Spherical nucleic acids as an infectious disease vaccine platform.

SignificanceUsing SARS-CoV-2 as a relevant case study for infectious disease, we investigate the structure-function relationships that dictate antiviral spherical nucleic acid (SNA) vaccine efficacy. We show that the SNA architecture can be rapidly employed to target COVID-19 through incorporation of the receptor-binding domain, and that the resulting vaccine potently activates human cells in vitro and mice in vivo. Furthermore, when challenged with a lethal viral infection, only mice treated with the SNA vaccine survived. Taken together, this work underscores the importance of rational vaccine design for infectious disease to yield vaccines that elicit more potent immune responses to effectively fight disease.

Temporal correlation between postreperfusion complement deposition and severe primary graft dysfunction in lung allografts.

Growing evidence implicates complement in the pathogenesis of primary graft dysfunction (PGD). We hypothesized that early complement activation postreperfusion could predispose to severe PGD grade 3 (PGD-3) at 72 hours, which is associated with worst posttransplant outcomes. Consecutive lung transplant patients (n = 253) from January 2018 through June 2023 underwent timed open allograft biopsies at the end of cold ischemia (internal control) and 30 minutes postreperfusion. PGD-3 at 72 hours occurred in 14% (35/253) of patients; 17% (44/253) revealed positive C4d staining on postreperfusion allograft biopsy, and no biopsy-related complications were encountered. Significantly more patients with PGD-3 at 72 hours had positive C4d staining at 30 minutes postreperfusion compared with those without (51% vs 12%, P < .001). Conversely, patients with positive C4d staining were significantly more likely to develop PGD-3 at 72 hours (41% vs 8%, P < .001) and experienced worse long-term outcomes. In multivariate logistic regression, positive C4d staining remained highly predictive of PGD-3 (odds ratio 7.92, 95% confidence interval 2.97-21.1, P < .001). Hence, early complement deposition in allografts is highly predictive of PGD-3 at 72 hours. Our data support future studies to evaluate the role of complement inhibition in patients with early postreperfusion complement activation to mitigate PGD and improve transplant outcomes.

A Novel Soluble ACE2 Protein Provides Lung and Kidney Protection in Mice Susceptible to Lethal SARS-CoV-2 Infection.

BACKGROUND: Severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) uses full-length angiotensin converting enzyme 2 (ACE2) as a main receptor to enter target cells. The goal of this study was to demonstrate the preclinical efficacy of a novel soluble ACE2 protein with increased duration of action and binding capacity in a lethal mouse model of COVID-19. METHODS: A human soluble ACE2 variant fused with an albumin binding domain (ABD) was linked via a dimerization motif hinge-like 4-cysteine dodecapeptide (DDC) to improve binding capacity to SARS-CoV-2. This novel soluble ACE2 protein (ACE2-1-618-DDC-ABD) was then administered intranasally and intraperitoneally to mice before intranasal inoculation of SARS-CoV-2 and then for two additional days post viral inoculation. RESULTS: Untreated animals became severely ill, and all had to be humanely euthanized by day 6 or 7 and had pulmonary alveolar hemorrhage with mononuclear infiltrates. In contrast, all but one mouse infected with a lethal dose of SARS-CoV-2 that received ACE2-1-618-DDC-ABD survived. In the animals inoculated with SARS-CoV-2 that were untreated, viral titers were high in the lungs and brain, but viral titers were absent in the kidneys. Some untreated animals, however, had variable degrees of kidney proximal tubular injury as shown by attenuation of the proximal tubular brush border and increased NGAL and TUNEL staining. Viral titers in the lung and brain were reduced or nondetectable in mice that received ACE2-1-618-DDC-ABD, and the animals developed only moderate disease as assessed by a near-normal clinical score, minimal weight loss, and improved lung and kidney injury. CONCLUSIONS: This study demonstrates the preclinical efficacy of a novel soluble ACE2 protein, termed ACE2-1-618-DDC-ABD, in a lethal mouse model of SARS-CoV-2 infection that develops severe lung injury and variable degrees of moderate kidney proximal tubular injury.

Clinical Characteristics and Outcomes of Patients With COVID-19-Associated Acute Respiratory Distress Syndrome Who Underwent Lung Transplant.

Importance: Lung transplantation is a potentially lifesaving treatment for patients who are critically ill due to COVID-19-associated acute respiratory distress syndrome (ARDS), but there is limited information about the long-term outcome. Objective: To report the clinical characteristics and outcomes of patients who had COVID-19-associated ARDS and underwent a lung transplant at a single US hospital. Design, Setting, and Participants: Retrospective case series of 102 consecutive patients who underwent a lung transplant at Northwestern University Medical Center in Chicago, Illinois, between January 21, 2020, and September 30, 2021, including 30 patients who had COVID-19-associated ARDS. The date of final follow-up was November 15, 2021. Exposures: Lung transplant. Main Outcomes and Measures: Demographic, clinical, laboratory, and treatment data were collected and analyzed. Outcomes of lung transplant, including postoperative complications, intensive care unit and hospital length of stay, and survival, were recorded. Results: Among the 102 lung transplant recipients, 30 patients (median age, 53 years [range, 27 to 62]; 13 women [43%]) had COVID-19-associated ARDS and 72 patients (median age, 62 years [range, 22 to 74]; 32 women [44%]) had chronic end-stage lung disease without COVID-19. For lung transplant recipients with COVID-19 compared with those without COVID-19, the median lung allocation scores were 85.8 vs 46.7, the median time on the lung transplant waitlist was 11.5 vs 15 days, and preoperative venovenous extracorporeal membrane oxygenation (ECMO) was used in 56.7% vs 1.4%, respectively. During transplant, patients who had COVID-19-associated ARDS received transfusion of a median of 6.5 units of packed red blood cells vs 0 in those without COVID-19, 96.7% vs 62.5% underwent intraoperative venoarterial ECMO, and the median operative time was 8.5 vs 7.4 hours, respectively. Postoperatively, the rates of primary graft dysfunction (grades 1 to 3) within 72 hours were 70% in the COVID-19 cohort vs 20.8% in those without COVID-19, the median time receiving invasive mechanical ventilation was 6.5 vs 2.0 days, the median duration of intensive care unit stay was 18 vs 9 days, the median post-lung transplant hospitalization duration was 28.5 vs 16 days, and 13.3% vs 5.5% required permanent hemodialysis, respectively. None of the lung transplant recipients who had COVID-19-associated ARDS demonstrated antibody-mediated rejection compared with 12.5% in those without COVID-19. At follow-up, all 30 lung transplant recipients who had COVID-19-associated ARDS were alive (median follow-up, 351 days [IQR, 176-555] after transplant) vs 60 patients (83%) who were alive in the non-COVID-19 cohort (median follow-up, 488 days [IQR, 368-570] after lung transplant). Conclusions and Relevance: In this single-center case series of 102 consecutive patients who underwent a lung transplant between January 21, 2020, and September 30, 2021, survival was 100% in the 30 patients who had COVID-19-associated ARDS as of November 15, 2021.

Lung donation following SARS-CoV-2 infection.

There have been over 177 million cases of COVID-19 worldwide, many of whom could be organ donors. Concomitantly, there is an anticipated increase in the need for donor lungs due to expanding indications. Given that the respiratory tract is most commonly affected by COVID-19, there is an urgent need to develop donor assessment criteria while demonstrating safety and "efficacy" of lung donation following COVID-19 infection. Accordingly, we report an intentional transplant using lungs from a donor with recent, microbiologically confirmed, COVID-19 infection into a recipient suffering from COVID-19 induced ARDS and pulmonary fibrosis. In addition to the standard clinical assays, both donor and recipient lungs were analyzed using RNAscope, which confirmed that tissues were negative for SARS-CoV-2. Immunohistochemistry demonstrated colocalized KRT17+ basaloid-like epithelium and COL1A1+ fibroblasts, a marker suggestive of lung fibrosis in COVID-19 associated lung disease, in the explanted recipient lungs but absent in the donor lungs. We demonstrate that following a thorough assessment, lung donation following resolved COVID-19 infection is safe and feasible.

Mitochondrial 8-oxoguanine DNA glycosylase mitigates alveolar epithelial cell PINK1 deficiency, mitochondrial DNA damage, apoptosis, and lung fibrosis.

Alveolar epithelial cell (AEC) apoptosis, arising from mitochondrial dysfunction and mitophagy defects, is important in mediating idiopathic pulmonary fibrosis (IPF). Our group established a role for the mitochondrial (mt) DNA base excision repair enzyme, 8-oxoguanine-DNA glycosylase 1 (mtOGG1), in preventing oxidant-induced AEC mtDNA damage and apoptosis and showed that OGG1-deficient mice have increased lung fibrosis. Herein, we determined whether mice overexpressing the mtOGG1 transgene (mtOgg1tg) are protected against lung fibrosis and whether AEC mtOGG1 preservation of mtDNA integrity mitigates phosphatase and tensin homolog-induced putative kinase 1 (PINK1) deficiency and apoptosis. Compared with wild type (WT), mtOgg1tg mice have diminished asbestos- and bleomycin-induced pulmonary fibrosis that was accompanied by reduced lung and AEC mtDNA damage and apoptosis. Asbestos and H2O2 promote the MLE-12 cell PINK1 deficiency, as assessed by reductions in the expression of PINK1 mRNA and mitochondrial protein expression. Compared with WT, Pink1-knockout (Pink1-KO) mice are more susceptible to asbestos-induced lung fibrosis and have increased lung and alveolar type II (AT2) cell mtDNA damage and apoptosis. AT2 cells from Pink1-KO mice and PINK1-silenced (siRNA) MLE-12 cells have increased mtDNA damage that is augmented by oxidative stress. Interestingly, mtOGG1 overexpression attenuates oxidant-induced MLE-12 cell mtDNA damage and apoptosis despite PINK1 silencing. mtDNA damage is increased in the lungs of patients with IPF as compared with controls. Collectively, these findings suggest that mtOGG1 maintenance of AEC mtDNA is crucial for preventing PINK1 deficiency that promotes apoptosis and lung fibrosis. Given the key role of AEC apoptosis in pulmonary fibrosis, strategies aimed at preserving AT2 cell mtDNA integrity may be an innovative target.

Pulmonary Mucormycosis: Risk Factors, Radiologic Findings, and Pathologic Correlation.

Pulmonary mucormycosis (PM) is an uncommon fungal infection most often seen in immunocompromised patients. The fungus grows on decaying food, soil, and animal excrement. Patients usually become infected by inhalation of spores. The most common risk factors include diabetes mellitus, hematologic malignancy, and solid organ or stem cell transplant. PM can have a nonspecific appearance at imaging. For example, early imaging may show peribronchial ground-glass opacity. Later, the disease progresses to consolidation, nodules, or masses. Because patients are usually immunocompromised, the differential diagnosis often includes invasive pulmonary aspergillosis (IPA). Various radiologic findings suggestive of PM have been identified to help differentiate it from IPA. For example, the reverse halo sign is more closely associated with PM than with IPA. The reverse halo sign is an area of ground-glass opacity surrounded by a rim of consolidation. In addition, the presence of pleural effusions and more than 10 nodules is more suggestive of PM than it is of IPA. PM can progress rapidly in neutropenic patients. Identification of the hyphae in tissue by using endobronchial or percutaneous sampling can allow differentiation from IPA and help confirm the diagnosis of mucormycosis. Because of the high mortality rate associated with PM, early identification of the disease is critical for an improved likelihood of survival. A multimodality treatment approach with antifungal agents and surgical débridement has been shown to improve outcomes. The authors review the risk factors for PM, describe its imaging appearance and disease process, and describe the treatment of the disease. ©RSNA, 2020.

Single-Cell Transcriptomic Analysis of Human Lung Provides Insights into the Pathobiology of Pulmonary Fibrosis.

Rationale: The contributions of diverse cell populations in the human lung to pulmonary fibrosis pathogenesis are poorly understood. Single-cell RNA sequencing can reveal changes within individual cell populations during pulmonary fibrosis that are important for disease pathogenesis. Objectives: To determine whether single-cell RNA sequencing can reveal disease-related heterogeneity within alveolar macrophages, epithelial cells, or other cell types in lung tissue from subjects with pulmonary fibrosis compared with control subjects. Methods: We performed single-cell RNA sequencing on lung tissue obtained from eight transplant donors and eight recipients with pulmonary fibrosis and on one bronchoscopic cryobiospy sample from a patient with idiopathic pulmonary fibrosis. We validated these data using in situ RNA hybridization, immunohistochemistry, and bulk RNA-sequencing on flow-sorted cells from 22 additional subjects. Measurements and Main Results: We identified a distinct, novel population of profibrotic alveolar macrophages exclusively in patients with fibrosis. Within epithelial cells, the expression of genes involved in Wnt secretion and response was restricted to nonoverlapping cells. We identified rare cell populations including airway stem cells and senescent cells emerging during pulmonary fibrosis. We developed a web-based tool to explore these data. Conclusions: We generated a single-cell atlas of pulmonary fibrosis. Using this atlas, we demonstrated heterogeneity within alveolar macrophages and epithelial cells from subjects with pulmonary fibrosis. These results support the feasibility of discovery-based approaches using next-generation sequencing technologies to identify signaling pathways for targeting in the development of personalized therapies for patients with pulmonary fibrosis.

The Sphingosine Kinase 1 Inhibitor, PF543, Mitigates Pulmonary Fibrosis by Reducing Lung Epithelial Cell mtDNA Damage and Recruitment of Fibrogenic Monocytes.

Idiopathic pulmonary fibrosis (IPF) is a chronic disease for which novel approaches are urgently required. We reported increased sphingosine kinase 1 (SPHK1) in IPF lungs and that SPHK1 inhibition using genetic and pharmacologic approaches reduces murine bleomycin-induced pulmonary fibrosis. We determined whether PF543, a specific SPHK1 inhibitor post bleomycin or asbestos challenge mitigates lung fibrosis by reducing mitochondrial (mt) DNA damage and pro-fibrotic monocyte recruitment-both are implicated in the pathobiology of pulmonary fibrosis. Bleomycin (1.5 U/kg), crocidolite asbestos (100 µg/50 µL) or controls was intratracheally instilled in Wild-Type (C57Bl6) mice. PF543 (1 mg/kg) or vehicle was intraperitoneally injected once every two days from day 7-21 following bleomycin and day 14-21 or day 30-60 following asbestos. PF543 reduced bleomycin- and asbestos-induced pulmonary fibrosis at both time points as well as lung expression of profibrotic markers, lung mtDNA damage, and fibrogenic monocyte recruitment. In contrast to human lung fibroblasts, asbestos augmented lung epithelial cell (MLE) mtDNA damage and PF543 was protective. Post-exposure PF543 mitigates pulmonary fibrosis in part by reducing lung epithelial cell mtDNA damage and monocyte recruitment. We reason that SPHK1 signaling may be an innovative therapeutic target for managing patients with IPF and other forms of lung fibrosis.

SIRT3 deficiency promotes lung fibrosis by augmenting alveolar epithelial cell mitochondrial DNA damage and apoptosis.

Alveolar epithelial cell (AEC) mitochondrial dysfunction and apoptosis are important in idiopathic pulmonary fibrosis and asbestosis. Sirtuin 3 (SIRT3) detoxifies mitochondrial reactive oxygen species, in part, by deacetylating manganese superoxide dismutase (MnSOD) and mitochondrial 8-oxoguanine DNA glycosylase. We reasoned that SIRT3 deficiency occurs in fibrotic lungs and thereby augments AEC mtDNA damage and apoptosis. Human lungs were assessed by using immunohistochemistry for SIRT3 activity via acetylated MnSODK68 Murine AEC SIRT3 and cleaved caspase-9 (CC-9) expression were assayed by immunoblotting with or without SIRT3 enforced expression or silencing. mtDNA damage was measured by using quantitative PCR and apoptosis via ELISA. Pulmonary fibrosis after asbestos or bleomycin exposure was evaluated in 129SJ/wild-type and SIRT3-knockout mice (Sirt3-/- ) by using fibrosis scoring and lung collagen levels. Idiopathic pulmonary fibrosis lung alveolar type II cells have increased MnSODK68 acetylation compared with controls. Asbestos and H2O2 diminished AEC SIRT3 protein expression and increased mitochondrial protein acetylation, including MnSODK68 SIRT3 enforced expression reduced oxidant-induced AEC OGG1K338/341 acetylation, mtDNA damage, and apoptosis, whereas SIRT3 silencing promoted these effects. Asbestos- or bleomycin-induced lung fibrosis, AEC mtDNA damage, and apoptosis in wild-type mice were amplified in Sirt3-/- animals. These data suggest a novel role for SIRT3 deficiency in mediating AEC mtDNA damage, apoptosis, and lung fibrosis.-Jablonski, R. P., Kim, S.-J., Cheresh, P., Williams, D. B., Morales-Nebreda, L., Cheng, Y., Yeldandi, A., Bhorade, S., Pardo, A., Selman, M., Ridge, K., Gius, D., Budinger, G. R. S., Kamp, D. W. SIRT3 deficiency promotes lung fibrosis by augmenting alveolar epithelial cell mitochondrial DNA damage and apoptosis.

Klotho, an antiaging molecule, attenuates oxidant-induced alveolar epithelial cell mtDNA damage and apoptosis.

Alveolar epithelial cell (AEC) apoptosis and inadequate repair resulting from "exaggerated" lung aging and mitochondrial dysfunction are critical determinants promoting lung fibrosis. α-Klotho, which is an antiaging molecule that is expressed predominantly in the kidney and secreted in the blood, can protect lung epithelial cells against hyperoxia-induced apoptosis. We reasoned that Klotho protects AEC exposed to oxidative stress in part by maintaining mitochondrial DNA (mtDNA) integrity and mitigating apoptosis. We find that Klotho levels are decreased in both serum and alveolar type II (AT2) cells from asbestos-exposed mice. We show that oxidative stress reduces AEC Klotho mRNA and protein expression, whereas Klotho overexpression is protective while Klotho silencing augments AEC mtDNA damage. Compared with wild-type, Klotho heterozygous hypomorphic allele (kl/+) mice have increased asbestos-induced lung fibrosis due in part to increased AT2 cell mtDNA damage. Notably, we demonstrate that serum Klotho levels are reduced in wild-type but not mitochondrial catalase overexpressing (MCAT) mice 3 wk following exposure to asbestos and that EUK-134, a MnSOD/catalase mimetic, mitigates oxidant-induced reductions in AEC Klotho expression. Using pharmacologic and genetic silencing studies, we show that Klotho attenuates oxidant-induced AEC mtDNA damage and apoptosis via mechanisms dependent on AKT activation arising from upstream fibroblast growth factor receptor 1 activation. Our findings suggest that Klotho preserves AEC mtDNA integrity in the setting of oxidative stress necessary for preventing apoptosis and asbestos-induced lung fibrosis. We reason that strategies aimed at augmenting AEC Klotho levels may be an innovative approach for mitigating age-related lung diseases.

Lung-Restricted Antibodies Mediate Primary Graft Dysfunction and Prevent Allotolerance after Murine Lung Transplantation.

Over one-third of lung recipients have preexisting antibodies against lung-restricted antigens: collagen (Col) type V and K-α1 tubulin (KAT). Although clinical studies have shown association of these antibodies with primary graft dysfunction (PGD), their biological significance remains unclear. We tested whether preexisting lung-restricted antibodies can mediate PGD and prevent allotolerance. A murine syngeneic (C57BL/6) or allogeneic (C57BL/6 to BALB/c) left lung transplantation model was used. Rabbit polyclonal antibodies were produced against KAT and Col-V and injected pretransplantation. T cell frequency was analyzed using enzyme-linked immunospot, whereas alloantibodies were determined using flow cytometry. Wet:dry ratio, arterial oxygenation, and histology were used to determine PGD. Preexisting Col-V or KAT, but not isotype control, antibodies lead to dose-dependent development of PGD after syngeneic lung transplantation, as evidenced by poor oxygenation and increased wet:dry ratio. Histology confirmed alveolar and capillary edema. The native right lung remained unaffected. Epitope spreading was observed where KAT antibody treatment led to the development of IL-17-producing CD4+ T cells and humoral response against Col-V, or vice versa. In contrast, isotype control antibody failed to induce Col-V- or KAT-specific cellular or humoral immunity. In addition, none of the mice developed immunity against a non-lung antigen, collagen type II. Preexisting lung-restricted antibodies, but not isotype control, prevented development of allotolerance using the MHC-related 1 and cytotoxic T-lymphocyte-associated protein 4-Ig regimen. Lung-restricted antibodies can induce both early and delayed lung graft dysfunction. These antibodies can also cause spreading of lung-restricted immunity and promote alloimmunity. Antibody-directed therapy to treat preexisting lung-restricted antibodies might reduce PGD after lung transplantation.

Asbestos-induced pulmonary fibrosis is augmented in 8-oxoguanine DNA glycosylase knockout mice.

Asbestos causes asbestosis and malignancies by mechanisms that are not fully established. Alveolar epithelial cell (AEC) injury and repair are crucial determinants of the fibrogenic potential of noxious agents such as asbestos. We previously showed that mitochondrial reactive oxygen species mediate asbestos-induced AEC intrinsic apoptosis and that mitochondrial human 8-oxoguanine-DNA glycosylase 1 (OGG1), a DNA repair enzyme, prevents oxidant-induced AEC apoptosis. We reasoned that OGG1 deficiency augments asbestos-induced pulmonary fibrosis. Compared with intratracheal instillation of PBS (50 µl) or titanium dioxide (100 µg/50 µl), crocidolite or Libby amphibole asbestos (100 µg/50 µl) each augmented pulmonary fibrosis in wild-type C57BL/6J (WT) mice after 3 weeks as assessed by histology, fibrosis score, lung collagen via Sircol, and type 1 collagen expression; these effects persisted at 2 months. Compared with WT mice, Ogg1 homozygous knockout (Ogg1(-/-)) mice exhibit increased pulmonary fibrosis after crocidolite exposure and apoptosis in cells at the bronchoalveolar duct junctions as assessed via cleaved caspase-3 immunostaining. AEC involvement was verified by colocalization studies using surfactant protein C. Asbestos increased endoplasmic reticulum stress in the lungs of WT and Ogg1(-/-) mice. Compared with WT, alveolar type 2 cells isolated from Ogg1(-/-) mice have increased mtDNA damage, reduced mitochondrial aconitase expression, and increased P53 and cleaved caspase-9 expression, and these changes were enhanced 3 weeks after crocidolite exposure. These findings suggest an important role for AEC mtDNA integrity maintained by OGG1 in the pathogenesis of pulmonary fibrosis that may represent a novel therapeutic target.

Uncommon molecular alterations in follicular-derived thyroid carcinoma: A single institution study.

Thyroid carcinomas are the most common endocrine malignancy and commonly have alterations in the mitogen-activated protein kinase (MAPK) and phosphatidylinositol-3 kinase (PI3K)/AKT signaling pathways in well-differentiated tumors. Alternative molecular alterations driving thyroid carcinomas have been identified rarely in the literature and are more likely to occur in poorly differentiated or anaplastic cases. In this study, uncommon genetic alterations such as MLH1, MSH2, NSD3::NUTM1, RET::SPECC1L, and G3BP2::FGFR2 were identified in patients with papillary thyroid carcinoma, poorly differentiated thyroid carcinoma, and differentiated high-grade thyroid carcinoma. Most of these tumors demonstrated an aggressive biological behavior. Atypical driver mutations in thyroid carcinomas can occur in patients with cancer predisposition syndromes as demonstrated by an NTRK1::TPM3 fusion in a patient with Li Fraumeni syndrome. In these settings of more aggressive disease, molecular testing targeting actionable fusions and mutations is important. As demonstrated in our case cohort, 100% of cases diagnosed as high-grade follicular-derived thyroid carcinoma had a mutation or fusion that is associated with worse prognosis, has a germline syndrome association requiring further work up, or an actionable mutation. This high yield seen in this cohort for molecular testing in patients with high-grade follicular-derived thyroid carcinoma suggests more routine molecular testing in this population would be a beneficial clinical practice.

Multimodal imaging of an acute presentation of ocular histoplasmosis syndrome in an immunocompetent patient.

Purpose: Presumed ocular histoplasmosis syndrome (POHS) is a posterior segment disorder that is usually subclinical unless choroidal neovascular membrane (CNVM) develops. It is thought to be the sequela of a prior systemic infection with Histoplasma capsulatum, and evidence supporting this association is based on epidemiologic, animal, and few enucleation studies. Acute presentation of chorioretinal involvement during an initial histoplasmosis systemic infection in immunocompetent patients is rarely reported, presumably due to the usual lack of or minimal symptoms of both the systemic and ocular disease. We report on an immunocompetent male with choroidal lesions detected during disseminated histoplasmosis infection and characterize the lesions using multimodal imaging. Observations: A 17-year-old male presented when routine optometry screening detected two deep, yellowish-white lesions in the left fundus. Optical coherence tomography (OCT) imaging confirmed a choroidal mass with extension through Bruch's membrane into the subretinal space and a small amount of subretinal fluid. Fluorescein angiography was suggestive of CNVM. There were no clinical findings of intraocular inflammation, and the patient was initially lost to follow-up. Eight weeks after last follow-up, the patient presented to the emergency department with fatigue, mild respiratory symptoms, and abdominal pain for the last month. Imaging revealed a mediastinal mass with hilar extension and innumerable nodules throughout the lung and spleen. Serum Histoplasma IgM/IgG were positive, and biopsy of the mediastinal mass revealed Histoplasma organisms. The patient was treated with antifungals and discharged. The patient underwent an extensive immunologic evaluation while admitted, which did not reveal an underlying immunodeficiency. On last follow-up, the choroidal lesions were smaller and more consolidated, and the subretinal fluid had resolved. Conclusions and Importance: We present a patient with choroidal lesions in the setting of disseminated systemic histoplasmosis infection and characterize a lesion using multimodal imaging. The presentation of acute chorioretinal lesions in the setting of biopsy proven systemic Histoplasma infection supports H. capsulatum as the etiology of POHS.

PLG nanoparticles target fibroblasts and MARCO+ monocytes to reverse multiorgan fibrosis.

Systemic sclerosis (SSc) is a chronic, multisystem orphan disease with a highly variable clinical course, high mortality rate, and a poorly understood complex pathogenesis. We have identified an important role for a subpopulation of monocytes and macrophages characterized by surface expression of the scavenger receptor macrophage receptor with collagenous structure (MARCO) in chronic inflammation and fibrosis in SSc and in preclinical disease models. We show that MARCO+ monocytes and macrophages accumulate in lesional skin and lung in topographic proximity to activated myofibroblasts in patients with SSc and in the bleomycin-induced mouse model of SSc. Short-term treatment of mice with a potentially novel nanoparticle, poly(lactic-co-glycolic) acid (PLG), which is composed of a carboxylated, FDA-approved, biodegradable polymer and modulates activation and trafficking of MARCO+ inflammatory monocytes, markedly attenuated bleomycin-induced skin and lung inflammation and fibrosis. Mechanistically, in isolated cells in culture, PLG nanoparticles inhibited TGF-dependent fibrotic responses in vitro. Thus, MARCO+ monocytes are potent effector cells of skin and lung fibrosis and can be therapeutically targeted in SSc using PLG nanoparticles.

IL-1ß-dependent extravasation of preexisting lung-restricted autoantibodies during lung transplantation activates complement and mediates primary graft dysfunction.

Preexisting lung-restricted autoantibodies (LRAs) are associated with a higher incidence of primary graft dysfunction (PGD), although it remains unclear whether LRAs can drive its pathogenesis. In syngeneic murine left lung transplant recipients, preexisting LRAs worsened graft dysfunction, which was evident by impaired gas exchange, increased pulmonary edema, and activation of damage-associated pathways in lung epithelial cells. LRA-mediated injury was distinct from ischemia-reperfusion injury since deletion of donor nonclassical monocytes and host neutrophils could not prevent graft dysfunction in LRA-pretreated recipients. Whole LRA IgG molecules were necessary for lung injury, which was mediated by the classical and alternative complement pathways and reversed by complement inhibition. However, deletion of Fc receptors in donor macrophages or mannose-binding lectin in recipient mice failed to rescue lung function. LRA-mediated injury was localized to the transplanted lung and dependent on IL-1ß-mediated permeabilization of pulmonary vascular endothelium, which allowed extravasation of antibodies. Genetic deletion or pharmacological inhibition of IL-1R in the donor lungs prevented LRA-induced graft injury. In humans, preexisting LRAs were an independent risk factor for severe PGD and could be treated with plasmapheresis and complement blockade. We conclude that preexisting LRAs can compound ischemia-reperfusion injury to worsen PGD for which complement inhibition may be effective.

Fibroblast A20 governs fibrosis susceptibility and its repression by DREAM promotes fibrosis in multiple organs.

In addition to autoimmune and inflammatory diseases, variants of the TNFAIP3 gene encoding the ubiquitin-editing enzyme A20 are also associated with fibrosis in systemic sclerosis (SSc). However, it remains unclear how genetic factors contribute to SSc pathogenesis, and which cell types drive the disease due to SSc-specific genetic alterations. We therefore characterize the expression, function, and role of A20, and its negative transcriptional regulator DREAM, in patients with SSc and disease models. Levels of A20 are significantly reduced in SSc skin and lungs, while DREAM is elevated. In isolated fibroblasts, A20 mitigates ex vivo profibrotic responses. Mice haploinsufficient for A20, or harboring fibroblasts-specific A20 deletion, recapitulate major pathological features of SSc, whereas DREAM-null mice with elevated A20 expression are protected. In DREAM-null fibroblasts, TGF-ß induces the expression of A20, compared to wild-type fibroblasts. An anti-fibrotic small molecule targeting cellular adiponectin receptors stimulates A20 expression in vitro in wild-type but not A20-deficient fibroblasts and in bleomycin-treated mice. Thus, A20 has a novel cell-intrinsic function in restraining fibroblast activation, and together with DREAM, constitutes a critical regulatory network governing the fibrotic process in SSc. A20 and DREAM represent novel druggable targets for fibrosis therapy.

A novel soluble ACE2 protein totally protects from lethal disease caused by SARS-CoV-2 infection.

Severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) uses full-length angiotensin converting enzyme 2 (ACE2), which is membrane bound, as its initial cell contact receptor preceding viral entry. Here we report a human soluble ACE2 variant fused with a 5kD albumin binding domain (ABD) and bridged via a dimerization motif hinge-like 4-cysteine dodecapeptide, which we term ACE2 1-618-DDC-ABD. This protein is enzymatically active, has increased duration of action in vivo conferred by the ABD-tag, and displays 20-30-fold higher binding affinity to the SARS-CoV-2 receptor binding domain than its des-DDC monomeric form (ACE2 1-618-ABD) due to DDC-linked dimerization. ACE2 1-618-DDC-ABD was administered for 3 consecutive days to transgenic k18-hACE2 mice, a model that develops lethal SARS-CoV-2 infection, to evaluate the preclinical preventative/ therapeutic value for COVID-19. Mice treated with ACE2 1-618-DDC-ABD developed a mild to moderate disease for the first few days assessed by a clinical score and modest weight loss. The untreated control animals, by contrast, became severely ill and had to be sacrificed by day 6/7 and lung histology revealed extensive pulmonary alveolar hemorrhage and mononuclear infiltrates. At 6 days, mortality was totally prevented in the treated group, lung histopathology was improved and viral titers markedly reduced. This demonstrates for the first time in vivo the preventative/ therapeutic potential of a novel soluble ACE2 protein in a preclinical animal model.

Liver Pathology and SARS-CoV-2 Detection in Formalin-Fixed Tissue of Patients With COVID-19.

OBJECTIVES: The novel coronavirus, severe acute respiratory syndrome coronavirus 2, causing coronavirus disease 2019 (COVID-19) remains a global health threat and a significant source of human morbidity and mortality. While the virus primarily induces lung injury, it also has been reported to cause hepatic sequelae. METHODS: We aimed to detect the virus in formalin-fixed tissue blocks and document the liver injury patterns in patients with COVID-19 compared with a control group. RESULTS: We were able to detect viral RNA in the bronchioalveolar cell blocks (12/12, 100%) and formalin-fixed, paraffin-embedded tissue of the lung (8/8, 100%) and liver (4/9, 44%) of patients with COVID-19. Although the peak values of the main liver enzymes and bilirubin were higher in the patients with COVID-19 compared with the control group, the differences were not significant. The main histologic findings were minimal to focal mild portal tract chronic inflammation (7/8, 88%, P < .05) and mild focal lobular activity (6/8, 75%, P = .06). CONCLUSIONS: We found that most patients who died of COVID-19 had evidence of mild focal hepatitis clinically and histologically; however, the virus was detected in less than half of the cases.