SARS-CoV-2 infection of human ACE2-transgenic mice causes severe lung inflammation and impaired function.

Although animal models have been evaluated for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, none have fully recapitulated the lung disease phenotypes seen in humans who have been hospitalized. Here, we evaluate transgenic mice expressing the human angiotensin I-converting enzyme 2 (ACE2) receptor driven by the cytokeratin-18 (K18) gene promoter (K18-hACE2) as a model of SARS-CoV-2 infection. Intranasal inoculation of SARS-CoV-2 in K18-hACE2 mice results in high levels of viral infection in lungs, with spread to other organs. A decline in pulmonary function occurs 4 days after peak viral titer and correlates with infiltration of monocytes, neutrophils and activated T cells. SARS-CoV-2-infected lung tissues show a massively upregulated innate immune response with signatures of nuclear factor-κB-dependent, type I and II interferon signaling, and leukocyte activation pathways. Thus, the K18-hACE2 model of SARS-CoV-2 infection shares many features of severe COVID-19 infection and can be used to define the basis of lung disease and test immune and antiviral-based countermeasures.

Publisher Correction: SARS-CoV-2 infection of human ACE2-transgenic mice causes severe lung inflammation and impaired function.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

AI-guided histopathology predicts brain metastasis in lung cancer patients.

Brain metastases can occur in nearly half of patients with early and locally advanced (stage I-III) non-small cell lung cancer (NSCLC). There are no reliable histopathologic or molecular means to identify those who are likely to develop brain metastases. We sought to determine if deep learning (DL) could be applied to routine H&E-stained primary tumor tissue sections from stage I-III NSCLC patients to predict the development of brain metastasis. Diagnostic slides from 158 patients with stage I-III NSCLC followed for at least 5 years for the development of brain metastases (Met+, 65 patients) versus no progression (Met-, 93 patients) were subjected to whole-slide imaging. Three separate iterations were performed by first selecting 118 cases (45 Met+, 73 Met-) to train and validate the DL algorithm, while 40 separate cases (20 Met+, 20 Met-) were used as the test set. The DL algorithm results were compared to a blinded review by four expert pathologists. The DL-based algorithm was able to distinguish the eventual development of brain metastases with an accuracy of 87% (p < 0.0001) compared with an average of 57.3% by the four pathologists and appears to be particularly useful in predicting brain metastases in stage I patients. The DL algorithm appears to focus on a complex set of histologic features. DL-based algorithms using routine H&E-stained slides may identify patients who are likely to develop brain metastases from those who will remain disease free over extended (>5 year) follow-up and may thus be spared systemic therapy. © 2024 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Smoking exposure-induced bronchus-associated lymphoid tissue in donor lungs does not prevent tolerance induction after transplantation.

The presence of bronchus-associated lymphoid tissue (BALT) in donor lungs has been suggested to accelerate graft rejection after lung transplantation. Although chronic smoke exposure can induce BALT formation, the impact of donor cigarette use on alloimmune responses after lung transplantation is not well understood. Here, we show that smoking-induced BALT in mouse donor lungs contains Foxp3+ T cells and undergoes dynamic restructuring after transplantation, including recruitment of recipient-derived leukocytes to areas of pre-existing lymphoid follicles and replacement of graft-resident donor cells. Our findings from mouse and human lung transplant data support the notion that a donor's smoking history does not predispose to acute cellular rejection or prevent the establishment of allograft acceptance with comparable outcomes to nonsmoking donors. Thus, our work indicates that BALT in donor lungs is plastic in nature and may have important implications for modulating proinflammatory or tolerogenic immune responses following transplantation.

Ischemia reperfusion injury facilitates lung allograft acceptance through IL-33-mediated activation of donor-derived IL-5 producing group 2 innate lymphoid cells.

Pathways regulating lung alloimmune responses differ from most other solid organs and remain poorly explored. Based on our recent work identifying the unique role of eosinophils in downregulating lung alloimmunity, we sought to define pathways contributing to eosinophil migration and homeostasis. Using a murine lung transplant model, we have uncovered that immunosuppression increases eosinophil infiltration into the allograft in an IL-5-dependent manner. IL-5 production depends on immunosuppression-mediated preservation of donor-derived group 2 innate lymphoid cells (ILC2). We further describe that ischemia reperfusion injury upregulates the expression of IL-33, which functions as the dominant and nonredundant mediator of IL-5 production by graft-resident ILC2. Our work thus identifies unique cellular mechanisms that contribute to lung allograft acceptance. Notably, ischemia reperfusion injury, widely considered to be solely deleterious to allograft survival, can also downregulate alloimmune responses by initiating unique pathways that promote IL-33/IL-5/eosinophil-mediated tolerance.

Bacterial products in donor airways prevent the induction of lung transplant tolerance.

Although postoperative bacterial infections can trigger rejection of pulmonary allografts, the impact of bacterial colonization of donor grafts on alloimmune responses to transplanted lungs remains unknown. Here, we tested the hypothesis that bacterial products present within donor grafts at the time of implantation promote lung allograft rejection. Administration of the toll-like receptor 2 (TLR2) agonist Pam3 Cys4 to Balb/c wild-type grafts triggered acute cellular rejection after transplantation into B6 wild-type recipients that received perioperative costimulatory blockade. Pam3 Cys4 -triggered rejection was associated with an expansion of CD8+ T lymphocytes and CD11c+ CD11bhi MHC (major histocompatibility complex) class II+ antigen-presenting cells within the transplanted lungs. Rejection was prevented when lungs were transplanted into TLR2-deficient recipients but not when MyD88-deficient donors were used. Adoptive transfer of B6 wild-type monocytes, but not T cells, following transplantation into B6 TLR2-deficient recipients restored the ability of Pam3 Cys4 to trigger acute cellular rejection. Thus, we have demonstrated that activation of TLR2 by a bacterial lipopeptide within the donor airways prevents the induction of lung allograft tolerance through a process mediated by recipient-derived monocytes. Our work suggests that donor lungs harboring bacteria may precipitate an inflammatory response that can facilitate allograft rejection.

IL-22 is required for the induction of bronchus-associated lymphoid tissue in tolerant lung allografts.

Long-term survival after lung transplantation remains profoundly limited by graft rejection. Recent work has shown that bronchus-associated lymphoid tissue (BALT), characterized by the development of peripheral nodal addressin (PNAd)-expressing high endothelial venules and enriched in B and Foxp3+ T cells, is important for the maintenance of allograft tolerance. Mechanisms underlying BALT induction in tolerant pulmonary allografts, however, remain poorly understood. Here, we show that the development of PNAd-expressing high endothelial venules within intragraft lymphoid follicles and the recruitment of B cells, but not Foxp3+ cells depends on IL-22. We identify graft-infiltrating gamma-delta (γδ) T cells and Type 3 innate lymphoid cells (ILC3s) as important producers of IL-22. Reconstitution of IL-22 at late time points through retransplantation into wildtype hosts mediates B cell recruitment into lymphoid follicles within the allograft, resulting in a significant increase in their size, but does not induce PNAd expression. Our work has identified cellular and molecular requirements for the induction of BALT in pulmonary allografts during tolerance induction and may provide a platform for the development of new therapies for lung transplant patients.

Clinicopathologic characteristics of de novo nodular regenerative hyperplasia in pediatric liver transplant.

Liver NRH is seen in all patients age; however, more frequently in those over the age of 60 years and associated with multiple systemic diseases. In liver allograft recipients, the development of DnNRH has been linked with the use of azathioprine or vascular abnormalities. We present the clinicopathologic characteristics of 17 pediatric patients who underwent liver transplantation and subsequently developed DnNRH. The patients were divided into early and late onset depending if DnNRH was diagnosed within or beyond 4 years after transplant. Eight patients (47%) presented as early onset, of which two had normal ultrasound at time of diagnosis. One patient (12.5%) with early onset lost the graft secondary to DnNRH. Nine patients (53%) presented as late onset, of which two (22%) had normal ultrasound at time of diagnosis. Two patients (25%) of the late onset lost their graft secondary to chronic rejection and DnNRH. Two patients (12%) died secondary to cytomegalovirus pneumonitis and pancolitis. Furthermore, both groups presented with symptoms differing from the adult population in prior studies and were not associated with the use of azathioprine or vascular abnormalities. Interestingly, episodes of acute cellular rejection were more common in the early-onset group compared to the late-onset group. In conclusion, DnNRH in the pediatric age group has a different clinical presentation, possibly reflecting a different pathogenesis compared to the adult population.

Pediatric Pulmonary Emboli at Autopsy: An Update and Case Series Review.

OBJECTIVES: Identify and characterize pediatric pulmonary emboli present at autopsy. DESIGN: Retrospective single institution observational study with clinicopathologic correlation. SETTING: Tertiary medical center. PATIENTS: All autopsy cases performed at Washington University from 1997 to 2017 in pediatric patients (≤ 18 yr old). MAIN RESULTS: Of 1,763 pediatric autopsies, 13 cases of pulmonary emboli were identified, including thromboemboli (6/13, 46.1%), septic emboli (3/13, 23.1%), fat emboli, and foreign body emboli. CONCLUSIONS: Pulmonary embolus is a relatively rare but potentially fatal cause of death in pediatric age patients and is often associated with congenital abnormalities, malignancy, or recent surgical procedures. Half of the fatal pulmonary emboli found in our series (3/6) show microscopic and diffuse, rather than large central or saddle emboli, potentially make a clinicoradiographic diagnosis more difficult. This series is also the first to report a case of hemostatic matrix pulmonary embolism in a pediatric age patient.

Pulmonary and pleural pathology: Contributions of Dr. Louis "Pepper" Dehner.

Dr. Louis Dehner is an internationally renowned surgical pathologist who has published multiple textbooks and has authored or co-authored nearly 400 original articles in the medical literature. While many think of him as a pediatric pathologist, he has contributed to the literature across virtually the entire breadth of surgical pathology, and the lung and pleura is no exception. This review will highlight Dr. Dehner׳s contributions to the pulmonary and pleural pathology literature in the areas of infectious disease, medical lung disease and transplant pathology, and a number of neoplasms of the lung and pleura, with the remainder of this manuscript dedicated to the still evolving story of the pleuropulmonary blastoma as the signature contribution of his long and distinguished career.

Emergency granulopoiesis promotes neutrophil-dendritic cell encounters that prevent mouse lung allograft acceptance.

The mechanisms by which innate immune signals regulate alloimmune responses remain poorly understood. In the present study, we show by intravital 2-photon microscopy direct interactions between graft-infiltrating neutrophils and donor CD11c(+) dendritic cells (DCs) within orthotopic lung allografts immediately after reperfusion. Neutrophils isolated from the airways of lung transplantation recipients stimulate donor DCs in a contact-dependent fashion to augment their production of IL-12 and expand alloantigen-specific IFN-γ(+) T cells. DC IL-12 expression is largely regulated by degranulation and induced by TNF-α associated with the neutrophil plasma membrane. Extended cold ischemic graft storage enhances G-CSF-mediated granulopoiesis and neutrophil graft infiltration, resulting in exacerbation of ischemia-reperfusion injury after lung transplantation. Ischemia reperfusion injury prevents immunosuppression-mediated acceptance of mouse lung allografts unless G-CSF-mediated granulopoiesis is inhibited. Our findings identify granulopoiesis-mediated augmentation of alloimmunity as a novel link between innate and adaptive immune responses after organ transplantation.

Development and validation of a decision tree for distinguishing pulmonary adenocarcinomas with mucinous features and metastatic colorectal adenocarcinoma.

BACKGROUND: Diagnosis of mucinous carcinomas in the lung on transbronchial biopsy or fine-needle aspiration (FNA) samples can be difficult for the pathologist, because primary and metastatic tumors can have similar morphological, immunohistochemical, and molecular characteristics. Correct diagnosis is key to determine appropriate therapy and to distinguish primary from metastatic disease. This distinction often falls to the pathologist in patients with a history of mucinous adenocarcinoma of the colon. Despite its drawbacks, immunohistochemistry is often employed to help assign a primary site for mucinous adenocarcinomas in the lung. However, the published data in this regard is limited to studies that use only a handful of markers. METHODS: The authors examined the staining characteristics and heterogeneity of CK7, TTF-1, NapsinA, CK20, CDX2, and SATB2 in resection specimens of pulmonary adenocarcinomas with mucinous features and metastatic colorectal adenocarcinoma. RESULTS: Based on the heterogeneity, sensitivity, and specificity in this cohort, the authors developed a decision tree based on TTF-1, SATB2, CDX2, and CK7 to categorize tumors as primary or metastatic lesions. Validation of the decision tree in FNA specimens from the lungs and lung-draining lymph nodes showed 84% concurrence in cases from the lung and 100% concurrence in cases from the lymph node. In cases where the algorithm assigned a primary site, it was 95% accurate compared to the multidisciplinary diagnosis. CONCLUSIONS: This method holds promise in distinguishing primary versus metastatic lesions in resection, biopsy, and FNA samples from the lungs.

Clinical and Histopathologic Characteristics of Recurrent Sarcoidosis in Posttransplant Lungs: 25 Years of Experience.

Lung transplantation is the definitive therapy for end-stage pulmonary sarcoidosis. While recurrent sarcoidosis in allografts has been described in several case reports, the incidence and clinicopathologic characteristics remain unclear. In this study, we characterize the clinical and histopathologic features of recurrent sarcoidosis diagnosed in posttransplant lung surveillance transbronchial biopsies (TBBx). We identified 35 patients who underwent lung transplant for pulmonary sarcoidosis during the study period. Among them, 18 patients (51%) experienced recurrent sarcoidosis posttransplant. These included 7 females and 11 males with mean age at recurrence of 51.6 years. The average time interval from transplant to recurrence was 252 days (22 to 984 d). All TBBx contained >4 pieces of alveolated lung tissue with no evidence of International Society for Heart and Lung Transplantation (ISHLT) grade A2, A3, or A4 acute cellular rejection; chronic rejection; or antibody-mediated rejection. There were 33 surveillance TBBx that contained granulomatous inflammation with a mean of 3.6 well-formed granulomas per TBBx (range: 1 to >20). Multinucleated giant cells were identified in 11 TBBx (33.3%), with 1 case containing asteroid bodies. While most of the granulomas were "naked granulomas," 5 cases (15.2%) showed prominent lymphoid cuffing. Two cases showed evidence of fibrosis. One of the granulomas had focal necrosis; however, no infectious organisms were identified by special stains and clinical correlation suggested this case represented recurrent sarcoidosis. Biopsies of recurrent sarcoidosis usually show multiple well-formed granulomas with giant cells in more than half of the cases, while lymphoid cuffing, fibrosis, asteroid bodies, and necrotizing granulomas are uncommon findings. Pathologists should be aware of these features, as recurrence of sarcoidosis following lung transplant occurs in more than half of patients.

Artificial Intelligence-Powered Assessment of Pathologic Response to Neoadjuvant Atezolizumab in Patients With NSCLC: Results From the LCMC3 Study.

INTRODUCTION: Pathologic response (PathR) by histopathologic assessment of resected specimens may be an early clinical end point associated with long-term outcomes with neoadjuvant therapy. Digital pathology may improve the efficiency and precision of PathR assessment. LCMC3 (NCT02927301) evaluated neoadjuvant atezolizumab in patients with resectable NSCLC and reported a 20% major PathR rate. METHODS: We determined PathR in primary tumor resection specimens using guidelines-based visual techniques and developed a convolutional neural network model using the same criteria to digitally measure the percent viable tumor on whole-slide images. Concordance was evaluated between visual determination of percent viable tumor (n = 151) performed by one of the 47 local pathologists and three central pathologists. RESULTS: For concordance among visual determination of percent viable tumor, the interclass correlation coefficient was 0.87 (95% confidence interval [CI]: 0.84-0.90). Agreement for visually assessed 10% or less viable tumor (major PathR [MPR]) in the primary tumor was 92.1% (Fleiss kappa = 0.83). Digitally assessed percent viable tumor (n = 136) correlated with visual assessment (Pearson r = 0.73; digital/visual slope = 0.28). Digitally assessed MPR predicted visually assessed MPR with outstanding discrimination (area under receiver operating characteristic curve, 0.98) and was associated with longer disease-free survival (hazard ratio [HR] = 0.30; 95% CI: 0.09-0.97, p = 0.033) and overall survival (HR = 0.14, 95% CI: 0.02-1.06, p = 0.027) versus no MPR. Digitally assessed PathR strongly correlated with visual measurements. CONCLUSIONS: Artificial intelligence-powered digital pathology exhibits promise in assisting pathologic assessments in neoadjuvant NSCLC clinical trials. The development of artificial intelligence-powered approaches in clinical settings may aid pathologists in clinical operations, including routine PathR assessments, and subsequently support improved patient care and long-term outcomes.

Are we overtreating papillomas diagnosed on core needle biopsy?

BACKGROUND: Breast papillomas often are diagnosed with core needle biopsy (CNB). Most studies support excision for atypical papillomas, because as many as one half will be upgraded to malignancy on final pathology. The literature is less clear on the management of papillomas without atypia on CNB. Our goal was to determine factors associated with pathology upgrade on excision. METHODS: Our pathology database was searched for breast papillomas diagnosed by CNB during the past 10 years. We identified 277 charts and excluded lesions associated with atypia or malignancy on CNB. Two groups were identified: papillomas that were surgically excised (group 1) and those that were not (group 2). Charts were reviewed for the subsequent diagnosis of cancer or high-risk lesions. Appropriate statistical tests were used to analyze the data. RESULTS: A total of 193 papillomas were identified. Eighty-two lesions were excised (42%). Caucasian women were more likely to undergo excision (p = 0.03). Twelve percent of excised lesions were upgraded to malignancy. Increasing age was a predictor of upgrading, but this was not significant. Clinical presentation, lesion location, biopsy technique, and breast cancer history were not associated with pathology upgrade. Two lesions in group 2 ultimately required excision due to enlargement, and both were upgraded to malignancy. CONCLUSIONS: Twenty-four percent of papillomas diagnosed on CNB have upgraded pathology on excision--half to malignancy. All of the cancers diagnosed were stage 0 or I. For patients in whom excision was not performed, 2 of 111 papillomas were later excised and upgraded to malignancy.

Emphysema quantification in inflation-fixed lungs using low-dose computed tomography and 3He magnetic resonance imaging.

OBJECTIVE: To evaluate the use of inflation-fixed lung tissue for emphysema quantification with computed tomography (CT) and He magnetic resonance (MR) diffusion imaging. METHODS: Fourteen subjects representing a range of chronic obstructive pulmonary disease severity who underwent complete or lobar lung resection were studied. Computed tomographic measurements of lung attenuation and MR measurements of the hyperpolarized 3He apparent diffusion coefficient (ADC) in resected specimens fixed in inflation with heated formalin vapor were compared with measurements obtained before fixation. RESULTS: The mean (SD) CT emphysema indices were 56% (17%) before and 58% (19%) after fixation (P = 0.77; R = 0.76). Index differences correlated with differences in lung volume (R = 0.47). The mean (SD) 3He ADCs were 0.40 (0.15) cm/s before and 0.39 (0.14) cm/s after fixation (P = 0.03, R = 0.98). The CT emphysema index and the 3He ADC were correlated before (R = 0.89) and after fixation (R = 0.79). CONCLUSIONS: Concordance of CT and 3He MR imaging measurements in unfixed and inflation-fixed lungs supports the use of inflation-fixed lungs for quantitative imaging studies in emphysema.

Lymphatic drainage from bronchus-associated lymphoid tissue in tolerant lung allografts promotes peripheral tolerance.

Tertiary lymphoid organs are aggregates of immune and stromal cells including high endothelial venules and lymphatic vessels that resemble secondary lymphoid organs and can be induced at nonlymphoid sites during inflammation. The function of lymphatic vessels within tertiary lymphoid organs remains poorly understood. During lung transplant tolerance, Foxp3+ cells accumulate in tertiary lymphoid organs that are induced within the pulmonary grafts and are critical for the local downregulation of alloimmune responses. Here, we showed that tolerant lung allografts could induce and maintain tolerance of heterotopic donor-matched hearts through pathways that were dependent on the continued presence of the transplanted lung. Using lung retransplantation, we showed that Foxp3+ cells egressed from tolerant lung allografts via lymphatics and were recruited into donor-matched heart allografts. Indeed, survival of the heart allografts was dependent on lymphatic drainage from the tolerant lung allograft to the periphery. Thus, our work indicates that cellular trafficking from tertiary lymphoid organs regulates immune responses in the periphery. We propose that these findings have important implications for a variety of disease processes that are associated with the induction of tertiary lymphoid organs.

SARS-CoV-2 infection in the lungs of human ACE2 transgenic mice causes severe inflammation, immune cell infiltration, and compromised respiratory function.

Severe Acute Respiratory Syndrome Coronavirus -2 (SARS-CoV-2) emerged in late 2019 and has spread worldwide resulting in the Coronavirus Disease 2019 (COVID-19) pandemic. Although animal models have been evaluated for SARS-CoV-2 infection, none have recapitulated the severe lung disease phenotypes seen in hospitalized human cases. Here, we evaluate heterozygous transgenic mice expressing the human ACE2 receptor driven by the epithelial cell cytokeratin-18 gene promoter (K18-hACE2) as a model of SARS-CoV-2 infection. Intranasal inoculation of SARS-CoV-2 in K18-hACE2 mice results in high levels of viral infection in lung tissues with additional spread to other organs. Remarkably, a decline in pulmonary function, as measured by static and dynamic tests of respiratory capacity, occurs 4 days after peak viral titer and correlates with an inflammatory response marked by infiltration into the lung of monocytes, neutrophils, and activated T cells resulting in pneumonia. Cytokine profiling and RNA sequencing analysis of SARS-CoV-2-infected lung tissues show a massively upregulated innate immune response with prominent signatures of NF-kB-dependent, type I and II interferon signaling, and leukocyte activation pathways. Thus, the K18-hACE2 model of SARS-CoV-2 infection recapitulates many features of severe COVID-19 infection in humans and can be used to define the mechanistic basis of lung disease and test immune and antiviral-based countermeasures.

Saline-linked surface radiofrequency ablation: a safe and effective method of surface ablation of hepatic metastatic colorectal cancer.

OBJECTIVE: To determine the safety and efficacy of saline-linked surface radiofrequency ablation (SLSRFA) in a clinical setting. SUMMARY BACKGROUND DATA: We have previously identified safe and effective parameters for use of SLSRFA in a porcine model. METHODS: An initial study was conducted to determine if parameters defined in the porcine model were safe and effective in human livers. In 16 patients undergoing liver resection, normal areas of liver were treated with SLSRFA using various power/diameter combinations (10 W/1 cm; 15 W/2 cm; 45 W/4 cm) for 9 minutes with and without inflow occlusion. In a second study, superficial hepatic colorectal cancer (CRC) metastases were treated at 45 W/4 cm for 9 minutes without inflow occlusion in 11 patients. Ablation depth was measured and samples were examined for cell viability by nicotine adenine dinucleotide stain. This study was registered in the ClinicalTrials.gov database and has the following ID number, NCT00869843. RESULTS: Ablation depth in normal liver varied from 3 to 20 mm. Depth was significantly dependent on power, lesion size, and inflow occlusion. Nicotine adenine dinucleotide stains showed total cell necrosis to the full depth of ablation. In the second study, large hepatic CRC metastases showed total cell necrosis to a mean depth of 12 mm. Two tumors less than 7 mm in depth showed complete necrosis. Metastases were more susceptible to SLSRFA than normal liver. CONCLUSION: SLSRFA completely and safely ablates normal liver to a depth of at least 4 mm at 45 W/4 cm treatment parameters. Remarkably, it is even more effective in ablating metastatic CRC. SLSRFA is an effective tool for extending resection margins and for ablating superficial small tumors or superficial parts of large tumors.