Proteomic profiling reveals autoimmune targets in sarcoidosis.

RATIONALE: There is a need to further characterize the antibody repertoire in relation to sarcoidosis and potentially related autoantigens. OBJECTIVES: We investigated bronchoalveolar lavage (BAL) and serum samples from patients with sarcoidosis and healthy and diseased control subjects to discover sarcoidosis-associated autoantigens. METHODS: Antigen microarrays built on 3,072 protein fragments were used to screen for IgG reactivity in 73 BAL samples from subjects with sarcoidosis, subjects with asthma, and healthy subjects. A set of 131 targets were selected for subsequent verification on suspension bead arrays using 272 additional BAL samples and 141 paired sera. Reactivity to four antigens was furthermore analyzed in 22 unprocessed BAL samples from patients with fibrosis and 269 plasma samples from patients diagnosed with myositis. MEASUREMENTS AND MAIN RESULTS: Reactivity toward zinc finger protein 688 and mitochondrial ribosomal protein L43 were discovered with higher frequencies in patients with sarcoidosis, for mitochondrial ribosomal protein L43 especially in patients with non-Löfgren syndrome. Increased reactivity toward nuclear receptor coactivator 2 was also observed in patients with non-Löfgren syndrome as compared with patients with Löfgren syndrome. The antigen representing adenosine diphosphate-ribosylation factor GTPase activating protein 1 revealed high reactivity frequency in all sample groups but with significantly higher level of IgG reactivities in patients with sarcoidosis. CONCLUSIONS: Autoantigen reactivity was present in most BAL and serum samples analyzed, and the results revealed high interindividual heterogeneity, with most of the reactivities observed in single individuals only. Four proteins are here proposed as sarcoidosis-associated autoimmune targets and of interest for further validation in independent cohorts.

Proteome profiling of home-sampled dried blood spots reveals proteins of SARS-CoV-2 infections.

BACKGROUND: Self-sampling of dried blood spots (DBS) offers new routes to gather valuable health-related information from the general population. Yet, the utility of using deep proteome profiling from home-sampled DBS to obtain clinically relevant insights about SARS-CoV-2 infections remains largely unexplored. METHODS: Our study involved 228 individuals from the general Swedish population who used a volumetric DBS sampling device and completed questionnaires at home during spring 2020 and summer 2021. Using multi-analyte COVID-19 serology, we stratified the donors by their response phenotypes, divided them into three study sets, and analyzed 276 proteins by proximity extension assays (PEA). After normalizing the data to account for variances in layman-collected samples, we investigated the association of DBS proteomes with serology and self-reported information. RESULTS: Our three studies display highly consistent variance of protein levels and share associations of proteins with sex (e.g., MMP3) and age (e.g., GDF-15). Studying seropositive (IgG+) and seronegative (IgG-) donors from the first pandemic wave reveals a network of proteins reflecting immunity, inflammation, coagulation, and stress response. A comparison of the early-infection phase (IgM+IgG-) with the post-infection phase (IgM-IgG+) indicates several proteins from the respiratory system. In DBS from the later pandemic wave, we find that levels of a virus receptor on B-cells differ between seropositive (IgG+) and seronegative (IgG-) donors. CONCLUSIONS: Proteome analysis of volumetric self-sampled DBS facilitates precise analysis of clinically relevant proteins, including those secreted into the circulation or found on blood cells, augmenting previous COVID-19 reports with clinical blood collections. Our population surveys support the usefulness of DBS, underscoring the role of timing the sample collection to complement clinical and precision health monitoring initiatives.

The COVID-19 pandemic has posed multiple challenges to healthcare systems. A significant gap that remains is a lack of understanding of the impact of SARS-CoV-2 on individuals who did not seek or require hospitalization. To address this, we distribute self-sampling devices to random citizens, aiming to analyze how blood protein levels are affected in people who have had COVID-19 but had no or mild symptoms. Conducting multiple molecular measurements in dried blood, our study confirms clinically known markers and their relationship to infection stages, even if the donors themselves collect the sample. Our work highlights the potential of combining self-sampling with laboratory methods to provide useful information on human health. This convenient patient-centric sampling approach may potentially be useful when studying other diseases.

Adipose tissue IL-8 is increased in normal weight women after menopause and reduced after gastric bypass surgery in obese women.

OBJECTIVE: The menopausal transition is characterized by increased body fat accumulation, including redistribution from peripheral to central fat depots. This distribution is associated with an increased risk of type 2 diabetes and cardiovascular disease that are linked to low-grade inflammation. We determined whether postmenopausal women have higher levels of inflammatory markers, compared with premenopausal women. We also wanted to determine whether these markers are reduced by stable weight loss in obese women. DESIGN AND METHODS: Anthropometric data, blood samples and subcutaneous adipose tissue biopsies were collected from normal weight premenopausal and postmenopausal women and obese women before and 2 years after gastric bypass (GBP) surgery. Serum protein levels and adipose tissue gene expression of inflammatory markers were investigated. RESULTS: IL-8 expression in adipose tissue and circulating levels were higher in postmenopausal vs premenopausal women. IL-8 expression was associated with waist circumference, independent of menopausal status. IL-6 expression and serum levels of monocyte chemoattractant protein (MCP)-1 were higher in postmenopausal vs premenopausal women. Two years after GBP surgery, adipose expression of IL-8, tumour necrosis factor-α and MCP-1 decreased significantly. Serum insulin levels were associated with inflammation-related gene expression before GBP surgery, but these associations disappeared after surgery. CONCLUSION: Postmenopausal women have an increased inflammatory response in the subcutaneous fat and circulation. Inflammatory markers in adipose tissue decreased significantly after surgery-induced weight loss. This effect may be beneficial for metabolic control and reduced cardiovascular risk after weight loss.

Bead-Based Assays for Validating Proteomic Profiles in Body Fluids.

Protein biomarkers in biological fluids represent an important resource for improving the clinical management of diseases. Current proteomics technologies are capable of performing high-throughput and multiplex profiling in different types of fluids, often leading to the shortlisting of tens of candidate biomarkers per study. However, before reaching any clinical setting, these discoveries require thorough validation and an assay that would be suitable for routine analyses. In the path from biomarker discovery to validation, the performance of the assay implemented for the intended protein quantification is extremely critical toward achieving reliable and reproducible results. Development of robust sandwich immunoassays for individual candidates is challenging and labor and resource intensive, and multiplies when evaluating a panel of interesting candidates at the same time. Here we describe a versatile pipeline that facilitates the systematic and parallel development of multiple sandwich immunoassays using a bead-based technology.

Multianalyte serology in home-sampled blood enables an unbiased assessment of the immune response against SARS-CoV-2.

Serological testing is essential to curb the consequences of the COVID-19 pandemic. However, most assays are still limited to single analytes and samples collected within healthcare. Thus, we establish a multianalyte and multiplexed approach to reliably profile IgG and IgM levels against several versions of SARS-CoV-2 proteins (S, RBD, N) in home-sampled dried blood spots (DBS). We analyse DBS collected during spring of 2020 from 878 random and undiagnosed individuals from the population in Stockholm, Sweden, and use classification approaches to estimate an accumulated seroprevalence of 12.5% (95% CI: 10.3%-14.7%). This includes 5.4% of the samples being IgG+IgM+ against several SARS-CoV-2 proteins, as well as 2.1% being IgG-IgM+ and 5.0% being IgG+IgM- for the virus' S protein. Subjects classified as IgG+ for several SARS-CoV-2 proteins report influenza-like symptoms more frequently than those being IgG+ for only the S protein (OR = 6.1; p < 0.001). Among all seropositive cases, 30% are asymptomatic. Our strategy enables an accurate individual-level and multiplexed assessment of antibodies in home-sampled blood, assisting our understanding about the undiagnosed seroprevalence and diversity of the immune response against the coronavirus.

Quantitative analysis of RU38486 (mifepristone) by HPLC triple quadrupole mass spectrometry.

A sensitive liquid chromatography-mass spectrometric method was validated for the quantification of RU38486 (mifepristone) in human and murine plasma. The analyte and internal standard (alfaxolone) were extracted by liquid-liquid extraction with diethyl ether, resolved on a C18 column using gradient elution with methanol and ammonium acetate and detected after positive electrospray ionization (m/z 430-->372; m/z 333-->297, respectively). Quantification was linear over the range 0.5-500ng (r(2)>0.997), precise and accurate (intra-assay RSD< or =7.2%, RME< or =8.2%; inter-assay RSD< or =15.7% RME< or =10.2%). The limit of quantification (LOQ) was 50pg injected on column, permitting reproducible analysis of RU38486 in small volumes of plasma.

Individual and stable autoantibody repertoires in healthy individuals.

In the era towards precision medicine, we here present the individual specific autoantibody signatures of 193 healthy individuals. The self-reactive IgG signatures are stable over time in a way that each individual profile is recognized in longitudinal sampling. The IgG autoantibody reactivity towards an antigen array comprising 335 protein fragments, representing 204 human proteins with potential relevance to autoimmune disorders, was measured in longitudinal plasma samples from 193 healthy individuals. This analysis resulted in unique autoantibody barcodes for each individual that were maintained over one year's time. The reactivity profiles, or signatures, are person specific in regards to the number of reactivities and antigen specificity. Two independent data sets were consistent in that each healthy individual displayed reactivity towards 0-16 antigens, with a median of six. Subsequently, four selected individuals were profiled on in-house produced high-density protein arrays containing 23,000 protein fragments representing 14,000 unique protein coding genes. Based on a unique, broad and deep longitudinal profiling of autoantibody reactivities, our results demonstrate a unique autoreactive profile in each analyzed healthy individual. The need and interest for broad-ranged and high-resolution molecular profiling of healthy individuals is rising. We have here generated and assessed an initial perspective on the global distribution of the self-reactive IgG repertoire in healthy individuals, by investigating 193 well-characterized healthy individuals. Highlights A unique longitudinal profiling of autoantibody repertoires in healthy individuals Autoantibody profiles are highly individual and stable over time All individuals display IgG binding to human protein fragments The specificity of disease associated autoantigens needs to be thoroughly characterized The identification of a small set of highly reactive autoantigens Importance of stringent antigen and sample specific cut-offs for defining reactivity.

High-Density Antigen Microarrays for the Assessment of Antibody Selectivity and Off-Target Binding.

With the increasing availability of collections of antibodies, their evaluation in terms of binding selectivity becomes an important but challenging task. Planar antigen microarrays are very suitable tools to address this task and provide a powerful proteomics platform for the characterization of the binding selectivity of antibodies toward thousands of antigens in parallel. In this chapter, we describe our in-house developed procedures for the generation of high-density planar antigen microarrays with over 21,000 features. We also provide the details of the assay protocol, which we routinely use for the assessment of binding selectivity of the polyclonal antibodies generated within the Human Protein Atlas.

Hippocampal 11beta-hydroxysteroid dehydrogenase type 1 messenger ribonucleic acid expression has a diurnal variability that is lost in the obese Zucker rat.

Circulating levels of glucocorticoids show a circadian rhythm. Obesity is associated with a flattening of the diurnal rhythm; plasma cortisol levels are slightly increased during the trough, although they are normal or low in the morning. Studies in humans and in leptin-resistant Zucker rats suggest that tissue-specific alterations in glucocorticoid exposure might play a key role for development of obesity and obesity-associated dysregulation of the hypothalamic-pituitary-adrenal axis. We hypothesized that there is a circadian rhythm in prereceptor metabolism of glucocorticoids exerted by 11beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD1) in brain and/or peripheral tissues (liver, fat, and muscle) that might be abrogated in obesity. The present study demonstrates a circadian rhythm in 11beta-HSD1 mRNA expression (35-45% increase at morning vs. evening, P < 0.05) in dentate gyrus granular layer and CA1 subregions of the hippocampus in lean Zucker rats that was lost in the obese rats. Sprague Dawley rats also revealed a diurnal rhythm in hippocampal 11beta-HSD1 mRNA expression. There was no circadian variation in 11beta-HSD enzyme activity in peripheral tissues, although obese Zucker rats had a decreased enzyme activity in liver and epididymal fat (by approximately 40%, P < 0.05) compared with lean rats. In Sprague Dawley rats, 11beta-HSD activity in adipose tissue was higher in retroperitoneal and epididymal vs. sc fat (P < 0.001). In summary, obese Zucker rats lack a circadian rhythm of 11beta-HSD1 gene expression in the hippocampus, which may contribute to increased activity of the hypothalamic-pituitary-adrenal axis and altered diurnal variation of circulating corticosterone levels.

Estrogens and glucocorticoid hormones in adipose tissue metabolism.

Women have a higher percentage of body fat than men, and there is a gender-specific difference in fat distribution: Females tend to accumulate fat around the hips, buttocks, and thighs while men have a larger intra-abdominal (visceral) fat mass. After menopause, there is a redistribution of fat depots, and post-menopausal women develop increased amounts of visceral fat. The risk of developing obesity-related diseases is significantly lower in pre-menopausal women compared to men, a difference that is abolished after menopause, suggesting that the female sex steroid estrogen influences adipogenesis and adipose metabolism. Experimentally, estrogen increases the size and number of subcutaneous adipocytes and attenuates lipolysis. Post-menopausal women also develop a more atherogenic lipid pattern and decreased levels of the prothrombotic protein plasminogen activator inhibitor-1, which attenuates fibrinolysis. Pathologically increased circulating cortisol concentration is associated with dysmetabolic features e.g., central obesity, elevated blood pressure, insulin resistance, and dyslipidemia. In "simple obesity," glucocorticoid production is elevated. Peak levels of circulating cortisol are however low or normal, possibly because of increased clearance and/or tissue-specific changes in cortisol production. In addition to the adrenal production of cortisol, cortisol is also generated in adipose tissue by the enzyme 11beta-hydroxysteroid dehydrogenase type 1 (11betaHSD1) which converts inactive cortisone to active cortisol. The enzyme activity in subcutaneous fat increases with increasing body weight. Estrogen seems to have a tissue-specific influence on 11betaHSD1 enzyme activity, attenuating it in liver, kidney, and testis but upregulating 11betaHSD1 mRNA expression in preadipocytes from women. In the present review, we summarize and discuss the interaction between glucocorticoids and sex steroids and their influence on adipocyte metabolism.

Exploration of high-density protein microarrays for antibody validation and autoimmunity profiling.

High-density protein microarrays of recombinant human protein fragments, representing 12,412 unique Ensembl Gene IDs, have here been produced and explored. These protein microarrays were used to analyse antibody off-target interactions, as well as for profiling the human autoantibody repertoire in plasma against the antigens represented by the protein fragments. Affinity-purified polyclonal antibodies produced within the Human Protein Atlas (HPA) were analysed on microarrays of three different sizes, ranging from 384 antigens to 21,120 antigens, for evaluation of the antibody validation criteria in the HPA. Plasma samples from secondary progressive multiple sclerosis patients were also screened in order to explore the feasibility of these arrays for broad-scale profiling of autoantibody reactivity. Furthermore, analysis on these near proteome-wide microarrays was complemented with analysis on HuProt™ Human Proteome protein microarrays. The HPA recombinant protein microarray with 21,120 antigens and the HuProt™ Human Proteome protein microarray are currently the largest protein microarray platforms available to date. The results on these arrays show that the Human Protein Atlas antibodies have few off-target interactions if the antibody validation criteria are kept stringent and demonstrate that the HPA-produced high-density recombinant protein fragment microarrays allow for a high-throughput analysis of plasma for identification of possible autoantibody targets in the context of various autoimmune conditions.

Affinity proteomics discovers decreased levels of AMFR in plasma from Osteoporosis patients.

PURPOSE: Affinity proteomic approaches by antibody bead arrays enable multiplexed analysis of proteins in body fluids. In the presented study, we investigated blood plasma within osteoporosis to discovery differential protein profiles and to propose novel biomarkers candidates for subsequent studies. EXPERIMENTAL DESIGN: Starting with 4608 antibodies and plasma samples from 22 women for an untargeted screening, a set of 72 proteins were suggested for further analysis. Complementing these with targets from literature and other studies, a targeted bead array of 180 antibodies was built to profile for 92 proteins in plasma samples of 180 women from two independent population-based studies. RESULTS: Differential profiles between osteoporosis patients and matched controls were discovered for 12 proteins in at least one of the two study sets. Among these targets, the levels of autocrine motility factor receptor (AMFR) were concordantly lower in plasma of female osteoporosis patients. Subsequently, verification of anti-AMFR antibody selectivity was conducted using high-density peptide and protein arrays, and Western blotting. CONCLUSIONS AND CLINICAL RELEVANCE: Further validation in additional study sets will be needed to determine the clinical value of the observed decrease in AMFR plasma levels in osteoporosis patients, but AMFR may aid our understanding of disease mechanisms and could support existing tools for diagnosis and monitoring of patient mobility within osteoporosis.

Obese Zucker rats have reduced mineralocorticoid receptor and 11beta-hydroxysteroid dehydrogenase type 1 expression in hippocampus-implications for dysregulation of the hypothalamic-pituitary-adrenal axis in obesity.

Obese Zucker rats have elevated basal corticosterone levels and an increased stress response suggestive of an increased activity of the hypothalamic-pituitary-adrenal (HPA) axis. We hypothesized that altered central expression of glucocorticoid receptors (GR), mineralocorticoid receptors (MR), and/or 11beta-hydroxysteroid dehydrogenase type 1 (11betaHSD1) contribute to these changes. In brains from young adult male rats, in situ hybridization and Western blotting showed that obese rats had normal hippocampal GR mRNA and protein levels. In contrast, in obese rats, 11betaHSD1 mRNA levels were reduced in a subpopulation of hippocampal cells in the main neuronal layers (by 37-47%, P < 0.05), whereas 11betaHSD1 levels in sparse high-expressing cells did not differ. MR mRNA was decreased in all regions of the hippocampus (by 37-49%, P < 0.05 for CA1-2 and P < 0.01 for dentate gyrus) and in frontal cortex (by 16%, P < 0.05) in obese rats. In whole hippocampal homogenates, however, neither the protein concentration of MR by Western blot nor activity of 11betaHSD1 was measurably different between the phenotypes. To test the functional importance of lower central MR expression, groups of lean and obese rats were given spironolactone before restraint stress. In vehicle-treated animals, obese rats had higher plasma corticosterone levels than lean rats after stress (by ANOVA, P < 0.05). Spironolactone markedly increased the corticosterone response in both groups, but the incremental rise was smaller in the obese rats, so that spironolactone abolished the differences between groups. We conclude that lower levels of MR, but not GR, contribute to the increased HPA activity in the obese Zucker rats and that this seems more influential during stress than in the basal state. This may be exacerbated by impaired local regeneration of corticosterone by 11betaHSD1. These abnormalities could contribute to the subtle changes in the HPA axis in rodent and human obesity.

First forty-eight hours of developing otitis media: an experimental study.

The early inflammatory changes in the tympanic membrane were explored in 2 rat models. Acute otitis media was induced by instillation of Streptococcus pneumoniae type 3 into the middle ear cavity, and otitis media with effusion was induced by blockage of the eustachian tube. Otomicroscopic examination was performed before the rats were painlessly sacrificed at 3, 6, 9, 12, 18, 24, or 48 hours after initiation of the otitis media conditions. The tympanic membrane was studied by light and electron microscopy. Both acute otitis media and otitis media with effusion caused early inflammatory changes of the tympanic membrane, and the pars flaccida was the portion that reacted first. The inflammatory alterations were most pronounced in the acute otitis media model. The course of inflammation showed a bimodal pattern with an early deposition of a filamentous material with a band pattern, typical of fibrin. Despite a fluid-filled middle ear cavity, the inflammatory changes in the otitis media with effusion model were moderate, as was consistent with the clinical appearance of the tympanic membrane.

Myringotomized tympanic membranes cultured in vitro do not develop myringosclerosis.

The aim of this study was to evaluate the development of myringosclerosis in in vitro-cultured tympanic membranes. Sprague-Dawley rats were myringotomized bilaterally and the tympanic membranes were excised after sacrifice. The explants were placed in inserts in wells filled with a nutrient medium. Every second day the tympanic membranes were photodocumented and after 9 days the explants were prepared for histology. On the 4th day the explants had attached to the bottom of the inserts and the specimens had thickened. From the perforation borders and the dissection edges a thin outgrowth was extending across the surface. By Day 9 the perforation had clearly diminished in size when examined in a stereomicroscope. In a light microscope the keratin layer was seen to protrude towards the centre of the perforation and, at the borders, epithelial cells were bridging the gap. Neither the pars tensa nor the pars flaccida showed any sclerotic lesions. The pars flaccida had thickened and the basal cells of the outer keratinized epithelium had invaded the connective tissue. Inflammatory cells were sparse in both the pars tensa and pars flaccida. The in vitro-cultured myringotomized tympanic membrane therefore shows a similar healing pattern to that in vivo. However, inflammatory reactions are sparse and there is no development of myringosclerosis.

A novel series of 6-substituted 3-(pyrrolidin-1-ylmethyl)chromen-2-ones as selective monoamine oxidase (MAO) A inhibitors.

A series of 6-substituted 3-(pyrrolidin-1-ylmethyl)chromen-2-ones (coumarins) have been synthesized and their inhibitory activity to human monoamine oxidase A (MAO A) and B (MAO B) determined. Incorporation of a basic amino function in the C3 position together with substitution at the C6 position produced novel coumarin compounds with selectivity for the MAO A subtype. Substitution in the C6 position with small hydrophilic groups such as hydroxy (19, IC50 = 1.46 µM) or amino (18, IC50 = 3.77 µM) gave the most potent and selective compounds for MAO A. These compounds also showed excellent aqueous solubility properties. Compound 18 [6-amino-3-(pyrrolidin-1-ylmethyl)chromen-2-one] administrated in vivo induced in rat brain a neurotransmitter metabolite profile typical of MAO A inhibition: decreased 3,4-dihydroxyphenylacetic acid (DOPAC) and 5-hydroxyindoleacetic acid (5-HIAA) but increased 3-methoxytyramine (3-MT) levels.

Structure-activity relationship of 5-chloro-2-methyl-3-(1,2,3,6-tetrahydropyridin-4-yl)-1H-indole analogues as 5-HT(6) receptor agonists.

To further investigate the structure-activity relationship (SAR) of the 5-hydroxytryptamine type 6 (5-HT6) receptor agonist 5-chloro-2-methyl-3-(1,2,3,6-tetrahydropyridin-4-yl)-1H-indole (EMD386088, 6), a series of 2-methyl-3-(1,2,3,6-tetrahydropyridin-4-yl)-1H-indoles were synthesized, and in vitro affinity to, and functional activity at 5-HT6 receptors was tested. We focused on substituents made at the indole N(1)-, 2- and 5-positions and these were found to not only influence the affinity at 5-HT6 receptors but also the intrinsic activity leading to antagonists, partial agonists and full agonists. In order for a compound to demonstrate potent 5-HT6 receptor agonist properties, the indole N(1) should be unsubstituted, an alkyl group such as 2-methyl is needed and finally halogen substituents in the indole 5-position (fluoro, chloro or, bromo) were essential requirements. However, the introduction of a benzenesulfonyl group at N(1)-position switched the full agonist 6 to be a 5-HT6 receptor antagonist (30). A few compounds within the 2-methyl-3-(1,2,3,6-tetrahydropyridin-4-yl)-1H-indoles were also screened for off-targets and generally they displayed low affinity for other 5-HT subtypes and serotonin transporter protein (SERT).

Systematic in vivo screening of a series of 1-propyl-4-arylpiperidines against dopaminergic and serotonergic properties in rat brain: a scaffold-jumping approach.

A series of 1-propyl-4-arylpiperidines were synthesized and their effects on the dopaminergic and serotonergic systems tested in vivo and in vitro. Scaffold jumping among five- and six-membered bicyclic aryl rings attached to the piperidine ring had a marked impact on these effects. Potent and selective dopamine D(2) receptor antagonists were generated from 3-indoles, 3-benzoisoxazoles, 3-benzimidazol-2-one, and 3-benzothiophenes. In contrast, 3-benzofuran was a potent and selective inhibitor of monoamine oxidase (MAO) A. The effects of the synthesized compounds on 3,4-dihydroxyphenylacetic acid (DOPAC) levels correlated very well with their affinity for dopamine D(2) receptors and MAO A. In the 4-arylpiperidine series, the most promising compound for development was the 6-chloro-3-(1-propyl-4-piperidyl)-1H-benzimidazol-2-one (19), which displayed typical dopamine D(2) receptor antagonist properties in vivo but produced only a partial reduction on spontaneous locomotor activity. This indicates that the compound may have a lower propensity to induce parkinsonism in patients.