, the decrease in the level of the vulnerability requires global, coordinated action. The value of the VCC3 index, reflecting, including changes in green energy production (X15), electricity consumption/inhabitant (X38), and green-house gas emissions (X14), exhibited more favorably the impact of these indicators on vulnerability to climate change, compared to the VCC1 reference value. In eleven poviats, the VCC3 index decreased between 1 and 4%. In seven of these poviats, green energy production increased, resulting in an average 10% decrease in the X15 indicator, the X14 indicator representing green-house gas emissions decreased by an average of 7%, while the X38 indicator describing electricity consumption/per capita decreased by an average of 16%. Third, harmonized and inclusive action by the population holds the potential to be the clue to reducing vulnerability to climate change.

Impact of the COVID-19 Pandemic Era on Residential Property Features: Pilot Studies in Poland.

Flats/houses in the COVID-19 pandemic era became the central place for living, working, learning, studying and entertainment. According to Maslow's pyramid, all the basic needs had to be satisfied within a single space, which caused a change in the importance of certain locational and physical features of the flat/house. This study aimed to investigate how the COVID-19 pandemic changed the perception of the environmental features and the physical features of flats/houses. The research material was obtained from a questionnaire study disseminated through different online channels. The study was conducted in Poland, and citizens' preferences are linked to the prevailing spatial and socio-economic determinants. A group of respondents were presented with 23 features describing the location and 17 features describing the physical features of flats/houses. They were also asked questions about the level of satisfaction with the current location and housing features. The results were analysed, and the statistical significance of the difference in the perception of the location features and the physical features of the flat/house was verified using a Chi-squared test. The results demonstrated a change in the importance of certain attributes concerning both external and internal factors. The physical features of the flat/house appeared to be more important (from the respondents' perspective) than the features related to the location, as most changes occurred in that group. The respondents indicated that access to medical care facilities had gained importance (+8%), while good access to public transport had declined (-9%). For the physical features of flats/houses, respondents from other countries also indicated the importance of other attributes, i.e., the floor area (+12%), number of rooms (+14%), additional rooms (+14%), and access to broadband Internet and digital platforms (+28%). The study showed that over 30% of respondents would change their flats/houses if their financial means permitted.

A mixed-methods feasibility and external pilot study to inform a large pragmatic randomised controlled trial of the effects of surgical wound dressing strategies on surgical site infections (Bluebelle Phase B): study protocol for a randomised controlled trial.

BACKGROUND: Surgical site infections (SSIs) are common, occurring in up to 25% of > 4 million operations performed in England each year. Previous trials of the effect of wound dressings on the risk of developing a SSI are of poor quality and underpowered. METHODS/DESIGN: This study is a feasibility and pilot trial to examine the feasibility of a full trial that will compare simple dressings, no dressing and tissue-glue as a dressing. It is examining the overall acceptability of trial participation, identifying opportunities for refinement, testing the feasibility of and validating new outcome tools to assess SSI, wound management issues and patients' wound symptom experiences. It is also exploring methods for avoiding performance bias and blinding outcome assessors by testing the feasibility of collecting wound photographs taken in theatre immediately after wound closure and, at 4-8 weeks after surgery, taken by participants themselves or their carers. Finally, it is identifying the main cost drivers for an economic evaluation of dressing types. Integrated qualitative research is exploring acceptability and reasons for non-adherence to allocation. Adults undergoing primary elective or unplanned abdominal general surgery or Caesarean section are eligible. The main exclusion criteria are abdominal or other major surgery less than three months before the index operation or contraindication to dressing allocation. The trial is scheduled to recruit for nine months. The findings will be used to inform the design of a main trial. DISCUSSION: This pilot trial is the first pragmatic study to randomise participants to no dressing or tissue-glue as a dressing versus a simple dressing. Early evidence from the ongoing pilot shows that recruitment is proceeding well and that the interventions are acceptable to participants. Combined with the qualitative findings, the findings will inform whether a main, large trial is feasible and, if so, how it should be designed. TRIAL REGISTRATION: ISRCTN49328913 . Registered on 20 October 2015.

Next generation sequencing for molecular diagnosis of neurological disorders using ataxias as a model.

Many neurological conditions are caused by immensely heterogeneous gene mutations. The diagnostic process is often long and complex with most patients undergoing multiple invasive and costly investigations without ever reaching a conclusive molecular diagnosis. The advent of massively parallel, next-generation sequencing promises to revolutionize genetic testing and shorten the 'diagnostic odyssey' for many of these patients. We performed a pilot study using heterogeneous ataxias as a model neurogenetic disorder to assess the introduction of next-generation sequencing into clinical practice. We captured 58 known human ataxia genes followed by Illumina Next-Generation Sequencing in 50 highly heterogeneous patients with ataxia who had been extensively investigated and were refractory to diagnosis. All cases had been tested for spinocerebellar ataxia 1-3, 6, 7 and Friedrich's ataxia and had multiple other biochemical, genetic and invasive tests. In those cases where we identified the genetic mutation, we determined the time to diagnosis. Pathogenicity was assessed using a bioinformatics pipeline and novel variants were validated using functional experiments. The overall detection rate in our heterogeneous cohort was 18% and varied from 8.3% in those with an adult onset progressive disorder to 40% in those with a childhood or adolescent onset progressive disorder. The highest detection rate was in those with an adolescent onset and a family history (75%). The majority of cases with detectable mutations had a childhood onset but most are now adults, reflecting the long delay in diagnosis. The delays were primarily related to lack of easily available clinical testing, but other factors included the presence of atypical phenotypes and the use of indirect testing. In the cases where we made an eventual diagnosis, the delay was 3-35 years (mean 18.1 years). Alignment and coverage metrics indicated that the capture and sequencing was highly efficient and the consumable cost was ∼£400 (€460 or US$620). Our pathogenicity interpretation pathway predicted 13 different mutations in eight different genes: PRKCG, TTBK2, SETX, SPTBN2, SACS, MRE11, KCNC3 and DARS2 of which nine were novel including one causing a newly described recessive ataxia syndrome. Genetic testing using targeted capture followed by next-generation sequencing was efficient, cost-effective, and enabled a molecular diagnosis in many refractory cases. A specific challenge of next-generation sequencing data is pathogenicity interpretation, but functional analysis confirmed the pathogenicity of novel variants showing that the pipeline was robust. Our results have broad implications for clinical neurology practice and the approach to diagnostic testing.

N-methyl-D-aspartate antibody encephalitis: temporal progression of clinical and paraclinical observations in a predominantly non-paraneoplastic disorder of both sexes.

Antibodies to the N-methyl-d-aspartate subtype of glutamate receptor have been associated with a newly-described encephalopathy that has been mainly identified in young females with ovarian tumours. However, the full clinical spectrum and treatment responses are not yet clear. We established a sensitive cell-based assay for detection of N-methyl-d-aspartate receptor antibodies in serum or cerebrospinal fluid, and a quantitative fluorescent immunoprecipitation assay for serial studies. Although there was marked intrathecal synthesis of N-methyl-d-aspartate receptor antibodies, the absolute levels of N-methyl-d-aspartate receptor antibodies were higher in serum than in cerebrospinal fluid. N-methyl-d-aspartate receptor antibodies were of the immunoglobulin G1 subclass and were able to activate complement on N-methyl d-aspartate receptor-expressing human embryonic kidney cells. From questionnaires returned on 44 N-methyl-d-aspartate receptor antibody-positive patients, we identified a high proportion without a detected tumour (35/44, 80%: follow-up 3.6-121 months, median 16 months). Among the latter were 15 adult females (43%), 10 adult males (29%) and 10 children (29%), with four in the first decade of life. Overall, there was a high proportion (29%) of non-Caucasians. Good clinical outcomes, as defined by reductions in modified Rankin scores, correlated with decreased N-methyl-d-aspartate receptor antibody levels and were associated with early (<40 days) administration of immunotherapies in non-paraneoplastic patients (P < 0.0001) and earlier tumour removal in paraneoplastic patients (P = 0.02). Ten patients (23%) who were first diagnosed during relapses had no evidence of tumours but had received minimal or no immunotherapy during earlier episodes. Temporal analysis of the onset of the neurological features suggested progression through two main stages. The time of onset of the early features, characterized by neuropsychiatric symptoms and seizures preceded by a median of 10-20 days, the onset of movement disorders, reduction in consciousness and dysautonomia. This temporal dichotomy was also seen in the timing of cerebrospinal fluid, electroencephalographic and in the rather infrequent cerebral imaging changes. Overall, our data support a model in which the early features are associated with cerebrospinal fluid lymphocytosis, and the later features with appearance of oligoclonal bands. The immunological events and neuronal mechanisms underlying these observations need to be explored further, but one possibility is that the early stage represents diffusion of serum antibodies into the cortical grey matter, whereas the later stage results from secondary expansion of the immunological repertoire within the intrathecal compartment acting on subcortical neurons. Four patients, who only had temporal lobe epilepsy without oligoclonal bands, may represent restriction to the first stage.

Cleavage in the {gamma}-subunit of the epithelial sodium channel (ENaC) plays an important role in the proteolytic activation of near-silent channels.

The mechanisms by which proteases activate the epithelial sodium channel (ENaC) are not yet fully understood. We investigated the effect of extracellular proteases on rat ENaC heterologously expressed in Xenopus laevis oocytes. Application of trypsin increased ENaC whole-oocyte currents by about 8-fold without a concomitant increase in channel surface expression. The stimulatory effect of trypsin was preserved in oocytes expressing alphagamma-ENaC, but was abolished in oocytes expressing alphabeta-ENaC. Thus, the gamma-subunit appears to be essential for channel activation by extracellular proteases. Site-directed mutagenesis of a putative prostasin cleavage site in the extracellular loop of the gamma-subunit revealed that mutating the 181Lys residue to alanine (gammaK181A) increases ENaC baseline whole-oocyte currents, decreases channel surface expression, and largely reduces the stimulatory effect of extracellular proteases (trypsin, chymotrypsin and human neutrophil elastase). In single-channel recordings from outside-out patches we demonstrated that the gammaK181A mutation essentially abolishes the activation of near-silent channels by trypsin, while a stimulatory effect of trypsin on channel gating is preserved. This apparent dual effect of trypsin on channel gating and on the recruitment of near-silent channels was confirmed by experiments using the beta518C mutant ENaC which can be converted to a channel with an open probability of nearly one by exposure to a sulfhydryl reagent. Interestingly, the gammaK181A mutation results in the spontaneous appearance of a 67 kDa fragment of the gamma-subunit in the plasma membrane which can be prevented by a furin inhibitor and also occurs after channel activation by extracellular trypsin. This suggests that the mutation promotes channel cleavage and activation by endogenous proteases. This would lower the pool of near-silent channels and explain the constitutive activation and reduced responsiveness of the mutant channel to extracellular proteases. We conclude that the mutated site (K181A) affects a region in the gamma-subunit of ENaC that is functionally important for the activation of near-silent channels by extracellular proteases.