Iron dysregulation and inflammatory stress erythropoiesis associates with long-term outcome of COVID-19.

Persistent symptoms following SARS-CoV-2 infection are increasingly reported, although the drivers of post-acute sequelae (PASC) of COVID-19 are unclear. Here we assessed 214 individuals infected with SARS-CoV-2, with varying disease severity, for one year from COVID-19 symptom onset to determine the early correlates of PASC. A multivariate signature detected beyond two weeks of disease, encompassing unresolving inflammation, anemia, low serum iron, altered iron-homeostasis gene expression and emerging stress erythropoiesis; differentiated those who reported PASC months later, irrespective of COVID-19 severity. A whole-blood heme-metabolism signature, enriched in hospitalized patients at month 1-3 post onset, coincided with pronounced iron-deficient reticulocytosis. Lymphopenia and low numbers of dendritic cells persisted in those with PASC, and single-cell analysis reported iron maldistribution, suggesting monocyte iron loading and increased iron demand in proliferating lymphocytes. Thus, defects in iron homeostasis, dysregulated erythropoiesis and immune dysfunction due to COVID-19 possibly contribute to inefficient oxygen transport, inflammatory disequilibrium and persisting symptomatology, and may be therapeutically tractable.

Disturbed sex hormone milieu in males and females with major depressive disorder and low-grade inflammation.

Sex hormones have biological effects on inflammation, and these might contribute to the sex-specific features of depression. C-reactive protein (CRP) is the most widely used inflammatory biomarker and consistent evidence shows a significant proportion (20-30 %) of patients with major depressive disorder (MDD) have CRP levels above 3 mg/L, a threshold indicating at least low-grade inflammation. Here, we investigate the interplay between sex hormones and CRP in the cross-sectional, observational Biomarkers in Depression Study. We measured serum high-sensitivity (hs-)CRP, in 64 healthy controls and 178 MDD patients, subdivided into those with hs-CRP below 3 mg/L (low-CRP; 53 males, 72 females) and with hs-CRP above 3 mg/L (high-CRP; 19 males, 34 females). We also measured interleukin-6, testosterone, 17-ß-estradiol (E2), progesterone, sex-hormone binding globulin (SHBG), follicle-stimulating and luteinising hormones, and calculated testosterone-to-E2 ratio (T/E2), free androgen and estradiol indexes (FAI, FEI), and testosterone secretion index. In males, high-CRP patients had lower testosterone than controls (p = 0.001), and lower testosterone (p = 0.013), T/E2 (p < 0.001), and higher FEI (p = 0.015) than low-CRP patients. In females, high-CRP patients showed lower SHGB levels than controls (p = 0.033) and low-CRP patients (p = 0.034). The differences in testosterone, T/E2 ratio, and FEI levels in males survived the Benjamini-Hochberg FDR correction. In linear regression analyses, testosterone (ß = -1.069 p = 0.033) predicted CRP concentrations (R2 = 0.252 p = 0.002) in male patients, and SHBG predicted CRP levels (ß = -0.628 p = 0.009, R2 = 0.172 p = 0.003) in female patients. These findings may guide future research investigating interactions between gonadal and immune systems in depression, and the potential of hormonal therapies in MDD with inflammation.

Higher immune-related gene expression in major depression is independent of CRP levels: results from the BIODEP study.

Compelling evidence demonstrates that some individuals suffering from major depressive disorder (MDD) exhibit increased levels of inflammation. Most studies focus on inflammation-related proteins, such as serum or plasma C-reactive protein (CRP). However, the immune-related modifications associated with MDD may be not entirely captured by CRP alone. Analysing mRNA gene expression levels, we aimed to identify broader molecular immune-related phenotypes of MDD. We examined 168 individuals from the non-interventional, case-control, BIODEP study, 128 with a diagnosis of MDD and 40 healthy controls. Individuals with MDD were further divided according to serum high-sensitivity (hs)CRP levels (n = 59 with CRP <1, n = 33 with CRP 1-3 and n = 36 with CRP >3 mg/L). We isolated RNA from whole blood and performed gene expression analyses using RT-qPCR. We measured the expression of 16 immune-related candidate genes: A2M, AQP4, CCL2, CXCL12, CRP, FKBP5, IL-1-beta, IL-6, ISG15, MIF, GR, P2RX7, SGK1, STAT1, TNF-alpha and USP18. Nine of the 16 candidate genes were differentially expressed in MDD cases vs. controls, with no differences between CRP-based groups. Only CRP mRNA was clearly associated with serum CRP. In contrast, plasma (proteins) IL-6, IL-7, IL-8, IL-10, IL-12/IL-23p40, IL-16, IL-17A, IFN-gamma and TNF-alpha, and neutrophils counts, were all differentially regulated between CRP-based groups (higher in CRP >3 vs. CRP <1 and/or controls), reflecting the gradient of CRP values. Secondary analyses on MDD individuals and controls with CRP values <1 mg/L (usually interpreted as 'no inflammation') confirmed MDD cases still had significantly different mRNA expression of immune-related genes compared with controls. These findings corroborate an immune-related molecular activation in MDD, which appears to be independent of serum CRP levels. Additional biological mechanisms may then be required to translate this mRNA signature into inflammation at protein and cellular levels. Understanding these mechanisms will help to uncover the true immune abnormalities in depression, opening new paths for diagnosis and treatment.

A patient-centric modeling framework captures recovery from SARS-CoV-2 infection.

The biology driving individual patient responses to severe acute respiratory syndrome coronavirus 2 infection remains ill understood. Here, we developed a patient-centric framework leveraging detailed longitudinal phenotyping data and covering a year after disease onset, from 215 infected individuals with differing disease severities. Our analyses revealed distinct 'systemic recovery' profiles, with specific progression and resolution of the inflammatory, immune cell, metabolic and clinical responses. In particular, we found a strong inter-patient and intra-patient temporal covariation of innate immune cell numbers, kynurenine metabolites and lipid metabolites, which highlighted candidate immunologic and metabolic pathways influencing the restoration of homeostasis, the risk of death and that of long COVID. Based on these data, we identified a composite signature predictive of systemic recovery, using a joint model on cellular and molecular parameters measured soon after disease onset. New predictions can be generated using the online tool http://shiny.mrc-bsu.cam.ac.uk/apps/covid-19-systemic-recovery-prediction-app , designed to test our findings prospectively.

Peripheral blood cellular immunophenotype in depression: a systematic review and meta-analysis.

INTRODUCTION: Meta-analyses implicate immune dysfunction in depression confirming increased levels of circulating immune proteins (e.g., cytokines) in depression cases compared to controls. White blood cells (WBC) both produce and are influenced by cytokines, and play key roles in orchestrating innate and adaptive immune responses, but their role in depression remains unclear. Therefore, a systematic review of studies of various WBC subsets in depression is required for a greater understanding of the nature of immune dysfunction in this illness. METHODS: We searched PubMed and PsycINFO databases (inception to 5th April 2022) and conducted a systematic review and meta-analysis of identified studies comparing absolute count and/or relative percentage of flow cytometry-derived WBC subsets between depression cases and controls. Selected studies were quality assessed. Random-effect meta-analysis was performed. RESULTS: Thirty-three studies were included and 27 studies (n = 2277) were meta-analysed. We report an increase in mean absolute counts of WBC (seven studies; standardised mean difference [SMD] = 1.07; 95% CI, 0.61-1.53; P < 0.01; I2 = 64%), granulocytes (two studies; SMD = 2.07; 95% CI, 1.45-2.68; P < 0.01; I2 = 0%), neutrophils (four studies; SMD = 0.91; 95% CI, 0.23-1.58; P < 0.01; I2 = 82%), monocytes (seven studies; SMD = 0.60; 95% CI, 0.19-1.01; P < 0.01; I2 = 66%), CD4+ helper T cells (11 studies; SMD = 0.30; 95% CI, 0.15-0.45; P < 0.01; I2 = 0%), natural killer cells (11 studies; SMD = 1.23; 95% CI, 0.38-2.08; P < 0.01; I2 = 95%), B cells (10 studies; SMD = 0.30; 95% CI, 0.03-0.57; P = 0.03; I2 = 56%), and activated T cells (eight studies; SMD = 0.45; 95% CI, 0.24-0.66; P < 0.01; I2 = 0%) in depression, compared to controls. Fewer studies reported relative percentage, indicating increased neutrophils and decreased total lymphocytes, Th1, and Th2 cells in depression. CONCLUSIONS: Depression is characterised by widespread alterations in circulating myeloid and lymphoid cells, consistent with dysfunction in both innate and adaptive immunity. Immune cells could be useful biomarkers for illness subtyping and patient stratification in future immunotherapy trials of depression, along with cytokines, other biomarkers, and clinical measures.

The impact of hypoxia on B cells in COVID-19.

BACKGROUND: Prominent early features of COVID-19 include severe, often clinically silent, hypoxia and a pronounced reduction in B cells, the latter important in defence against SARS-CoV-2. This presentation resembles the phenotype of mice with VHL-deficient B cells, in which Hypoxia-Inducible Factors are constitutively active, suggesting hypoxia might drive B cell abnormalities in COVID-19. METHODS: Detailed B cell phenotyping was undertaken by flow-cytometry on longitudinal samples from patients with COVID-19 across a range of severities (NIHR Cambridge BioResource). The impact of hypoxia on the transcriptome was assessed by single-cell and whole blood RNA sequencing analysis. The direct effect of hypoxia on B cells was determined through immunisation studies in genetically modified and hypoxia-exposed mice. FINDINGS: We demonstrate the breadth of early and persistent defects in B cell subsets in moderate/severe COVID-19, including reduced marginal zone-like, memory and transitional B cells, changes also observed in B cell VHL-deficient mice. These findings were associated with hypoxia-related transcriptional changes in COVID-19 patient B cells, and similar B cell abnormalities were seen in mice kept in hypoxic conditions. INTERPRETATION: Hypoxia may contribute to the pronounced and persistent B cell pathology observed in acute COVID-19 pneumonia. Assessment of the impact of early oxygen therapy on these immune defects should be considered, as their correction could contribute to improved outcomes. FUNDING: Evelyn Trust, Addenbrooke's Charitable Trust, UKRI/NIHR, Wellcome Trust.

Coagulation factor V is a T-cell inhibitor expressed by leukocytes in COVID-19.

Clotting Factor V (FV) is primarily synthesized in the liver and when cleaved by thrombin forms pro-coagulant Factor Va (FVa). Using whole blood RNAseq and scRNAseq of peripheral blood mononuclear cells, we find that FV mRNA is expressed in leukocytes, and identify neutrophils, monocytes, and T regulatory cells as sources of increased FV in hospitalized patients with COVID-19. Proteomic analysis confirms increased FV in circulating neutrophils in severe COVID-19, and immunofluorescence microscopy identifies FV in lung-infiltrating leukocytes in COVID-19 lung disease. Increased leukocyte FV expression in severe disease correlates with T-cell lymphopenia. Both plasma-derived and a cleavage resistant recombinant FV, but not thrombin cleaved FVa, suppress T-cell proliferation in vitro. Anticoagulants that reduce FV conversion to FVa, including heparin, may have the unintended consequence of suppressing the adaptive immune system.

B cell receptor repertoire kinetics after SARS-CoV-2 infection and vaccination.

B cells are important in immunity to both severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection and vaccination, but B cell receptor (BCR) repertoire development in these contexts has not been compared. We analyze serial samples from 171 SARS-CoV-2-infected individuals and 63 vaccine recipients and find the global BCR repertoire differs between them. Following infection, immunoglobulin (Ig)G1/3 and IgA1 BCRs increase, somatic hypermutation (SHM) decreases, and, in severe disease, IgM and IgA clones are expanded. In contrast, after vaccination, the proportion of IgD/M BCRs increase, SHM is unchanged, and expansion of IgG clones is prominent. VH1-24, which targets the N-terminal domain (NTD) and contributes to neutralization, is expanded post infection except in the most severe disease. Infection generates a broad distribution of SARS-CoV-2-specific clones predicted to target the spike protein, while a more focused response after vaccination mainly targets the spike's receptor-binding domain. Thus, the nature of SARS-CoV-2 exposure differentially affects BCR repertoire development, potentially informing vaccine strategies.

Dysconnectivity of a brain functional network was associated with blood inflammatory markers in depression.

OBJECTIVE: There is increasing evidence for a subgroup of major depressive disorder (MDD) associated with heightened peripheral blood inflammatory markers. In this study, we aimed to understand the mechanistic brain-immune axis in inflammation-linked depression by investigating associations between functional connectivity (FC) of brain networks and peripheral blood immune markers in depression. METHODS: Resting-state functional magnetic resonance imaging (fMRI) and peripheral blood inflammatory markers (C-reactive protein; CRP, interleukin-6; IL-6 and immune cells) were collected on N = 46 healthy controls (HC; CRP ≤ 3 mg/L) and N = 83 cases of depression, stratified further into low CRP cases (loCRP cases; ≤ 3 mg/L; N = 50) and high CRP cases (hiCRP cases; > 3 mg/L; N = 33). In a two-part analysis, network-based statistics (NBS) was firstly used to ascertain whole-brain FC differences in HC vs hiCRP cases. Secondly, we investigated the association between this network of interconnected brain regions and continuous measures of peripheral CRP (N = 83), IL-6 (N = 72), neutrophils and CD4+ T-cells (N = 36) in depression cases only. RESULTS: Case-control NBS testing revealed a single network of abnormally attenuated FC in the high CRP depression cases compared to healthy controls. Connections within this network were mainly between brain regions located in the left insula/frontal operculum and posterior cingulate cortex, which were assigned to ventral attention and default mode canonical fMRI networks respectively. Within-group analysis across all depression cases, secondarily demonstrated that FC within the identified network significantly negatively scaled with CRP, IL-6 and neutrophils. CONCLUSIONS: The findings suggest that inflammation is associated with disruption of functional connectivity within a brain network deemed critical for interoceptive signalling, e.g. accurate communication of peripheral bodily signals such as immune states to the brain, with implications for the pathogenesis of inflammation-linked depression.

Longitudinal analysis reveals that delayed bystander CD8+ T cell activation and early immune pathology distinguish severe COVID-19 from mild disease.

The kinetics of the immune changes in COVID-19 across severity groups have not been rigorously assessed. Using immunophenotyping, RNA sequencing, and serum cytokine analysis, we analyzed serial samples from 207 SARS-CoV2-infected individuals with a range of disease severities over 12 weeks from symptom onset. An early robust bystander CD8+ T cell immune response, without systemic inflammation, characterized asymptomatic or mild disease. Hospitalized individuals had delayed bystander responses and systemic inflammation that was already evident near symptom onset, indicating that immunopathology may be inevitable in some individuals. Viral load did not correlate with this early pathological response but did correlate with subsequent disease severity. Immune recovery is complex, with profound persistent cellular abnormalities in severe disease correlating with altered inflammatory responses, with signatures associated with increased oxidative phosphorylation replacing those driven by cytokines tumor necrosis factor (TNF) and interleukin (IL)-6. These late immunometabolic and immune defects may have clinical implications.

RAISE study protocol: a cross-sectional, multilevel, neurobiological study of resilience after individual stress exposure.

INTRODUCTION: This paper describes the protocol for an ongoing project funded by the Royal Society, the Resilience After Individual Stress Exposure (RAISE) study; which aims to examine the factors and mechanisms that facilitate resilient functioning after childhood adversity (CA). METHODS AND ANALYSIS: We aim to recruit up to 200 participants. We will use dimension reduction techniques (principal component analysis) on standard-normally transformed individual parameters of mental health, social functioning and CA to calculate a composite measure of adaptive (ie, 'resilient') psychosocial functioning. To examine the neuroimmune responses to stress and their relationship with the brain and social environment, we will use a well validated functional MRI task; the Montreal imaging stress task and venepuncture. We will run group or dimensional comparisons in multiple levels of biological and psychological outcomes, as well as mediation and moderation analyses to study how key biological systems (ie, the hypothalamic-pituitary-adrenal axis and the immune system) interrelate and interact with brain function and social influences in order to facilitate resilient functioning after CA. We hypothesise that resilient functioning will be facilitated by reduced morning cortisol and cytokine levels before and after the stressor and improved neural responses to such stress, as well as increased gray matter volume in the hippocampus and prefrontal cortex, enhanced inhibitory control and emotion regulation, and more friendship and family support. ETHICS AND DISSEMINATION: This study has been reviewed and given favourable opinion by the National Research Ethics Service, NRES Committee East of England-Cambridge Central and external reviewers from the Royal Society (RGF\R1\180064 and RGF\EA\180029). The results of the RAISE study will be disseminated through (1) publications in scientific peer reviewed journals, (2) presentations on relevant scientific conferences and meetings, (3) publications and presentations for the general public and (4) through social media.

Treatment of COVID-19 with remdesivir in the absence of humoral immunity: a case report.

The response to the coronavirus disease 2019 (COVID-19) pandemic has been hampered by lack of an effective severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antiviral therapy. Here we report the use of remdesivir in a patient with COVID-19 and the prototypic genetic antibody deficiency X-linked agammaglobulinaemia (XLA). Despite evidence of complement activation and a robust T cell response, the patient developed persistent SARS-CoV-2 pneumonitis, without progressing to multi-organ involvement. This unusual clinical course is consistent with a contribution of antibodies to both viral clearance and progression to severe disease. In the absence of these confounders, we take an experimental medicine approach to examine the in vivo utility of remdesivir. Over two independent courses of treatment, we observe a temporally correlated clinical and virological response, leading to clinical resolution and viral clearance, with no evidence of acquired drug resistance. We therefore provide evidence for the antiviral efficacy of remdesivir in vivo, and its potential benefit in selected patients.

Correction: Whole-blood expression of inflammasome- and glucocorticoid-related mRNAs correctly separates treatment-resistant depressed patients from drug-free and responsive patients in the BIODEP study.

We have corrected this Article post-publication, because Dr. Cattaneo's affiliation details were originally incorrect (she was affiliated with three institutions but is in fact only linked to one: Biological Psychiatry Unit, IRCCS Istituto Centro San Giovanni di Dio Fatebenefratelli, Brescia). These changes reflect in both the PDF and HTML versions of this Article.

Whole-blood expression of inflammasome- and glucocorticoid-related mRNAs correctly separates treatment-resistant depressed patients from drug-free and responsive patients in the BIODEP study.

The mRNA expression signatures associated with the 'pro-inflammatory' phenotype of depression, and the differential signatures associated with depression subtypes and the effects of antidepressants, are still unknown. We examined 130 depressed patients (58 treatment-resistant, 36 antidepressant-responsive and 36 currently untreated) and 40 healthy controls from the BIODEP study, and used whole-blood mRNA qPCR to measure the expression of 16 candidate mRNAs, some never measured before: interleukin (IL)-1-beta, IL-6, TNF-alpha, macrophage inhibiting factor (MIF), glucocorticoid receptor (GR), SGK1, FKBP5, the purinergic receptor P2RX7, CCL2, CXCL12, c-reactive protein (CRP), alpha-2-macroglobulin (A2M), acquaporin-4 (AQP4), ISG15, STAT1 and USP-18. All genes but AQP4, ISG15 and USP-18 were differentially regulated. Treatment-resistant and drug-free depressed patients had both increased inflammasome activation (higher P2RX7 and proinflammatory cytokines/chemokines mRNAs expression) and glucocorticoid resistance (lower GR and higher FKBP5 mRNAs expression), while responsive patients had an intermediate phenotype with, additionally, lower CXCL12. Most interestingly, using binomial logistics models we found that a signature of six mRNAs (P2RX7, IL-1-beta, IL-6, TNF-alpha, CXCL12 and GR) distinguished treatment-resistant from responsive patients, even after adjusting for other variables that were different between groups, such as a trait- and state-anxiety, history of childhood maltreatment and serum CRP. Future studies should replicate these findings in larger, longitudinal cohorts, and test whether this mRNA signature can identify patients that are more likely to respond to adjuvant strategies for treatment-resistant depression, including combinations with anti-inflammatory medications.

Immune dysregulation among students exposed to exam stress and its mitigation by mindfulness training: findings from an exploratory randomised trial.

Psychological distress persisting for weeks or more promotes pro-inflammatory immune dysregulation, a risk factor for a range of chronic diseases. We have recently shown that mindfulness training reduces distress among university students. Here we present an exploratory trial to study immune dysregulation in a cohort of students who were exposed to progressively greater stress as the exam period approached, and to explore whether mindfulness training mitigated this dysregulation. Healthy University of Cambridge students were randomised to join an 8-week mindfulness course (N = 27), or to mental health support as usual (N = 27). Psychological distress, immune cell proportions, cytokines, CRP and serum cortisol were measured at baseline and during the exam period. Increased distress was associated with statistically significant increases in the proportion of B cells, regardless of trial arm (*p = 0.027). There were no other associations between any of the measured parameters, distress or mindfulness. Our finding that the proportion of B cells increases with psychological distress supports the findings of other studies. However, we found no evidence that mindfulness training is able to buffer the effects of psychological distress on healthy participants' immune system. In order to detect these effects, should they exist, larger randomised trials will be required.

Peripheral Blood Cell-Stratified Subgroups of Inflamed Depression.

BACKGROUND: Depression has been associated with increased inflammatory proteins, but changes in circulating immune cells are less well defined. METHODS: We used multiparametric flow cytometry to count 14 subsets of peripheral blood cells in 206 depression cases and 77 age- and sex-matched controls (N = 283). We used univariate and multivariate analyses to investigate the immunophenotypes associated with depression and depression severity. RESULTS: Depression cases, compared with controls, had significantly increased immune cell counts, especially neutrophils, CD4+ T cells, and monocytes, and increased inflammatory proteins (C-reactive protein and interleukin-6). Within-group analysis of cases demonstrated significant associations between the severity of depressive symptoms and increased myeloid and CD4+ T-cell counts. Depression cases were partitioned into 2 subgroups by forced binary clustering of cell counts: the inflamed depression subgroup (n = 81 out of 206; 39%) had increased monocyte, CD4+, and neutrophil counts; increased C-reactive protein and interleukin-6; and more severe depression than the uninflamed majority of cases. Relaxing the presumption of a binary classification, data-driven analysis identified 4 subgroups of depression cases, 2 of which (n = 38 and n = 100; 67% collectively) were associated with increased inflammatory proteins and more severe depression but differed in terms of myeloid and lymphoid cell counts. Results were robust to potentially confounding effects of age, sex, body mass index, recent infection, and tobacco use. CONCLUSIONS: Peripheral immune cell counts were used to distinguish inflamed and uninflamed subgroups of depression and to indicate that there may be mechanistically distinct subgroups of inflamed depression.

Helicobacter pylori Outer Membrane Vesicle Size Determines Their Mechanisms of Host Cell Entry and Protein Content.

Gram-negative pathogens ubiquitously shed outer membrane vesicles (OMVs) that play a central role in initiating and regulating pathogenesis in the host. Due to their highly inflammatory nature, OMVs are extensively being examined for their role in mediating disease in addition to their applications in innovative vaccines. A key mechanism whereby OMVs mediate inflammation and disease progression is dependent on their ability to enter host cells. Currently, the role of OMV size on determining their mechanism of cellular entry and their protein composition remains unknown. In this study, we examined the mechanisms whereby OMV size regulates their mode of entry into epithelial cells, in addition to their protein cargo and composition. We identified that a heterogeneous sized population of Helicobacter pylori OMVs entered epithelial cells via macropinocytosis, clathrin, and caveolin-dependent endocytosis. However, smaller OMVs ranging from 20 to 100 nm in size preferentially entered host cells via caveolin-mediated endocytosis. Whereas larger OMVs ranging between 90 and 450 nm in size entered host epithelial cells via macropinocytosis and endocytosis. Most importantly, we identified the previously unknown contribution that OMV size has on determining their protein content, as fewer and less diverse bacterial proteins were contained within small OMVs compared to larger OMVs. Collectively, these findings identify the importance of OMV size in determining the mechanisms of OMV entry into host cells, in addition to regulating their protein cargo, composition, and subsequent immunogenicity. These findings have significant implications in broadening our understanding of the bacterial regulation of virulence determinants and immunogenic proteins associated with OMVs, their role in mediating pathogenesis and in refining the design and development of OMV-based vaccines.

A Helicobacter pylori Homolog of Eukaryotic Flotillin Is Involved in Cholesterol Accumulation, Epithelial Cell Responses and Host Colonization.

The human pathogen Helicobacter pylori acquires cholesterol from membrane raft domains in eukaryotic cells, commonly known as "lipid rafts." Incorporation of this cholesterol into the H. pylori cell membrane allows the bacterium to avoid clearance by the host immune system and to resist the effects of antibiotics and antimicrobial peptides. The presence of cholesterol in H. pylori bacteria suggested that this pathogen may have cholesterol-enriched domains within its membrane. Consistent with this suggestion, we identified a hypothetical H. pylori protein (HP0248) with homology to the flotillin proteins normally found in the cholesterol-enriched domains of eukaryotic cells. As shown for eukaryotic flotillin proteins, HP0248 was detected in detergent-resistant membrane fractions of H. pylori. Importantly, H. pylori HP0248 mutants contained lower levels of cholesterol than wild-type bacteria (P < 0.01). HP0248 mutant bacteria also exhibited defects in type IV secretion functions, as indicated by reduced IL-8 responses and CagA translocation in epithelial cells (P < 0.05), and were less able to establish a chronic infection in mice than wild-type bacteria (P < 0.05). Thus, we have identified an H. pylori flotillin protein and shown its importance for bacterial virulence. Taken together, the data demonstrate important roles for H. pylori flotillin in host-pathogen interactions. We propose that H. pylori flotillin may be required for the organization of virulence proteins into membrane raft-like structures in this pathogen.

Peripheral Immune Cell Populations Associated with Cognitive Deficits and Negative Symptoms of Treatment-Resistant Schizophrenia.

BACKGROUND: Hypothetically, psychotic disorders could be caused or conditioned by immunological mechanisms. If so, one might expect there to be peripheral immune system phenotypes that are measurable in blood cells as biomarkers of psychotic states. METHODS: We used multi-parameter flow cytometry of venous blood to quantify and determine the activation state of 73 immune cell subsets for 18 patients with chronic schizophrenia (17 treated with clozapine), and 18 healthy volunteers matched for age, sex, BMI and smoking. We used multivariate methods (partial least squares) to reduce dimensionality and define populations of differentially co-expressed cell counts in the cases compared to controls. RESULTS: Schizophrenia cases had increased relative numbers of NK cells, naïve B cells, CXCR5+ memory T cells and classical monocytes; and decreased numbers of dendritic cells (DC), HLA-DR+ regulatory T-cells (Tregs), and CD4+ memory T cells. Likewise, within the patient group, more severe negative and cognitive symptoms were associated with decreased relative numbers of dendritic cells, HLA-DR+ Tregs, and CD4+ memory T cells. Motivated by the importance of central nervous system dopamine signalling for psychosis, we measured dopamine receptor gene expression in separated CD4+ cells. Expression of the dopamine D3 (DRD3) receptor was significantly increased in clozapine-treated schizophrenia and covaried significantly with differentiated T cell classes in the CD4+ lineage. CONCLUSIONS: Peripheral immune cell populations and dopaminergic signalling are disrupted in clozapine-treated schizophrenia. Immuno-phenotypes may provide peripherally accessible and mechanistically specific biomarkers of residual cognitive and negative symptoms in this treatment-resistant subgroup of patients.

Increased Outer Membrane Vesicle Formation in a Helicobacter pylori tolB Mutant.

BACKGROUND: Multiple studies have established the importance of the tol-pal gene cluster in bacterial cell membrane integrity and outer membrane vesicle (OMV) formation in Escherichia coli. In contrast, the functions of Tol-Pal proteins in pathogenic organisms, including those of the Epsilonproteobacteria, remain poorly if at all defined. The aim of this study was to characterize the roles of two key components of the Tol-Pal system, TolB and Pal, in OMV formation in the pathogenic bacterium, Helicobacter pylori. METHODS: H. pylori ΔtolB, Δpal and ΔtolBpal mutants, as well as complemented strains, were generated and assessed for changes in morphology and OMV production by scanning electron microscopy and enzyme-linked immunoassay (ELISA), respectively. The protein content and pro-inflammatory properties of OMVs were determined by mass spectroscopy and interleukin-8 (IL-8) ELISA on culture supernatants from OMV-stimulated cells, respectively. RESULTS: H. pylori ΔtolB and Δpal bacteria exhibited aberrant cell morphology and/or flagella biosynthesis. Importantly, the disruption of H. pylori tolB but not pal resulted in a significant increase in OMV production. The OMVs from H. pylori ΔtolB and Δpal bacteria harbored many of the major outer membrane and virulence proteins observed in wild-type (WT) OMVs. Interestingly, ΔtolB, Δpal and ΔtolBpal OMVs induced significantly higher levels of IL-8 production by host cells, compared with WT OMVs. CONCLUSIONS: This work demonstrates that TolB and Pal are important for membrane integrity in H. pylori. Moreover, it shows how H. pylori tolB-pal genes may be manipulated to develop "hypervesiculating" strains for vaccine purposes.