Objective In a previous pilot study, we have shown that intraarticular gold micro-particles can reduce knee osteoarthritis (KOA) pain at two years follow-up. Proteomic changes in serum and synovial fluid within eight weeks were associated with multiple inflammatory and immunological processes. The relation between the different biomarkers and the outcome measures is not known. We hypothesized that improvement in pain and function were associated with specific groups of biomarkers. We present the integrative analyses between proteomic biomarkers and outcomes. Design A cohort of 30 patients, with moderate KOA, were included. Using the patients’ synovial fluid as the carrier, 20 mg gold microparticles were injected intraarticularly. Clinical outcome measures at inclusion, 8 weeks, and 2 years, were the PainDetect questionnaire, WOMAC pain, stiffness, and function. In addition, Quantitative Sensory Testing, Pain Pressure Threshold, Temporal Summation, Conditioned Pain Modulation, and pain diary were assessed at inclusion and after 8 weeks. Proteomic analysis was performed on SF and blood samples before and after 8 weeks of treatment. Results Linear combinations of serum or synovial biomarkers changed significantly alongside the effect measures and PainDetect scores following gold micro-particle treatment. Of particular interest was identifying multiple members of a molecular complex that is suggestive of neural tissue regeneration and modulation following gold micro-particle treatment. Conclusions Gold microparticles are a possible future option for the treatment of knee osteoarthritis. The treatment triggers putative regenerative and inflammation-modulating molecular mechanisms.

Abstract Background Recently, in an open pilot study, we found up to two years, a potential pain-relieving effect of intra-articular gold micro-particles using the patient’s synovial fluid for patients with knee osteoarthritis (KOA). During the study the excluded group of patients, due to multisite pain, co-morbidities, and other exclusion criteria., received intra-articular gold micro-particles using hyaluronic acid,. We aimed to identify if pre-treatment characteristics influence the global outcome two years after intra-articular treatment for painful KOA with gold microparticles using hyaluronic acid. Methods Using hyaluronic acid as the carrier, 136 patients with KOA received intraarticular injections with 20 mg gold microparticles (72.000 particles, 20–40 μm in diameter). In the analysis, we included the Global Rating of Change Scale, Pain Detect Questionnaire (PDQ), Body Mass Index (BMI), and Kellgren & Lawrence score at the inclusion, Western Ontario, and McMaster Universities Osteoarthritis Index (WOMAC) sub-scores for pain, stiffness, and function at inclusion and two years. Results On the Global Rating Change Scale, 69.1% of patients reported a positive effect, 28.7% no effect, and 2.2% worse. PDQ and the three WOMAC subscores all improved at two years of follow-up. PDQ ≥ 13 (P = 0.028), BMI (P = 0.022) and Kellgren & Lawrence grade 4 (P = 0.028) at inclusion reduced the effect with a minor odds ratio compared to the baseline effect of treatment (P = 0.025). WOMAC subscores at inclusion did not influence the outcome (P > 0.5). Conclusions Severe osteoarthritis, obesity, and neuropathic pain, reduced the effect of intra-articular gold microparticles for knee OA. Trial registration The study followed the principles of the Declaration of Helsinki and was approved by the local ethics committee of the North Denmark Region by 27/07/2016 (N-20,160,045). The regional data protection agency approved the project by 06/07/2016 (2008-58-0028, ID 2016 − 116) and registered in ClinicalTrial.Gov by 04/01/2018 (NCT03389906).

Introduction: Pleural effusions of unknown etiology frequently occur in lung transplant (LTX) recipients and are associated with reduced survival. The pulmonary microbiome is increasingly recognized in LTX recipients. The pleural microbiome however, has not yet been investigated and several factors, e. g. open chest surgery, severe immunosuppression and long-term macrolide therapy might alter the pleural microbiome in LTX recipients. Methods: We analyzed a cohort of 52 pleural effusions of lung transplant recipients and non-transplant patients and 14 matched bronchoalveolar lavage samples of lung transplant recipients by 16S rRNA sequencing. Results: Significant differences in pleural cytology as well as alpha- and beta-diversity were found between early and late pleural effusions after LTX. Late pleural effusions after LTX showed a trend towards a higher alpha- and beta-diversity compared to non-transplant effusions. The pulmonary microbiome of BAL samples showed a correlation to the pleural microbiome, which was associated with an exudative composition of the pleural effusion. Conclusions: Lung transplantation and time after lung transplantation seem to affect the pleural microbiome. As the pulmonary and pleural microbiome directly correlated, we suggest a communication of the microbiome of both compartments.

Polymyalgia rheumatica (PMR) is an inflammatory disorder of unknown etiology, sharing symptoms with giant cell arthritis (GCA) and rheumatoid arthritis (RA). The pathogenic inflammatory roots are still not well understood, and there is a lack of extensive biomarker studies to explain the disease debut and post-acute phase. This study aimed to deeply analyze the serum proteome and inflammatory response of PMR patients before and after glucocorticoid treatment. We included treatment-naïve PMR patients, collecting samples before and after 3 months of treatment. For comparison, disease-modifying antirheumatic drug (DMARD)-naïve RA patients were included and matched to healthy controls (CTL). The serum proteome was examined using label-free quantitative mass spectrometry, while inflammation levels were assessed using multiplex inflammatory cytokine and cell-free DNA assays. The serum proteomes of the four groups comprised acute phase reactants, coagulation factors, complement proteins, immunoglobulins, and apolipoproteins. Serum amyloid A (SAA1) was significantly reduced by active PMR treatment. Cell-free DNA levels in PMR and RA groups were significantly higher than in healthy controls due to acute inflammation. Complement factors had minimal changes post-treatment. The individual serum proteome in PMR patients showed over 100 abundantly variable proteins, emphasizing the systemic impact of PMR disease debut and the effect of treatment. Interleukin (IL)-6 and interferon-gamma (IFN-γ) were significantly impacted by glucocorticoid treatment. Our study defines the PMR serum proteome during glucocorticoid treatment and highlights the role of SAA1, IL-6, and IFN-γ in treatment responses. An involvement of PGLYRP2 in acute PMR could indicate a response to bacterial infection, highlighting its role in the acute phase of the immune response. The results suggest that PMR may be an aberrant response to a bacterial infection with an exacerbated IL-6 and acute phase inflammatory response and molecular attempts to limit the inflammation.

Inflammatory Bowel Disease (IBD) affects approximately 0.3% of the global population, with incidence rates rising dramatically worldwide. Emerging evidence points to an interplay between exposome factors such as diet and gut microbiota, host genetics, and the immune system as crucial elements in IBD development. ATP-binding cassette (ABC) transporters, including human p-glycoprotein encoded by the Abcb1 gene, influence intestinal inflammation, and their expression may interact with environmental factors such as diet and gut microbes. Our study aimed to examine the impact of protein sources on a genetic colitis mouse model.MethodsAbcb1a-deficient colitis mice were fed either casein or red meat-supplemented diets to investigate potential colitis-aggravating components in red meat and their effects on host-microbiota interactions. We conducted deep label free quantitative proteomic inflammation profiling of gastrointestinal tissue (colon, ileum) and urine, and determined the overall microbiome in feces using 16S rRNA gene sequencing. Microbiota shifts by diet and protein transporter impairment were addressed by multivariate statistical analysis. Colon and systemic gut inflammation were validated through histology and immune assays, respectively.ResultsA quantitative discovery based proteomic analysis of intestinal tissue and urine revealed associations between ileum and urine proteomes in relation to Abcb1a deficiency. The absence of Abcb1a efflux pump function and diet-induced intestinal inflammation impacted multiple systemic immune processes, including extensive neutrophil extracellular trap (NET) components observed in relation to neutrophil degranulation throughout the gastrointestinal tract. The colitis model’s microbiome differed significantly from that of wild-type mice, indicating the substantial influence of efflux transporter deficiency on microbiota.ConclusionThe proteomic and microbiota analyzes of a well-established murine model enabled the correlation of gastrointestinal interactions not readily identifiable in human cohorts. Insights into dysregulated biological pathways in this disease model might offer translational biomarkers based on NETs and improved understanding of IBD pathogenesis in human patients. Our findings demonstrate that drug transporter deficiency induces substantial changes in the microbiota, leading to increased levels of IBD-associated strains and resulting in intestinal inflammation.GRAPHICAL ABSTRACT

Background: Blood–barrier (BBB) breakdown and active inflammation are hallmarks of relapsing multiple sclerosis (RMS), but the molecular events contributing to the development of new lesions are not well explored. Leaky endothelial junctions are associated with increased production of endothelial-derived extracellular microvesicles (EVs) and result in the entry of circulating immune cells into the brain. MRI with intravenous gadolinium (Gd) can visualize acute blood–barrier disruption as the initial event of the evolution of new lesions. Methods: Here, weekly MRI with Gd was combined with proteomics, multiplex immunoassay, and endothelial stress-optimized EV array to identify early markers related to BBB disruption. Five patients with RMS with no disease-modifying treatment were monitored weekly using high-resolution 3T MRI scanning with intravenous gadolinium (Gd) for 8 weeks. Patients were then divided into three groups (low, medium, or high MRI activity) defined by the number of new, total, and maximally enhancing Gd-enhancing lesions and the number of new FLAIR lesions. Plasma samples taken at each MRI were analyzed for protein biomarkers of inflammation by quantitative proteomics, and cytokines using multiplex immunoassays. EVs were characterized with an optimized endothelial stress EV array based on exosome surface protein markers for the detection of soluble secreted EVs. Results: Proteomics analysis of plasma yielded quantitative information on 208 proteins at each patient time point (n = 40). We observed the highest number of unique dysregulated proteins (DEPs) and the highest functional enrichment in the low vs. high MRI activity comparison. Complement activation and complement/coagulation cascade were also strongly overrepresented in the low vs. high MRI activity comparison. Activation of the alternative complement pathway, pathways of blood coagulation, extracellular matrix organization, and the regulation of TLR and IGF transport were unique for the low vs. high MRI activity comparison as well, with these pathways being overrepresented in the patient with high MRI activity. Principal component analysis indicated the individuality of plasma profiles in patients. IL-17 was upregulated at all time points during 8 weeks in patients with high vs. low MRI activity. Hierarchical clustering of soluble markers in the plasma indicated that all four MRI outcomes clustered together with IL-17, IL-12p70, and IL-1β. MRI outcomes also showed clustering with EV markers CD62E/P, MIC A/B, ICAM-1, and CD42A. The combined cluster of these cytokines, EV markers, and MRI outcomes clustered also with IL-12p40 and IL-7. All four MRI outcomes correlated positively with levels of IL-17 (p < 0.001, respectively), and EV-ICAM-1 (p < 0.0003, respectively). IL-1β levels positively correlated with the number of new Gd-enhancing lesions (p < 0.01), new FLAIR lesions (p < 0.001), and total number of Gd-enhancing lesions (p < 0.05). IL-6 levels positively correlated with the number of new FLAIR lesions (p < 0.05). Random Forests and linear mixed models identified IL-17, CCL17/TARC, CCL3/MIP-1α, and TNF-α as composite biomarkers predicting new lesion evolution. Conclusions: Combination of serial frequent MRI with proteome, neuroinflammation markers, and protein array data of EVs enabled assessment of temporal changes in inflammation and endothelial dysfunction in RMS related to the evolution of new and enhancing lesions. Particularly, the Th17 pathway and IL-1β clustered and correlated with new lesions and Gd enhancement, indicating their importance in BBB disruption and initiating acute brain inflammation in MS. In addition to the Th17 pathway, abundant protein changes between MRI activity groups suggested the role of EVs and the coagulation system along with innate immune responses including acute phase proteins, complement components, and neutrophil degranulation.

Abstract Background: Gold microparticles injected intra-articular in knee osteoarthritic joints (OA) may provide pain relief. The present open, exploratory study sought to identify the efficacy and factors associated with the outcomes. Methods: This study included thirty patients with knee OA who received intraarticular injections with 20 mg gold microparticles (72.000 particles, 20–40 µm in diameter) using the patient’s synovial fluid as the carrier, and 136 patients with knee OA who received 20 mg gold microparticles using hyaluronic acid as the carrier. In the analysis, we included the Global Rating of Change Scale, PainDetectQuestionnaire (PDQ), the use of a carrier, Body Mass Index (BMI), and Kellgren Lawrence score at the inclusion, Western Ontario, and McMaster Universities Osteoarthritis Index (WOMAC) sub-scores for pain, stiffness, and function at inclusion and two years. Results: On the Global Rating Change Scale, 72.9% of patients reported a positive effect, 23.5% no effect, and 2.4% worse. PDQ and the three WOMAC subscores all improved at two years of follow-up. The use of carrier did not influence the effect at two years of follow-ups corrected for the other selected co-variables (P = 0.36). PainDetect ≥ 13 (P = 0.0027), BMI (P = 0.008) and Kellgren-Lawrence grade 4 (P = 0.049) at inclusion reduced the effect with a minor odds ratio compared to the baseline effect of treatment (P = 0.03). WOMAC subscores at inclusion did not influence the effect (P > 0.5). Conclusions: A single intra-articular injection of gold microparticles was associated with pain reduction and improved function in patients with knee OA. Neuropathic pain, obesity, and severe osteoarthritis, to a minor degree, reduced the effect of intra-articular gold microparticles for knee OA. Trial registration The study followed the principles of the Declaration of Helsinki and was approved by the local ethics committee of the North Denmark Region by 27/07/2016 (N-20160045). The regional data protection agency approved the project by 06/07/2016 (2008-58-0028, ID 2016-116) and registered in ClinicalTrial.Gov by 04/01/2018 (NCT03389906).

BACKGROUND:This exploratory study investigates if intra-articular injected gold microparticles in knee osteoarthritis (KOA) reduce immunomodulatory-based pain via proteomic changes in the synovial fluid (SF) and serum.METHODS:Thirty patients with moderate KOA were included. Intraarticular injections with 20 mg gold microparticles (72.000 particles, 20-40 µm in diameter) using the patient's synovial fluid (SF) as carrier were performed. Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC) subscores for pain, stiffness, and function were assessed at inclusion, 8 weeks and 2 years The PainDetect questionnaire, pain pressure threshold (PPT), temporal summation (TS), and conditioned pain modulation (CPM), and pain diary were assessed at inclusion and 8 weeks. Proteome analysis was performed on SF and blood samples before and after 8 weeks of treatment.RESULTS:A decrease in WOMAC scores (pain (p = 0.0001), stiffness (p = 0.0088), activity (p = 0.0001)), PainDetect (p = 0.0002) and increase in PPT (p = 0.001) and CPM (p = 0.021) and a decrease in TS (p = 0.03) were found after 8 weeks compared to inclusion assessments. At 2 years follow-up compared to baseline there was a decrease in WOMAC scores (pain (p = 0.0001), stiffness (p = 0.007), activity (p = 0.0001)) and PainDetect (p = 0.0001). In SF, 28 different proteins were downregulated and 11 upregulated (p < 0.05) mainly associated immune response. Similarly, 31 proteins were downregulated and 1 upregulated in serum (p < 0.05) reflecting key immune response and anatomical structure development processes. No adverse effects related to the treatment were recorded.CONCLUSIONS:Gold microparticles injected intra-articular in KOA joints may provide pain relief and an inflammatory modulatory effect based on proteome changes found in SF and serum. A randomized, controlled, double-blind study is needed to infer a conclusion.SIGNIFICANCE:This study indicates that intra-articular gold may provide advantages in clinical practice for managing knee osteoarthritic pain. The use of intraarticular gold can add new knowledge to the treatment of inflammation and pain.

Introduction Immune-checkpoint inhibitors (ICI) works by blocking inhibitory signals of T cells. This produces an effective anti-tumor response but can also cause immune-related adverse events (irAEs). Most irAEs are transient, but ICI-induced inflammatory arthritis (ICI-IIA) might become chronic and affect the quality-of-life, or even necessitate treatment discontinuation. However, there exist no tools to identify patients that are susceptible to develop ICI-IIA.Areas covered This non-systematic review briefly presents a sparse number of studies, that have tried to identify circulating biomarkers for early prediction of ICI-IIA. Challenges, recommendations, and possibilities related to biomarker discovery in the context of ICI-IIA are then covered.Expert opinion Improved diagnosis adapted from rheumatological settings is needed for future studies to avoid a major pitfall of bad endpoints. Synovial tissue biopsies, omics technologies and particularly integration of multiple omics data is useful when searching for biomarkers of ICI-IIA and can also help unravel underlying biological mechanisms. Future biomarkers could ultimately aid clinical decision-making and facilitate early prophylaxis, pave the way for new treatment or even translational models to study autoimmune arthritis.

Background: MiR-146a is an important regulator of innate inflammatory responses and is also implicated in cell death and survival. Methods: By sorting CNS resident cells, microglia were the main cellular source of miR-146a. Therefore, we investigated microglia function and phenotype in miR-146a knock-out (KO) mice, analyzed the proteome of KO and wild-type (WT) microglia by LC-MS/MS, and examined miR-146a expression in different brain lesions of patients with multiple sclerosis (MS). Results: When stimulated with LPS or myelin in vitro, microglia from KO mice expressed higher levels of IL-1β, TNF, IL-6, IL-10, CCL3, and CCL2 compared to WT. Stimulation increased migration and phagocytosis of WT but not KO microglia. CD11c+ microglia were induced by cuprizone (CPZ) in the WT mice but less in the KO. The proteome of ex vivo microglia was not different in miR-146a KO compared to WT mice, but CPZ treatment induced differential and reduced protein responses in the KO: GOT1, COX5b, CRYL1, and cystatin-C were specifically changed in KO microglia. We explored discriminative features of microglia proteomes: sparse Partial Least Squares-Discriminant Analysis showed the best discrimination when control and CPZ-treated conditions were compared. Cluster of ten proteins separated WT and miR-146a KO microglia after CPZ: among them were sensomes allowing to perceive the environment, Atp1a3 that belongs to the signature of CD11c+ microglia, and proteins related to inflammatory responses (S100A9, Ppm1g). Finally, we examined the expression of miR-146a and its validated target genes in different brain lesions of MS patients. MiR-146 was upregulated in all lesion types, and the highest expression was in active lesions. Nineteen of 88 validated target genes were significantly changed in active lesions, while none were changed in NAWM. Conclusion: Our data indicated that microglia is the major source of miR-146a in the CNS. The absence of miR-146a differentially affected microglia function and proteome, and miR-146a may play an important role in gene regulation of active MS lesions.

INTRODUCTION:The development of companion diagnostics (CDx) will increase efficacy and cost-benefit markedly, compared to the currently prevailing trial-and-error approach for treatment. Recent improvements in high-throughput protein technology have resulted in large amounts of predictive biomarkers that are potentially useful components of future CDx assays. Current high multiplex protein arrays are suitable for discovery-based approaches, while low-density and more simple arrays are suitable for use in point-of-care facilities.AREA COVERED:This review discusses the technical platforms available for protein array focused CDx, explains the technical details of the platforms and provide examples of clinical use, ranging from multiplex arrays to low-density clinically applicable arrays. We thereafter highlight recent predictive biomarkers within different disease areas, such as oncology and autoimmune diseases. Lastly, we discuss some of the challenges connected to the implementation of CDx assays as point-of-care tests.EXPERT OPINION:Recent advances in the field of protein arrays have enabled high-density arrays permitting large biomarker discovery studies, which are beneficial for future CDx assays. The density of protein arrays range from a single protein to proteome-wide arrays, allowing the discovery of protein signatures that may correlate with drug response. Protein arrays will undoubtedly play a key role in future CDx assays.