Parasitic, bacterial, viral, immune-mediated, metabolic, and nutritional factors associated with Nodding syndrome

Nodding syndrome is a neglected, disabling and potentially fatal epileptic disorder of unknown aetiology affecting thousands of individuals mostly confined to Eastern sub-Saharan Africa. Previous studies have identified multiple associations – including O. volvulus, antileiomodin-1 antibodies, vitamin B6 deficiency, and measles virus infection – yet none is proven causal. We conducted a case-control study of children with early-stage Nodding syndrome (symptom onset <1 year). Cases and controls were identified through a household survey in the Greater Mundri area in South Sudan. A wide range of parasitic, bacterial, viral, immune-mediated, metabolic, and nutritional risk factors was investigated using conventional and state-of-the-art untargeted assays. Associations were examined by multiple logistic regression analysis and a hypothetical causal model was constructed using structural equation modelling. From 607 children with Nodding syndrome, 72 with early-stage disease were included as cases and matched to 65 household- and 44 community controls. Mansonella perstans infection (odds ratio [OR] 7.04, 95% confidence interval [CI] 2.28-21.7), Necator americanus infection (OR 2.33, 95% CI 1.02-5.3), higher antimalarial seroreactivity (OR 1.75, 95% CI 1.20-2.57), higher vitamin E concentration (OR 1.53 per standard deviation [SD] increase, 95% CI 1.07-2.19) and lower vitamin B12 concentration (OR 0.56 per SD increase, 95% CI 0.36-0.87) were associated with higher odds of NS. In a structural equation model, we hypothesized that M. perstans infection, higher vitamin E concentration and fewer viral exposures increased the risk of Nodding syndrome while lower vitamin B12 concentration, N. americanus and malaria infections resulted from having Nodding syndrome. We found no evidence that O. volvulus, antileiomodin-1 antibodies, vitamin B6 and other factors were associated with Nodding syndrome. Our results argue against several previous causal hypotheses including O. volvulus. Instead, Nodding syndrome may be caused by a complex interplay between multiple pathogens and nutrient levels. Further studies need to confirm these associations and determine the direction of effect

Genomic investigations of unexplained acute hepatitis in children

Since its first identification in Scotland, over 1,000 cases of unexplained paediatric hepatitis in children have been reported worldwide, including 278 cases in the UK1. Here we report an investigation of 38 cases, 66 age-matched immunocompetent controls and 21 immunocompromised comparator participants, using a combination of genomic, transcriptomic, proteomic and immunohistochemical methods. We detected high levels of adeno-associated virus 2 (AAV2) DNA in the liver, blood, plasma or stool from 27 of 28 cases. We found low levels of adenovirus (HAdV) and human herpesvirus 6B (HHV-6B) in 23 of 31 and 16 of 23, respectively, of the cases tested. By contrast, AAV2 was infrequently detected and at low titre in the blood or the liver from control children with HAdV, even when profoundly immunosuppressed. AAV2, HAdV and HHV-6 phylogeny excluded the emergence of novel strains in cases. Histological analyses of explanted livers showed enrichment for T cells and B lineage cells. Proteomic comparison of liver tissue from cases and healthy controls identified increased expression of HLA class 2, immunoglobulin variable regions and complement proteins. HAdV and AAV2 proteins were not detected in the livers. Instead, we identified AAV2 DNA complexes reflecting both HAdV-mediated and HHV-6B-mediated replication. We hypothesize that high levels of abnormal AAV2 replication products aided by HAdV and, in severe cases, HHV-6B may have triggered immune-mediated hepatic disease in genetically and immunologically predisposed children

An act of revision: IgA in health and disease

In humans, IgA is expressed in three different forms: monomeric IgA, dimeric IgA (dIgA), and secretory IgA (SIgA). On the mucosal sites, mainly dIgA is produced by local plasma cells, which is composed of two IgA molecules that are connected tail-to-tail by a so-called J chain. Binding of dIgA to the polymeric Ig receptor (pIgR), present on epithelial cells on mucosal membranes, causes dIgA to be secreted in secretions such as mucus and breast milk. A portion of the pIgR, referred to as the secretory component (SC), remains attached to dIgA to form SIgA. In contrast to the mucosal sites, the blood circulation mainly contains monomeric IgA produced by plasma cells in the bone marrow, spleen and lymph nodes. In the circulation, IgA is the second most abundant antibody after IgG. Based on the characteristics of IgA on mucosal sites, IgA in circulation is thought to play mainly a passive and anti-inflammatory role in systemic immunity. However, elevated blood IgA levels or the presence of IgA autoantibodies have been detected in multiple diseases, including rheumatoid arthritis, IgA nephropathy, IgA vasculitis, dermatitis herpetiformis, celiac disease, inflammatory bowel disease, Sjögren's syndrome, ankylosing spondylitis, and alcoholic liver syndrome acquired immune cirrhosis. The exact contribution of IgA in the pathology of these diseases is insufficiently known to date. In this thesis, I investigated the development of IgA and its inflammatory potential after vaccination, during persistent infection and during autoimmunity or chronic inflammation in order to gain a better understanding of the role of IgA in health and disease. With this thesis, I refute the current belief that IgA is a redundant antibody in circulation. It is important to emphasize that the role of IgA in circulation has mainly been studied using different mouse models. Due to the lack of the IgA receptor, FcαRI, in mice, the inflammatory potential of IgA may have been overlooked and the results of these studies should be interpreted carefully. Using a human FcαRI/IgA transgenic mouse model, we demonstrated that FcαRI-mediated neutrophil activation by IgA exacerbates the pathology of DSS-induced intestinal inflammation. FcαRI-mediated cell activation may play an important role in defense against pathogens, however, the flip side of the coin is that abnormal or excessive IgA inflammatory responses can result in tissue damage and thereby contribute to the pathology of inflammatory diseases. On the one hand, increasing antigen-specific IgA levels in the circulation through vaccination may protect against infectious diseases. On the other hand, blocking the interaction between IgA and FcαRI through monoclonal antibodies or peptides can dampen inflammation and disease. It is therefore of great importance that future serological research on vaccination, infectious and inflammatory diseases also includes IgA- and FcαRI-mediated cell activation

An act of revision: IgA in health and disease

In humans, IgA is expressed in three different forms: monomeric IgA, dimeric IgA (dIgA), and secretory IgA (SIgA). On the mucosal sites, mainly dIgA is produced by local plasma cells, which is composed of two IgA molecules that are connected tail-to-tail by a so-called J chain. Binding of dIgA to the polymeric Ig receptor (pIgR), present on epithelial cells on mucosal membranes, causes dIgA to be secreted in secretions such as mucus and breast milk. A portion of the pIgR, referred to as the secretory component (SC), remains attached to dIgA to form SIgA. In contrast to the mucosal sites, the blood circulation mainly contains monomeric IgA produced by plasma cells in the bone marrow, spleen and lymph nodes. In the circulation, IgA is the second most abundant antibody after IgG. Based on the characteristics of IgA on mucosal sites, IgA in circulation is thought to play mainly a passive and anti-inflammatory role in systemic immunity. However, elevated blood IgA levels or the presence of IgA autoantibodies have been detected in multiple diseases, including rheumatoid arthritis, IgA nephropathy, IgA vasculitis, dermatitis herpetiformis, celiac disease, inflammatory bowel disease, Sjögren's syndrome, ankylosing spondylitis, and alcoholic liver syndrome acquired immune cirrhosis. The exact contribution of IgA in the pathology of these diseases is insufficiently known to date. In this thesis, I investigated the development of IgA and its inflammatory potential after vaccination, during persistent infection and during autoimmunity or chronic inflammation in order to gain a better understanding of the role of IgA in health and disease. With this thesis, I refute the current belief that IgA is a redundant antibody in circulation. It is important to emphasize that the role of IgA in circulation has mainly been studied using different mouse models. Due to the lack of the IgA receptor, FcαRI, in mice, the inflammatory potential of IgA may have been overlooked and the results of these studies should be interpreted carefully. Using a human FcαRI/IgA transgenic mouse model, we demonstrated that FcαRI-mediated neutrophil activation by IgA exacerbates the pathology of DSS-induced intestinal inflammation. FcαRI-mediated cell activation may play an important role in defense against pathogens, however, the flip side of the coin is that abnormal or excessive IgA inflammatory responses can result in tissue damage and thereby contribute to the pathology of inflammatory diseases. On the one hand, increasing antigen-specific IgA levels in the circulation through vaccination may protect against infectious diseases. On the other hand, blocking the interaction between IgA and FcαRI through monoclonal antibodies or peptides can dampen inflammation and disease. It is therefore of great importance that future serological research on vaccination, infectious and inflammatory diseases also includes IgA- and FcαRI-mediated cell activation

IgA Immune Complexes Induce Osteoclast-Mediated Bone Resorption

Objective: Autoantibodies are detected in most patients with rheumatoid arthritis (RA) and can be of the IgM, IgG or IgA subclass. Correlations between IgA autoantibodies and more severe disease activity have been previously reported, but the functional role of IgA autoantibodies in the pathogenesis of RA is ill understood. In this study, we explored the effect of IgA immune complexes on osteoclast mediated bone resorption. Methods: Anti-citrullinated peptide antibody (ACPA) and anti-carbamylated protein (anti-CarP) antibody levels of the IgA and IgG isotype and rheumatoid factor (RF) IgA were determined in synovial fluid (SF) of RA patients. Monocytes, neutrophils, and osteoclasts were stimulated with precipitated immune complexes from SF of RA patients or IgA- and IgG-coated beads. Activation was determined by neutrophil extracellular trap (NET) release, cytokine secretion, and bone resorption. Results: NET formation by neutrophils was enhanced by SF immune complexes compared to immune complexes from healthy or RA serum. Monocytes stimulated with isolated SF immune complexes released IL-6 and IL-8, which correlated with the levels of ACPA IgA levels in SF. Osteoclasts cultured in the presence of supernatant of IgA-activated monocytes resorbed significantly more bone compared to osteoclasts that were cultured in supernatant of IgG-activated monocytes (p=0.0233). Osteoclasts expressed the Fc receptor for IgA (FcαRI; CD89) and Fc gamma receptors. IgA-activated osteoclasts however produced significantly increased levels of IL-6 (p<0.0001) and IL-8 (p=0.0007) compared to IgG-activated osteoclasts. Both IL-6 (p=0.03) and IL-8 (p=0.0054) significantly enhanced bone resorption by osteoclasts. Conclusion: IgA autoantibodies induce release of IL-6 and IL-8 by immune cells as well as osteoclasts, which enhances bone resorption by osteoclasts. We anticipate that this will result in more severe disease activity in RA patients. Targeting IgA-FcαRI interactions therefore represents a promising novel therapeutic strategy for RA patients with IgA autoantibodies

Fcα Receptor-1-Activated Monocytes Promote B Lymphocyte Migration and IgA Isotype Switching.

Patients with inflammatory bowel disease (IBD) produce enhanced immunoglobulin A (IgA) against the microbiota compared to healthy individuals, which has been correlated with disease severity. Since IgA complexes can potently activate myeloid cells via the IgA receptor FcαRI (CD89), excessive IgA production may contribute to IBD pathology. However, the cellular mechanisms that contribute to dysregulated IgA production in IBD are poorly understood. Here, we demonstrate that intestinal FcαRI-expressing myeloid cells (i.e., monocytes and neutrophils) are in close contact with B lymphocytes in the lamina propria of IBD patients. Furthermore, stimulation of FcαRI-on monocytes triggered production of cytokines and chemokines that regulate B-cell differentiation and migration, including interleukin-6 (IL6), interleukin-10 (IL10), tumour necrosis factor-α (TNFα), a proliferation-inducing ligand (APRIL), and chemokine ligand-20 (CCL20). In vitro, these cytokines promoted IgA isotype switching in human B cells. Moreover, when naïve B lymphocytes were cultured in vitro in the presence of FcαRI-stimulated monocytes, enhanced IgA isotype switching was observed compared to B cells that were cultured with non-stimulated monocytes. Taken together, FcαRI-activated monocytes produced a cocktail of cytokines, as well as chemokines, that stimulated IgA switching in B cells, and close contact between B cells and myeloid cells was observed in the colons of IBD patients. As such, we hypothesize that, in IBD, IgA complexes activate myeloid cells, which in turn can result in excessive IgA production, likely contributing to disease pathology. Interrupting this loop may, therefore, represent a novel therapeutic strategy

Fcα Receptor-1-Activated Monocytes Promote B Lymphocyte Migration and IgA Isotype Switching

Patients with inflammatory bowel disease (IBD) produce enhanced immunoglobulin A (IgA) against the microbiota compared to healthy individuals, which has been correlated with disease severity. Since IgA complexes can potently activate myeloid cells via the IgA receptor FcαRI (CD89), excessive IgA production may contribute to IBD pathology. However, the cellular mechanisms that contribute to dysregulated IgA production in IBD are poorly understood. Here, we demonstrate that intestinal FcαRI-expressing myeloid cells (i.e., monocytes and neutrophils) are in close contact with B lymphocytes in the lamina propria of IBD patients. Furthermore, stimulation of FcαRI-on monocytes triggered production of cytokines and chemokines that regulate B-cell differentiation and migration, including interleukin-6 (IL6), interleukin-10 (IL10), tumour necrosis factor-α (TNFα), a proliferation-inducing ligand (APRIL), and chemokine ligand-20 (CCL20). In vitro, these cytokines promoted IgA isotype switching in human B cells. Moreover, when naïve B lymphocytes were cultured in vitro in the presence of FcαRI-stimulated monocytes, enhanced IgA isotype switching was observed compared to B cells that were cultured with non-stimulated monocytes. Taken together, FcαRI-activated monocytes produced a cocktail of cytokines, as well as chemokines, that stimulated IgA switching in B cells, and close contact between B cells and myeloid cells was observed in the colons of IBD patients. As such, we hypothesize that, in IBD, IgA complexes activate myeloid cells, which in turn can result in excessive IgA production, likely contributing to disease pathology. Interrupting this loop may, therefore, represent a novel therapeutic strategy

Fcα Receptor-1-Activated Monocytes Promote B Lymphocyte Migration and IgA Isotype Switching

Patients with inflammatory bowel disease (IBD) produce enhanced immunoglobulin A (IgA) against the microbiota compared to healthy individuals, which has been correlated with disease severity. Since IgA complexes can potently activate myeloid cells via the IgA receptor FcαRI (CD89), excessive IgA production may contribute to IBD pathology. However, the cellular mechanisms that contribute to dysregulated IgA production in IBD are poorly understood. Here, we demonstrate that intestinal FcαRI-expressing myeloid cells (i.e., monocytes and neutrophils) are in close contact with B lymphocytes in the lamina propria of IBD patients. Furthermore, stimulation of FcαRI-on monocytes triggered production of cytokines and chemokines that regulate B-cell differentiation and migration, including interleukin-6 (IL6), interleukin-10 (IL10), tumour necrosis factor-α (TNFα), a proliferation-inducing ligand (APRIL), and chemokine ligand-20 (CCL20). In vitro, these cytokines promoted IgA isotype switching in human B cells. Moreover, when naïve B lymphocytes were cultured in vitro in the presence of FcαRI-stimulated monocytes, enhanced IgA isotype switching was observed compared to B cells that were cultured with non-stimulated monocytes. Taken together, FcαRI-activated monocytes produced a cocktail of cytokines, as well as chemokines, that stimulated IgA switching in B cells, and close contact between B cells and myeloid cells was observed in the colons of IBD patients. As such, we hypothesize that, in IBD, IgA complexes activate myeloid cells, which in turn can result in excessive IgA production, likely contributing to disease pathology. Interrupting this loop may, therefore, represent a novel therapeutic strategy

Targeting the MAPK and PI3K pathways in combination with PD1 blockade in melanoma

Immunotherapy of advanced melanoma with CTLA-4 or PD-1/PD-L1 checkpoint blockade induces in a proportion of patients long durable responses. In contrast, targeting the MAPK-pathway by selective BRAF and MEK inhibitors induces high response rates, but most patients relapse. Combining targeted therapy with immunotherapy is proposed to improve the long-term outcomes of patients. Preclinical data endorsing this hypothesis are accumulating. Inhibition of the PI3K-Akt-mTOR pathway may be a promising treatment option to overcome resistance to MAPK inhibition and for additional combination with immunotherapy. We therefore evaluated to which extent dual targeting of the MAPK and PI3K-Akt-mTOR pathways affects tumor immune infiltrates and whether it synergizes with PD-1 checkpoint blockade in a BRAFV600E/PTEN-/--driven melanoma mouse model. Short-term dual BRAF + MEK inhibition enhanced tumor immune infiltration and improved tumor control when combined with PD-1 blockade in a CD8+ T cell dependent manner. Additional PI3K inhibition did not impair tumor control or immune cell infiltration and functionality. Analysis of on-treatment samples from melanoma patients treated with BRAF or BRAF + MEK inhibitors indicates that inhibitor-mediated T cell infiltration occurred in all patients early after treatment initiation but was less frequent found in on-treatment biopsies beyond day 15. Our findings provide a rationale for clinical testing of short-term BRAF + MEK inhibition in combination with immune checkpoint blockade, currently implemented at our institutes. Additional PI3K inhibition could be an option for BRAF + MEK inhibitor resistant patients that receive targeted therapy in combination with immune checkpoint blockade