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Background: Immunoglobulin G subclass deficiencies (IgGsd) comprise a wide clinical spectrum from no symptoms to repeated respiratory infections and risk for the development of lung damage. Our aims were to investigate whether the immunological phenotype of IgGsd patients on and off immunoglobulin replacement therapy (IgRT) was reflected in the clinical features of IgGsd., Method: Thirty patients with IgGsd were included in this prospective study of 18 months of IgRT, followed by 7-18 months of IgRT discontinuation. Blood samples were collected when patients were on and off IgRT and compared with samples from 34 cross-sectional healthy controls. An in-depth lymphocyte phenotyping was performed by flow cytometry and plasma levels of immune checkpoints were assessed., Results: IgG3 subclass deficiency was most common. Patients with IgGsd had decreased levels of activated T cells and B cells and plasma levels of negative immune checkpoint molecules correlated negatively with T cell and B cell activation. The decreased T cell activation level was unaffected by IgRT, while the B cell activation was partly restored. Of note, decreased levels of activated regulatory T cells (Tregs) were found in IgGsd patients and was partly restored during IgRT. The profile of comorbidities did not associate with Treg levels., Discussion: IgGsd is associated with decreased B cell and T cell activation including Tregs, and increased plasma levels of negative immune checkpoint molecules. The consequence of reduced activated Tregs in IgGsd remains unclear. Decreased immune cell activation was partly restored during IgRT, demonstrating that IgRT may contribute to improved immune function in patients with IgGsd., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Wågström, Hjorth, Appelgren, Björkander, Dahle, Nilsson, Nilsdotter-Augustinsson, Ernerudh and Nyström.)

The objective of this study was to investigate the immune mechanisms involved in preterm labor (PTL), preterm prelabor rupture of the membranes (PPROM), and normal pregnancies. The second objective was to explore immune profiles in PTL for association with early ( < 34 gestational weeks (gw)) or instant ( < 48 h) delivery. This prospective observational multi-center study included women with singleton pregnancies with PTL (n = 80) or PPROM (n = 40) before 34 gw, women with normal pregnancies scheduled for antenatal visits (n = 44), and women with normal pregnancies in active labor at term (n = 40). Plasma samples obtained at admission were analyzed for cytokine and chemokine quantification using a multiplex bead assay in order to compare the immune profiles between PTL, PPROM, and normal pregnancies. In PTL, CXCL1 and CCL17 were significantly higher compared to gestational age-matched women at antenatal visits, whereas for PPROM, CXCL1 and IL-6 were increased. Women in term labor had a more pronounced inflammatory pattern with higher levels of CXCL1, CXCL8, and IL-6 compared with PTL (p = 0.007, 0.003, and 0.013, respectively), as well as higher levels of CCL17, CXCL1 and IL-6 (all p < 0.001) compared with the women at antenatal visits. In PTL, CXCL8 was higher in women with delivery before 34 gw, whereas CXCL8, GM-CSF, and IL-6 were significantly higher in women with delivery within 48 h. To conclude, PTL and PPROM were associated with a complex pattern of inflammation, both involving Th17 (CXCL1) responses. Although further studies are needed, CXCL8, GM-CSF, and IL-6 may be potential candidates for predicting preterm birth in PTL., Competing Interests: Declaration of Competing Interest None, (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Background: In order to identify and possibly offer prophylactic treatment to women at risk for preterm birth (PTB), novel prediction models for PTB are needed. Our objective was to utilize high-sensitive plasma protein profiling to investigate whether early prediction of spontaneous PTB (sPTB) before 34 gestational weeks (gw) was possible in a low-risk population., Methods: A case-control study was conducted on 46 women with sPTB before 34 gw and 46 women with normal pregnancies and term deliveries. Prospectively collected plasma sampled at gw 11 (range 7-16) and gw 25 (range 23-30) was analyzed with a high-sensitivity Proximity Extension Assay for levels of 177 inflammation-associated proteins, and statistically processed with multivariate logistic regression analysis., Results: In the first trimester, higher levels of hepatocyte growth factor (HGF) were associated with sPTB <34 gw (OR 1.49 (1.03-2.15)). In the second trimester, higher levels of interleukin (IL)-10 (OR 2.15 (1.18-3.92)), IL-6 (OR 2.59 (1.34-4.99)), and the receptor activator of nuclear factor κB (RANK) (OR 2.18 (1.26-3.77)) were associated with sPTB <34 gw. The area under the curve for the prediction models including these proteins was 0.653 (0.534-0.759) in the first trimester and 0.854 (0.754-0.925) in the second trimester., Conclusion: A combination of inflammation-associated plasma proteins from the second trimester of pregnancy showed a good predictive ability regarding sPTB before 34 gw, suggesting it could be a valuable supplement for the assessment of the clinical risk of sPTB. However, although a high number (n=177) of plasma proteins were analyzed with a high-sensitivity method, the prediction of sPTB in the first trimester remains elusive., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Svenvik, Raffetseder, Brudin, Berg, Hellberg, Blomberg, Jenmalm and Ernerudh.)

Objective: Ovarian removal prior to spontaneous/natural menopause (SM) is associated with increased risk of late life dementias including Alzheimer's disease. This increased risk may be related to the sudden and early loss of endogenous estradiol. Women with breast cancer gene mutations (BRCAm) are counseled to undergo oophorectomy prior to SM to significantly reduce their risk of developing breast, ovarian, and cervical cancers. There is limited evidence of the neurological effects of ovarian removal prior to the age of SM showing women without the BRCAm had cortical thinning in medial temporal lobe structures. A second study in women with BRCAm and bilateral salpingo-oophorectomy (BSO) noted changes in cognition., Methods: The present, cross-sectional study examined whole-brain differences in gray matter (GM) volume using high-resolution, quantitative magnetic resonance imaging in women with BRCAm and intact ovaries (BRCA-preBSO [study cohort with BRCA mutation prior to oophorectomy]; n = 9) and after surgery with (BSO + estradiol-based therapy [ERT]; n = 10) and without (BSO; n = 10) postsurgical estradiol hormone therapy compared with age-matched women (age-matched controls; n = 10) with their ovaries., Results: The BRCA-preBSO and BSO groups showed significantly lower GM volume in the left medial temporal and frontal lobe structures. BSO + ERT exhibited few areas of lower GM volume compared with age-matched controls. Novel to this study, we also observed that all three BRCAm groups exhibited significantly higher GM volume compared with age-matched controls, suggesting continued plasticity., Conclusions: The present study provides evidence, through lower GM volume, to support both the possibility that the BRCAm, alone, and early life BSO may play a role in increasing the risk for late-life dementia. At least for BRCAm with BSO, postsurgical ERT seems to ameliorate GM losses., Competing Interests: Financial disclosure/conflicts of interest: None reported., (Copyright © 2024 by The Menopause Society.)

Common variable immunodeficiency (CVID) is an inborn error of immunity characterized by low levels of antibodies. In addition to infections, many patients also suffer from T-helper 1-driven immune dysregulation, which is associated with increased mortality. The aim of this study was to perform in-depth characterization of the T and the B cell compartments in a well-defined cohort of patients affected by CVID and correlate the findings to the level of clinical immune dysregulation. We used mass cytometry, targeted proteomics, flow cytometry and functional assays to delineate the immunological phenotype of 15 CVID-affected patients with different levels of immune dysregulation. Unbiased clustering of T cell mass cytometry data correlated with CVID-related immune dysregulation and plasma protein profiles. Expanded CXCR3 + T-bet-expressing B cells correlated with effector memory CD4 + T cell clusters, and increased plasma levels of CXCR3-ligands. Our findings indicate an interplay between B cells and T cells in CVID-related immune dysregulation and provide a better understanding of the underlying pathological mechanisms., Competing Interests: Declaration of Competing Interest The authors have no financial or non-financial interests to disclose., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: Dimethyl fumarate (DMF) is a common treatment for multiple sclerosis (MS), but its mechanisms of action are not fully understood. Targeted proteomics offers insights into effects of DMF and biomarkers for treatment responses., Objectives: To assess influence of DMF on inflammation- and neuro-associated proteins in plasma and cerebrospinal fluid (CSF) in MS and to reveal biomarkers for predicting treatment responses., Methods: Using the high-sensitivity and high-specificity method of proximity extension assay (PEA), we measured 182 inflammation- and neuro-associated proteins in paired plasma (n = 28) and CSF (n = 12) samples before and after one year of DMF treatment. Disease activity was evaluated through clinical examination and MRI. Statistical tests, network analysis, and regression models were used., Results: Several proteins including T-helper 1 (Th1)-associated proteins (CXCL10, CXCL11, granzyme A, IL-12p70, lymphotoxin-alpha) were consistently decreased in CSF, while IL-7 was increased after one year of treatment. The changes in plasma protein levels did not follow the same pattern as in CSF. Logistic regression models identified potential biomarker candidates (including plexins and neurotrophins) for prediction of treatment response., Conclusions: DMF treatment induced prominent changes in CSF proteins, consistently reducing Th1-associated pro-inflammatory proteins. Neurodegeneration-related CSF proteins were able to predict treatment response. Protein biomarkers hold promise for personalized medicine., Competing Interests: Declaration of Competing Interest JM has received honoraria for Advisory boards for Sanofi Genzyme and Merck and lecture honorarium from Merck. All other authors declare no competing interests., (Copyright © 2023. Published by Elsevier B.V.)

The pro-inflammatory and regulatory roles of T lymphocytes in atherosclerosis are well established but less is known about natural killer (NK) cells and natural killer T (NKT)-like cells. The effects of cardiovascular risk management on the phenotypes of these cells are unknown. To assess changes in NK cell and lymphocyte phenotypes and circulating inflammatory proteins in response to cardiovascular risk management in patients with carotid atherosclerosis. Fifty patients were included in a prospective clinical study. Measurements were at baseline and after 12 months of cardiovascular risk management. Circulating NK, NKT-like and T lymphocyte subpopulations were phenotyped by multi-colour flow cytometry. Proximity extension assay was performed for 176 plasma proteins associated with inflammation and cardiovascular disease. At 12 months there were significant reductions in LDL (P = 0.001) and blood pressure (P = 0.028). NK cells responded with a reduction in pro-inflammatory (NKG2C + ) cells (P = 0.0003), an increase in anti-inflammatory (NKG2A + ) cells (P = 0.032), and a reduction in terminally differentiated (CD57 + ) NK cells. NKT-like cells showed a similar decrease in terminally differentiated subpopulations (P = 0.000002). Subpopulations of T helper cells exhibited a significant reduction in central memory (P = 1.09 × 10 -8 ) and a significant increase in CD4 + naïve- (P = 0.0008) and effector memory T cells (P = 0.006). The protein analysis indicated that cardiovascular risk management affects proteins involved in the inflammatory NF-κB pathway. The consistent decrease in senescent phenotypes of NK, NKT-like and CD4 + cells with a concomitant increase in more naïve, phenotypes suggests a change towards a less pro-inflammatory lymphocyte profile in response to cardiovascular risk management.Trial registry name: CARotid MRI of Atherosclerosis (CARMA). ClinicalTrials.gov identifier NCT04835571 (08/04/2021). https://www.clinicaltrials.gov/study/NCT04835571 ., (© 2023. The Author(s).)

Sensitive and reliable protein biomarkers are needed to predict disease trajectory and personalize treatment strategies for multiple sclerosis (MS). Here, we use the highly sensitive proximity-extension assay combined with next-generation sequencing (Olink Explore) to quantify 1463 proteins in cerebrospinal fluid (CSF) and plasma from 143 people with early-stage MS and 43 healthy controls. With longitudinally followed discovery and replication cohorts, we identify CSF proteins that consistently predicted both short- and long-term disease progression. Lower levels of neurofilament light chain (NfL) in CSF is superior in predicting the absence of disease activity two years after sampling (replication AUC = 0.77) compared to all other tested proteins. Importantly, we also identify a combination of 11 CSF proteins (CXCL13, LTA, FCN2, ICAM3, LY9, SLAMF7, TYMP, CHI3L1, FYB1, TNFRSF1B and NfL) that predict the severity of disability worsening according to the normalized age-related MS severity score (replication AUC = 0.90). The identification of these proteins may help elucidate pathogenetic processes and might aid decisions on treatment strategies for persons with MS., (© 2023. The Author(s).)

Preeclampsia (PE) is the leading cause of maternal and fetal mortality globally and may trigger dementia later in life in mothers and their offspring. However, the etiological drivers remain elusive. Cis P-tau is an early etiological driver and blood biomarker in pre-clinical Alzheimer's and after vascular or traumatic brain injury, which can be targeted by stereo-specific antibody, with clinical trials ongoing. Here we find significant cis P-tau in the placenta and serum of PE patients, and in primary human trophoblasts exposed to hypoxia or sera from PE patients due to Pin1 inactivation. Depletion of cis P-tau from PE patient sera by the antibody prevents their ability to disrupt trophoblast invasion and endovascular activity and to cause the PE-like pathological and clinical features in pregnant humanized tau mice. Our studies uncover that cis P-tau is a central circulating etiological driver and its stereo-specific antibody is valuable for early PE diagnosis and treatment., (© 2023. Springer Nature Limited.)

Background: Fatty degeneration of thymus (or thymus involution) has long been considered a normal ageing process. However, there is emerging evidence that thymic involution is linked to T cell aging, chronic inflammation and increased morbidity. Other factors, aside from chronological age, have been proposed to affect the involution rate. In the present study, we investigated the imaging characteristics of thymus on computed tomography (CT) in a Swedish middle-aged population. The major aims were to establish the prevalence of fatty degeneration of thymus and to determine its associations with demographic, lifestyle and clinical factors, as well as inflammation, T cell differentiation and thymic output., Results: In total, 1 048 randomly invited individuals (aged 50-64 years, 49% females) were included and thoroughly characterized. CT evaluation of thymus included measurements of attenuation, size and a 4-point scoring system, with scale 0-3 based on the ratio of fat and soft tissue. A majority, 615 (59%) showed complete fatty degeneration, 259 (25%) predominantly fatty attenuation, 105 (10%) half fatty and half soft-tissue attenuation, while 69 (6.6%) presented with a solid thymic gland with predominantly soft-tissue attenuation. Age, male sex, high BMI, abdominal obesity and low dietary intake of fiber were independently associated with complete fatty degeneration of thymus. Also, fatty degeneration of thymus as well as low CT attenuation values were independently related to lower proportion of naïve CD8 + T cells, which in turn was related to lower thymic output, assessed by T-cell receptor excision circle (TREC) levels., Conclusion: Among Swedish middle-aged subjects, nearly two-thirds showed complete fatty degeneration of thymus on CT. This was linked to depletion of naïve CD8 + T cells indicating that CT scans of thymus might be used to estimate immunological aging. Furthermore, our findings support the intriguing concept that obesity as well as low fiber intake contribute to immunological aging, thereby raising the possibility of preventive strategies., (© 2023. BioMed Central Ltd., part of Springer Nature.)

Regulatory B (Breg) cells can dampen inflammation, autoreactivity, and transplant rejection. We investigated the frequencies, phenotypes, and function of Breg cells in granulomatosis with polyangiitis (GPA) to gain further knowledge as to whether there are numerical alterations or limitations of their ability to regulate T-cell function. Frequencies and phenotypes of CD24hiCD27+ and CD24hiCD38hi B-cells in the blood were determined with flow cytometry in 37 GPA patients (22 in remission and 15 with active disease) and 31 healthy controls (HC). A co-culture model was used to study the capacity of Breg cells to regulate T-cell activation and proliferation in cells from 10 GPA patients in remission and 12 HC. T-cell cytokine production in vitro and levels in plasma were determined with enzyme-linked immunosorbent assay. Frequencies of CD24hiCD27+ B-cells were reduced both during active disease and remission compared with HC (P = 0.005 and P = 0.010, respectively), whereas CD24hiCD38hi B-cells did not differ. Patient CD24hiCD27+ B-cells exhibited decreased expression of CD25 but increased expression of PD-L1 and PD-L2 during remission. B-cells from GPA patients regulated T-cell proliferation but failed to regulate interferon (IFN)-γ production (median T-cells alone 222 ng/ml vs. T-cells + B-cells 207 ng/ml, P = 0.426). IFN-γ was also elevated in patient plasma samples (P = 0.016). In conclusion, GPA patients exhibit altered numbers and phenotypes of CD24hiCD27+ B-cells. This is accompanied by a disability to control T-cell production of Th1-type cytokines during remission, which might be of fundamental importance for the granulomatous inflammation that characterizes the chronic phase of this disease., (© The Author(s) 2023. Published by Oxford University Press on behalf of the British Society for Immunology.)

Lyme neuroborreliosis (LNB) is associated with increased levels of pro-inflammatory cytokines and chemokines in the cerebrospinal fluid (CSF). Residual symptoms after antibiotic treatment can have deleterious effects on patients and knowledge regarding the pathogenesis linked to prolonged recovery is lacking. In this prospective follow-up study, we investigated the B cell-associated and T helper (Th) cell-associated immune responses in well-characterized patients with LNB and controls. The aims were to assess the kinetics of selected cytokines and chemokines involved in the inflammatory response and to identify potential prognostic markers. We investigated 13 patients with LNB according to a standardized clinical protocol before antibiotic treatment and after 1, 6 and 12 months of follow-up. CSF and blood samples were obtained at baseline and after 1 month. As controls, we used CSF samples from 37 patients who received spinal anesthesia during orthopedic surgery. The CSF samples were analyzed for CXCL10 (Th1-related), CCL22 (Th2-related) and IL-17A, CXCL1 and CCL20 (Th17-related), as well as for the B cell-related cytokines of a proliferation-inducing ligand (APRIL), B cell-activating factor (BAFF) and CXCL13. The CSF levels of all the cytokines and chemokines, with the exception of APRIL, were significantly higher at baseline in patients with LNB compared with controls. All the cytokines and chemokines, except for IL-17A were significantly reduced at 1-month follow-up. Patients with quick recovery (< 1 month, n = 3) had significantly lower levels of CCL20 at baseline and lower levels of IL-17A at 1-month follow-up. Patients with time of recovery > 6 months (n = 7) had significantly higher levels of IL-17A at the one-month follow-up. No other cytokines or chemokines were associated with prolonged recovery. Dominating residual symptoms were fatigue, myalgia, radiculitis and/or arthralgia. In this prospective follow-up study of patients with LNB, we found significantly lower levels of CCL20 in those who recovered rapidly, and increased levels of IL-17A in patients with delayed recovery post-treatment. Our findings indicate persistent Th17-driven inflammation in the CSF, possibly contributing to a longer convalescence, and suggest IL-17A and CCL20 as potential biomarker candidates for patients with LNB., (© 2023. The Author(s).)

Background: Multiple sclerosis (MS) is a neuroinflammatory disease in which pregnancy leads to a temporary amelioration in disease activity as indicated by the profound decrease in relapses rate during the 3rd trimester of pregnancy. CD4 + and CD8 + T cells are implicated in MS pathogenesis as being key regulators of inflammation and brain lesion formation. Although Tcells are prime candidates for the pregnancy-associated improvement of MS, the precise mechanisms are yet unclear, and in particular, a deep characterization of the epigenetic and transcriptomic events that occur in peripheral T cells during pregnancy in MS is lacking., Methods: Women with MS and healthy controls were longitudinally sampled before, during (1st, 2nd and 3rd trimesters) and after pregnancy. DNA methylation array and RNA sequencing were performed on paired CD4 +  and CD8 +  T cells samples. Differential analysis and network-based approaches were used to analyze the global dynamics of epigenetic and transcriptomic changes., Results: Both DNA methylation and RNA sequencing revealed a prominent regulation, mostly peaking in the 3rd trimester and reversing post-partum, thus mirroring the clinical course with improvement followed by a worsening in disease activity. This rebound pattern was found to represent a general adaptation of the maternal immune system, with only minor differences between MS and controls. By using a network-based approach, we highlighted several genes at the core of this pregnancy-induced regulation, which were found to be enriched for genes and pathways previously reported to be involved in MS. Moreover, these pathways were enriched for in vitro stimulated genes and pregnancy hormones targets., Conclusion: This study represents, to our knowledge, the first in-depth investigation of the methylation and expression changes in peripheral CD4 +  and CD8 +  T cells during pregnancy in MS. Our findings indicate that pregnancy induces profound changes in peripheral T cells, in both MS and healthy controls, which are associated with the modulation of inflammation and MS activity., (© 2023. The Author(s).)

During pregnancy, maternal blood circulates through the intervillous space of the placenta and the reciprocal interactions between foetal tissues and maternal immune cells makes the intervillous space a unique immunological niche. Labour is characterised by a proinflammatory response in the myometrium, but the relationship between local and systemic changes during the onset of labour remains elusive. We here aimed to investigate how the systemic and intervillous circulatory systems are affected during labour from an immunological point of view. We report that the proportion of monocytes is dramatically higher in peripheral (PB), intervillous blood (IVB) and decidua in labouring (n = 14) compared to non-labouring women (n = 15), suggesting that labour leads to both a systemic and local mobilisation of monocytes. Labour was associated with a relative increase of effector memory T cells in the intervillous space compared to the periphery, and MAIT cells and T cells showed an elevated expression of activation markers both in PB and IVB. Intervillous monocytes consisted to a higher degree of CD14 + CD16 + intermediate monocytes compared to peripheral monocytes, independently of mode of delivery, and displayed an altered phenotypic expression pattern. A proximity extension assay analysis of 168 proteins revealed that several proteins associated to myeloid cell migration and function, including CCL2 and M-CSF, were upregulated in IVB plasma in labouring women. Thus, the intervillous space could be a bridging site for the communication between the placenta and the periphery, which contribute to monocyte mobilisation and generation of inflammatory reactions during spontaneous labour., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Vikberg, Lindau, Solders, Raffetseder, Budhwar, Ernerudh, Tiblad and Kaipe.)

During human pregnancy, placenta-derived extravillous trophoblasts (EVTs) invade the decidua and communicate with maternal immune cells. The decidua distinguishes into basalis (decB) and parietalis (decP). The latter remains unaffected by EVT invasion. By defining a specific gating strategy, we report the accumulation of macrophages in decB. We describe a decidua basalis-associated macrophage (decBAM) population with a differential transcriptome and secretome compared with decidua parietalis-associated macrophages (decPAMs). decBAMs are CD11c hi and efficient inducers of Tregs, proliferate in situ, and secrete high levels of CXCL1, CXCL5, M-CSF, and IL-10. In contrast, decPAMs exert a dendritic cell-like, motile phenotype characterized by induced expression of HLA class II molecules, enhanced phagocytosis, and the ability to activate T cells. Strikingly, EVT-conditioned media convert decPAMs into a decBAM phenotype. These findings assign distinct macrophage phenotypes to decidual areas depending on placentation and further highlight a critical role for EVTs in the induction of decB-associated macrophage polarization., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Epigenetics may play a central, yet unexplored, role in the profound changes that the maternal immune system undergoes during pregnancy and could be involved in the pregnancy-induced modulation of several autoimmune diseases. We investigated changes in the methylome in isolated circulating CD4 +  T-cells in non-pregnant and pregnant women, during the 1 st  and 2 nd  trimester, using the Illumina Infinium Human Methylation 450K array, and explored how these changes were related to autoimmune diseases that are known to be affected during pregnancy. Pregnancy was associated with several hundreds of methylation differences, particularly during the 2 nd  trimester. A network-based modular approach identified several genes,  e.g., CD28, FYN, VAV1  and pathways related to T-cell signalling and activation, highlighting T-cell regulation as a central component of the observed methylation alterations. The identified pregnancy module was significantly enriched for disease-associated methylation changes related to multiple sclerosis, rheumatoid arthritis and systemic lupus erythematosus. A negative correlation between pregnancy-associated methylation changes and disease-associated changes was found for multiple sclerosis and rheumatoid arthritis, diseases that are known to improve during pregnancy whereas a positive correlation was found for systemic lupus erythematosus, a disease that instead worsens during pregnancy. Thus, the directionality of the observed changes is in line with the previously observed effect of pregnancy on disease activity. Our systems medicine approach supports the importance of the methylome in immune regulation of T-cells during pregnancy. Our findings highlight the relevance of using pregnancy as a model for understanding and identifying disease-related mechanisms involved in the modulation of autoimmune diseases. Abbreviations : BMIQ: beta-mixture quantile dilation; DMGs: differentially methylated genes; DMPs: differentially methylated probes; FE: fold enrichment; FDR: false discovery rate; GO: gene ontology; GWAS: genome-wide association studies; MDS: multidimensional scaling; MS: multiple sclerosis; PBMC: peripheral blood mononuclear cells; PBS: phosphate buffered saline; PPI; protein-protein interaction; RA: rheumatoid arthritis; SD: standard deviation; SLE: systemic lupus erythematosus; SNP: single nucleotide polymorphism; T H : CD4 + T helper cell; VIStA: diVIsive Shuffling Approach.

Profiling of mRNA expression is an important method to identify biomarkers but complicated by limited correlations between mRNA expression and protein abundance. We hypothesised that these correlations could be improved by mathematical models based on measuring splice variants and time delay in protein translation. We characterised time-series of primary human naïve CD4 + T cells during early T helper type 1 differentiation with RNA-sequencing and mass-spectrometry proteomics. We performed computational time-series analysis in this system and in two other key human and murine immune cell types. Linear mathematical mixed time delayed splice variant models were used to predict protein abundances, and the models were validated using out-of-sample predictions. Lastly, we re-analysed RNA-seq datasets to evaluate biomarker discovery in five T-cell associated diseases, further validating the findings for multiple sclerosis (MS) and asthma. The new models significantly out-performing models not including the usage of multiple splice variants and time delays, as shown in cross-validation tests. Our mathematical models provided more differentially expressed proteins between patients and controls in all five diseases. Moreover, analysis of these proteins in asthma and MS supported their relevance. One marker, sCD27, was validated in MS using two independent cohorts for evaluating response to treatment and disease prognosis. In summary, our splice variant and time delay models substantially improved the prediction of protein abundance from mRNA expression in three different immune cell types. The models provided valuable biomarker candidates, which were further validated in MS and asthma., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Magnusson, Rundquist, Kim, Hellberg, Na, Benson, Gomez-Cabrero, Kockum, Tegnér, Piehl, Jagodic, Mellergård, Altafini, Ernerudh, Jenmalm, Nestor, Kim and Gustafsson.)

Multiple sclerosis (MS) is a chronic autoimmune neuroinflammatory and neurodegenerative disorder of the central nervous system. Pregnancy represents a natural modulation of the disease course, where the relapse rate decreases, especially in the 3 rd trimester, followed by a transient exacerbation after delivery. Although the exact mechanisms behind the pregnancy-induced modulation are yet to be deciphered, it is likely that the immune tolerance established during pregnancy is involved. In this study, we used the highly sensitive and specific proximity extension assay technology to perform protein profiling analysis of 92 inflammation-related proteins in MS patients (n=15) and healthy controls (n=10), longitudinally sampled before, during, and after pregnancy. Differential expression analysis was performed using linear models and p-values were adjusted for false discovery rate due to multiple comparisons. Our findings reveal gradual dynamic changes in plasma proteins that are most prominent during the 3 rd trimester while reverting post-partum. Thus, this pattern reflects the disease activity of MS during pregnancy. Among the differentially expressed proteins in pregnancy, several proteins with known immunoregulatory properties were upregulated, such as PD-L1, LIF-R, TGF-β1, and CCL28. On the other hand, inflammatory chemokines such as CCL8, CCL13, and CXCL5, as well as members of the tumor necrosis factor family, TRANCE and TWEAK, were downregulated. Further in-depth studies will reveal if these proteins can serve as biomarkers in MS and whether they are mechanistically involved in the disease amelioration and worsening. A deeper understanding of the mechanisms involved may identify new treatment strategies mimicking the pregnancy milieu., Competing Interests: IT has served at advisory board for Pfizer Inc. JM has received honoraria for Advisory boards for Sanofi Genzyme and Merck and lecture honorarium from Merck. TO has received grant support from the Swedish Research Council, the Swedish Brain foundation, The Knut and Wallenberg foundation and Margaretha af Ugglas foundation and has received compensation for lectures/advisory boards, and unrestricted MS research grants from Biogen, Novartis, Sanofi and Merck. JE has received compensation for lectures from AbbVie, Biogen and Merck. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The reviewer SS declared a past co-authorship with one of the authors JE to the handling editor., (Copyright © 2022 Papapavlou Lingehed, Hellberg, Huang, Khademi, Kockum, Carlsson, Tjernberg, Svenvik, Lind, Blomberg, Vrethem, Mellergård, Gustafsson, Jenmalm, Olsson and Ernerudh.)

Objectives: The purpose of this study was to evaluate levels and kinetics of cerebrospinal fluid (CSF) markers of inflammation and brain injury in patients with Lyme neuroborreliosis (LNB)., Methods: Adult patients with clinically suspected LNB were enrolled, in a prospective clinical study in the South East of Sweden. Patients were classified according to the European Federation of Neurological Societies' guidelines. Definite cases of LNB were re-examined one month later including a repeat CSF investigation. Routine laboratory parameters were investigated along with CSF levels of neurodegenerative markers glial fibrillary acidic protein (GFAp), total tau (t-tau) and neurofilament light protein (NFL), as well as neuroinflammatory markers soluble triggering receptor expressed on myeloid cells 2 (sTREM2), YKL-40 and CXCL13. Non-LNB served as controls. An additional comparison group consisted of spinal anesthesia subjects (SAS) without known central nervous system conditions., Results: CSF levels of sTREM2 and CXCL13 were elevated in definite LNB patients at diagnosis compared with non-LNB patients (p<0.001) and SAS (p≤0.01). In addition, CSF levels of sTREM2, YKL-40 and CXCL13 rapidly declined in at follow-up after antibiotic treatment. In contrast, CSF levels of GFAp and t-tau did not differ across LNB groups, and did not change after treatment., Conclusions: Although in a limited number of LNB patients, the results indicate a predominance of microglial and neuroinflammatory involvement rather than parenchymal CNS injury in CSF at diagnosis of LNB with a prompt decline after antibiotic treatment. The findings provide pathogenetic insights and may be of value in differential diagnosis of CSF findings., (© 2022 Walter de Gruyter GmbH, Berlin/Boston.)

Estradiol (E2) and progesterone (P4) are steroid hormones important for the regulation of immune responses during pregnancy. Their increasing levels coincide with an improvement of T cell-mediated diseases such as multiple sclerosis (MS). Although immune-endocrine interactions are involved in this phenomenon, the relative contribution of hormones is not known. We here report a direct comparison of E2- and P4-mediated effects on human CD4 + T cells, key cells in immune regulation. T cells were stimulated to obtain different activation levels and exposed to a broad range of hormone concentrations. Activation level was assessed by CD69/CD25 expression by flow cytometry, and secreted proteins (n = 196) were measured in culture supernatants using proximity extension assay and electrochemiluminescence immunoassay. We found that in low activated cells, pregnancy-relevant E2 concentrations increased activation and the secretion of several immune- and inflammation-related proteins. P4, on the other hand, showed a biphasic pattern, where serum-related concentrations upregulated activation and protein secretion while placenta-relevant concentrations induced a prominent dampening irrespective of the initial activation level. Our results demonstrate the importance of P4 as a major hormone in the immune modulation of T cells during pregnancy and emphasize the need to further evaluate its potency in the treatment of diseases like MS., (© 2021 The Authors. European Journal of Immunology published by Wiley-VCH GmbH.)

Mucosal-associated invariant T (MAIT) cells are an innate-like T cell subset with proinflammatory and cytotoxic effector functions. During pregnancy, modulation of the maternal immune system, both at the fetal-maternal interface and systemically, is crucial for a successful outcome and manifests through controlled enhancement of innate and dampening of adaptive responses. Still, immune defenses need to efficiently protect both the mother and the fetus from infection. So far, it is unknown whether MAIT cells are subjected to immunomodulation during pregnancy, and characterization of decidual MAIT cells as well as their functional responses during pregnancy are mainly lacking. We here characterized the presence and phenotype of Vα7.2 + CD161 + MAIT cells in blood and decidua (the uterine endometrium during pregnancy) from women pregnant in the 1 st trimester, i.e. , the time point when local immune tolerance develops. We also assessed the phenotype and functional responses of MAIT cells in blood of women pregnant in the 3 rd trimester, i.e. , when systemic immunomodulation is most pronounced. Multi-color flow cytometry panels included markers for MAIT subsets, and markers of activation (CD69, HLA-DR, Granzyme B) and immunoregulation (PD-1, CTLA-4). MAIT cells were numerically decreased at the fetal-maternal interface and showed, similar to other T cells in the decidua, increased expression of immune checkpoint markers compared with MAIT cells in blood. During the 3 rd trimester, circulating MAIT cells showed a higher expression of CD69 and CD56, and their functional responses to inflammatory (activating anti-CD3/CD28 antibodies, and IL-12 and IL-18) and microbial stimuli ( Escherichia coli , group B streptococci and influenza A virus) were generally increased compared with MAIT cells from non-pregnant women, indicating enhanced antimicrobial defenses during pregnancy. Taken together, our findings indicate dual roles for MAIT cells during pregnancy, with an evidently well-adapted ability to balance the requirements of immune tolerance in parallel with maintained antimicrobial defenses. Since MAIT cells are easily activated, they need to be strictly regulated during pregnancy, and failure to do so could contribute to pregnancy complications., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Raffetseder, Lindau, van der Veen, Berg, Larsson and Ernerudh.)

A non-invasive and sensitive blood test has long been a goal for early stage disease diagnosis and treatment for Alzheimer's disease (AD) and other proteinopathy diseases. We previously reported that preeclampsia (PE), a severe pregnancy complication, is another proteinopathy disorder with impaired autophagy. We hypothesized that induced autophagy deficiency would promote accumulation of pathologic protein aggregates. Here, we describe a novel, sensitive assay that detects serum protein aggregates from patients with PE (n = 33 early onset and 33 late onset) and gestational age-matched controls (n = 77) as well as AD in both dementia and prodromal mild cognitive impairment (MCI, n = 24) stages with age-matched controls (n = 19). The assay employs exposure of genetically engineered, autophagy-deficient human trophoblasts (ADTs) to serum from patients. The aggregated protein complexes and their individual components, including transthyretin, amyloid β-42, α-synuclein, and phosphorylated tau231, can be detected and quantified by co-staining with ProteoStat, a rotor dye with affinity to aggregated proteins, and respective antibodies. Detection of protein aggregates in ADTs was not dependent on transcriptional upregulation of these biomarkers. The ROC curve analysis validated the robustness of the assay for its specificity and sensitivity (PE; AUC: 1, CI: 0.949-1.00; AD; AUC: 0.986, CI: 0.832-1.00). In conclusion, we have developed a novel, noninvasive diagnostic and predictive assay for AD, MCI and PE., (© 2021. The Author(s).)

Background: Environmental exposures may alter DNA methylation patterns of T helper cells. As T helper cells are instrumental for allergy development, changes in methylation patterns may constitute a mechanism of action for allergy preventive interventions. While epigenetic effects of separate perinatal probiotic or ω-3 fatty acid supplementation have been studied previously, the combined treatment has not been assessed. We aimed to investigate epigenome-wide DNA methylation patterns from a sub-group of children in an on-going randomised double-blind placebo-controlled allergy prevention trial using pre- and postnatal combined Lactobacillus reuteri and ω-3 fatty acid treatment. To this end, > 866000 CpG sites (MethylationEPIC 850K array) in cord blood CD4+ T cells were examined in samples from all four study arms (double-treatment: n = 18, single treatments: probiotics n = 16, ω-3 n = 15, and double placebo: n = 14). Statistical and bioinformatic analyses identified treatment-associated differentially methylated CpGs and genes, which were used to identify putatively treatment-induced network modules. Pathway analyses inferred biological relevance, and comparisons were made to an independent allergy data set., Results: Comparing the active treatments to the double placebo group, most differentially methylated CpGs and genes were hypermethylated, possibly suggesting induction of transcriptional inhibition. The double-treated group showed the largest number of differentially methylated CpGs, of which many were unique, suggesting synergy between interventions. Clusters within the double-treated network module consisted of immune-related pathways, including T cell receptor signalling, and antigen processing and presentation, with similar pathways revealed for the single-treatment modules. CpGs derived from differential methylation and network module analyses were enriched in an independent allergy data set, particularly in the double-treatment group, proposing treatment-induced DNA methylation changes as relevant for allergy development., Conclusion: Prenatal L. reuteri and/or ω-3 fatty acid treatment results in hypermethylation and affects immune- and allergy-related pathways in neonatal T helper cells, with potentially synergistic effects between the interventions and relevance for allergic disease. Further studies need to address these findings on a transcriptional level, and whether the results associate to allergy development in the children. Understanding the role of DNA methylation in regulating effects of perinatal probiotic and ω-3 interventions may provide essential knowledge in the development of efficacious allergy preventive strategies. Trial registration ClinicalTrials.gov, ClinicalTrials.gov-ID: NCT01542970. Registered 27th of February 2012-Retrospectively registered, https://clinicaltrials.gov/ct2/show/NCT01542970 .

The changes in progesterone (P4) levels during and after pregnancy coincide with the temporary improvement and worsening of several autoimmune diseases like multiple sclerosis (MS) and rheumatoid arthritis (RA). Most likely immune-endocrine interactions play a major role in these pregnancy-induced effects. In this study, we used next generation sequencing to investigate the direct effects of P4 on CD4 + T cell activation, key event in pregnancy and disease. We report profound dampening effects of P4 on T cell activation, altering the gene and protein expression profile and reversing many of the changes induced during the activation. The transcriptomic changes induced by P4 were significantly enriched for genes associated with diseases known to be modulated during pregnancy such as MS, RA and psoriasis. STAT1 and STAT3 were significantly downregulated by P4 and their downstream targets were significantly enriched among the disease-associated genes. Several of these genes included well-known and disease-relevant cytokines, such as IL-12β, CXCL10 and OSM, which were further validated also at the protein level using proximity extension assay. Our results extend the previous knowledge of P4 as an immune regulatory hormone and support its importance during pregnancy for regulating potentially detrimental immune responses towards the semi-allogenic fetus. Further, our results also point toward a potential role for P4 in the pregnancy-induced disease immunomodulation and highlight the need for further studies evaluating P4 as a future treatment option., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Hellberg, Raffetseder, Rundquist, Magnusson, Papapavlou, Jenmalm, Ernerudh and Gustafsson.)

Problem: While there are several known causes for recurrent pregnancy loss (RPL), about 50% are unexplained (uRPL), and in these cases, an aberrant immune regulation seems to be involved. Although fetally derived trophoblast cells have a key role in immune regulation, it is difficult to study their immune function during pregnancy, and it is not known whether trophoblast function may be an inherent aberration in uRPL or whether it is associated with the outcome of the current pregnancy., Method of Study: Chorionic villus sampling (CVS) was performed for clinical indications at 12 weeks of gestation. Superfluous materials, divided in small explants, were cultured for 20-24 hours, and supernatants (conditioned medium) were collected from 36 women with singleton normal pregnancies, of whom 9 women had a history of RPL. The secreted immune protein profile was measured by proximity extension assay, and the conditioned medium was further used in functional ex vivo models to assess ability to polarize blood monocytes and CD4 + T cells into immune regulatory phenotypes, as detected by flow cytometry., Results: Conditioned medium from chorionic villi, human fetally derived placental tissue, was able to induce a decidual-type of M2-like macrophages, as well as an expansion of Treg cells ex vivo, both in women with uRPL and in control women. The preserved immunological properties were confirmed by a maintained immune protein profile in RPL compared with controls., Conclusion: Trophoblasts in an ex vivo model maintain tolerogenic and proteomic profile features in successful pregnancies, despite a previous history of RPL., (© 2020 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Polymyositis and inclusion body myositis are idiopathic inflammatory myopathies, with a pathology characterized by partial invasion of non-necrotic muscle fibres by CD8+ cytotoxic T-cells, leading to fibre degeneration. Although the main effector pathway of CD8+ T-cells is to induce apoptosis of target cells, it has remained unclear if apoptosis occurs in these diseases, and if so, if it is mediated by CD8+ T-cells. In consecutive biopsy sections from 10 patients with partial invasion, muscle fibres and inflammatory cells were assessed by immunohistochemistry and apoptotic nuclei by the TUNEL assay. Analysis of muscle fibre morphology, staining pattern and quantification were performed on digital images, and they were compared with biopsies from 10 dermatomyositis patients and 10 controls without muscle disease. Apoptotic myonuclei were found in muscle with partial invasion, but not in the invaded fibres. Fibres with TUNEL positive nuclei were surrounded by CD8+ T-cells, granzyme B+ cells and macrophages, but lacked FAS receptor expression. In contrast, apoptotic myonuclei were rare in dermatomyositis and absent in controls. The findings confirm that apoptosis occurs in idiopathic inflammatory myopathies and support that it is mediated by CD8+ cytotoxic T- cells, acting in parallel to the process of partial invasion., Competing Interests: The authors have declared that no competing interests exist.

One of the main functions of the human placenta is to provide a barrier between the fetal and maternal blood circulations, where gas exchange and transfer of nutrients to the developing fetus take place. Despite being a barrier, there is a multitude of crosstalk between maternal immune cells and fetally derived semi-allogeneic trophoblast cells. Therefore, the maternal immune system has a difficult task to both tolerate the fetus but at the same time also defend the mother and the fetus from infections. Mucosal-associated invariant T (MAIT) cells are an increasingly recognized subset of T cells with anti-microbial functions that get activated in the context of non-polymorphic MR1 molecules, but also in response to inflammation. MAIT cells accumulate at term pregnancy in the maternal blood that flows into the intervillous space inside the placenta. Chemotactic factors produced by the placenta may be involved in recruiting and retaining particular immune cell subsets, including MAIT cells. In this Mini-Review, we describe what is known about MAIT cells during pregnancy and discuss the potential biological functions of MAIT cells at the fetal-maternal interface. Since MAIT cells have anti-microbial and tissue-repairing functions, but lack alloantigen reactivity, they could play an important role in protecting the fetus from bacterial infections and maintaining tissue homeostasis without risks of mediating harmful responses toward semi-allogenic fetal tissues., (Copyright © 2020 Kaipe, Raffetseder, Ernerudh, Solders and Tiblad.)

Background: Common variable immunodeficiency (CVID) is a disorder characterized by antibody deficiency. A significant fraction of the patients suffer from immune dysregulation, which leads to increased morbidity and mortality. The pathogenesis of this condition is poorly understood., Objective: Our aim was to find out whether the plasma protein signature in CVID is associated with clinical characteristics and lymphocyte aberrations., Methods: A highly sensitive proximity extension assay was used for targeted profiling of 145 plasma proteins in 29 patients with CVID. Phenotyping of peripheral lymphocytes was done by flow cytometry. The findings were correlated with the burden of immune dysregulation., Results: Unsupervised clustering of plasma protein profiles identified 2 distinct groups of patients with CVID that differed significantly in terms of the degree of complications due to immune dysregulation and in terms of the frequency of activated B- and T-cell subpopulations. Pathway analysis identified IFN-γ and IL-1β as the top enriched upstream regulators associated with higher grade of immune dysregulation. In addition, CVID was found to be associated with increased plasma levels of the B-cell-attracting chemokine CXCL13., Conclusion: Clustering based on plasma protein profiles delineated a subgroup of patients with CVID with activated T cells and clinical complications due to immune dysregulation. Thus, data indicate that CVID-associated immune dysregulation is a T H 1-mediated inflammatory process driven by the IFN-γ pathway., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: Perinatal childhood exposures, including probiotic supplementation, may affect epigenetic modifications and impact on immune maturation and allergy development. The aim of this study was to assess the effects of pre- and postnatal Lactobacillus reuteri supplementation on DNA methylation in relation to immune maturation and allergy development., Methods: DNA methylation patterns were investigated for allergy-related T helper subsets using a locus-specific method and at a genome-wide scale using the Illumina 450K array. From a randomised, double-blind, placebo-controlled allergy prevention trial with pre- and postnatal probiotic supplementation, CD4+ T helper cells were obtained at birth (from cord blood), and 12 and 24 months of age (total (placebo/probiotics); locus-specific method: CB = 32 (17/15), 12 months = 24 (9/15), 24 months = 35 (15/20); Illumina: CB = 19 (10/9), 12 months = 10 (6/4), 24 months = 19(11/8))., Results: Comparing probiotics to placebo, the greatest genome-wide differential DNA methylation was observed at birth, where the majority of sites were hypomethylated, indicating transcriptional accessibility in the probiotic group. Bioinformatic analyses, including network analyses, revealed a module containing 91 genes, enriched for immune-related pathways such as chemotaxis, PI3K-Akt, MAPK and TGF-β signalling. A majority of the module genes were associated with atopic manifestations (OR = 1.43, P = 2.4 × 10 -6 ), and a classifier built on this model could predict allergy development (AUC = 0.78, P = 3.0 × 10 e-3 ). Pathways such as IFN-γ signalling and T-cell activation were more hypermethylated at birth compared with later in life in both intervention groups over time, in line with DNA methylation patterns in the IFNG locus obtained by the locus-specific methodology., Conclusion: Maternal L. reuteri supplementation during pregnancy alters DNA methylation patterns in CD4+ T cells towards enhanced immune activation at birth, which may affect immune maturation and allergy development., (© 2020 The Authors. Pediatric Allergy and Immunology published by John Wiley & Sons Ltd.)

The aim of this study was to analyse the dynamics of tissue damage and inflammatory response markers perioperatively and whether these differ between women operated with robotic and abdominal hysterectomy in treating early-stage endometrial cancer. At a Swedish university hospital fifty women with early-stage low-risk endometrial cancer were allocated to robotic or abdominal hysterectomy in a randomiszed controlled trial. Blood samples reflecting inflammatory responses (high sensitivity CRP, white blood cells (WBC), thrombocytes, IL-6, cortisol) and tissue damage (creatine kinase (CK), high-mobility group box 1 protein (HMGB1)) were collected one week preoperatively, just before surgery, postoperatively at two, 24 and 48 hours, and one and six weeks postoperatively. High sensitivity CRP (p = 0.03), WBC (p < 0.01), IL-6 (p = 0.03) and CK (p = 0.03) were significantly lower in the robotic group, but fast transitory. Cortisol returned to baseline two hours after robotic hysterectomy but remained elevated in the abdominal group comparable to the preoperative high levels for both groups just before surgery (p < 0.0001). Thrombocytes and HMGB1 were not affected by the mode of surgery. Postoperative inflammatory response and tissue damage were lower after robotic hysterectomy compared to abdominal hysterectomy. A significant remaining cortisol elevation two hours after surgery may reflect a higher stress response in the abdominal group.

An amendment to this paper has been published and can be accessed via the original article.

Prolactin is known to have immune modulatory effects acting through the prolactin receptor, which is present on a variety of immune cells. Certain chemokines contribute to form the type of T helper (Th) preponderance in the immune response. The objective of this work was to assess if hyperprolactinemia not related to pregnancy is associated with changes in circulating levels of chemokines and other immunological markers. In this cross sectional study, 35 patients with hyperprolactinemia (5 men), and 102 healthy blood donors (19 men) were included. Serum levels of Th1- Th2- and Th17-associated chemokines, C-reactive protein, immunoglobulins, and the B cell attracting chemokine CXCL13 were assessed. The hyperprolactinemic group had significantly higher levels of Th2 associated CCL22 (p=0.022), Th17 associated CXCL1 (p=0.001), B cell attracting CXCL13 (p=0.003), and C-reactive protein (p<0.001) compared to controls, and these proteins were also positively correlated with prolactin levels. While differences in CCL22, CXCL1, CXCL13, and C-reactive protein were present in patients with low or moderate hyperprolactinemia, no differences were observed at high (>3600 mU/l) prolactin levels. To evaluate a possible dose-associated response to prolactin, an in vitro model was used, showing prolactin-induced increase in T-helper cell activation at moderate levels, while activation decreased at higher levels. Hyperprolactinemia seems to have several immunomodulatory effects and was associated with increased levels of chemokines associated with Th2 and Th17 responses and B cell attraction. However, patients with greatly increased prolactin had normal levels of chemokines, and in vitro, high levels of prolactin decreased T-helper cell activation., Competing Interests: The authors declare that they have no conflict of interest., (© Georg Thieme Verlag KG Stuttgart · New York.)

To assess if markers of complement activation are associated with disease activity, C1q, C3, C3a and sC5b-9 levels in plasma and cerebrospinal fluid (CSF) were determined in 41 patients with clinically isolated syndrome (CIS) or remitting multiple sclerosis (RRMS), in a prospective longitudinal four-year cohort study. C1q in CSF (CSF-C1q) was significantly higher in patients than in controls. Baseline CSF-C1q and CSF-C3a correlated with several neuroinflammatory markers and neurofilament light chain levels. Baseline CSF-C3a correlated with the number of T2 lesions at baseline and new T2 lesions during follow-up. Baseline CSF-C3a was also significantly higher in patients with (n = 21) than in patients without (n = 20) signs of disease activity according to the NEDA-3 concept during one year of follow-up (p ≤ .01) Study results support that complement activation is involved in MS pathophysiology and that CSF-C3a carries prognostic information., Competing Interests: Declaration of Competing interest IH and KNE declare that they have no competing interests. JE has received honoraria for lectures from Biogen. MV has received unrestricted research grants from Biogen and Novartis, honoraria for lectures from Biogen and Genzyme and for advisory boards from Roche and Novartis. CD has received honoraria for lectures from Biogen, Teva, Genzyme and Novartis and for advisory boards from Roche, Novartis and Biogen., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Background: Fatigue is common in multiple sclerosis and is associated with reduced quality of life. This study aimed to assess the correlation between fatigue scores and data from other self-assessment questionnaires, neuropsychological tests and neuroimaging, as well as data on neuroimmunological markers in cerebrospinal fluid (CSF) and serum/plasma, in clinically isolated syndrome (CIS) and relapsing remitting MS (RRMS)., Methods: Modified fatigue impact scale (MFIS) scores were determined in 38 patients with newly diagnosed CIS or RRMS at baseline and after one year in a prospective longitudinal cohort study. Non-parametric correlation analyses were used to assess associations between MFIS scores and other self-assessment questionnaire data (Hospital Anxiety and Depression scale (HAD), Multiple Sclerosis Impact Scale 29 (MSIS-29) and Short Form 36 (SF-36)), as well as with neuropsychological test performances (e.g. Auditory Consonant Trigram Test (ACTT)), clinical parameters (e.g. disease duration and expanded disability status scale (EDSS)), magnetic resonance imaging (MRI) data (number of T2 lesions in brain MRI and total brain volume) and several neurodegenerative/neuroinflammatory markers in CSF and serum/plasma (IL-1β, IL-6, CXCL1, CXCL10, CXCL13, CCL-22 in plasma; neurofilament light chain (NFL) in serum; IL-6, CXCL1, CXCL10, CXCL13, CCL22, NFL and chitinase-3-like-1 (CHI3L1) in CSF. CSF and serum/plasma from 21 age- and sex-matched healthy controls were available for comparison., Results: At both baseline and one-year follow-up, fatigue scores correlated significantly with HAD, MSIS-29 and SF-36 scores and ACTT performance (Spearman´s rho 0.45-0.78, all p ≤ 0.01) but not with the other neuropsychological test results, disease duration, EDSS ratings, number of T2 lesions, total brain volume or neurodegenerative/neuroinflammatory markers, including neurofilament light chain levels in CSF and serum. In group comparisons, MFIS scores were similar in patients fulfilling no evidence of disease activity-3 (NEDA-3) (n = 18) and patients not fulfilling NEDA-3 (n = 20) during one year of follow-up (p > 0.01)., Conclusions: In this cohort of patients with newly diagnosed CIS and RRMS, fatigue scores were associated with mood, disease impact on daily life and quality of life as well as with alterations of attentive functions. Study results indicate that subjective fatigue scores are not well reflected by some commonly used and objectively measurable disease parameters like EDSS, T2 lesions and NFL levels., (Copyright © 2019 Elsevier B.V. All rights reserved.)

There are only few studies with specific focus on predictors of survival in nursing home residents (NHRs). The aim was to study whether 1-year changes in complete blood count (including hemoglobin, red blood cells, erythrocyte volume fraction, mean corpuscular volume, mean corpuscular hemoglobin concentration, white blood cells count and platelet count), C-reactive protein and interleukin-1β (IL-1β), IL-1Ra, IL-6, IL-8 and IL-10, are associated with 8-year survival in elderly NHRs, aged ≥ 80 years. Complete blood count, C-reactive protein and interleukins were measured at baseline, after 6 and 12 months from 167 NHRs aged 80-101 years, mean age 88 ± 4.5 years, 75% of whom were women. Dates of death were collected from the National Death Register 8 years after baseline. Levels of hemoglobin, red blood cells and mean corpuscular hemoglobin concentration were lower after 1-year, but higher for mean corpuscular volume and IL-1β, compared to baseline or 6 month follow-up. In the Cox regression model with a time-dependent covariate, raised levels of C-reactive protein and IL-8 were associated with reduced survival time. Elevated levels of C-reactive protein and IL-8 during 1-year follow-up were related to reduce lengths of survival in elderly NHRs.

Background: Genomic medicine has paved the way for identifying biomarkers and therapeutically actionable targets for complex diseases, but is complicated by the involvement of thousands of variably expressed genes across multiple cell types. Single-cell RNA-sequencing study (scRNA-seq) allows the characterization of such complex changes in whole organs., Methods: The study is based on applying network tools to organize and analyze scRNA-seq data from a mouse model of arthritis and human rheumatoid arthritis, in order to find diagnostic biomarkers and therapeutic targets. Diagnostic validation studies were performed using expression profiling data and potential protein biomarkers from prospective clinical studies of 13 diseases. A candidate drug was examined by a treatment study of a mouse model of arthritis, using phenotypic, immunohistochemical, and cellular analyses as read-outs., Results: We performed the first systematic analysis of pathways, potential biomarkers, and drug targets in scRNA-seq data from a complex disease, starting with inflamed joints and lymph nodes from a mouse model of arthritis. We found the involvement of hundreds of pathways, biomarkers, and drug targets that differed greatly between cell types. Analyses of scRNA-seq and GWAS data from human rheumatoid arthritis (RA) supported a similar dispersion of pathogenic mechanisms in different cell types. Thus, systems-level approaches to prioritize biomarkers and drugs are needed. Here, we present a prioritization strategy that is based on constructing network models of disease-associated cell types and interactions using scRNA-seq data from our mouse model of arthritis, as well as human RA, which we term multicellular disease models (MCDMs). We find that the network centrality of MCDM cell types correlates with the enrichment of genes harboring genetic variants associated with RA and thus could potentially be used to prioritize cell types and genes for diagnostics and therapeutics. We validated this hypothesis in a large-scale study of patients with 13 different autoimmune, allergic, infectious, malignant, endocrine, metabolic, and cardiovascular diseases, as well as a therapeutic study of the mouse arthritis model., Conclusions: Overall, our results support that our strategy has the potential to help prioritize diagnostic and therapeutic targets in human disease.

Background: The objectives of this study were to 1) evaluate the influence of treatment with praziquantel on the inflammatory milieu in maternal, placental, and cord blood, 2) assess the extent to which proinflammatory signatures in placental and cord blood impacts birth outcomes, and 3) evaluate the impact of other helminths on the inflammatory micro environment., Methods/findings: This was a secondary analysis of samples from 369 mother-infant pairs participating in a randomized controlled trial of praziquantel given at 12-16 weeks' gestation. We performed regression analysis to address our study objectives. In maternal peripheral blood, the concentrations of CXCL8, and TNF receptor I and II decreased from 12 to 32 weeks' gestation, while IL-13 increased. Praziquantel treatment did not significantly alter the trajectory of the concentration of any of the cytokines examined. Hookworm infection was associated with elevated placental IL-1, CXCL8 and IFN-γ. The risk of small-for-gestational age increased with elevated IL-6, IL-10, and CXCL8 in cord blood. The risk of prematurity was increased when cord blood sTNFRI and placental IL-5 were elevated., Conclusions: Our study suggests that fetal cytokines, which may be related to infectious disease exposures, contribute to poor intrauterine growth. Additionally, hookworm infection influences cytokine concentrations at the maternal-fetal interface., Clinical Trial Registry Number and Website: ClinicalTrials.gov (NCT00486863)., Competing Interests: The authors have declared that no competing interests exist.

Genome amplification and cellular senescence are commonly associated with pathological processes. While physiological roles for polyploidization and senescence have been described in mouse development, controversy exists over their significance in humans. Here, we describe tetraploidization and senescence as phenomena of normal human placenta development. During pregnancy, placental extravillous trophoblasts (EVTs) invade the pregnant endometrium, termed decidua, to establish an adapted microenvironment required for the developing embryo. This process is critically dependent on continuous cell proliferation and differentiation, which is thought to follow the classical model of cell cycle arrest prior to terminal differentiation. Strikingly, flow cytometry and DNAseq revealed that EVT formation is accompanied with a genome-wide polyploidization, independent of mitotic cycles. DNA replication in these cells was analysed by a fluorescent cell-cycle indicator reporter system, cell cycle marker expression and EdU incorporation. Upon invasion into the decidua, EVTs widely lose their replicative potential and enter a senescent state characterized by high senescence-associated (SA) β-galactosidase activity, induction of a SA secretory phenotype as well as typical metabolic alterations. Furthermore, we show that the shift from endocycle-dependent genome amplification to growth arrest is disturbed in androgenic complete hydatidiform moles (CHM), a hyperplastic pregnancy disorder associated with increased risk of developing choriocarinoma. Senescence is decreased in CHM-EVTs, accompanied by exacerbated endoreduplication and hyperploidy. We propose induction of cellular senescence as a ploidy-limiting mechanism during normal human placentation and unravel a link between excessive polyploidization and reduced senescence in CHM., Competing Interests: The authors have declared that no competing interests exist.

In mice, B1 and marginal zone (MZ) B-cells play an important role in prevention of autoimmunity through production of regulatory cytokines and natural antibodies. There is limited knowledge about the human counterparts of these cells. We therefore investigated functions of MZ-like B-cells and the frequency of circulating MZ-like and B1-like B-cells in healthy controls (HC), as well as in patients with autoimmune vasculitis to learn more about the role of these cells in autoimmune disease. After stimulation with CpG oligodeoxynucleotides (ODN) of class B in vitro , MZ-like B-cells were the main producers of IgM whereas switched memory B-cells primarily produced IgG and IgA. TNF and IL-10 were produced by both MZ-like and switched memory B-cells. Neither antibody nor TNF/IL-10 production by the B-cell subsets differed between patients and HC. Patients with autoimmune vasculitis, irrespective of disease activity, had lower percentage and absolute numbers of circulating MZ-like B-cells, and lower absolute numbers of B1-like B-cells. The percentage of B1-like B-cells was reduced during active disease. These findings remained significant when the analysis was confined to active treatment-naïve patients (disease onset).Our results suggest that human innate-like B-cells might have a physiological role in prevention of autoimmunity.

Although oxylipins are involved in inflammation, data on these lipid mediators in multiple sclerosis are sparse. In this study, a panel of oxylipins were analysed swith liquid chromatography tandem mass spectrometry in cerebrospinal fluid (CSF) from 41 treatment naïve patients with clinically isolated syndrome (CIS) or relapsing remitting MS (RRMS) and 22 healthy controls. CSF levels of 9-hydroxyoctadecadienoic acid (9-HODE) and 13-hydroxyoctadecadienoic acid (13-HODE) were significantly higher in patients than in healthy controls (9-HODE median 380 nM (interquartile range 330-450 nM) in patients and 290 nM (interquartile range 250-340 nM) in controls, 13-HODE median 930 nM (interquartile range 810-1080 nM) in patients and 690 nM (interquartile range 570-760 nM) in controls, p < 0.001 in Mann-Whitney U tests). 9-HODE and 13-HODE performed well for separation of patients and healthy controls (AUC 0.85 and 0.88, respectively, in ROC curve analysis). However, baseline CSF levels of the oxylipins did not differ between patients with signs of disease activity during one, two and four years of follow-up and patients without. In conclusion, this study indicates that 9-HODE and 13-HODE levels are increased in CSF from CIS and RRMS patients compared with healthy controls, but does not support 9-HODE or 13-HODE as prognostic biomarkers of disease activity in patients during follow-up., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Background: There is a need for clinically useful biomarkers of disease activity in clinically isolated syndrome (CIS) and relapsing remitting MS (RRMS). The aim of this study was to assess the correlation between neurofilament light chain (NFL) in cerebrospinal fluid (CSF) and serum and the relationship between NFL and other biomarkers, subsequent disease activity, and brain volume loss in CIS and RRMS., Methods: A panel of neurodegenerative and neuroinflammatory markers were analyzed in repeated CSF samples from 41 patients with CIS or RRMS in a prospective longitudinal cohort study and from 22 healthy controls. NFL in serum was analyzed using a single-molecule array (Simoa) method. "No evidence of disease activity-3" (NEDA-3) status and brain volume (brain parenchymal fraction calculated using SyMRI®) were recorded during 4 years of follow-up., Results: NFL levels in CSF and serum correlated significantly (all samples, n = 63, r 0.74, p < 0.001), but CSF-NFL showed an overall stronger association profile with NEDA-3 status, new T2 lesions, and brain volume loss. CSF-NFL was associated with both new T2 lesions and brain volume loss during follow-up, whereas CSF-CHI3L1 was associated mainly with brain volume loss and CXCL1, CXCL10, CXCL13, CCL22, and MMP-9 were associated mainly with new T2 lesions., Conclusions: Serum and CSF levels of NFL correlate, but CSF-NFL predicts and reflects disease activity better than S-NFL. CSF-NFL levels are associated with both new T2 lesions and brain volume loss. Our findings further add to the accumulating evidence that CSF-NFL is a clinically useful biomarker in CIS and RRMS and should be considered in the expanding NEDA concept. CSF-CXCL10 and CSF-CSF-CHI3L1 are potential markers of disease activity and brain volume loss, respectively.

To prospectively study systemic in vivo immunological effects of sex hormones, using different phases of oral combined hormonal contraceptives (CHC), and the natural menstrual cycles in both healthy women and in women with multiple sclerosis (MS), blood samples from sixty female MS patients and healthy controls with and without CHC were drawn in high and low estrogenic/progestogenic phases. Expression of Th-associated genes in blood cells was determined by qPCR and a panel of cytokines and chemokines was measured in plasma. High hormone level phases were associated with increases in Th1 (TBX21) and Th2 (GATA3) associated markers, as well as the B cell-associated chemokine CXCL13, while the inhibitory regulator CTLA-4 was decreased. These changes were not observed in MS patients, of whom most were treated with immunomodulatory drugs. Our data indicate immune activating properties in vivo of high steroid sex hormone levels during both CHC and normal menstrual cyclicity., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Low molecular weight heparin (LMWH) is widely used in recurrent miscarriage treatment. The anti-coagulant effects are established, while immunological effects are not fully known. Our aim was to assess LMWH effects on activation and polarization of central regulatory immune cells from healthy women, and on placenta tissues from women undergoing elective abortions. Isolated blood monocytes and T helper (Th) cells under different activation and polarizing conditions were cultured with or without LMWH. Flow cytometry showed that LMWH exposure induced increased expression of HLA-DR and CD206 in macrophages. This phenotype was associated with increased secretion of Th17-associated CCL20, and decreased secretion of CCL2 (M2-associated) and CCL22 (Th2), as measured by multiplex bead array. In accordance, LMWH exposure to Th cells reduced the proportion of CD25highFoxp3+ regulatory T-cells, intensified IFN-γ secretion and showed a tendency to increase the lymphoblast proportions. Collectively, a mainly pro-inflammatory effect was noted on two essential tolerance-promoting cells. Although the biological significancies of these in vitro findings are uncertain and need to be confirmed in vivo, they suggest the possibility that immunological effects of LMWH may be beneficial mainly at an earlier gestational age to provide an appropriate implantation process in women with recurrent miscarriage.

Background: To our knowledge, no studies have addressed whether maternal anemia of inflammation (AI) affects newborn iron status, and few have addressed risk factors for specific etiologies of maternal anemia., Objectives: The study aims were to evaluate 1) the contribution of AI and iron deficiency anemia (IDA) to newborn iron endowment, 2) hepcidin as a biomarker to distinguish AI from IDA among pregnant women, and 3) risk factors for specific etiologies of maternal anemia., Methods: We measured hematologic biomarkers in maternal blood at 12 and 32 wk of gestation and in cord blood from a randomized trial of praziquantel in 358 pregnant women with Schistosoma japonicum in The Philippines. IDA was defined as anemia with serum ferritin <30 ng/mL and non-IDA (NIDA), largely due to AI, as anemia with ferritin ≥30 ng/mL. We identified cutoffs for biomarkers to distinguish IDA from NIDA by using area under the curve (AUC) analyses and examined the impact of different causes of anemia on newborn iron status (primary outcome) by using multivariate regression modeling., Results: Of the 358 mothers, 38% (n = 136) had IDA and 9% (n = 32) had NIDA at 32 wk of gestation. At 32 wk of gestation, serum hepcidin performed better than soluble transferrin receptor (sTfR) in identifying women with NIDA compared with the rest of the cohort (AUCs: 0.75 and 0.70, respectively) and in identifying women with NIDA among women with anemia (0.73 and 0.72, respectively). The cutoff that optimally distinguished women with NIDA from women with IDA in our cohort was 6.1 µg/L. Maternal IDA, but not NIDA, was associated with significantly lower newborn ferritin (114.4 ng/mL compared with 148.4 µg/L; P = 0.042)., Conclusions: Hepcidin performed better than sTfR in identifying pregnant women with NIDA, but its cost may limit its use. Maternal IDA, but not NIDA, is associated with decreased newborn iron stores, emphasizing the need to identify this cause and provide iron therapy. This trial was registered at www.clinicaltrials.gov as NCT00486863.

Background: Previous studies showed that women with breast cancer treated in anthroposophic clinic versus conventional care had increased quality of life (QoL) parameters, fighting spirit, and anxiety coping. We have now analyzed immune and QoL factors in these 2 groups for possible differences during the first 6 months after admission, prompted by anthroposophic studies, including mistletoe extracts, showing beneficial immune system effects., Patients and Methods: Fourteen immunological variables, including leukocyte count, lymphocyte count, activated T cells (CD4+ and CD8+), NK cells, B cells, IL1β, IL6, IL10, and oxytocin, were longitudinally analyzed in both groups (n = 2 × 26). A panel of QoL parameters were analyzed using 3 different instruments. Statistical evaluation included that each patient was its own control., Results: Cytotoxic CD8+ T cell frequency (percent of lymphocytes analyzed by flow-cytometry) significantly decreased over time in the anthroposophic group versus the conventional group (repeated measures ANOVA, p = 0.05). No major differences were observed in other immunological parameters, whereas QoL variables, anxiety decreased and physical symptoms increased/improved significantly in the anthroposophic group (p = 0.04 and p = 0.05, respectively)., Conclusion: Overall, women with breast cancer in anthroposophic or conventional therapy did not differ in their immune profiles over time, with exception of decreased cytotoxic T cells in the anthroposophic group. Improvement in physical symptoms along with less anxiety in this group may have influenced the brain-immune axis resulting in lower frequency of CD8+ T cells, a feature associated with less aggressive cancer stages. To evaluate whether this observation is associated with good or bad prognosis, further detailed analyses of memory and naïve CD8+ T cells at tumor site and in blood circulation are essential., (© 2018 S. Karger GmbH, Freiburg.)

Background: The immunological environment during pregnancy may differ between allergic and non-allergic women. This study investigates the effect of maternal allergy on the allergen-induced cytokine and chemokine levels and whether pregnancy modulates these immune responses differently in allergic and non-allergic women., Methods: The birch-, cat-, phytohemagglutinin- and tetanus toxoid-induced interferon-γ (IFN-γ), interleukin (IL)-4, IL-5, IL-10, IL-13, the T-helper 1 (Th1)-associated chemokine CXCL10 and the Th2-associated chemokine CCL17 levels were quantified in 20 women with allergic symptoms (sensitized, n = 13) and 36 women without allergic symptoms (non-sensitized, n = 30) at gestational weeks 10-12, 15-16, 25, 35 and 2 and 12 months post-partum., Results: Birch-, but not cat-induced, IL-5, IL-13 and CCL17 levels were increased during pregnancy as compared to post-partum in the sensitized women with allergic symptoms. In contrast, cat-, but not birch-induced, IL-5 and IL-13 levels were increased during pregnancy as compared to post-partum in the non-sensitized women without allergic symptoms. Furthermore, IFN-γ secretion was increased in the first and decreased in the second and third trimesters in response to birch and decreased in the third trimester in response to cat as compared to post-partum in the non-sensitized women without allergic symptoms. Increased allergen-induced IL-4, IL-5 and IL-13 levels were associated with allergic symptoms and sensitization., Conclusions: Pregnancy had a clear effect on the allergen-induced IL-5, IL-13, CCL17, IFN-γ and CXCL10 production, with distinct enhanced Th2-responses to birch in the allergic group and to cat in the non-allergic group., (© 2017 EAACI and John Wiley and Sons A/S. Published by John Wiley and Sons Ltd.)

Background: During early tendon healing, the cells within the regenerating tissue are, to a large part, inflammatory leukocytes (CD45 + ). In a rat Achilles tendon healing model, the inflammation resolves between 5 and 10 days. In the same model, Cox inhibitors (NSAIDs) impair healing when given during the first 5 days, but have a positive effect if given later. We tested the hypothesis that a Cox inhibitor would exert these effects by influencing inflammation, and thereby the composition of the inflammatory cell subpopulations., Methods: Achilles tendon transection was performed in 44 animals. Animals were randomized to either parecoxib or saline injections. Healing was evaluated by mechanical testing day 7 after surgery and by flow cytometry day 3 and 10., Results: Cross-sectional area, peak force and stiffness were reduced by parecoxib 31, 33, and 25% respectively (p=0.005, p=0.002, and p=0.005). By flow cytometry, there was a strong effect of time (p<0.001) on virtually all inflammatory cell subpopulations (CD45, CD11b, CD68, CCR7, CD163, CD206, CD3, CD4), but no significant effect of parecoxib at any time point., Conclusion: The results suggest that the negative effects of Cox inhibitors on tendon healing might be exerted mainly via mechanisms not directly related to inflammatory cells., Competing Interests: Conflict of interest No conflicts of interest, financial or otherwise, are declared by the Author(s).