Developmental Dysfunction of the Central Nervous System Lymphatics Modulates the Adaptive Neuro-Immune Response in the Perilesional Cortex in a Mouse Model of Traumatic Brain Injury

Rationale The recently discovered meningeal lymphatic vessels (mLVs) have been proposed to be the missing link between the immune and the central nervous system. The role of mLVs in modulating the neuro-immune response following a traumatic brain injury (TBI), however, has not been analyzed. Parenchymal T lymphocyte infiltration has been previously reported as part of secondary events after TBI, suggestive of an adaptive neuro-immune response. The phenotype of these cells has remained mostly uncharacterized. In this study, we identified subpopulations of T cells infiltrating the perilesional areas 30 days post-injury (an early-chronic time point). Furthermore, we analyzed how the lack of mLVs affects the magnitude and the type of T cell response in the brain after TBI. Methods TBI was induced in K14-VEGFR3-Ig transgenic (TG) mice or in their littermate controls (WT; wild type), applying a controlled cortical impact (CCI). One month after TBI, T cells were isolated from cortical areas ipsilateral or contralateral to the trauma and from the spleen, then characterized by flow cytometry. Lesion size in each animal was evaluated by MRI. Results In both WT and TG-CCI mice, we found a prominent T cell infiltration in the brain confined to the perilesional cortex and hippocampus. The majority of infiltrating T cells were cytotoxic CD8+ expressing a CD44(hi)CD69+ phenotype, suggesting that these are effector resident memory T cells. K14-VEGFR3-Ig mice showed a significant reduction of infiltrating CD4+ T lymphocytes, suggesting that mLVs could be involved in establishing a proper neuro-immune response. Extension of the lesion (measured as lesion volume from MRI) did not differ between the genotypes. Finally, TBI did not relate to alterations in peripheral circulating T cells, as assessed one month after injury. Conclusions Our results are consistent with the hypothesis that mLVs are involved in the neuro-immune response after TBI. We also defined the resident memory CD8+ T cells as one of the main population activated within the brain after a traumatic injury.Peer reviewe

Mimicking superinfection exclusion disrupts alphavirus infection and transmission in the yellow fever mosquito Aedes aegypti

Multiple viruses, including pathogenic viruses, bacteriophages, and even plant viruses, cause a phenomenon termed superinfection exclusion whereby a currently infected cell is resistant to secondary infection by the same or a closely related virus. In alphaviruses, this process is thought to be mediated, at least in part, by the viral protease (nsP2) which is responsible for processing the nonstructural polyproteins (P123 and P1234) into individual proteins (nsP1–nsP4), forming the viral replication complex. Taking a synthetic biology approach, we mimicked this naturally occurring phenomenon by generating a superinfection exclusion-like state in Aedes aegypti mosquitoes, rendering them refractory to alphavirus infection. By artificially expressing Sindbis virus (SINV) and chikungunya virus (CHIKV) nsP2 in mosquito cells and transgenic mosquitoes, we demonstrated a reduction in both SINV and CHIKV viral replication rates in cells following viral infection as well as reduced infection prevalence, viral titers, and transmission potential in mosquitoes.</p

An electrostatic potassium channel opener targeting the final voltage sensor transition

Free polyunsaturated fatty acids (PUFAs) modulate the voltage dependence of voltage-gated ion channels. As an important consequence thereof, PUFAs can suppress epileptic seizures and cardiac arrhythmia. However, molecular details for the interaction between PUFA and ion channels are not well understood. In this study, we have localized the site of action for PUFAs on the voltage-gated Shaker K channel by introducing positive charges on the channel surface, which potentiated the PUFA effect. Furthermore, we found that PUFA mainly affects the final voltage sensor movement, which is closely linked to channel opening, and that specific charges at the extracellular end of the voltage sensor are critical for the PUFA effect. Because different voltage-gated K channels have different charge profiles, this implies channel-specific PUFA effects. The identified site and the pharmacological mechanism will potentially be very useful in future drug design of small-molecule compounds specifically targeting neuronal and cardiac excitability

Optimizing CRE and PhiC31 mediated recombination in Aedes aegypti

Introduction: Genetic manipulation of Aedes aegypti is key to developing a deeper understanding of this insects’ biology, vector-virus interactions and makes future genetic control strategies possible. Despite some advances, this process remains laborious and requires highly skilled researchers and specialist equipment.Methods: Here we present two improved methods for genetic manipulation in this species. Use of transgenic lines which express Cre recombinase and a plasmid-based method for expressing PhiC31 when injected into early embryos.Results: Use of transgenic lines which express Cre recombinase allowed, by simple crossing schemes, germline or somatic recombination of transgenes, which could be utilized for numerous genetic manipulations. PhiC31 integrase based methods for site-specific integration of genetic elements was also improved, by developing a plasmid which expresses PhiC31 when injected into early embryos, eliminating the need to use costly and unstable mRNA as is the current standard.Discussion: Here we have expanded the toolbox for synthetic biology in Ae. aegypti. These methods can be easily transferred into other mosquito and even insect species by identifying appropriate promoter sequences. This advances the ability to manipulate these insects for fundamental studies, and for more applied approaches for pest control

Bathing Adaptations in the Homes of Older Adults (BATH-OUT-2) : study protocol for a randomised controlled trial, economic evaluation and process evaluation

Background The onset of disability in bathing is particularly important for older adults as it can be rapidly followed by disability in other daily activities; this may represent a judicious time point for intervention in order to improve health, well-being and associated quality of life. An important environmental and preventative intervention is housing adaptation, but there are often lengthy waiting times for statutory provision. In this randomised controlled trial (RCT), we aim to evaluate the effectiveness and cost-effectiveness of bathing adaptations compared to no adaptations and to explore the factors associated with routine and expedited implementation of bathing adaptations. Methods BATH-OUT-2 is a multicentre, two-arm, parallel-group RCT. Adults aged 60 and over who are referred to their local authority for an accessible level access shower will be randomised, using pairwise randomisation, 1:1, to receive either an expedited provision of an accessible shower via the local authority or a usual care control waiting list. Participants will be followed up for a maximum of 12 months and will receive up to four follow-ups in this duration. The primary outcome will be the participant’s physical well-being, assessed by the Physical Component Summary score of the Short Form-36 (SF-36), 4 weeks after the intervention group receives the accessible shower. The secondary outcomes include the Mental Component Summary score of the SF-36, self-reported falls, health and social care resource use, health-related quality of life (EQ-5D-5L), social care-related quality of life (Adult Social Care Outcomes Toolkit (ASCOT)), fear of falling (Short Falls Efficacy Scale), independence in bathing (Barthel Index bathing question), independence in daily activities (Barthel Index) and perceived difficulty in bathing (0–100 scale). A mixed-methods process evaluation will comprise interviews with stakeholders and a survey of local authorities with social care responsibilities in England. Discussion The BATH-OUT-2 trial is designed so that the findings will inform future decisions regarding the provision of bathing adaptations for older adults. This trial has the potential to highlight, and then reduce, health inequalities associated with waiting times for bathing adaptations and to influence policies for older adults. Trial registration ISRCTN Registry ISRCTN48563324. Prospectively registered on 09/04/2021

Allele-Specific HLA Loss and Immune Escape in Lung Cancer Evolution

Immune evasion is a hallmark of cancer. Losing the ability to present neoantigens through human leukocyte antigen (HLA) loss may facilitate immune evasion. However, the polymorphic nature of the locus has precluded accurate HLA copy-number analysis. Here, we present loss of heterozygosity in human leukocyte antigen (LOHHLA), a computational tool to determine HLA allele-specific copy number from sequencing data. Using LOHHLA, we find that HLA LOH occurs in 40% of non-small-cell lung cancers (NSCLCs) and is associated with a high subclonal neoantigen burden, APOBEC-mediated mutagenesis, upregulation of cytolytic activity, and PD-L1 positivity. The focal nature of HLA LOH alterations, their subclonal frequencies, enrichment in metastatic sites, and occurrence as parallel events suggests that HLA LOH is an immune escape mechanism that is subject to strong microenvironmental selection pressures later in tumor evolution. Characterizing HLA LOH with LOHHLA refines neoantigen prediction and may have implications for our understanding of resistance mechanisms and immunotherapeutic approaches targeting neoantigens. Video Abstract [Figure presented] Development of the bioinformatics tool LOHHLA allows precise measurement of allele-specific HLA copy number, improves the accuracy in neoantigen prediction, and uncovers insights into how immune escape contributes to tumor evolution in non-small-cell lung cancer