We have designed a versatile and sensitive liquid chromatographic (LC) system, featuring a monolithic trap column and a very narrow (10 μm ID) fused silica open tubular liquid chromatography (OTLC) separation column functionalized with C18-groups, for separating a wide range of molecules (from small metabolites to intact proteins). Compared to today’s capillary/nanoLC approaches, our system provides significantly enhanced sensitivity (up to several orders) with matching or improved separation efficiency, and highly repeatable chromatographic performance. The chemical properties of the trap column and the analytical column were fine-tuned to obtain practical sample loading capacities (above 2 μg), an earlier bottleneck of OTLC. Using the OTLC system (combined with Orbitrap mass spectrometry), we could perform targeted metabolomics of sub-μg amounts of exosomes with 25 attogram detection limit of a breast cancer-related hydroxylated cholesterol. With the same set-up, sensitive bottom-up proteomics (targeted and untargeted) was possible, and high-resolving intact protein analysis. In contrast to state-of-the-art packed columns, our platform performs chromatography with very little dilution and is “fit-for-all”, well suited for comprehensive analysis of limited samples, and has potential as a tool for challenges in diagnostics.

The polymorphism L412F in TLR3 has been associated with several infectious diseases. However, the mechanism underlying this association is still unexplored. Here, we show that the L412F polymorphism in TLR3 is a marker of severity in COVID-19. This association increases in the sub-cohort of males. Impaired macroautophagy/autophagy and reduced TNF/TNFα production was demonstrated in HEK293 cells transfected with TLR3 L412F -encoding plasmid and stimulated with specific agonist poly(I:C). A statistically significant reduced survival at 28 days was shown in L412F COVID-19 patients treated with the autophagy-inhibitor hydroxychloroquine (p = 0.038). An increased frequency of autoimmune disorders such as co-morbidity was found in L412F COVID-19 males with specific class II HLA haplotypes prone to autoantigen presentation. Our analyses indicate that L412F polymorphism makes males at risk of severe COVID-19 and provides a rationale for reinterpreting clinical trials considering autophagy pathways. Abbreviations: AP: autophagosome; AUC: area under the curve; BafA1: bafilomycin A1; COVID-19: coronavirus disease-2019; HCQ: hydroxychloroquine; RAP: rapamycin; ROC: receiver operating characteristic; SARS-CoV-2: severe acute respiratory syndrome coronavirus 2; TLR: toll like receptor; TNF/TNF-α: tumor necrosis factor

Background: Underlying causes of adrenal insufficiency include congenital adrenal hyperplasia (CAH) and autoimmune adrenocortical destruction leading to autoimmune Addison's disease (AAD). Here, we report a patient with a homozygous stop-gain mutation in 3β-hydroxysteroid dehydrogenase type 2 (3βHSD2), in addition to impaired steroidogenesis due to AAD.Case Report: Whole exome sequencing revealed an extremely rare homozygous nonsense mutation in exon 2 of the HSD3B2 gene, leading to a premature stop codon (NM_000198.3: c.15C>A, p.Cys5Ter) in a patient with AAD and premature ovarian insufficiency. Scrutiny of old medical records revealed that the patient was initially diagnosed with CAH with hyperandrogenism and severe salt-wasting shortly after birth. However, the current steroid profile show complete adrenal insufficiency including low production of pregnenolone, dehydroepiandrosterone (DHEA) and DHEA sulfate (DHEA-S), without signs of overtreatment with steroids.Conclusion: To the best of our knowledge, this is the first description of autoimmune adrenalitis in a patient with 3βHSD2 deficiency and suggests a possible association between AAD and inborn errors of the steroidogenesis.

The polymorphism L412F in TLR3 has been associated with several infectious diseases. However, the mechanism underlying this association is still unexplored. Here, we show that the L412F polymorphism in TLR3 is a marker of severity in COVID-19. This association increases in the sub-cohort of males. Impaired autophagy and reduced TNFα production was demonstrated in HEK293 cells transfected with TLR3-L412F plasmid and stimulated with specific agonist poly(I:C). A statistically significant reduced survival at 28 days was shown in L412F COVID-19 patients treated with the autophagy-inhibitor hydroxychloroquine (P=0.038). An increased frequency of autoimmune disorders as co-morbidity was found in L412F COVID-19 males with specific class II HLA haplotypes prone to autoantigen presentation. Our analyses indicate that L412F polymorphism makes males at risk of severe COVID-19 and provides a rationale for reinterpreting clinical trials considering autophagy pathways.

Autoimmune Addison's disease (AAD) is a classic organ-specific autoimmune disease characterized by an immune-mediated attack on the adrenal cortex. As most autoimmune diseases, AAD is believed to be caused by a combination of genetic and environmental factors, and probably interactions between the two. Persistent viral infections have been suggested to play a triggering role, by invoking inflammation and autoimmune destruction. The inability of clearing infections can be due to aberrations in innate immunity, including mutations in genes involved in the recognition of conserved microbial patterns. In a whole exome sequencing study of anonymized AAD patients, we discovered several rare variants predicted to be damaging in the gene encoding Toll-like receptor 3 (TLR3). TLR3 recognizes double stranded RNAs, and is therefore a major factor in antiviral defense. We here report the occurrence and functional characterization of five rare missense variants in TLR3 of patients with AAD. Most of these variants occurred together with a common TLR3 variant that has been associated with a wide range of immunopathologies. The biological implications of these variants on TLR3 function were evaluated in a cell-based assay, revealing a partial loss-of-function effect of three of the rare variants. In addition, rare mutations in other members of the TLR3-interferon (IFN) signaling pathway were detected in the AAD patients. Together, these findings indicate a potential role for TLR3 and downstream signaling proteins in the pathogenesis in a subset of AAD patients.