Vassallo, Robert

Peroxisomes are vital but often overlooked metabolic organelles. We found that excessive interferon signaling remodeled macrophage peroxisomes. This loss of peroxisomes impaired inflammation resolution and lung repair during severe respiratory viral infections. Peroxisomes were found to modulate lipid metabolism and mitochondrial health in a macrophage type-specific manner and enhanced alveolar macrophage-mediated tissue repair and alveolar regeneration after viral infection. Peroxisomes also prevented excessive macrophage inflammasome activation and IL-1β release, limiting accumulation of KRT8 high dysplastic epithelial progenitors following viral injury. Pharmacologically enhancing peroxisome biogenesis mitigated both acute symptoms and post-acute sequelae of COVID-19 (PASC) in animal models. Thus, macrophage peroxisome dysfunction contributes to chronic lung pathology and fibrosis after severe acute respiratory syndrome coronavirus 2 infection.

Background: Erdheim-Chester disease (ECD) manifests as multi-organ infiltration of clonal histiocytes with mutations primarily in the mitogen-activated protein kinase (MAPK) pathway. The revised classification of histiocytosis (2016) categorizes ECD with typical bilateral, symmetric osteosclerosis of the lower extremity long bones as classical (C) and as non-classical (NC) if otherwise. The clinical and biological significance of this classification is unclear. This study compared the clinical and genotypic findings of the 2 subtypes within a large cohort of ECD patients. Methods: We included patients seen at our institution since 1990 meeting the following criteria: A) had an ECD diagnosis based on the revised criteria by Haroche J, et al. (Blood 2020;135:1311-1318); B) completed whole-body imaging studies including long bones of the legs. Classification as C-ECD required symmetric osteosclerosis of the distal femur and/or proximal tibia/fibula. Results: A total of 145 patients were enrolled, comprising a male-to-female ratio of 2:1 (66.2% male). C-ECD was diagnosed in 77.2% of cases, and NC-ECD in 22.8%. The median age at diagnosis was similar with 58.5 years (range, 18-82) in C-ECD and 55 years (range, 20-81) in NC-ECD, (p=0.65). The most frequently affected systems included skeletal (81.4%), renal (60.0%), vascular (45.5%), cardiac (40.0%), central nervous (38.6%), sinus (37.2%), pulmonary (36.6%), and adrenal (32.4%). The numbers of organs involved were significantly higher in C-ECD compared to NC-ECD (median 5 vs 3, p<0.01). Higher rates of skeletal (100% vs. 18.2%, p<0.01), adrenal gland (37.5% vs. 15.2%, p=0.02), and sinus (44.6% vs. 12.1%, p<0.01) involvement were observed in C-ECD. Arginine vasopressin deficiency (AVP-D) was exclusively observed in C-ECD (20.7% vs. 0%, p<0.01). NC-ECD had a higher rate of cardiac involvement (69.7% vs. 31.3%, p<0.01). No significant differences were found in other systems. The overall median time between onset of the first symptoms/signs to diagnosis was 15 months with no significant difference between C-ECD and NC-ECD (17.5 vs. 12 months, p=0.18). The median time to diagnosis of ECD in patients with AVP-D was 38.5 months, compared to 12 months without AVP-D (p=0.09). Genetic testing was conducted in 86.2% of patients and B-Raf proto-oncogene (BRAF) alterations were identified in 59.2% of the tested cases. BRAFV600E was more prevalent in C-ECD (61.3% vs. 21.9%, p<0.01), whereas non-V600 mutations were more commonly found in NC-ECD (12.5% vs. 1.1%, p<0.01). There was no significant difference in the frequency of BRAF fusions. NC-ECD patients exhibited a higher rate of MAP2K1 mutations (25.0% vs. 5.4%, p<0.01). Other frequently mutated genes included KRAS (4.0%), NRAS (2.4%), and CSF1R (2.4%), although no significant difference was observed in their frequency between C-ECD and NC-ECD. Testicular involvement appeared to be more common in patients with MAP2K1 mutations (23.1% vs. 8%, p=0.08). Two patients with pathogenic CSF1R mutations both had central nervous system (CNS) involvement. Among C-ECD patients, BRAFV600E conferred a higher rate of CNS involvement (50.9% vs. 25%, p=0.01). No significant difference was observed in NC-ECD patients (71.4% vs 36%, p=0.09). Among NC-ECD cases, MAP2K1 mutations conferred a reduced risk of cardiac involvement (37.5% vs. 79.2%, p=0.03) and no such difference was observed in patients with C-ECD (40% vs 34.1%, p=0.79). The median numbers of organs involved in patients with BRAF, MAP2K1, and KRAS/NRAS mutations were 5, 3, and 3, respectively, with no significant difference. Conclusions: C-ECD affects more organs and more frequently involves bones, adrenal glands, and sinuses. AVP-D was exclusively observed in C-ECD. Conversely, NC-ECD showed a higher prevalence of cardiac involvement. BRAFV600E was more commonly identified in C-ECD, whereas non-V600 mutations and MAP2K1 mutations were more prevalent in NC-ECD. Among C-ECD patients, BRAFV600E was correlated with a greater likelihood of CNS involvement. Conversely, NC-ECD patients with MAP2K1 mutations had a reduced risk of cardiac involvement. This study highlights the distinct clinical and molecular features of C-ECD and NC-ECD, underscoring the importance of subclassifying ECD into classical and non-classical forms. Further studies are needed to explore differences in the clinical outcomes between the 2 forms.

ABSTRACTInflammatory cell infiltration is a characteristic feature of COPD and correlates directly with the severity of the disease. Interleukin‐23 (IL‐23) is a pro‐inflammatory cytokine that regulates Th‐17 inflammation, which mediates many pathophysiological events in COPD. The primary goal of this study was to determine the role of IL‐23 as a mediator of key pathologic processes in cigarette smoke‐induced COPD. In this study, we report an increase in IL23 gene expression in the lung biopsies of COPD patients compared to controls and identified a positive correlation between IL23 gene expression and disease severity. In a cigarette smoke‐induced murine emphysema model, the suppression of IL‐23 with a monoclonal blocking antibody reduced the severity of cigarette smoke‐induced murine emphysema. Mechanistically, the suppression of IL‐23 was associated with a reduction in immune cell infiltration, oxidative stress injury, and apoptosis, suggesting a role for IL‐23 as an essential immune mediator of the inflammatory processes in the pathogenesis of CS‐induced emphysema.

Postacute sequelae of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection (PASC) represent an urgent public health challenge and are estimated to affect more than 60 million individuals globally. Although a growing body of evidence suggests that dysregulated immune reactions may be linked with PASC symptoms, most investigations have primarily centered around blood-based studies, with few focusing on samples derived from affected tissues. Furthermore, clinical studies alone often provide correlative insights rather than causal mechanisms. Thus, it is essential to compare clinical samples with relevant animal models and conduct functional experiments to understand the etiology of PASC. In this study, we comprehensively compared bronchoalveolar lavage fluid single-cell RNA sequencing data derived from clinical PASC samples and a mouse model of PASC. This revealed a pro-fibrotic monocyte-derived macrophage response in respiratory PASC, as well as abnormal interactions between pulmonary macrophages and respiratory resident T cells, in both humans and mice. Interferon-γ (IFN-γ) emerged as a key node mediating the immune anomalies in respiratory PASC. Neutralizing IFN-γ after the resolution of acute SARS-CoV-2 infection reduced lung inflammation and tissue fibrosis in mice. Together, our study underscores the importance of performing comparative analysis to understand the cause of PASC and suggests that the IFN-γ signaling axis might represent a therapeutic target.

AbstractObjectiveAnalyze the duration of symptom‐free intervals following laser wedge excision (LWE) for recurrent idiopathic subglottic stenosis (iSGS). Secondary aim includes evaluating the influence of patient‐related or disease factors.Study DesignRetrospective review.SettingTertiary center.MethodsReview of iSGS patients who underwent LWE between 2002 and 2021. LWE patients without prior airway surgery were labeled LWE primary (LWEP) and those with prior history of dilation were labeled LWE secondary (LWES). A conditional frailty repeated events model was used to analyze the median time to recurrence (MTR) for each nth recurrence. Secondary analysis included stratification by use of medical therapy and initial preoperative characteristics of scar (Myer‐Cotton grade, distance between the glottis and superior‐most aspect of scar, DGS; length of scar, DL).ResultsTwo hundred and ten iSGS patients underwent LWE (131 LWEP, 79 LWES). The proportion of patients experiencing at least 1, 3, 6, and 12 recurrences, respectively, was 68.0% (n = 143), 40.7% (n = 85), 20.0% (n = 42), and 5.2% (n = 11). There was exponential time‐shortening from the 1st to 12th recurrence (P < .0001). While MTR was 4.1 years after the first LWE, this fell to 2.8, 1.7, 1.0, and 0.7 years for the 2nd, 3rd, 6th, and 12th recurrences. Furthermore, LWEP patients experienced longer MTR than LWES counterparts within the first 6 recurrences (P < .01). There was no significant relationship between intersurgical interval and medication adherence, DL, DGS, or grade for recurrences beyond the first (P = .207, P = .20, P = .43, P = .16).ConclusionSymptom‐free intervals in iSGS shorten with each subsequent recurrence and LWE. The difference in MTR between LWEP and LWES groups was significant within the first 6 recurrences with LWEP having longer MTR.Level of Evidence3.

Introduction The discovery of MAPK/ERK pathway mutations has revolutionized the care for patients with histiocytic neoplasms. However, mutational analysis on biopsy specimens is often limited by low tumor cellularity leading to testing failure or disease involvement of difficult-to-reach sites (e.g., central nervous system). Recently, molecular analysis of cell-free DNA (cfDNA) has shown promise as a viable tool for discovering driver mutations in multiple cancers. A recent study of mainly BRAF-mutated non-Langerhans cell histiocytosis (LCH) showed 50% concordance between tumor and plasma cfDNA analysis for BRAF-V600E. However, it is unknown whether cfDNA has a role in the evaluation of cases that harbor mutations beyond BRAF-V600E. We conducted this study to address this unanswered question using a large cohort of individuals with diverse histiocytic neoplasms from a tertiary-care institution. Methods We included 40 cases with histiocytic neoplasms with stored plasma and germline DNA in this study. Tissue molecular analysis was conducted using a targeted next generation sequencing (NGS) using commercial assays or whole exome sequencing (WES) using an in-house assay. A custom panel of 226 genes, including exonic and intronic regions, was used for the cfDNA assay in this study. To avoid cfDNA false positives, germline mutations identified from normal DNA sequencing were filtered using a BostonGene ®-developed error reduction algorithm.The total length of the analyzed genomic regions was 1.56 MB. For samples with an input greater than 30 ng, a 0.2% variant allele frequency (VAF) limit of detection (LOD) was used; a 0.4% VAF LOD was used for samples less than 30 ng. Results The final analysis cohort included 38 patients with histiocytic neoplasms after exclusion of 2 cases (failure of germline DNA library preparation and mismatch of germline and somatic DNA, respectively). Disease types included Erdheim-Chester disease (ECD; n=13, 34%), Langerhans cell histiocytosis (LCH; n=13, 34%), and Rosai-Dorfman disease (RDD; n=12, 32%). 24 cases were multisystem (63%), and the remaining were single-system. Commonly involved organs included bone (n=25, 66%), kidney (n=12, 32%), skin (n=8, 21%), central nervous system (n=7, 18%), and pituitary (n=7, 18%). Most cases (n=31, 82%) underwent plasma collection pre-treatment, 5 cases underwent collection after histiocytosis-directed therapy, and 2 while on treatment. Tissue NGS/WES data were available for 28 (74%) cases; 5 cases (13%) had insufficient tissue cellularity, and 5 (13%) did not undergo any tissue sequencing. Among those who underwent successful tissue sequencing assays, the most common common driver alterations was BRAF V600E (n=5, 26%). Other alterations included those in MAP2K1 (n=4, 21%), BRAF-fusions (n=3, 10%; partners UBDT2, RNF11, and LMTK2, respectively), and single cases of KRAS, NRAS and BRAF indel, respectively. cfDNA analysis showed at least one variant in 23 cases (60%). Correlation between tissue and cfDNA for known driver mutations was poor and concordant findings were only seen in one case with ECD harboring BRAF V600E in tissue as well as plasma (VAF 0.5%). Among the 5 cases where tissue mutational analysis had failed, cfDNA showed mutations in MUTYH, PTCH2, NOTCH3 SDHD, and TMEM127, respectively (Figure 1). Among the 5 cases where tissue molecular assessment could not be undertaken due to various reasons, cfDNA showed alterations in 4 cases, encompassing NOTCH1, TET2, BRCA1, MSH6, PIK3CD, and FUS genes. Conclusions In a large cohort of adults with diverse histiocytic neoplasms, we found poor correlation between tumor and plasma cfDNA molecular findings. The reason for the lack of correlation may be related to low tumor shedding especially among non- BRAF V600E cases. cfDNA analysis may have a role to identify potential drivers of pathogenesis among cases that are unable to successfully undergo tissue molecular analysis. Further studies are underway to characterize other novel alterations that were discovered in the cfDNA analysis.

Purpose: The understanding of cardiac-involved ECD (ECD-CV) and how this affects outcomes and pre-existing diseases remains limited. We aimed to determine the association, burden, and severity of pre-existing and de novo cardiovascular (CV) comorbidities with ECD-CV compared to patients (pts) without cardiac-involved ECD (ECD-noCV). Methods: Records of pts with ECD diagnosed from Jan-1990 to Dec-2021, consecutively seen at Mayo Clinic, AZ, FL, and MN were reviewed. Cardiac involvement was diagnosed by imaging. CV comorbidities were captured before and after ECD diagnosis. CTCAE v3.0 based grading system was used to assess CV comorbidities which graded the conditions as mild (grade 1), moderate (grade 2), severe (grade 3), and life-threatening or disabling (grade 4). Chi-square test was used to determine if having ECD-CV worsens pre-existing CV comorbidities in pts with ECD. As per the established methods, the cumulative CV disease burden was defined after considering 8 CV morbidities that are known to have direct structural and functional implications on the heart, including hypertension (HTN), coronary artery disease (CAD), myocardial infarction (MI), congestive heart failure (CHF), atrial fibrillation (afib), pericardial effusion, valvular disease, and conduction disorders. A composite CV morbidity grade was assigned based on the highest grade in one of the 8 CV comorbidities (Oeffinger, NEJM, 2006). Lastly, a multivariant analysis was conducted using the presence of BRAFV600E mutation, smoking, age ≥ 60 years (yr), conventional vs targeted therapy, and ECD-CV in pts with a composite grade of 4. Results: Among 106 ECD pts, 38% (n=40) had cardiac involvement. The median age at diagnosis of ECD was 57 yr (range: 49-67); 61% were males. The median follow-up was 5 yr (95% CI: 3.5-6.1), and 4.1 yr (95% CI: 3.2-6.5) for ECD-CV and ECD-noCV pts, respectively p=0.92. The median time of CV diagnoses for the entire cohort before and after ECD diagnosis was 4.8 yr (range: 0.06-42.5) and 3.4 yr (range: 0.05-18.7), respectively. BRAFV600E mutation was present in 65% (n=26) of ECD-CV vs 42% (n=28) of ECD-noCV, p=0.02. Eight pts with ECD-CV died; one from sudden cardiac death, while 9 ECD-noCV pts died, p=0.39. Smoking was present in 43% (n=18) of ECD-CV pts compared to 24% with ECD-noCV (n=16), p=0.05. In comparison to ECD-noCV pts, increased prevalence of pre-existing HTN [59% (n=23) vs 35% (n=23), p=0.016] and MI [10% (n=4) vs 2% (n=1), p=0.046] was seen in ECD-CV. There was no difference in hyperlipidemia, diabetes mellitus, CAD, CHF, valvular disease, afib, and conduction disorders among the two cohorts. After ECD diagnosis, de novo diagnosis of hyperlipidemia [67% (n=26) vs 38% (n=25); p=0.004] and CHF [35% (n=14) vs 7% (n=5); p=&lt;0.001] was seen more frequently in ECD-CV pts compared to ECD-noCV. Following the diagnosis of ECD, the severity of pre-existing conditions did not change in pts with or without ECD-CV. ECD-CV pts had a greater cumulative CV disease burden as the total number of CV morbidities was greater in ECD-CV pts both before and after ECD diagnosis (Table 1). Additionally, pts with ECD-CV had greater severity of CV morbidity (composite grade ≥4) than ECD-noCV before (35% v 3%; p=&lt;0.001) and after (42.5% vs 7.6%; p=&lt;0.001) ECD diagnosis. The composite grade of 4 before ECD diagnosis was most attributable to pericardial effusion (42% in pts with ECD-CV vs 0% in pts with ECD-noCV) and CAD (50% in pts with ECD-CV vs 100% in pts with ECD-noCV); after ECD diagnosis pericardial effusion (42% in pts with ECD-CV vs 0% in pts with ECD-noCV) and CAD (41% in pts with ECD-CV vs 100% in pts with ECD-noCV). After considering BRAFV600E mutation, age ≥60, smoking, ECD-CV, and first-line therapy (conventional vs targeted therapy), ECD-CV was the only factor independently associated with a composite grade of ≥4, before (OR 25.6, 95% CI: 4.8-137) and after (OR 9.7, 95% CI: 3.0-30.8) ECD diagnosis. Nonetheless, smoking, age &gt;60, and ECD-CV were independent factors associated when considered a composite grade of ≥3, before and after ECD diagnosis. Conclusion: ECD-CV did not prognosticate pts and did not worsen the pre-existing CV conditions compared to pts with ECD-noCV. However, ECD-CV pts had a higher cumulative CV disease burden and a higher grade of pre-existing and de novo CV morbidities compared to ECD-noCV. The presence of cardiac involvement in ECD was independently associated with a severe cumulative CV disease burden grade.