Disruption of membrane potential (Vmem) can activate pathways associated with cancer proliferation. Manipulating ion channels may therefore present an effective strategy for treating cancers that fail to respond to conventional therapies. One approach to target these channels is to manipulate the membrane charge, which involves the use of wireless bipolar electrodes such as carbon nanotube porins (CNTPs) inserted into cell membranes to modulate membrane charge and ionic flux. By utilizing membrane dyes, we observed alterations in Vmem induced by CNTPs and externally applied voltages. Analyses of cellular behaviors and processes indicated that Vmem is more receptive to stimuli in invasive cancers, while it leads to increased metabolism in less invasive cancers, with notable changes in the cell cycle occurring at approximately 48 h post-treatment in Glioblastoma (GB) cell lines. This work shows that CNTPs, in combination and with externally applied voltages, can modulate Vmem and alter cancer cell processes, supporting their potential as a therapeutic.

Bronchial epithelial cell-derived extracellular vesicles (EVs) are central to airway immune responses to inhaled particulate stimuli, as well as regulating respiratory diseases. Through a complex bioactive cargo, EVs can influence inflammatory signalling in the epithelium. Typically, a variety of technologies are used to analyse EVs derived from biofluids, such as nanoparticle tracking analysis, western blotting, and transmission electron microscopy. But recent advances in flow cytometers (FCs) potentially provide a single technology that can rapidly enumerate, size and phenotype epithelial cell-derived EVs, without the absolute need for purification. With multiple FCs available, this study aimed to describe methods and discuss considerations for analysing epithelial cell-derived EV on different FCs. Thus, supernatants containing EVs from primary human bronchial epithelial cells were stained with calcein-AM, in combination with anti-fluorescently conjugated tetraspanin antibodies, before analysing on a CytoFLEX S, ImageStream X MKII, and CytoFLEX nano. NIST traceable polystyrene particle size standards or synthetic EV size standards were used for EV size calibration, and antibody capture microspheres were used to measure the limit of detection for tetraspanin antibodies. We demonstrated that epithelial cell-derived EVs can be sized, enumerated, and phenotyped using all tested FC technologies, with varying sizing sensitivities and considerations for each FC. These findings provide a guidance for selecting suitable FC technologies for EV characterisation and highlight their potential to dissect epithelial EV heterogeneity in the context of airway immune responses and inflammation.

Atopic allergy is rising globally and placing a significant strain on healthcare systems, yet the understanding of the underpinning mechanisms of allergic sensitization remains incomplete. Extracellular vesicles (EVs) have recently emerged as important mediators of immune modulation, due to their diverse cargo, and therefore may play a mechanistic role in allergic sensitization development. Thus, this study investigated whether EVs released by activated dendritic cells (DCs) contribute to allergic sensitization of the common egg allergen, ovalbumin (OVA). DCs were generated from human monocytes cultured with GM-CSF and IL-4, then stimulated with LPS and/or OVA. EVs were subsequently isolated using size-exclusion chromatography and added to freshly isolated naive T cells at defined time points. T cell responses were then analyzed using spectral flow cytometry. The results highlight that EVs derived from LPS or LPS + OVA-stimulated DCs enhanced IL-4 production and reduced IFN-γ production in naive T cells from egg-allergic donors, indicating a shift toward a Th2 profile. In healthy donors, LPS-induced DC EVs also suppressed IFN-γ expression. Notably, EVs alone were insufficient to activate T cells without CD3/CD28 co-stimulation, suggesting that EVs may function as a "third signal" shaping T cell polarization. These findings highlight a potential role for DC-derived EVs in initiating allergic sensitization.

Our goal was to probe the potential transcriptomic basis for the relationship between plasma levels of the specialized pro-resolving precursor, 17-hydroxy-docosahexaenoic acid (17-HDHA) and chronic pain. Participants with osteoarthritis (average age of 62.3, 60% were female, n = 30) were stratified by levels of 17-HDHA and self-reported pain scores. RNAs from CD14++/CD16-/CD66b-/HLA-DR+ (classical) monocytes were sequenced and differentially expressed mRNAs were identified with DESeq2. QIAGEN ingenuity pathway analysis identified the top ranked canonical biological pathway to be eukaryotic initiation factor 2 (EIF2) signaling (lower activation level in the low 17-HDHA-high pain group compared to the high 17-HDHA-low pain group (Z score -3)), followed by EIF4 and P70S6K signaling pathways and mTOR signaling. Our approach provides insight into the biological pathways contributing to the association between 17-HDHA and chronic osteoarthritis (OA) pain, identifying EIF2 signaling, with known roles in osteoclast differentiation, OA pathology, and pain, as a potential downstream target.

Introduction HLH is a hyper-inflammatory syndrome characterised by fevers, cytopenias and hemophagocytosis. The IL1-antagonist anakinra is approved by NHS-England, although efficacy across different triggers is unclear. We used full spectral flow cytometry and cytokine analysis pre and post anakinra for adult HLH to describe immune cell subsets and identify potentially targetable immune cells or cytokines. Methods Patients were diagnosed using the H-score. Blood was taken pre- and 72 hours post-anakinra. Leukocytes were analysed using full spectrum flow cytometry, using both manual and unsupervised analysis. Cytokine, chemokine and growth factor levels were measured using 48-Plex Bio-Plex Pro-Human Cytokine Screening Panel. Results Patient 1: 44 year old with lupus and EBV reactivation - complete response to anakinra and rituximab. At baseline high IL1ra, IL2, IL3, IL5, IL6, IL13, CXCL10 and HGF were seen; post treatment, IL-1 family members IL1α , IL1β and IL-18 decreased with >20 fold drop in IL2, IL6, IL13, CCL2, CXCL9, CXCL10 and HGF. IFNγ, elevated at baseline further increased. High % of monocytes and low % of CD4+ T-cells were present. T-cells were activated (CD38+/HLADR+) with high PD1 expression. The balance of CD8 memory phenotype was perturbed with high (early) effector and TEMRA T-cells. Only 7.43% B-cells were naïve and only 7.84% CD56DIMCD16+ conventional NK-cells but very high (87.64%) unconventional CD56DIMCD16- NK-cells. Post-treatment there were reduced monocytes, increased % CD8+ cells and marked increases in CD4+ and CD8+ effector memory cells and activated PD1+ CD4+ and CD8+ cells. Patient 2: 71 year old with EBV+ve plasmablastic lymphoma - progression on anakinra. Baseline IL10 levels were very high with elevated IL1Rα, IL2, IL6, IL9, IL18, IFNγ, TNFα IL18, IL2ra and CXCL1. Post-treatment an 11-fold increase in IFN-γ was seen, and >2 fold increase in IL1α, IL2, IL4, IL7, IL12(p40). IL10 rose further. At baseline high monocytes and low total T-cells, particularly CD4+. Within CD8+ T-cells, low % of naive and central memory cells with high TEMRA cells, and high PD-1 expression on CD4+ and CD8+ cells were seen, increasing with treatment. High levels of CD56+ monocytes and unconventional NK-cells were seen. Post treatment increased T-regs and central memory CD4+ T-cells occurred. Patient 3: 74 year old with T-cell lymphoma - no response to anakinra. Baseline elevated IL1Rα, IL2, IL9, IL12(p40), IL18, IFNγ, TNFα, CXCL1 and CXCL9 was seen, and post-treatment a 7-fold increase in IFN-γ, and >2 fold increase in IL1α, IL4, IL7, IL12(p40), TNFα and b-NGF. Neutropenia was present but relatively normal % monocytes and T-cells. In CD8+ but not CD4+ there were high levels of TEMRA. CD4+ and CD8+ cells showed high activation levels and PD-1 expression. 8.65% monocytes expressed CD56 rising post-treatment with concomitant increase in intermediate monocytes and reduced classical monocytes. NK-cells were predominately conventional. Patient 4: 75 year old with EBV+ve Hodgkin lymphoma - no response to anakinra. Elevated IL1Rα, IL2, IL6, IL9, IL12(p40), IL18, MIF, IFNγ, TNFα, CXCL1 and CXCL9 was seen. Post treatment 8-fold increase in IFN-γ, and <2-fold increase in IL2 and b-NGF. As per patients 1 and 2, there were low monocytes and CD4+ T-cells and reduced % of naïve and central memory CD8+ T-cells and very high TEMRA. High activation and PD-1 expression were present in CD4+ and CD8+ cells. Very low baseline naïve B-cells were seen, partly reversing post treatment. High unconventional NK-cells were seen. There were more CD11c+ dendritic cells (DCs) and less plasmacytoid DCs than other patients which reversed post anakinra. Discussion Patient 1 had high levels of nearly all cytokines which, other than IFNγ, markedly reduced post-treatment. Patients 2-4 all had modestly raised IL-1 family and T-cell related cytokines and post-treatment increases in IL1α, ILβ, TNFα, IL4, IL17A, CCL2, LIF, bFGF and NGF. All patients had increased IFNγ post treatment. All patients displayed T-cell hyper-activation and exhaustion. EBV positive patients 1, 2 and 4 had a pattern of high % of monocytes, reduced CD4 T-cells, and CD8 compartment dominated by effector memory and TEMRA phenotype combined with a high % of unconventional NK-cells. We conclude there is marked variation in immune status in HLH. All patients had increased IFN-γ post anakinra, suggesting IFNγ or JAK1-2 inhibitors might allow combination strategies.

In brief:Male reproductive tract extracellular vesicles play a critical role in regulating sperm quality and male fertility. This study shows that extracellular vesicles from distinct regions of the male reproductive tract differ in their size, abundance and composition. Abstract:As sperm transit the male reproductive tract, they undergo a series of dynamic changes, gaining motility, modifying lipid and protein content and refining their epigenetic composition. Extracellular vesicles are central to this post-testicular maturation and changes in their composition could directly impact male reproductive health, sperm quality and post-fertilisation development. This study aimed to characterise and compare extracellular vesicles isolated from distinct regions of the male reproductive tract. Extracellular vesicles were isolated from adult, male C57BL/6J cauda and caput epididymis (epididymosomes) and seminal vesicle fluid by precipitation and size exclusion chromatography. Isolated vesicles were characterised using nanoparticle tracking analysis, transmission electron microscopy, Western blotting and imaging flow cytometry. Epididymosomes and seminal fluid vesicles ranged from 110.26 to 121.26 nm in diameter, had a concentration of 109 to 1010 particles/cm3 and had a typical round, cup-shaped morphology. The size and concentration of extracellular vesicles from the caput were significantly larger than those from the cauda and seminal fluid. Imaging flow cytometry revealed that all isolated extracellular vesicles expressed CD81 and CD9 tetraspanins; however, CD63 was detected only in caput epididymosomes. Furthermore, there were significantly fewer CD9+ vesicles in seminal fluid EVs compared to epididymosomes. Using a range of bulk- and single-vesicle analytical approaches, we show that different regions of the male reproductive tract display distinct vesicle compositional phenotypes. However, additional studies are warranted to define the significance of this heterogeneity, their roles in regulating male reproductive health and the development of their offspring.

The aim of this systematic review was to evaluate the current evidence for the involvement of epithelial-derived extracellular vesicles (EVs) in Immunoglobulin E (IgE)-mediated allergic sensitisation. Original clinical and research studies specifically examining the effect of epithelial-derived EVs in IgE-mediated allergic sensitisation were included. Non-IgE mediated allergies, abstracts and review articles were excluded. A total of 18 publications were identified from three databases (EMBASE, Web of Science and PubMed) that indicate epithelial-derived EVs have the potential to promote tolerance or allergic sensitisation. For example, epithelial-derived EVs have the potential to promote IgE-mediated allergic sensitisation by delivering mRNAs that promote T helper 2 (Th2) polarisation and cytokine secretion, or promote tolerance through the induction of T regulatory (Treg) cells. The results also indicate that the potential role of epithelial-derived EVs in IgE-mediated allergic sensitisation may be dependent on the barrier, with all publications related to intestinal epithelium driving tolerance, but publications on nasal and bronchial/alveolar epithelia gaving mixed effects. No publications were found on cutaneous epithelia. Taken together, the literature suggests that epithelial-derived EVs play a key role in influencing IgE-mediated allergic sensitisation. Further research examining all epithelial barriers, using both robust human in vitro models that give more biologically relevant information, as well as clinical studies, are required to further characterise the role of epithelial-derived EVs in IgE-mediated allergic sensitisation.

BACKGROUND AND OBJECTIVES:Mesenchymal stem cells (MSCs) represent a potential cellular therapy for multiple sclerosis (MS). It has been suggested that MSCs from patients with MS have lower immunomodulatory properties than those from healthy controls (HCs). The aims of this study were to compare the immunomodulatory abilities of MSCs from patients with MS and HCs against autologous immune cells and to identify potential targets affecting this ability. METHODS:MSCs were obtained by bone marrow aspiration from 5 people with MS and 7 HCs. Autologous peripheral blood mononuclear cells (PBMCs) were stimulated in monoculture or co-culture with MSCs and tested for T-cell expression of IL-17, IFN-γ, and granulocyte-macrophage colony-stimulating factor (GM-CSF) by flow cytometry. Supernatants of monoculture unstimulated MSCs were tested for a panel of cytokines and chemokines using multiplex array or individual ELISA. Gene expression profiling in MSCs was studied using Lexogen QuantSeq RNA-seq platform and subsequent Ingenuity Pathway Analysis. RESULTS:The MSCs from HCs reduced the proportion of autologous T cells expressing IL-17 (Th17, p = 0.046), IFN-γ (Th1, p = 0.03), and GM-CSF (p = 0.012), whereas MSCs from patients with MS had an opposite effect, increasing both autologous Th17 cells (p = 0.01) and GM-CSF-expressing T cells (p = 0.03), with no changes in Th1 cell abundance. Unstimulated MSCs from patients with MS produced less IL-10 (p = 0.03) and more osteopontin (OPN) (p = 0.002) than those from HCs. Gene expression profiling suggested an increase of ADAM28 in the MS MSCs. This was further confirmed at mRNA (by quantitative polymerase chain reaction [qPCR]) and protein (by flow cytometry) levels. Co-cultures of MS MSCs and autologous PBMCs in the presence of natalizumab, a monoclonal antibody that binds α4β1 and blocks its interaction with both ADAM28 and OPN, resulted in a reduction of Th17 cells. In addition, adding IL-10 to the co-cultures abrogated the increase in Th17 cells, potentially interfering with MS MSC inflammatory effect. DISCUSSION:Our results suggest that blocking ADAM-28 and OPN interaction with cognate receptors or increasing IL-10 reduces the proinflammatory potential of MSCs from people with MS. Similar approaches could be used in clinical MSC treatments.

The membrane potential (Vmem) and faradaic charge transfer, resulting from altered charge distribution due to ion channels, play a crucial role in cellular bioelectricity. Disruption of Vmem can activate pathways associated with cancer proliferation. Manipulating ion channels may therefore present an effective strategy for treating cancers that fail to respond to conventional therapies. One approach to target these channels, is to manipulate the membrane charge which involves the use of wireless bipolar electrodes such as carbon nanotube porins (CNTPs), which could be inserted into cell membranes to mimic these channels. By utilizing membrane dyes, we observed alterations in Vmem induced by CNTPs and externally applied electric fields. Analyses of cellular behaviors and processes indicated that Vmem is more receptive to stimuli in invasive cancers, while it leads to increased metabolism in less invasive cancers, with notable changes in the cell cycle occurring at approximately 48 hours post-treatment in GBM cell lines. This work shows that CNTPs and electric fields can be used to modulate Vmem and alter cancer cell processes, supporting their potential therapeutic capability. ### Competing Interest Statement The authors have declared no competing interest. Engineering and Physical Sciences Research Council, https://ror.org/0439y7842, EP/R004072/1

PURPOSE:Asymptomatic SARS-CoV-2 infections were widely reported during the COVID-19 pandemic, acting as a hidden source of infection. Many existing studies investigating asymptomatic immunity failed to recruit true asymptomatic individuals. Thus, we conducted a longitudinal cohort study to evaluate humoral- and cell-mediated responses to infection and vaccination in well-defined asymptomatic young adults (the Asymptomatic COVID-19 in Education [ACE] cohort). METHODS:Asymptomatic testing services located at three UK universities identified asymptomatic young adults who were subsequently recruited with age- and sex-matched symptomatic and uninfected controls. Blood and saliva samples were collected after SARS-CoV-2 Wuhan infection, and again after vaccination. 51 participant's anti-spike antibody titres, neutralizing antibodies, and spike-specific T-cell responses were measured, against both Wuhan and Omicron B.1.1.529.1. RESULTS:Asymptomatic participants exhibited reduced Wuhan-specific neutralization antibodies pre- and post-vaccination, as well as fewer Omicron-specific neutralization antibodies post-vaccination, compared to symptomatic participants. Lower Wuhan and Omicron-specific IgG titres in asymptomatic individuals were also observed pre- and post-vaccination, compared to symptomatic participants. There were no differences in salivary IgA levels. Conventional flow cytometry analysis and multi-dimensional clustering analysis indicated unvaccinated asymptomatic participants had significantly fewer Wuhan-specific IL-2 secreting CD4+ CD45RA+ T cells and activated CD8+ T cells than symptomatic participants, though these differences dissipated after vaccination. CONCLUSIONS:Asymptomatic infection results in decreased antibody and T cell responses to further exposure to SARS-CoV-2 variants, compared to symptomatic infection. Post-vaccination, antibody responses are still inferior, but T cell immunity increases to match symptomatic subjects, emphasising the importance of vaccination to help protect asymptomatic individuals against future variants.

Allergies affect approximately 10–30% of people worldwide, with an increasing number of cases each year; however, the underlying mechanisms are still poorly understood. In recent years, extracellular vesicles (EVs) have been suggested to play a role in allergic sensitization and skew to a T helper type 2 (Th2) response. The aim of this review is to highlight the existing evidence of EV involvement in allergies. A total of 22 studies were reviewed; 12 studies showed EVs can influence a Th2 response, while 10 studies found EVs promoted a Th1 or Treg response. EVs can drive allergic sensitization through up-regulation of pro-Th2 cytokines, such as IL-4 and IL-13. In addition, EVs from MRSA can induce IgE hypersensitivity in mice towards MRSA. On the other hand, EVs can induce tolerance in the immune system; for example, pre-exposing OVA-loaded EVs prevented OVA sensitization in mice. The current literature thus suggests that EVs play an essential role in allergy. Further research utilizing human in vitro models and clinical studies is needed to give a reliable account of the role of EVs in allergy.

Chronic inflammation dominates disease pathogenesis in Cystic Fibrosis (CF) and there is a need to characterise CF immunity. Whole blood cultures offer a cost-effective and non-invasive approach to investigate immune responses within the host environment. Here we used whole blood cultures to investigate the differentiation potential of monocytes (CD45+CD14+ cells) in CF (N=10) and controls (N=8) in the presence and absence of exogenous macrophage-colony stimulatory factor (M-CSF) or granulocyte-macrophage (GM)-CSF with and without interleukin (IL)-4. In CF and control cultures, CD45+CD14+ cells upregulated HLA-DR expression in all instances, and increased CD206 in the presence of GM-CSF with and without IL-4, and CD209 in the presence of GM-CSF and IL-4. In CF, we consistently observed reduced upregulation of CD206 in response to GM-CSF and a positive correlation between CD206 expression and lung function (FEV 1 ). This was unique to cultured monocytes, and not seen with any other marker. These results highlight the potential of whole blood cultures to reveal cellular characteristics in differentiating monocytes related to clinical parameters that could guide the identification of novel biomarkers in CF.

Natalizumab (NTZ), a monoclonal antibody against the integrin α4β1 (VLA-4) found on activated T cells and B cells, blocks the interaction of this integrin with adhesion molecules of central nervous system (CNS) endothelial cells and lymphocyte migration through the blood–brain barrier, effectively preventing new lesion formation and relapses in multiple sclerosis (MS). Whether NTZ treatment has additional effects on the peripheral immune system cells, and how its actions compare with other MS disease-modifying treatments, have not been extensively investigated. In particular, its effect on the proportions of circulating regulatory T cells (Treg) is unclear. In this study, we investigated the effect of NTZ treatment in 12 patients with relapsing MS, at 6 and 12 months after the start of treatment. We evaluated the proportions of regulatory T cells (Treg), defined by flow cytometry as CD4+ CD25++ FoxP3+ cells and CD4+ CD25++ CD127– cells at these intervals. As an exploratory study, we also investigated the NTZ effects on the proportions of bulk T and B lymphocyte populations, and of those expressing novel the markers CD195 (CCR5), CD196 (CCR6), or CD161 (KLRB1), which are involved in MS pathogenesis but have been studied less in the context of MS treatment. The effects of NTZ were compared to those obtained with 11 patients under interferon-beta-1a (IFN-β1a) treatment, and against 9 healthy volunteers. We observed a transient increment in the proportion of Treg cells at 6 months, which was not sustained at 12 months. We observed a reduction in the proportion of T cells expressing CD195 (CCR5) and CD161 (KLRB1) subsets of T cells. We conclude that NTZ does not have an effect on the proportion of Treg cells over 1 year, but it may affect the expression of molecules important for some aspects MS pathogenesis, in a manner that is not shared with IFN-β1a.

Drug sensitivity of NSCLC specimens to HDAC inhibitors and combination with standards of care.

HGF expression in CAFS and MSCs.

Growth of NSCLC specimens in the 3D-TGA.

Pharmacological modulation of cannabinoid receptor type 2 (CB2R) holds promise for the treatment of neuroinflammatory disorders, such as Alzheimer's disease. Despite the importance of CB2R, its expression and downstream signaling are insufficiently understood in disease- and tissue-specific contexts. Herein, we report the first ligand-directed covalent (LDC) labeling of CB2R enabled by a novel synthetic strategy and application of platform reagents. The LDC modification allows visualization and study of CB2R while maintaining its ability to bind other ligands at the orthosteric site. We employed in silico docking and molecular dynamics simulations to guide probe design and assess the feasibility of LDC labeling of CB2R. We demonstrate selective, covalent labeling of a peripheral lysine residue of CB2R by exploiting fluorogenic O-nitrobenzoxadiazole (O-NBD)-functionalized probes in a TR-FRET assay. The rapid proof-of-concept validation with O-NBD probes inspired incorporation of advanced electrophiles suitable for experiments in live cells. To this end, novel synthetic strategies toward N-sulfonyl pyridone (N-SP) and N-acyl-N-alkyl sulfonamide (NASA) LDC probes were developed, which allowed covalent delivery of fluorophores suitable for cellular studies. The LDC probes were characterized by a radioligand binding assay and TR-FRET experiments. Additionally, the probes were applied to specifically visualize CB2R in conventional and imaging flow cytometry as well as in confocal fluorescence microscopy using overexpressing and endogenously expressing microglial live cells.

A schematic of the patient derived 3D-TGA method.