Symptom Burden and Immune Dynamics 6 to 18 Months Following Mild Severe Acute Respiratory Syndrome Coronavirus 2 Infection (SARS-CoV-2): A Case-control Study.

BACKGROUND: The burden and duration of persistent symptoms after nonsevere coronavirus disease 2019 (COVID-19) remains uncertain. This study aimed to assess postinfection symptom trajectories in home-isolated COVID-19 cases compared with age- and time- matched seronegative controls, and investigate immunological correlates of long COVID. METHODS: A prospective case-control study included home-isolated COVID-19 cases between February 28 and April 4, 2020, and followed for 12 (n = 233) to 18 (n = 149) months, and 189 age-matched severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-naive controls. We collected clinical data at baseline, 6, 12, and 18 months postinfection, and blood samples at 2, 4, 6, and 12 months for analysis of SARS-CoV-2-specific humoral and cellular responses. RESULTS: Overall, 46% (108/233) had persisting symptoms 12 months after COVID-19. Compared with controls, adult cases had a high risk of fatigue (27% excess risk, sex, and comorbidity adjusted odds ratio [aOR] 5.86; 95% confidence interval [CI], 3.27-10.5), memory problems (21% excess risk; aOR 7.42; CI, 3.51-15.67), concentration problems (20% excess risk; aOR 8.88; 95% CI, 3.88-20.35), and dyspnea (10% excess risk; aOR 2.66; 95% CI, 1.22-5.79). The prevalence of memory problems increased overall from 6 to 18 months (excess risk 11.5%; 95% CI, 1.5-21.5; P = .024) and among women (excess risk 18.7%; 95% CI, 4.4-32.9; P = .010). Longitudinal spike immunoglobulin G was significantly associated with dyspnea at 12 months. The spike-specific clonal CD4+ T-cell receptor ß depth was significantly associated with both dyspnea and number of symptoms at 12 months. CONCLUSIONS: This study documents a high burden of persisting symptoms after mild COVID-19 and suggests that infection induced SARS-CoV-2-specific immune responses may influence long-term symptoms.

Live-Attenuated Influenza Vaccine Induces Tonsillar Follicular T Helper Cell Responses That Correlate With Antibody Induction.

BACKGROUND: Influenza remains a major threat to public health. Live-attenuated influenza vaccines (LAIV) have been shown to be effective, particularly in children. Follicular T helper (TFH) cells provide B-cell help and are crucial for generating long-term humoral immunity. However the role of TFH cells in LAIV-induced immune responses is unknown. METHODS: We collected tonsils, plasma, and saliva samples from children and adults receiving LAIV prior to tonsillectomy. We measured influenza-specific TFH-cell responses after LAIV by flow cytometry and immunohistochemistry. Systemic and local antibody responses were analysed by hemagglutination inhibition assay and enzyme-linked immunosorbent assay. RESULTS: We report that LAIV induced early (3-7 days post-vaccination) activation of tonsillar follicles and influenza-specific TFH-cell (CXCR5+CD57+CD4+ T cell) responses in children, and to a lesser extent in adults. Serological analyses showed that LAIV elicited rapid (day 14) and long-term (up to 1 year post-vaccination) antibody responses (hemagglutination inhibition, influenza-specific IgG) in children, but not adults. There was an inverse correlation between pre-existing influenza-specific salivary IgA concentrations and tonsillar TFH-cell responses, and a positive correlation between tonsillar TFH-cell and systemic IgG induction after LAIV. CONCLUSIONS: Our data, taken together, demonstrate an important role of tonsillar TFH cells in LAIV-induced immunity in humans.

Viral antigens elicit augmented immune responses in primary Sjögren's syndrome.

OBJECTIVES: Infections have been suggested in the pathogenesis of primary SS (pSS). Systematic studies of immune responses to microbial antigens in vivo may be performed during vaccination. In the present study, we therefore longitudinally followed patients with pSS and controls during split-virion influenza vaccination to identify pSS-specific cellular, transcriptomic and serological responses. METHODS: Patients without treatment (pSSUntr, n = 17), on hydroxychloroquine-treatment (pSSHCQ, n = 8), and healthy controls (n = 16) were included. Antibody titres were determined by ELISA. Plasma proteins were measured by proximity extension assay. Monocyte gene expression was assessed by Nanostring. Routine laboratory tests were performed and clinical disease symptoms were registered by questionnaires. RESULTS: pSSUntr developed higher vaccine-specific IgG titres compared with controls. Notably, anti-Ro52 autoantibody titres increased in pSSUntr but remained unchanged in pSSHCQ. No changes in disease symptoms including EULAR Sjögren's Syndrome Patient Reported Index score were registered. Twenty-four hours after vaccination, the leucocyte count in pSSUntr decreased, with a concomitant increase of CCL7 in plasma. Transcriptomic analysis in monocytes revealed differential vaccination-related expression of the NEMO/IKBKG gene, and its higher induced expression in pSSUntr associated with higher serological vaccine responses. Moreover, titres of vaccine-specific antibodies were associated with higher vaccination-induced NF-κB signalling and higher steady-state IFN signatures in monocytes, and with the levels of several plasma proteins with soluble PD-1 displaying the strongest association. CONCLUSION: We observed augmented innate and adaptive immune responses in pSS following viral antigen exposure suggesting an underlying hyper-responsiveness to immune challenges, supporting a role for infections driving the immunopathology and acting as environmental risk factor for pSS.

Small molecule promotes ß-catenin citrullination and inhibits Wnt signaling in cancer.

Wnt (wingless)/ß-catenin signaling is critical for tumor progression and is frequently activated in colorectal cancer as a result of the mutation of adenomatous polyposis coli (APC); however, therapeutic agents targeting this pathway for clinical use are lacking. Here we report that nitazoxanide (NTZ), a clinically approved antiparasitic drug, efficiently inhibits Wnt signaling independent of APC. Using chemoproteomic approaches, we have identified peptidyl arginine deiminase 2 (PAD2) as the functional target of NTZ in Wnt inhibition. By targeting PAD2, NTZ increased the deamination (citrullination) and turnover of ß-catenin in colon cancer cells. Replacement of arginine residues disrupted the transcriptional activity, and NTZ induced degradation of ß-catenin. In Wnt-activated colon cancer cells, knockout of either PAD2 or ß-catenin substantially increased resistance to NTZ treatment. Our data highlight the potential of NTZ as a modulator of ß-catenin citrullination for the treatment of cancer patients with Wnt pathway mutations.

Effects of low doses of fish and milk proteins on glucose regulation and markers of insulin sensitivity in overweight adults: a randomised, double blind study.

PURPOSE: To examine whether supplementation with low doses of fish or milk proteins would affect glucose regulation and circulating lipid concentrations in overweight healthy adults. METHODS: Ninety-three overweight adults were assigned to receive 2.5 g protein/day from herring (HER), salmon (SAL), cod (COD) or milk (CAS, a casein-whey mixture as positive control) as tablets for 8 weeks. RESULTS: Seventy-seven participants were included in the analyses. HER and SAL did not affect glucose and insulin concentrations. COD significantly reduced within-group changes in 90 and 120 min postprandial glucose concentrations but changes were not different from HER and SAL groups. CAS supplementation significantly reduced the area under the curve for glucose concentrations (- 7%), especially when compared to SAL group, and reduced postprandial insulin c-peptide concentration (- 23%). Reductions in acetoacetate (- 24%) and ß-hydroxybutyrate (- 29%) serum concentrations in HER group were more prominent compared to SAL and COD groups, with no differences between fish protein groups for α-hydroxybutyrate. Serum concentrations of α-hydroxybutyrate (- 23%), acetoacetate (- 39%) and ß-hydroxybutyrate (- 40%) were significantly reduced within CAS group, and the decreases were significantly more pronounced when compared to SAL group. Serum lipid concentrations were not altered in any of the intervention groups. CONCLUSION: Findings indicate that 2.5 g/day of proteins from fish or milk may be sufficient to improve glucose regulation in overweight adults. The effects were most pronounced after supplementation with proteins from cod, herring and milk, whereas salmon protein did not affect any of the measurements related to glucose regulation. CLINICAL TRAIL REGISTRATION: This trial was registered at clinicaltrials.gov as NCT01641055.

Axitinib blocks Wnt/ß-catenin signaling and directs asymmetric cell division in cancer.

Oncogenic mutations of the Wnt (wingless)/ß-catenin pathway are frequently observed in major cancer types. Thus far, however, no therapeutic agent targeting Wnt/ß-catenin signaling is available for clinical use. Here we demonstrate that axitinib, a clinically approved drug, strikingly blocks Wnt/ß-catenin signaling in cancer cells, zebrafish, and Apc(min/+) mice. Notably, axitinib dramatically induces Wnt asymmetry and nonrandom DNA segregation in cancer cells by promoting nuclear ß-catenin degradation independent of the GSK3ß (glycogen synthase kinase3ß)/APC (adenomatous polyposis coli) complex. Using a DARTS (drug affinity-responsive target stability) assay coupled to 2D-DIGE (2D difference in gel electrophoresis) and mass spectrometry, we have identified the E3 ubiquitin ligase SHPRH (SNF2, histone-linker, PHD and RING finger domain-containing helicase) as the direct target of axitinib in blocking Wnt/ß-catenin signaling. Treatment with axitinib stabilizes SHPRH and thereby increases the ubiquitination and degradation of ß-catenin. Our findings suggest a previously unreported mechanism of nuclear ß-catenin regulation and indicate that axitinib, a clinically approved drug, would provide therapeutic benefits for cancer patients with aberrant nuclear ß-catenin activation.

T follicular-like helper cells in the peripheral blood of patients with primary Sjögren's syndrome.

Primary Sjögren's syndrome (pSS) is a chronic autoimmune disease characterized by exocrine gland dysfunction, mainly causing sicca symptoms. B cells have a prominent role in SS, and the T follicular helper (TFH ) cells provide B cells with survival- and specialization signals in germinal centers. Here we investigate peripheral TFH cells in pSS. Sixteen pSS patients and healthy controls were enrolled in the study, with thirteen women and three men in each group. Whole blood was collected and separated into PBMC and plasma, followed by cryopreservation. Plasma samples were analyzed for Ro52, Ro60 and La48 autoantibodies by indirect ELISA. For flow cytometric analysis, we defined four subsets of TFH-like cells within the CD3+CD4+CXCR5+ population, namely the ICOS-PD1-, ICOS-PD-1+, ICOS+PD-1- and ICOS+PD-1+ ("TFH") cells. We also investigated four CD19+ B cell subsets, the CD20+CD27+CD38- memory B cells, CD20+CD27+CD38+ memory B cells, CD20-CD27+CD38++CD138- plasmablasts and CD20-CD27+CD38++CD138+ plasma cells. We observed higher fractions of ICOS+PD-1- cells, ICOS+PD-1+ ("TFH ") cells and plasmablasts in pSS patients compared to controls, and lower frequencies of both types of memory B cells. The number of TFH cells correlated positively with the levels of plasmablasts and plasma cells in the pSS patients, but not in the controls. The pSS patients were stratified according to Ro52/Ro60/La48 serology, and a positive association was found between autoantibody levels and increased level of TFH cells, plasmablasts and plasma cells and lowered levels of memory B cells. We observed a higher response to Ro/La stimulation in pSS patients compared to controls of the memory B cells, although only significantly for the CD38- memory B cells. Overall, a pathological relation between the ICOS+ T follicular-like helper cells and B cells in pSS was observed, but further work should be conducted to explore their overall impact upon disease progression. This article is protected by copyright. All rights reserved.

Current concepts on Sjögren's syndrome - classification criteria and biomarkers.

Sjögren's syndrome is a lymphoproliferative disease with autoimmune features characterized by mononuclear cell infiltration of exocrine glands, notably the lacrimal and salivary glands. These lymphoid infiltrations lead to dryness of the eyes (keratoconjunctivitis sicca), dryness of the mouth (xerostomia), and, frequently, dryness of other surfaces connected to exocrine glands. Sjögren's syndrome is associated with the production of autoantibodies because B-cell activation is a consistent immunoregulatory abnormality. The spectrum of the disease extends from an organ-specific autoimmune disorder to a systemic process and is also associated with an increased risk of B-cell lymphoma. Current treatments are mainly symptomatic. As a result of the diverse presentation of the syndrome, a major challenge remains to improve diagnosis and therapy. For this purpose an international set of classification criteria for primary Sjögren's syndrome has recently been developed and validated and seems well suited for enrolment in clinical trials. Salivary gland biopsies have been examined and histopathology standards have been developed, to be used in clinical trials and patient stratification. Finally, ultrasonography and saliva meet the need of non-invasive imaging and sampling methods for discovery and validation of disease biomarkers in Sjögren's syndrome.

Boosting of Cross-Reactive and Protection-Associated T Cells in Children After Live Attenuated Influenza Vaccination.

Background: Live attenuated influenza vaccines (LAIVs) stimulate a multifaceted immune response including cellular immunity, which may provide protection against newly emerging strains. This study shows proof of concept that LAIVs boost preexisting, cross-reactive T cells in children to genetically diverse influenza A virus (IAV) strains to which the children had not been exposed. Methods: We studied the long-term cross-reactive T-cell response in 14 trivalent LAIV-vaccinated children using the fluorescent immunospot assay (FluoroSpot) with heterologous H1N1 and H3N2 IAVs and CD8+ peptides from the internal proteins (matrix protein 1 [M1], nucleoprotein [NP], polymerase basic protein 1 [PB1]). Serum antibody responses were determined by means of hemagglutination inhibition assay. Blood samples were collected before vaccination and up to 1 year after vaccination. Results: Preexisting cross-reactive T cells to genetically diverse IAV strains were found in the majority of the children, which were further boosted in 50% of them after receipt of LAIV. Further analyses of these T cells showed significant increases in CD8+ T cells, mainly dominated by NP-specific responses. After vaccination with LAIV, the youngest children showed the highest increase in T-cell responses. Conclusion: LAIV boosts durable, cross-reactive T-cell responses in children and may have a clinically protective effect at the population level. LAIV may be a first step toward the desired universal influenza vaccine.

H1N1 vaccination in Sjögren's syndrome triggers polyclonal B cell activation and promotes autoantibody production.

OBJECTIVES: Vaccination of patients with rheumatic disease has been reported to result in lower antibody titres than in healthy individuals. However, studies primarily include patients on immunosuppressive therapy. Here, we investigated the immune response of treatment-naïve patients diagnosed with primary Sjögren's syndrome (pSS) to an H1N1 influenza vaccine. METHODS: Patients with Sjögren's syndrome without immunomodulatory treatment and age-matched and gender-matched healthy controls were immunised with an H1N1 influenza vaccine and monitored for serological and cellular immune responses. Clinical symptoms were monitored with a standardised form. IgG class switch and plasma cell differentiation were induced in vitro in purified naïve B cells of untreated and hydroxychloroquine-treated patients and healthy controls. Gene expression was assessed by NanoString technology. RESULTS: Surprisingly, treatment-naïve patients with Sjögren's syndrome developed higher H1N1 IgG titres of greater avidity than healthy controls on vaccination. Notably, off-target B cells were also triggered resulting in increased anti-EBV and autoantibody titres. Endosomal toll-like receptor activation of naïve B cells in vitro revealed a greater propensity of patient-derived cells to differentiate into plasmablasts and higher production of class switched IgG. The amplified plasma cell differentiation and class switch could be induced in cells from healthy donors by preincubation with type 1 interferon, but was abolished in hydroxychloroquine-treated patients and after in vitro exposure of naïve B cells to chloroquine. CONCLUSIONS: This comprehensive analysis of the immune response in autoimmune patients to exogenous stimulation identifies a mechanistic basis for the B cell hyperactivity in Sjögren's syndrome, and suggests that caution is warranted when considering vaccination in non-treated autoimmune patients.

High intake of fatty fish, but not of lean fish, improved postprandial glucose regulation and increased the n-3 PUFA content in the leucocyte membrane in healthy overweight adults: a randomised trial.

The prevalence of type 2 diabetes (T2D) is low in populations with a high fish intake; however prospective studies with fish intake have shown positive, negative or no association between fish intake and the risk for T2D. The aim of this study was to investigate the effects of high intake of lean or fatty fish on glucose tolerance, leucocyte membrane fatty acid composition and leucocyte function in overweight/obese adults. In this randomised clinical trial, sixty-eight healthy overweight/obese participants consumed 750 g/week of either lean or fatty fish as dinners, or were instructed to continue their normal eating habits but to avoid fish intake (control group), for 8 weeks. Energy and macronutrient intake and physical activity were not changed within the groups during the study period. High intake of fatty fish, but not of lean fish, significantly improved glucose regulation 120 min postprandially (P=0·012), but did not affect fasting glucose concentration. A smaller increase in fasting to 120 min postprandial insulin C-peptide concentration was seen after fatty fish intake (P=0·012). Lean fish increased the DHA content in leucocyte membranes (P=0·010), and fatty fish increased the total content of n-3 PUFA (P=0·00016) and reduced the content of n-6 PUFA (P=0·00057) in leucocyte membranes. Lean and fatty fish intake did not affect phagocytosis of bacteria ex vivo. The findings suggest that high intake of fatty fish, but not of lean fish, beneficially affected postprandial glucose regulation in overweight/obese adults, and may therefore prevent or delay the development of T2D in this population.

High intake of fatty fish, but not of lean fish, affects serum concentrations of TAG and HDL-cholesterol in healthy, normal-weight adults: a randomised trial.

The aim of the present study was to examine whether high intake of lean or fatty fish (cod and farmed salmon, respectively) by healthy, normal-weight adults would affect risk factors of type 2 diabetes and CVD when compared with lean meat (chicken). More knowledge is needed concerning the potential health effects of high fish intake (>300 g/week) in normal-weight adults. In this randomised clinical trial, thirty-eight young, healthy, normal-weight participants consumed 750 g/week of lean or fatty fish or lean meat (as control) for 4 weeks at dinner according to provided recipes to ensure similar ways of preparations and choices of side dishes between the groups. Energy and macronutrient intakes at baseline and end point were similar in all groups, and there were no changes in energy and macronutrient intakes within any of the groups during the course of the study. High intake of fatty fish, but not lean fish, significantly reduced TAG and increased HDL-cholesterol concentrations in fasting serum when compared with lean meat intake. When compared with lean fish intake, fatty fish intake increased serum HDL-cholesterol. No differences were observed between lean fish, fatty fish and lean meat groups regarding fasting and postprandial glucose regulation. These findings suggest that high intake of fatty fish, but not of lean fish, could beneficially affect serum concentrations of TAG and HDL-cholesterol, which are CVD risk factors, in healthy, normal-weight adults, when compared with high intake of lean meat.

Serum calprotectin is a biomarker of carotid atherosclerosis in patients with primary Sjögren's syndrome.

OBJECTIVES: We aimed to identify the association of carotid atherosclerosis with the traditional risk factors, disease features, cytokine profile, and calprotectin in patients with primary Sjögren's syndrome (pSS). METHODS: 63 primary pSS patients and 63 age- and sex-matched healthy controls underwent carotid ultrasound, clinical and laboratory examination. The presence of carotid plaques was taken as carotid atherosclerosis. The covariates of carotid atherosclerosis were identified in univariate and multivariate regressions. RESULTS: Patients with pSS had higher prevalence of carotid atherosclerosis (13% vs. 2%, p<0.05) and higher serum levels of calprotectin, tumour necrosis factor receptor 2 (TNF-R2), hepatocyte growth factor (HGF), and monocyte chemoattractant protein-1 (MCP-1) than controls. Sex, menopause, and the prevalence of traditional cardiovascular did not differ between groups (all p>0.05). In univariate analyses, serum calprotectin, most traditional cardiovascular (age, male sex, metabolic syndrome, hypertension, hypertriglyceridaemia, and serum creatinine), and some disease-associated risk factors (glucocorticoid or saliva substitute use, constitutional domain of Eular-Sjögren's syndrome disease activity index - EULAR) were associated with a higher risk for plaque. In a multivariate analysis, having pSS and higher serum calprotectin were associated with carotid atherosclerosis independent of traditional risk factors. CONCLUSIONS: pSS have a higher prevalence of carotid atherosclerosis, which is associated with higher serum calprotectin level independent of traditional cardiovascular risk factors. Our findings suggest calprotectin as a biomarker of subclinical atherosclerosis in pSS.

Longevity of B-cell and T-cell responses after live attenuated influenza vaccination in children.

BACKGROUND: The live attenuated influenza vaccine (LAIV) is the preferred vaccine for children, but the mechanisms behind protective immune responses are unclear, and the duration of immunity remains to be elucidated. This study reports on the longevity of B-cell and T-cell responses elicited by the LAIV. METHODS: Thirty-eight children (3-17 years old) were administered seasonal LAIV. Blood samples were collected before vaccination with sequential sampling up to 1 year after vaccination. Humoral responses were evaluated by a hemagglutination inhibition assay, and memory B-cell responses were evaluated by an enzyme-linked immunosorbent spot assay (ELISpot). T-cell responses were evaluated by interferon γ (IFN-γ) ELISpot analysis, and intracellular cytokine staining of CD4(+) T cells for detection of IFN-γ, interleukin 2, and tumor necrosis factor α was performed using flow cytometry. RESULTS: LAIV induced significant increases in B-cell and T-cell responses, which were sustained at least 1 year after vaccination. Strain variations were observed, in which the B strain elicited stronger responses. IFN-γ-expressing T cell counts increased significantly, and remained higher than prevaccination levels 1 year later. Expression of T-helper type 1 intracellular cytokines (interleukin 2, IFN-γ, and tumor necrosis factor α) increased after 1 dose and were boosted after the second dose. Hemagglutination inhibition titers were sustained for 1 year. Vaccine-induced memory B cell counts were significantly increased, and the response persisted for one year. CONCLUSIONS: LAIV elicited B-cell and T-cell responses that persisted for at least 1 year in children. This is a novel finding that will aid future vaccine policy.

Comparison of inflammatory markers in induced and spontaneous sputum in a cohort of COPD patients.

BACKGROUND: Sputum induction is a non-invasive method for obtaining measurements of inflammation in the airways. Whether spontaneously sampled sputum can be a valid surrogate is unknown. The aim of this study was to compare levels of six inflammatory markers in sputum pairs consisting of induced and spontaneous sputum sampled on the same consultation either in a stable state or during exacerbations of chronic obstructive pulmonary disease (COPD). METHODS: 433 COPD patients aged 40-76, Global initiative for chronic Obstructive Lung Disease (GOLD) stage II-IV were enrolled in 2006/07 and followed every six months for three years. 356 patients were followed for potential exacerbations. Interleukin-6, interleukin-8, interleukin-18, interferon gamma-inducible protein-10, monokine induced by gamma interferon and tumor necrosis factor-alpha (IL-6, IL-8, IL-18, IP-10, MIG and TNF-α) were measured by bead based multiplex immunoassay in 60 paired sputum samples from 45 patients. Albumin was measured by enzyme immunoassay, for concentration correction. Culturing for bacterial growth was performed on 24 samples. Bland-Altman plots were used to assess agreement. The paired non-parametric Wilcoxon signed-rank test, the non-parametric Spearman's rank correlation test and Kruskal-Wallis test were used for statistical analyses. For all analyses, a p-value < 0.05 was considered significant. RESULTS: Agreement between the two measurements was generally low for all six markers. TNF-α was significantly higher in spontaneous sputum at exacerbations (p = 0.002) and trending higher at the steady state (p = 0.06). Correlation coefficients between the levels of markers in induced and spontaneous sputum varied between 0.58 (IL-18) to 0.83 (IP-10). In spontaneous sputum IL-18 and MIG were higher in ex-smokers (p < 0.05). The levels of all markers were higher in GOLD stage III & IV except for IL-6 in spontaneous sputum and IL-18 in induced sputum, compared with GOLD stage II, although not statistically significant. In spontaneous sputum the levels of IL-6 were significantly higher if Haemophilus influenzae (HI) was not cultured. CONCLUSION: We observed a low agreement and significant differences in inflammatory markers between induced and spontaneous sputum, both at steady state and exacerbations. We recommend considering sampling method when reporting on inflammatory markers in sputum.

SARS-CoV-2 specific immune responses in overweight and obese COVID-19 patients.

Obesity is a known risk factor for severe respiratory tract infections. In this prospective study, we assessed the impact of being obese or overweight on longitudinal SARS-CoV-2 humoral and cellular responses up to 18 months after infection. 274 patients provided blood samples at regular time intervals up to 18 months including obese (BMI ≥30, n=32), overweight (BMI 25-29.9, n=103) and normal body weight (BMI 18.5-24.9, n=134) SARS-CoV-2 patients. We determined SARS-CoV-2 spike-specific IgG, IgA, IgM levels by ELISA and neutralising antibody titres by neutralisation assay. RBD- and spike-specific memory B cells were investigated by ELISpot, spike- and non-spike-specific IFN-γ, IL-2 and IFN-γ/IL-2 secreting T cells by FluoroSpot and T cell receptor (TCR) sequencing was performed. Higher BMI correlated with increased COVID-19 severity. Humoral and cellular responses were stronger in overweight and obese patients than normal weight patients and associated with higher spike-specific IgG binding titres relative to neutralising antibody titres. Linear regression models demonstrated that BMI, age and COVID-19 severity correlated independently with higher SARS-CoV-2 immune responses. We found an increased proportion of unique SARS-CoV-2 specific T cell clonotypes after infection in overweight and obese patients. COVID-19 vaccination boosted humoral and cellular responses irrespective of BMI, although stronger immune boosting was observed in normal weight patients. Overall, our results highlight more severe disease and an over-reactivity of the immune system in overweight and obese patients after SARS-CoV-2 infection, underscoring the importance of recognizing overweight/obese individuals as a risk group for prioritisation for COVID-19 vaccination.

Durable T-cellular and humoral responses in SARS-CoV-2 hospitalized and community patients.

BACKGROUND: Neutralizing antibodies are important for protection against the pandemic SARS-CoV-2 virus, and long-term memory responses determine the risk of re-infection or boosting after vaccination. T-cellular responses are considered important for partial protection against novel variants of concern. METHODS: A prospective cohort of hospitalized (n = 14) and community (n = 38) patients with rt-PCR confirmed SARS-CoV-2 infection were recruited. Blood samples and clinical data were collected when diagnosed and at 6 months. Serum samples were analyzed for SARS-CoV-2-spike specific antibodies using ELISA (IgG, IgA, IgM), pseudotype neutralization and microneutralization assays. Peripheral blood mononuclear cells were investigated for virus-specific T-cell responses in the interferon-γ and interleukin-2 fluorescent-linked immunosorbent spot (FluroSpot) assay. RESULTS: We found durable SARS-CoV-2 spike- and internal protein specific T-cellular responses in patients with persistent antibodies at 6 months. Significantly higher IL-2 and IFN-γ secreting T-cell responses as well as SARS-CoV-2 specific IgG and neutralizing antibodies were detected in hospitalized compared to community patients. The immune response was impacted by age, gender, comorbidity and severity of illness, reflecting clinical observations. CONCLUSIONS: SARS-CoV-2 specific T-cellular and antibody responses persisted for 6 months post confirmed infection. In previously infected patients, re-exposure or vaccination will boost long-term immunity, possibly providing protection against re-infection with variant viruses.

Variability of Primary Sjögren's Syndrome Is Driven by Interferon-α and Interferon-α Blood Levels Are Associated With the Class II HLA-DQ Locus.

OBJECTIVE: Primary Sjögren's syndrome (SS) is the second most frequent systemic autoimmune disease, affecting 0.1% of the general population. To characterize the molecular and clinical variabilities among patients with primary SS, we integrated transcriptomic, proteomic, cellular, and genetic data with clinical phenotypes in a cohort of 351 patients with primary SS. METHODS: We analyzed blood transcriptomes and genotypes of 351 patients with primary SS who were participants in a multicenter prospective clinical cohort. We replicated the transcriptome analysis in 3 independent cohorts (n = 462 patients). We determined circulating interferon-α (IFNα) and IFNγ protein concentrations using digital single molecular arrays (Simoa). RESULTS: Transcriptome analysis of the prospective cohort showed a strong IFN gene signature in more than half of the patients; this finding was replicated in the 3 independent cohorts. Because gene expression analysis did not discriminate between type I IFN and type II IFN, we used Simoa to demonstrate that the IFN transcriptomic signature was driven by circulating IFNα and not by IFNγ protein levels. IFNα protein levels, detectable in 75% of patients, were significantly associated with clinical and immunologic features of primary SS disease activity at enrollment and with increased frequency of systemic complications over the 5-year follow-up. Genetic analysis revealed a significant association between IFNα protein levels, a major histocompatibility (MHC) class II haplotype, and anti-SSA antibody. Additional cellular analysis revealed that an MHC class II HLA-DQ locus acts through up-regulation of HLA class II molecules on conventional dendritic cells. CONCLUSION: We identified the predominance of IFNα as a driver of primary SS variability, with IFNα demonstrating an association with HLA gene polymorphisms.

Cytokine and autoantibody profiling related to histopathological features in primary Sjogren's syndrome.

OBJECTIVE: To investigate a potential correlation between circulating cytokine and autoantibody levels and histopathological features in subgroups of patients with primary SS (pSS). METHODS: Minor salivary gland biopsies from a cohort of 141 patients fulfilling the American-European consensus classification criteria for pSS were re-examined and grouped according to focus score (FS) and germinal centre (GC) status; serum samples were analysed for autoantibodies, chemokines and cytokines. RESULTS: Of the 115 available biopsies, 18 (16%) lacked characteristic focal mononuclear cell infiltrates [FS < 1 (FS-)] but patients were positive for Ro/SSA and/or La/SSB. IL-17, IL-1RA, IL-15, macrophage inflammatory protein (MIP)-1alpha, MIP-1beta, eotaxin, IFN-alpha and IL-4 levels were significantly increased in the 27 (23%) patients with ectopic GC formation (GC+) in the salivary glands compared with the GC- patients (n = 70). In addition, minor differences in cytokine levels were found when comparing age groups. CONCLUSION: Degenerative changes observed in the minor salivary glands of patients with pSS may represent 'burned out' inflammation. The elevated levels of IL-4 found in these patients may influence the reduced salivary flow observed in GC+ patients. Increased titres of Th17-associated cytokines, IL-17, IL-1beta and the IL-23 subunit IL-12p40, may indicate a higher activity of these cells in GC+ patients. Differences in cytokine levels may be utilized when sub-grouping the SS patients into disease phases and may consequently have implications for treatment.

Allergic sensitisation in tuberculosis patients at the time of diagnosis and following chemotherapy.

BACKGROUND: It is still a matter of debate whether there is an association between infection with Mycobacterium tuberculosis (M. tuberculosis) and allergy. Previously, we have shown higher levels of specific IgE to different inhalant allergens and total IgE in tuberculosis (TB) patients compared to controls. The objectives of this study were to evaluate a possible change in allergic sensitisation after successful TB treatment and to confirm the finding of our previous study of enhanced allergic sensitisation in TB patients compared to controls in a more controlled setting. Additionally, we wanted to determine the cytokine profile in the same groups and finally to evaluate the association between the presence of Bacillus Calmette-Guérin vaccination (BCG) scar and allergic sensitisation among the controls. METHODS: Sera were analysed for specific IgE to inhalant allergens (Phadiatop) and total IgE by the use of ImmunoCAP 1000 (Pharmacia Diagnostics). Thirteen different cytokines were also analysed in the sera by multiplex bead immunoassay (Luminex 100, Luminex Corporation), and clinical symptoms of allergy and BCG scar were reported in a questionnaire. RESULTS: A reduction in levels of specific and total IgE were observed after successful TB treatment. TB patients also had higher levels of specific and total IgE compared to healthy controls. Both interleukin (IL)-6 and interferon (IFN)gamma were higher in TB patients compared to healthy controls. The levels of IL-6 were reduced after successful TB treatment. The presence of a BCG scar was associated with a reduced risk of developing allergic sensitisation. CONCLUSION: We observed a reduced level of allergic sensitisation after successful TB treatment. TB patients seem to be more allergically sensitised than healthy controls, confirming our previous finding. Furthermore, we observed an inverse association between allergic sensitisation and visible BCG scar, which adds additional support to the hygiene hypothesis.