Investigating cryopreserved PBMC functionality in an antigen-induced model of sarcoidosis granuloma formation.

Sarcoidosis, a systemic inflammatory disease, poses challenges in understanding its etiology and variable clinical courses. Despite ongoing uncertainty about causative agents and genetic predisposition, granuloma formation remains its hallmark feature. To address this, we developed a validated in vitro human granuloma model using patient-derived peripheral blood mononuclear cells (PBMCs), offering a dynamic platform for studying early granuloma formation and sarcoidosis pathogenesis. However, a current limitation of this model is its dependence on freshly isolated PBMCs obtained from whole blood. While cryopreservation is a common method for long-term sample preservation, the biological effects of freezing and thawing PBMCs on granuloma formation remain unclear. This study aimed to assess the viability and functionality of cryopreserved sarcoidosis PBMCs within the granuloma model, revealing similar granulomatous responses to fresh cells and highlighting the potential of cryopreserved PBMCs as a valuable tool for studying sarcoidosis and related diseases.

Application of laboratory models for sarcoidosis research.

This manuscript will review the implications and applications of sarcoidosis models towards advancing our understanding of sarcoidosis disease mechanisms, identification of biomarkers, and preclinical testing of novel therapies. Emerging disease models and innovative research tools will also be considered.

Impact of concurrent glomerulonephritis on outcomes of diffuse alveolar haemorrhage in antineutrophil cytoplasmic antibody-associated vasculitis.

OBJECTIVES: Antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV) commonly presents with diffuse alveolar haemorrhage (DAH) and/or glomerulonephritis. Patients who present with DAH but without kidney involvement have been understudied. METHODS: Patients with DAH diagnosed by bronchoscopy and attributed to AAV over 8.5 years were retrospectively identified through electronic medical records and bronchoscopy reporting software. Patients with end-stage kidney disease (ESKD) or prior kidney transplant were excluded. Characteristics, treatments, and outcomes were abstracted. RESULTS: 30 patients were identified with DAH secondary to AAV. Five with ESKD or prior kidney transplant, and one with concomitant anti-glomerular basement membrane disease, were excluded, leaving 24 patients for analysis. At the time of qualifying bronchoscopy, six patients had no apparent kidney involvement by AAV, while eight of 18 with kidney involvement required dialysis. Of the eight patients dialysed during the initial hospitalisation, four were declared to have ESKD and three died in the subsequent year (one of whom did both). None of the 16 patients without initial dialysis requirement developed kidney involvement requiring dialysis in the subsequent year, though three of the six without initial evidence of kidney involvement by AAV ultimately developed it. No patient without initial kidney involvement died during follow-up. CONCLUSIONS: In our cohort, patients with DAH due to AAV without initial kidney involvement did not develop kidney involvement requiring dialysis or die during the follow-up period, though half of patients without initial evidence of kidney involvement subsequently developed it. Larger studies are warranted to better characterise this population and guide medical management.

Leveraging in vitro and pharmacokinetic models to support bench to bedside investigation of XTMAB-16 as a novel pulmonary sarcoidosis treatment.

Background: Sarcoidosis is a chronic, multisystem inflammatory disorder characterized by non-caseating epithelioid granulomas; infiltration of mononuclear cells; and destruction of microarchitecture in the skin, eye, heart, and central nervous system, and the lung in >90% of cases. XTMAB-16 is a chimeric anti-tumor necrosis factor alpha (TNFα) antibody, distinct from other anti-TNF antibodies based on its molecular structure. The efficacy of XTMAB-16 has not been clinically demonstrated, and it is still undergoing clinical development as a potential treatment for sarcoidosis. The current study demonstrates the activity of XTMAB-16 in a well-established in vitro sarcoidosis granuloma model, although XTMAB-16 is not yet approved by the United States Food and Drug Administration (FDA) for treatment of sarcoidosis, or any other disease. Objective: To provide data to guide safe and efficacious dose selection for the ongoing clinical development of XTMAB-16 as a potential treatment for sarcoidosis. Methods: First, XTMAB-16 activity was evaluated in an established in vitro model of granuloma formation using peripheral blood mononuclear cells from patients with active pulmonary sarcoidosis to determine a potentially efficacious dose range. Second, data obtained from the first-in-human study of XTMAB-16 (NCT04971395) were used to develop a population pharmacokinetic (PPK) model to characterize the pharmacokinetics (PK) of XTMAB-16. Model simulations were performed to evaluate the sources of PK variability and to predict interstitial lung exposure based on concentrations in the in vitro granuloma model. Results: XTMAB-16 dose levels of 2 and 4 mg/kg, once every 2 weeks (Q2W) or once every 4 weeks (Q4W) for up to 12 weeks, were supported by data from the non-clinical, in vitro secondary pharmacology; the Phase 1 clinical study; and the PPK model developed to guide dose level and frequency assumptions. XTMAB-16 inhibited granuloma formation and suppressed interleukin-1ß (IL-1ß) secretion in the in vitro granuloma model with a half maximal inhibitory concentration (IC50) of 5.2 and 3.5 µg/mL, respectively. Interstitial lung concentrations on average, following 2 or 4 mg/kg administered Q2W or Q4W, are anticipated to exceed the in vitro IC50 concentrations. Conclusion: The data presented in this report provide a rationale for dose selection and support the continued clinical development of XTMAB-16 for patients with pulmonary sarcoidosis.

Rationale and design of a randomized controlled clinical trial; Titration of Oxygen Levels (TOOL) during mechanical ventilation.

BACKGROUND: Both hyperoxemia and hypoxemia are deleterious in critically ill patients. Targeted oxygenation is recommended to prevent both of these extremes, however this has not translated to the bedside. Hyperoxemia likely persists more than hypoxemia due to absence of immediate discernible adverse effects, cognitive biases and delay in prioritization of titration. METHODS: We present the methodology for the Titration Of Oxygen Levels (TOOL) trial, an open label, randomized controlled trial of an algorithm-based FiO2 titration with electronic medical record-based automated alerts. We hypothesize that the study intervention will achieve targeted oxygenation by curbing episodes of hyperoxemia while preventing hypoxemia. In the intervention arm, electronic alerts will be used to titrate FiO2 if SpO2 is ≥94% with FiO2 levels ≥0.4 over 45 min. FiO2 will be titrated per standard practice in the control arm. This study is being carried out with deferred consent. The sample size to determine efficacy is 316 subjects, randomized in a 1:1 ratio to the intervention vs. control arm. The primary outcome is proportion of time during mechanical ventilation spent with FiO2 ≥ 0.4 and SpO2 ≥ 94%. We will also assess proportion of time during mechanical ventilation spent with SpO2 < 88%, duration of mechanical ventilation, length of ICU and hospital stay, hospital mortality, and adherence to electronic alerts as secondary outcomes. CONCLUSION: This study is designed to evaluate the efficacy of a high fidelity, bioinformatics-based, electronic medical record derived electronic alert system to improve targeted oxygenation in mechanically ventilated patients by reducing excessive FiO2 exposure.

Early Titration of Oxygen During Mechanical Ventilation Reduces Hyperoxemia in a Pilot, Feasibility, Randomized Control Trial for Automated Titration of Oxygen Levels.

Timely regulation of oxygen (Fio2) is essential to prevent hyperoxemia or episodic hypoxemia. Exposure to excessive Fio2 is often noted early after onset of mechanical ventilation. In this pilot study, we examined the feasibility, safety, and efficacy of a clinical trial to prioritize Fio2 titration with electronic alerts to respiratory therapists. STUDY DESIGN: Open-labeled, randomized control pilot trial. SETTING: Medical ICU. SUBJECTS: Adults requiring mechanical ventilation. INTERVENTIONS: Protocolized oxygen titration was initiated one hour after initiation of mechanical ventilation. When Spo2 exceeded 92% while on Fio2 ≥ 0.5, an electronic alert to respiratory therapists was triggered at 30-minute intervals. In the control arm, respiratory therapists titrated Fio2 by standard physician's orders. MEASUREMENTS AND MAIN RESULTS: The primary end point was to determine if early Fio2 titration based on automated alerts was feasible in terms of reducing hyperoxemia. Secondary analyses included the number and frequency of alerts, mechanical ventilation duration, and ICU length of stay. Among 135 randomized patients, 72 were assigned to the intervention arm and 63 to the control arm. A total 877 alerts were sent. Exposure to hyperoxemia was significantly reduced in the intervention group by a median of 7.5 hours (13.7 [interquartile range (IQR), 2.9-31.1] vs 21.2 [IQR, 10.9-64.4]; p < 0.0004). Maximal Fio2 titration during the first quartile resulted in significant reduction in mechanical ventilation duration and ICU stay. Minor hypoxemic events (Spo2 < 88%) represented 12% of alerts, 9% were transient and responded to a single Fio2 increase, whereas 3% of alerts were associated with recurrent transient hypoxemia. CONCLUSIONS: Our pilot study indicates that early Fio2 titration driven by automated alerts is feasible in the ICU, as reflected by a statistically significant reduction of hyperoxemia exposure, limited consequential hypoxemia, and reduced ICU resource utilization. The encouraging results of this pilot study need to be validated in a larger ICU cohort.

The influence of age and sex in sarcoidosis.

PURPOSE OF REVIEW: This review aims to describe how the clinical manifestations of sarcoidosis may be shaped by the effects of sex hormones and by age dependent changes in immune functions and physiology This review is intended to highlight the need to consider the effects of sex and sex in future studies of sarcoidosis. RECENT FINDINGS: The clinical manifestations of sarcoidosis differ based on sex and gender There is emerging evidence that female and male hormones and X-linked genes are important determinants of immune responses to environmental antigens, which has important implications for granuloma formation in the context of sarcoidosis Furthermore, sex hormone levels predictably change throughout adolescence and adulthood, and this occurs in parallel with the onset immune senescence and changes in physiology with advanced age. SUMMARY: Recent studies indicate that sex and age are important variables shaping the immune response of humans to environmental antigens We posit herein that sex and age are important determinants of sarcoidosis clinical phenotypes Many gaps in our understanding of the roles played by sex and gender in sarcoidosis, and these need to be considered in future studies.

The landscape of transcriptomic and proteomic studies in sarcoidosis.

Multiple overlapping pathways are identified in tissue, BAL cells, PBMCs and a sarcoidosis in vitro granuloma model. Inferences from omic studies are constrained by small sample sizes. Studies comparing differences between sarcoidosis phenotypes are needed. https://bit.ly/30NaHz4.

Inflammasome Activation in an In Vitro Sepsis Model Recapitulates Increased Monocyte Distribution Width Seen in Patients With Sepsis.

OBJECTIVES: Increased monocyte distribution width (MDW) has recently been shown to be a reliable indicator of early sepsis detection. This study therefore sought to determine if inflammasome activation can be linked to monocyte size changes in sepsis. DESIGN: An in vitro sepsis model using bacterial endotoxin (lipopolysaccharide [LPS]) to study the effect of inflammasome activation on monocyte cell size distribution by microscopy and MDW measurements using a standard clinical hematology analyzer. SETTING: University research laboratory. SUBJECTS: Healthy adult volunteers and cultured human monocyte cells in wild-type state and after clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 knockout of key inflammasome components (apoptosis-associated speck-like protein containing a caspase recruitment domain, caspase-1, gasdermin-D). INTERVENTIONS: In vitro treatment of specimens with bacterial LPS. MEASUREMENTS AND MAIN RESULTS: Wild-type THP1 cells demonstrated a significant increase in cell area (207 µm2 [159-400 µm2] vs 160 µm2 [134-198 µm2]; p < 0.001) and distribution width (198 vs 55 µm2; p < 0.0001) by microscopy following treatment with LPS. Increased MDW correlated with inflammasome activation as demonstrated by release of interleukin (IL)-1ß and with the presence of large distended pyroptotic cells by microscopy. All of these effects were blocked in the inflammasome knockout cells. Whole blood samples treated similarly also demonstrated IL-1ß release and increased MDW (median 24.7 U [22.2-27.2 U] vs 16.3 U [15.1-17.6 U]; p = 0.008) as measured using the Beckman-Coulter Unicel DxH900 analyzer. When peripheral blood mononuclear cells were isolated prior to treatment with LPS, microscopy confirmed the presence of large pyroptotic cells correlating to IL-1ß release in the human subject samples as well. CONCLUSIONS: The increased MDW seen in patients with sepsis can be reproduced in an in vitro sepsis model and blocked using clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 technology to inactivate the inflammasome. These findings suggest that pyroptotic cellular swelling underlies changes in MDW in septic patients and connect MDW to early events in the inflammatory cascade of sepsis.

A Pilot Randomized Trial of Transdermal Nicotine for Pulmonary Sarcoidosis.

BACKGROUND: Tobacco smoking is associated with a reduced risk of developing sarcoidosis, and we previously reported that nicotine normalizes immune responses to environmental antigens in patients with active pulmonary sarcoidosis. The effects of nicotine on the progression of pulmonary sarcoidosis are unknown. RESEARCH QUESTION: Is nicotine treatment well tolerated, and will it improve lung function in patients with active pulmonary sarcoidosis? STUDY DESIGN AND METHODS: With local institutional review board approval, a randomized, double-blind, controlled pilot trial was conducted of daily nicotine transdermal patch treatment (21 mg daily) or placebo patch use for 24 weeks. The Ohio State University Wexner Medical Center and Cleveland Clinic enrolled 50 consecutive subjects aged ≥ 18 years with active pulmonary sarcoidosis, based on symptoms (ie, dyspnea, cough) and objective radiographic evidence of infiltrates consistent with nonfibrotic lung disease. Each study group was compared at 26 weeks based on repeated measures of FVC, FEV1, quantitative lung texture score based on CT texture analysis, Fatigue Assessment Score (FAS), St. George's Respiratory Questionnaire (SGRQ), and the Sarcoidosis Assessment Tool. RESULTS: Nicotine treatment was associated with a clinically significant, approximately 2.1% (70 mL) improvement in FVC from baseline to 26 weeks. FVC decreased by a similar amount (2.2%) in the placebo group, with a net increase of 140 mL (95% CI, 10-260) when comparing nicotine vs placebo groups at 26 weeks. FEV1 and FAS improved marginally in the nicotine-treated group, compared with those on placebo. No improvement was observed in lung texture score, FAS, St. George's Respiratory Questionnaire score, or the Sarcoidosis Assessment Tool. There were no reported serious adverse events or evidence of nicotine addiction. INTERPRETATION: Nicotine treatment was well tolerated in patients with active pulmonary sarcoidosis, and the preliminary findings of this pilot study suggest that it may reduce disease progression, based on FVC. CLINICAL TRIAL REGISTRATION: ClinicalTrials.gov; No.: NCT02265874; URL: www.clinicaltrials.gov.

Phase II Investigation of the Efficacy of Antimycobacterial Therapy in Chronic Pulmonary Sarcoidosis.

BACKGROUND: A Phase I, single-center investigation found that 8 weeks of antimycobacterial therapy improved sarcoidosis FVC. Safety and efficacy assessments have not been performed in a multicenter cohort. RESEARCH QUESTION: The objective of this study was to determine the safety and efficacy of antimycobacterial therapy on the physiological and immunologic end points of sarcoidosis. STUDY DESIGN AND METHODS: In a double-blind, placebo-controlled, multicenter investigation, patients with pulmonary sarcoidosis were randomly assigned to receive 16 weeks of concomitant levofloxacin, ethambutol, azithromycin, and rifabutin (CLEAR) or matching placebo to investigate the effect on FVC. The primary outcome was a comparison of change in percentage of predicted FVC among patients randomized to receive CLEAR or placebo in addition to their baseline immunosuppressive regimen. Secondary outcomes included 6-min walk distance (6MWD), St. George's Respiratory Questionnaire (SGRQ) score, adverse events, and decrease in mycobacterial early secreted antigenic target of 6 kDa (ESAT-6) immune responses. RESULTS: The intention-to-treat analysis revealed no significant differences in change in FVC among the 49 patients randomized to receive CLEAR (1.1% decrease) compared with the 48 randomized to receive placebo (0.02% increase) (P = .64). Physiological parameters such as the change in 6MWD were likewise similar (P = .91); change in SGRQ favored placebo (-8.0 for placebo vs -1.5 for CLEAR; P = .028). The per-protocol analysis revealed no significant change in FVC at 16 weeks between CLEAR and placebo. There was no significant change in 6MWD (36.4 m vs 6.3 m; P = .24) or SGRQ (-2.3 vs -7.0; P = .14). A decline in ESAT-6 immune responses at 16 weeks was noted among CLEAR-treated patients (P = .0003) but not patients receiving placebo (P = .24). INTERPRETATION: Despite a significant decline in ESAT-6 immune responses, a 16-week CLEAR regimen provided no physiological benefit in FVC or 6MWD among patients with sarcoidosis.

Circulating exosomal microRNA expression patterns distinguish cardiac sarcoidosis from myocardial ischemia.

OBJECTIVE: Cardiac sarcoidosis is difficult to diagnose, often requiring expensive and inconvenient advanced imaging techniques. Circulating exosomes contain genetic material, such as microRNA (miRNA), that are derived from diseased tissues and may serve as potential disease-specific biomarkers. We thus sought to determine whether circulating exosome-derived miRNA expression patterns would distinguish cardiac sarcoidosis (CS) from acute myocardial infarction (AMI). METHODS: Plasma and serum samples conforming to CS, AMI or disease-free controls were procured from the Biologic Specimen and Data Repository Information Coordinating Center repository and National Jewish Health. Next generation sequencing (NGS) was performed on exosome-derived total RNA (n = 10 for each group), and miRNA expression levels were compared after normalization using housekeeping miRNA. Quality assurance measures excluded poor quality RNA samples. Differentially expressed (DE) miRNA patterns, based upon >2-fold change (p < 0.01), were established in CS compared to controls, and in CS compared to AMI. Relative expression of several DE-miRNA were validated by qRT-PCR. RESULTS: Despite the advanced age of the stored samples (~5-30 years), the quality of the exosome-derived miRNA was intact in ~88% of samples. Comparing plasma exosomal miRNA in CS versus controls, NGS yielded 18 DE transcripts (12 up-regulated, 6 down-regulated), including miRNA previously implicated in mechanisms of myocardial injury (miR-92, miR-21) and immune responses (miR-618, miR-27a). NGS further yielded 52 DE miRNA in serum exosomes from CS versus AMI: 5 up-regulated in CS; 47 up-regulated in AMI, including transcripts previously detected in AMI patients (miR-1-1, miR-133a, miR-208b, miR-423, miR-499). Five miRNAs with increased DE in CS included two isoforms of miR-624 and miR-144, previously reported as markers of cardiomyopathy. CONCLUSIONS: MiRNA patterns of exosomes derived from CS and AMI patients are distinct, suggesting that circulating exosomal miRNA patterns could serve as disease biomarkers. Further studies are required to establish their specificity relative to other cardiac disorders.

Phagosome-regulated mTOR signalling during sarcoidosis granuloma biogenesis.

INTRODUCTION: Sarcoidosis and tuberculosis are granulomatous pulmonary diseases characterised by heightened immune reactivity to Mycobacterium tuberculosis antigens. We hypothesised that an unsupervised analysis comparing the molecular characteristics of granulomas formed in response to M. tuberculosis antigens in patients with sarcoidosis or latent tuberculosis infection (LTBI) would provide novel insights into the pathogenesis of sarcoidosis. METHODS: A genomic analysis identified differentially expressed genes in granuloma-like cell aggregates formed by sarcoidosis (n=12) or LTBI patients (n=5) in an established in vitro human granuloma model wherein peripheral blood mononuclear cells were exposed to M. tuberculosis antigens (beads coated with purified protein derivative) and cultured for 7 days. Pathway analysis of differentially expressed genes identified canonical pathways, most notably antigen processing and presentation via phagolysosomes, as a prominent pathway in sarcoidosis granuloma formation. The phagolysosomal pathway promoted mechanistic target of rapamycin complex 1 (mTORc1)/STAT3 signal transduction. Thus, granuloma formation and related immune mediators were evaluated in the absence or presence of various pre-treatments known to prevent phagolysosome formation (chloroquine) or phagosome acidification (bafilomycin A1) or directly inhibit mTORc1 activation (rapamycin). RESULTS: In keeping with genomic analyses indicating enhanced phagolysosomal activation and predicted mTORc1 signalling, it was determined that sarcoidosis granuloma formation and related inflammatory mediator release was dependent upon phagolysosome assembly and acidification and mTORc1/S6/STAT3 signal transduction. CONCLUSIONS: Sarcoidosis granulomas exhibit enhanced and sustained intracellular antigen processing and presentation capacities, and related phagolysosome assembly and acidification are required to support mTORc1 signalling to promote sarcoidosis granuloma formation.

Differential transcriptomics in sarcoidosis lung and lymph node granulomas with comparisons to pathogen-specific granulomas.

RATIONALE: Despite the availability of multi-"omics" strategies, insights into the etiology and pathogenesis of sarcoidosis have been elusive. This is partly due to the lack of reliable preclinical models and a paucity of validated biomarkers. As granulomas are a key feature of sarcoidosis, we speculate that direct genomic interrogation of sarcoid tissues, may lead to identification of dysregulated gene pathways or biomarker signatures. OBJECTIVE: To facilitate the development sarcoidosis genomic biomarkers by gene expression profiling of sarcoidosis granulomas in lung and lymph node tissues (most commonly affected organs) and comparison to infectious granulomas (coccidiodomycosis and tuberculosis). METHODS: Transcriptomic profiles of immune-related gene from micro-dissected sarcoidosis granulomas within lung and mediastinal lymph node tissues and compared to infectious granulomas from paraffin-embedded blocks. Differentially-expressed genes (DEGs) were profiled, compared among the three granulomatous diseases and analyzed for functional enrichment pathways. RESULTS: Despite histologic similarities, DEGs and pathway enrichment markedly differed in sarcoidosis granulomas from lymph nodes and lung. Lymph nodes showed a clear immunological response, whereas a structural regenerative response was observed in lung. Sarcoidosis granuloma gene expression data corroborated previously reported genomic biomarkers (STAB1, HBEGF, and NOTCH4), excluded others and identified new genomic markers present in lung and lymph nodes, ADAMTS1, NPR1 and CXCL2. Comparisons between sarcoidosis and pathogen granulomas identified pathway divergences and commonalities at gene expression level. CONCLUSION: These findings suggest the importance of tissue and disease-specificity evaluation when exploring sarcoidosis genomic markers. This relevant translational information in sarcoidosis and other two histopathological similar infections provides meaningful specific genomic-derived biomarkers for sarcoidosis diagnosis and prognosis.