Modulation of Salmonella virulence by a novel SPI-2 injectisome effector that interacts with the dystrophin-associated protein complex.

The injectisome encoded by Salmonella pathogenicity island 2 (SPI-2) had been thought to translocate 28 effectors. Here, we used a proteomic approach to characterize the secretome of a clinical strain of invasive non-typhoidal Salmonella enterica serovar Enteritidis that had been mutated to cause hyper-secretion of the SPI-2 injectisome effectors. Along with many known effectors, we discovered the novel SseM protein. sseM is widely distributed among the five subspecies of Salmonella enterica, is found in many clinically relevant serovars, and is co-transcribed with pipB2, a SPI-2 effector gene. The translocation of SseM required a functional SPI-2 injectisome. Following expression in human cells, SseM interacted with five components of the dystrophin-associated protein complex (DAPC), namely, ß-2-syntrophin, utrophin/dystrophin, α-catulin, α-dystrobrevin, and ß-dystrobrevin. The interaction between SseM and ß-2-syntrophin and α-dystrobrevin was verified in Salmonella Typhimurium-infected cells and relied on the postsynaptic density-95/discs large/zonula occludens-1 (PDZ) domain of ß-2-syntrophin and a sequence corresponding to a PDZ-binding motif (PBM) in SseM. A ΔsseM mutant strain had a small competitive advantage over the wild-type strain in the S. Typhimurium/mouse model of systemic disease. This phenotype was complemented by a plasmid expressing wild-type SseM from S. Typhimurium or S. Enteritidis and was dependent on the PBM of SseM. Therefore, a PBM within a Salmonella effector mediates interactions with the DAPC and modulates the systemic growth of bacteria in mice. Furthermore, the ΔsseM mutant strain displayed enhanced replication in bone marrow-derived macrophages, demonstrating that SseM restrains intracellular bacterial growth to modulate Salmonella virulence. IMPORTANCE: In Salmonella enterica, the injectisome machinery encoded by Salmonella pathogenicity island 2 (SPI-2) is conserved among the five subspecies and delivers proteins (effectors) into host cells, which are required for Salmonella virulence. The identification and functional characterization of SPI-2 injectisome effectors advance our understanding of the interplay between Salmonella and its host(s). Using an optimized method for preparing secreted proteins and a clinical isolate of the invasive non-typhoidal Salmonella enterica serovar Enteritidis strain D24359, we identified 22 known SPI-2 injectisome effectors and one new effector-SseM. SseM modulates bacterial growth during murine infection and has a sequence corresponding to a postsynaptic density-95/discs large/zonula occludens-1 (PDZ)-binding motif that is essential for interaction with the PDZ-containing host protein ß-2-syntrophin and other components of the dystrophin-associated protein complex (DAPC). To our knowledge, SseM is unique among Salmonella effectors in containing a functional PDZ-binding motif and is the first bacterial protein to target the DAPC.

Meeting report 'Microbiology 2023: from single cell to microbiome and host', an international interacademy conference in Würzburg.

On September 20-22 September 2023, the international conference 'Microbiology 2023: from single cell to microbiome and host' convened microbiologists from across the globe for a very successful symposium, showcasing cutting-edge research in the field. Invited lecturers delivered exceptional presentations covering a wide range of topics, with a major emphasis on phages and microbiomes, on the relevant bacteria within these ecosystems, and their multifaceted roles in diverse environments. Discussions also spanned the intricate analysis of fundamental bacterial processes, such as cell division, stress resistance, and interactions with phages. Organized by four renowned Academies, the German Leopoldina, the French Académie des sciences, the Royal Society UK, and the Royal Swedish Academy of Sciences, the symposium provided a dynamic platform for experts to share insights and discoveries, leaving participants inspired and eager to integrate new knowledge into their respective projects. The success of Microbiology 2023 prompted the decision to host the next quadrennial academic meeting in Sweden. This choice underscores the commitment to fostering international collaboration and advancing the frontiers of microbiological knowledge. The transition to Sweden promises to be an exciting step in the ongoing global dialogue and specific collaborations on microbiology, a field where researchers will continue to push the boundaries of knowledge, understanding, and innovation not only in health and disease but also in ecology.

A phase 2a open-label clinical trial to determine the effect of famciclovir on EBV activity as measured by EBV shedding in the saliva of patients with multiple sclerosis.

BACKGROUND: Despite increasing evidence that Epstein-Barr virus (EBV) plays a causal role in MS, no treatments have been shown to reduce EBV turnover. We studied the effect of famciclovir on salivary EBV shedding in people with MS (NCT05283551) in a pilot, proof-of-concept study. METHODS: People with MS receiving natalizumab provided weekly saliva samples for 12 weeks before starting famciclovir 500 mg twice daily for 12 weeks. Twelve saliva samples were provided on treatment and 12 following treatment. A real-time qPCR Taqman assay was used to detect EBV DNA in saliva. The proportion of saliva samples containing EBV DNA was compared using the Friedman test. RESULTS: Of 30 participants (19 F; mean age 41 years; median EDSS 3.5), 29 received famciclovir, and 24 completed the 12-week course. Twenty-one participants provided at least one usable saliva sample in all epochs. Ten of the 21 had shedding in at least one sample pre-drug; 7/21 when taking famciclovir (not significant). No difference in EBV DNA copy number was seen. There were no drug-related serious adverse events. CONCLUSION: No significant effect of famciclovir on EBV shedding was seen in this small pilot study. Given the low numbers, a small effect of famciclovir cannot be excluded. Salivary EBV shedding in this natalizumab-treated cohort was lower than in previous studies, which requires replication.

Disease activity 4.5 years after starting cladribine: experience in 264 patients with multiple sclerosis.

Background: Cladribine is an effective immunotherapy for people with multiple sclerosis (pwMS). Whilst most pwMS do not require re-treatment following standard dosing (two treatment courses), disease activity re-emerges in others. The characteristics of pwMS developing re-emerging disease activity remain incompletely understood. Objectives: To explore whether clinical and/or paraclinical baseline characteristics, including the degree of lymphocyte reduction, drug dose and lesions on magnetic resonance imaging (MRI) are associated with re-emerging disease activity. Design: Service evaluation in pwMS undergoing subcutaneous cladribine (SClad) treatment. Methods: Demographics, clinical, laboratory and MRI data of pwMS receiving two courses of SClad were extracted from health records. To assess associations of predictor variables with re-emerging disease activity, a series of Cox proportional hazards models was fitted (one for each predictor variable). Results: Of n = 264 pwMS 236 received two courses of SClad and were included in the analysis. Median follow-up was 4.5 years (3.9, 5.3) from the first, and 3.5 years (2.9, 4.3) from the last SClad administration. Re-emerging disease activity occurred in 57/236 pwMS (24%); 22/236 received further cladribine doses (SClad or cladribine tablets) at 36.7 months [median; interquartile range (IQR): 31.7, 42.1], and 22/236 other immunotherapies 18.9 months (13.0, 30.2) after their second course of SClad, respectively. Eligibility was based on MRI activity in 29, relapse in 5, both in 13, elevated cerebrospinal fluid neurofilament light chain level in 3, deterioration unrelated to relapse in 4 and other in 3. Only 36/57 of those eligible for additional immunotherapy had received a reduced dose of SClad for their second treatment course. Association was detected between re-emerging disease activity and (i) high baseline MRI activity and (ii) low second dose of SClad. Conclusion: Re-emerging disease activity was associated with baseline MRI activity and low dose second course of SClad.

The Salmonella Typhi SPI-2 injectisome enigma.

The Salmonella pathogenicity island 2 (SPI-2)-encoded type III secretion system (injectisome) is assembled following uptake of bacteria into vacuoles in mammalian cells. The injectisome translocates virulence proteins (effectors) into infected cells. Numerous studies have established the requirement for a functional SPI-2 injectisome for growth of Salmonella Typhimurium in mouse macrophages, but the results of similar studies involving Salmonella Typhi and human-derived macrophages are not consistent. It is important to clarify the functions of the S. Typhi SPI-2 injectisome, not least because an inactivated SPI-2 injectisome forms the basis for live attenuated S. Typhi vaccines that have undergone extensive trials in humans. Intracellular expression of injectisome genes and effector delivery take longer in the S. Typhi/human macrophage model than for S. Typhimurium and we propose that this could explain the conflicting results. Furthermore, strains of both S. Typhimurium and S. Typhi contain intact genes for several 'core' effectors. In S. Typhimurium these cooperate to regulate the vacuole membrane and contribute to intracellular bacterial replication; similar functions are therefore likely in S. Typhi.

Changing from face-to-face to virtual meetings due to the COVID-19 pandemic: protocol for a mixed-methods study exploring the impact on cancer multidisciplinary team (MDT) meetings.

INTRODUCTION: In the UK, the National Cancer Plan (2000) requires every cancer patient's care to be reviewed by a multidisciplinary team (MDT). Since the introduction of these guidelines, MDTs have faced escalating demands with increasing numbers and complexity of cases. The COVID-19 pandemic has presented MDTs with the challenge of running MDT meetings virtually rather than face-to-face.This study aims to explore how the change from face-to-face to virtual MDT meetings during the COVID-19 pandemic may have impacted the effectiveness of decision-making in cancer MDT meetings and to make recommendations to improve future cancer MDT working based on the findings. METHODS AND ANALYSIS: A mixed-methods study with three parallel phases:Semistructured remote qualitative interviews with ≤40 cancer MDT members.A national cross-sectional online survey of cancer MDT members in England, using a validated questionnaire with both multiple-choice and free-text questions.Live observations of ≥6 virtual/hybrid cancer MDT meetings at four NHS Trusts.Participants will be recruited from Cancer Alliances in England. Data collection tools have been developed in consultation with stakeholders, based on a conceptual framework devised from decision-making models and MDT guidelines. Quantitative data will be summarised descriptively, and χ2 tests run to explore associations. Qualitative data will be analysed using applied thematic analysis. Using a convergent design, mixed-methods data will be triangulated guided by the conceptual framework.The study has been approved by NHS Research Ethics Committee (London-Hampstead) (22/HRA/0177). The results will be shared through peer-reviewed journals and academic conferences. A report summarising key findings will be used to develop a resource pack for MDTs to translate learnings from this study into improved effectiveness of virtual MDT meetings.The study has been registered on the Open Science Framework (https://doi.org/10.17605/OSF.IO/D2NHW).

Transmembrane substrates of type three secretion system injectisomes.

The type three secretion system injectisome of Gram-negative bacterial pathogens injects virulence proteins, called effectors, into host cells. Effectors of mammalian pathogens carry out a range of functions enabling bacterial invasion, replication, immune suppression and transmission. The injectisome secretes two translocon proteins that insert into host cell membranes to form a translocon pore, through which effectors are delivered. A subset of effectors also integrate into infected cell membranes, enabling a unique range of biochemical functions. Both translocon proteins and transmembrane effectors avoid cytoplasmic aggregation and integration into the bacterial inner membrane. Translocated transmembrane effectors locate and integrate into the appropriate host membrane. In this review, we focus on transmembrane translocon proteins and effectors of bacterial pathogens of mammals. We discuss what is known about the mechanisms underlying their membrane integration, as well as the functions conferred by the position of injectisome effectors within membranes.

Effect of teriflunomide on Epstein-Barr virus shedding in relapsing-remitting multiple sclerosis patients: Outcomes from a real-world pilot cohort study.

BACKGROUND: Given its potential antiviral activity, we investigated the effect of teriflunomide on EBV in patients with relapsing-remitting MS (RRMS). METHODS: Saliva samples were collected at home and analysed for EBV DNA presence in patients with RRMS treated with teriflunomide for ≥3 months. RESULTS: The proportion of patients with detectable EBV in the teriflunomide cohort was lower than in the reference cohorts. The proportion of samples with EBV DNA or shedding from teriflunomide-treated patients was reduced relative to each reference cohort (P<0.0001; >5.8 virus copies/µL cut-off). CONCLUSION: This pilot study demonstrated the feasibility of at-home saliva sample collection and revealed a possible effect of teriflunomide on EBV shedding.

The Salmonella transmembrane effector SteD hijacks AP1-mediated vesicular trafficking for delivery to antigen-loading MHCII compartments.

SteD is a transmembrane effector of the Salmonella SPI-2 type III secretion system that inhibits T cell activation by reducing the amounts of at least three proteins -major histocompatibility complex II (MHCII), CD86 and CD97 -from the surface of antigen-presenting cells. SteD specifically localises at the trans-Golgi network (TGN) and MHCII compartments; however, the targeting, membrane integration and trafficking of SteD are not understood. Using systematic mutagenesis, we identify distinct regions of SteD that are required for these processes. We show that SteD integrates into membranes of the ER/Golgi through a two-step mechanism of membrane recruitment from the cytoplasm followed by integration. SteD then migrates to and accumulates within the TGN. From here it hijacks the host adaptor protein (AP)1-mediated trafficking pathway from the TGN to MHCII compartments. AP1 binding and post-TGN trafficking require a short sequence in the N-terminal cytoplasmic tail of SteD that resembles the AP1-interacting dileucine sorting signal, but in inverted orientation, suggesting convergent evolution.

The relationship of cerebrospinal fluid neurofilament levels with magnetic resonance imaging lesion location and disease activity in multiple sclerosis.

BACKGROUND AND PURPOSE: Neurofilament light chain (NfL) is an accepted biomarker of disease activity in multiple sclerosis (MS), but its relationship with magnetic resonance imaging (MRI) activity particularly in reference to lesion location and recurrent activity is not well understood. METHODS: In 139 MS patients who underwent lumbar punctures with follow-up in 25, the relationship between cerebrospinal fluid (CSF) NfL and cranial MRI based on lesion location and lesion number was evaluated. Spearman rank correlation was used to assess the association between CSF NfL and MRI lesion location and lesion counts at baseline and follow-up at 1 year. Multiple linear regression analysis was performed to assess which lesion location was most strongly associated with CSF NfL values. RESULTS: The associations between baseline CSF NfL and lesion location and follow-up lesions were modest, whilst those between baseline MRI and follow-up CSF NfL were greater: periventricular (r = 0.31, p = 0.141), juxtacortical (r = 0.47, p = 0.022), infratentorial (r = 0.71, p ≤ 0.001) and cord lesions (r = 0.60, p = 0.002). All associations, however, improved following adjustment for disease duration and type of MS. Modelling revealed 53% of (log) CSF NfL could be explained by variance in baseline MRI lesion location. CONCLUSIONS: Baseline CSF NfL did not correlate with current or future MRI activity and lesion location. However, baseline MRI activity explained around 53% of the variation in the follow-up CSF NfL, suggesting that the relationship between MRI and CSF NfL is mainly precedent rather than an association, that is one occurring before the other.

Factors contributing to CSF NfL reduction over time in those starting treatment for multiple sclerosis: An observational study.

BACKGROUND: In multiple sclerosis (MS) neurofilament light chain (NfL) is a marker of neuronal damage secondary to inflammation and neurodegeneration. NfL levels drop after commencement of disease-modifying treatment, especially the highly active ones. However, the factors that influence this drop are unknown. OBJECTIVE: To examine the patient and treatment-related factors that influence CSF NfL before and after starting treatment. METHODS: Eligible patients across two centres with two CSF NfL measurements, clinical and MRI data were included as part of an observational cohort study. RESULTS: Data were available in 61 patients, of which 40 were untreated at the first CSF sampling (T1) and treated at the second (T2; mean T1-T2: 19 months). CSF NfL reduction correlated with age (beta = 1.24 95%CI(1.07,1.43); R2 = 0.17; p = 0.005), Expanded Disability Status Scale (EDSS) (beta = 1.12 95%CI(1.00,1.25); R2 = 0.21; p = 0.05) and the type of MS (beta = 0.63 95%CI(0.43, 0.92); R2 = 0.12; p = 0.018; reference=relapsing MS). The treatment effect on a baseline NfL of 702 pg/mL was 451 pg/ml 95%CI(374,509) in a 30-year-old versus 228 pg/ml 95%CI(63,350) in a 60-year-old. There was no association in CSF NfL reduction with BMI, disease duration or sex. In cladribine- and alemtuzumab-treated patients, the CSF NfL T2/T1 ratio did not correlate with lymphocyte depletion rate at 23 weeks. CONCLUSIONS: In this observational study, we found that factors reflecting early disease stage, including a younger age, lower disability and relapsing MS were associated with treatment response in CSF NfL. Other factors were not found to be related, including lymphopaenia in highly-active treatments.

Global mapping of Salmonella enterica-host protein-protein interactions during infection.

Intracellular bacterial pathogens inject effector proteins to hijack host cellular processes and promote their survival and proliferation. To systematically map effector-host protein-protein interactions (PPIs) during infection, we generated a library of 32 Salmonella enterica serovar Typhimurium (STm) strains expressing chromosomally encoded affinity-tagged effectors and quantified PPIs in macrophages and epithelial cells. We identified 446 effector-host PPIs, 25 of which were previously described, and validated 13 by reciprocal co-immunoprecipitation. While effectors converged on the same host cellular processes, most had multiple targets, which often differed between cell types. We demonstrate that SseJ, SseL, and SifA modulate cholesterol accumulation at the Salmonella-containing vacuole (SCV) partially via the cholesterol transporter Niemann-Pick C1 protein. PipB recruits the organelle contact site protein PDZD8 to the SCV, and SteC promotes actin bundling by phosphorylating formin-like proteins. This study provides a method for probing host-pathogen PPIs during infection and a resource for interrogating STm effector mechanisms.

CD97 stabilises the immunological synapse between dendritic cells and T cells and is targeted for degradation by the Salmonella effector SteD.

The Salmonella enterica effector SteD depletes mature MHC class II (mMHCII) molecules from the surface of infected antigen-presenting cells through ubiquitination of the cytoplasmic tail of the mMHCII ß chain. This requires the Nedd4 family HECT E3 ubiquitin ligase Wwp2 and a tumor-suppressing transmembrane protein adaptor Tmem127. Here, through a proteomic screen of dendritic cells, we found that SteD targets the plasma membrane protein CD97 for degradation by a similar mechanism. SteD enhanced ubiquitination of CD97 on K555 and mutation of this residue eliminated the effect of SteD on CD97 surface levels. We showed that CD97 localises to and stabilises the immunological synapse between dendritic cells and T cells. Removal of CD97 by SteD inhibited dendritic cell-T cell interactions and reduced T cell activation, independently of its effect on MHCII. Therefore, SteD suppresses T cell immunity by two distinct processes.

Lipid and lipoprotein predictors of functional outcomes and long-term mortality after surgical sepsis.

RATIONALE: Sepsis is a life-threatening, dysregulated response to infection. Lipid biomarkers including cholesterol are dynamically regulated during sepsis and predict short-term outcomes. In this study, we investigated the predictive ability of lipid biomarkers for physical function and long-term mortality after sepsis. METHODS: Prospective cohort study of sepsis patients admitted to a surgical intensive-care unit (ICU) within 24 h of sepsis bundle initiation. Samples were obtained at enrollment for lipid biomarkers. Multivariate regression models determined independent risk factors predictive of poor performance status (Zubrod score of 3/4/5) or survival at 1-year follow-up. MEASUREMENTS AND MAIN RESULTS: The study included 104 patients with surgical sepsis. Enrollment total cholesterol and high-density lipoprotein (HDL-C) levels were lower, and myeloperoxidase (MPO) levels were higher for patients with poor performance status at 1 year. A similar trend was seen in comparisons based on 1-year mortality, with HDL-C and ApoA-I levels being lower and MPO levels being higher in non-survivors. However, multivariable logistic regression only identified baseline Zubrod and initial SOFA score as significant independent predictors of poor performance status at 1 year. Multivariable Cox regression modeling for 1-year survival identified high Charlson comorbidity score, low ApoA-I levels, and longer vasopressor duration as predictors of mortality over 1-year post-sepsis. CONCLUSIONS: In this surgical sepsis study, lipoproteins were not found to predict poor performance status at 1 year. ApoA-I levels, Charlson comorbidity scores, and duration of vasopressor use predicted 1 year survival. These data implicate cholesterol and lipoproteins as contributors to the underlying pathobiology of sepsis.

Structure of the cytoplasmic domain of SctV (SsaV) from the Salmonella SPI-2 injectisome and implications for a pH sensing mechanism.

Bacterial type III secretion systems assemble the axial structures of both injectisomes and flagella. Injectisome type III secretion systems subsequently secrete effector proteins through their hollow needle into a host, requiring co-ordination. In the Salmonella enterica serovar Typhimurium SPI-2 injectisome, this switch is triggered by sensing the neutral pH of the host cytoplasm. Central to specificity switching is a nonameric SctV protein with an N-terminal transmembrane domain and a toroidal C-terminal cytoplasmic domain. A 'gatekeeper' complex interacts with the SctV cytoplasmic domain in a pH dependent manner, facilitating translocon secretion while repressing effector secretion through a poorly understood mechanism. To better understand the role of SctV in SPI-2 translocon-effector specificity switching, we purified full-length SctV and determined its toroidal cytoplasmic region's structure using cryo-EM. Structural comparisons and molecular dynamics simulations revealed that the cytoplasmic torus is stabilized by its core subdomain 3, about which subdomains 2 and 4 hinge, varying the flexible outside cleft implicated in gatekeeper and substrate binding. In light of patterns of surface conservation, deprotonation, and structural motion, the location of previously identified critical residues suggest that gatekeeper binds a cleft buried between neighboring subdomain 4s. Simulations suggest that a local pH change from 5 to 7.2 stabilizes the subdomain 3 hinge and narrows the central aperture of the nonameric torus. Our results are consistent with a model of local pH sensing at SctV, where pH-dependent dynamics of SctV cytoplasmic domain affect binding of gatekeeper complex.

SteC and the intracellular Salmonella-induced F-actin meshwork.

Salmonella enterica serovars infect a broad range of mammalian hosts including humans, causing both gastrointestinal and systemic diseases. Following uptake into host cells, bacteria replicate within vacuoles (Salmonella-containing vacuoles; SCVs). Clusters of SCVs are frequently associated with a meshwork of F-actin. This meshwork is dependent on the Salmonella pathogenicity island 2 encoded type III secretion system and its effector SteC. SteC contains a region with weak similarity to conserved subdomains of eukaryotic kinases and has kinase activity that is required for the formation of the F-actin meshwork. Several substrates of SteC have been identified. In this mini-review, we attempt to integrate these findings and propose a more unified model to explain SCV-associated F-actin: SteC (i) phosphorylates the actin sequestering protein Hsp27, which increases the local G-actin concentration (ii) binds to and phosphorylates formin family FMNL proteins, which enables actin polymerisation and (iii) phosphorylates MEK, resulting in activation of the MEK/ERK/MLCK/Myosin II pathway, leading to F-actin bundling. We also consider the possible physiological functions of SCV-associated F-actin and similar structures produced by other intracellular bacterial pathogens.

Opioid Overdose Risk in Patients Returning to the Emergency Department for Pain.

OBJECTIVE: Using the Risk Index for Overdose or Serious Opioid-induced Respiratory Depression (CIP-RIOSORD) in patients returning to the emergency department (ED) for pain and discharged with an opioid prescription, we assessed overall opioid overdose risk and compared risk in opioid naive patients to those who are non-opioid naive. DESIGN: This was a secondary analysis from a prospective observational study of patients ≥ 18 years old returning to the ED within 30 days. Data were collected from patient interviews and chart reviews. Patients were categorized as Group 1 (not using prescription opioids) or Group 2 (consuming prescription opioids). Statistical analyses were performed using Fisher's exact and Wilcoxon's rank sum tests. Risk class and probability of overdose was determined using Risk Index for Overdose or Serious Opioid-induced Respiratory Depression (CIP-RIOSORD). RESULTS: Of the 389 enrollees who returned to the ED due to pain within 30 days of an initial visit, 67 (17%) were prescribed opioids. The majority of these patients were in Group 1 (60%). Both Group 1 (n = 40) and Group 2 (n = 27) held an average CIP-RIOSORD risk class of 3. Race significantly differed between groups; the majority of Group 1 self-identified as African American (80%) (P = .0267). There were no differences in age, gender, or CIP-RIOSORD risk class between groups. However, Group 2 had nearly double the number of predictive factors (median = 1.93) as Group 1 (median = 1.18) (P = .0267). CONCLUSIONS: A substantial proportion of patients (25%) were high risk for opioid overdose. CIP-RIOSORD may prove beneficial in risk stratification of patients discharged with prescription opioids from the ED.

CSF neurofilament light chain testing as an aid to determine treatment strategies in MS.

OBJECTIVE: To evaluate the use of CSF neurofilament light chain (NfL) measurements in clinical practice as well as their effect on treatment strategies and outcomes in patients with MS. METHODS: This was an observational cohort study of patients with MS who had a CSF NfL measurement between December 2015 and July 2018 as part of their routine clinical care. Treatment strategies were classified as "No Treatment/No Escalation" (no treatment or no escalation of treatment) or "Treatment/Escalation" (first-line injectable/oral disease-modifying therapies (DMTs), highly active DMTs, or treatment escalation). Change in Expanded Disability Status Scale (EDSS) scores was evaluated after 1-year follow-up. RESULTS: Of 203 patients with MS, 117 (58%) had relapsing-remitting MS. Disease activity was most frequently indicated by elevated CSF NfL (n = 85), followed by clinical (n = 81) and MRI activity (n = 65). CSF NfL measurements were independently associated with clinical (p = 0.02) and MRI activity (p < 0.001). Of those with elevated CSF NfL as the only evidence of disease activity (n = 22), 77% had progressive MS (PMS). In patients with PMS, 17 (20%) had elevated CSF NfL as the sole indicator of disease activity. Elevated CSF NfL resulted more frequently in Treatment/Escalation than normal CSF NfL (p < 0.001). Median EDSS change at follow-up was similar between patients receiving No Treatment/No Escalation and Treatment/Escalation decisions (p = 0.81). CONCLUSIONS: CSF NfL measurements informed treatment strategies, alongside clinical and MRI measures. CSF NfL levels were the only indicator of disease activity in a subset of patients, which was more pronounced in patients with PMS. Elevated CSF NfL was associated with more Treatment/Escalation strategies, which had an impact on EDSS outcomes at 1 year.

The Tumour Suppressor TMEM127 Is a Nedd4-Family E3 Ligase Adaptor Required by Salmonella SteD to Ubiquitinate and Degrade MHC Class II Molecules.

The Salmonella enterica effector SteD depletes mature MHC class II (mMHCII) molecules from the surface of infected antigen-presenting cells through ubiquitination of the cytoplasmic tail of the mMHCII ß chain. Here, through a genome-wide mutant screen of human antigen-presenting cells, we show that the NEDD4 family HECT E3 ubiquitin ligase WWP2 and a tumor-suppressing transmembrane protein of unknown biochemical function, TMEM127, are required for SteD-dependent ubiquitination of mMHCII. Although evidently not involved in normal regulation of mMHCII, TMEM127 was essential for SteD to suppress both mMHCII antigen presentation in mouse dendritic cells and MHCII-dependent CD4+ T cell activation. We found that TMEM127 contains a canonical PPxY motif, which was required for binding to WWP2. SteD bound to TMEM127 and enabled TMEM127 to interact with and induce ubiquitination of mature MHCII. Furthermore, SteD also underwent TMEM127- and WWP2-dependent ubiquitination, which both contributed to its degradation and augmented its activity on mMHCII.

Persistently increased cell-free DNA concentrations only modestly contribute to outcome and host response in sepsis survivors with chronic critical illness.

BACKGROUND: Although early survival from sepsis has improved with timely resuscitation and source control, survivors frequently experience persistent inflammation and develop chronic critical illness. We examined whether increased copy number of endogenous alarmins, mitochondrial DNA, and nuclear DNA are associated with the early "genomic storm" in blood leukocytes and the development of chronic critical illness in hospitalized patients with surgical sepsis. METHODS: A prospective, observational, cohort study of critically ill septic patients was performed at a United States tertiary health care center. Blood samples were obtained at multiple time points after the onset of sepsis. Droplet Digital polymerase chain reaction (Bio-Rad Laboratories, Hercules, CA) was performed to quantify RHO (nuclear DNA) and MT-CO2 (mitochondrial DNA) copies in plasma. Leukocyte transcriptomic expression of 63 genes was also measured in whole blood. RESULTS: We enrolled 112 patients with surgical sepsis. Two experienced early death, 69 recovered rapidly, and 41 developed chronic critical illness. Both mitochondrial DNA and nuclear DNA copy number were increased in all sepsis survivors, but early nuclear DNA, and not mitochondrial DNA, copy number was further increased in patients who developed chronic critical illness. Cell-free DNA copy number was associated with in-hospital but not long-term (180-day and 365-day) mortality and were only weakly correlated with leukocyte transcriptomics. CONCLUSION: Increased cell-free DNA copy number persists in survivors of sepsis but is not strongly associated with leukocyte transcriptomics. Nuclear DNA but not mitochondrial DNA copy number is associated with adverse, short-term, clinical trajectories and outcomes.