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.

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.

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.

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.

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.

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.

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.

Asbestosis: demonstration of distinctive interstitial fibroelastosis: a pilot study.

Asbestosis has long been defined as a diffuse interstitial "fibrotic" process, in similarity to other chronic interstitial pulmonary diseases. To address the hypothesis (which was based on morphological nuances) that the interstitial connective tissue response in asbestosis may be fibroelastotic rather than fibrotic, a comparative characterization of the connective response in cases of asbestosis and other forms of interstitial lung disease was performed. Archival open lung biopsies or autopsy specimens of pulmonary diseases featuring interstitial connective tissue abnormalities (15 of asbestosis, 21 of organizing pneumonia, 15 usual interstitial pneumonitis/idiopathic pulmonary fibrosis [IPF], 9 organizing diffuse alveolar damage, 9 "nonspecific" interstitial pneumonitis, 4 sarcoidosis, 3 each of desquamative interstitial pneumonia and chronic amiodarone toxicity, 2 cryptogenic organizing pneumonias, and 1 each of chronic hypersensitivity pneumonitis and chronic eosinophilic pneumonitis [85 total]) were stained histochemically with hematoxylin and eosin, Perl's method, Gomori's trichrome procedure, and the Verhoeff-van Gieson technique. Representative subsets of the cases (n = 20) were also studied immunohistologically using an antibody to elastin. Fibroelastosis in each of the samples was assessed for the degree of response and its location using a 3-tiered scale. The degree of fibroelastosis in the 15 cases of asbestosis was variable, with the pattern being peribronchial and perivascular in all instances; at least 2 asbestos bodies were identified in fibroelastotic foci in each of the 15 cases as highlighted with Perl's stain. Forty-seven cases of nonasbestotic lung disease (71%) showed interstitial fibrosis with a variable (usually modest) amount of admixed elastic tissue; when present, elastic fibers were distributed in a diffuse interstitial pattern, with or without perivascular accentuation. All cases of IPF also showed areas of fibroelastosis, but those foci were confined to regions of overt "honeycomb" change. No asbestos bodies were seen in any disease except asbestosis, and a predominantly peribronchial pattern of fibroelastosis was not identified in any nonasbestotic interstitial lung disease in this study. The authors conclude that the types and patterns of pulmonary connective tissue response in interstitial lung diseases may provide additional diagnostic clues to the presence of asbestosis.

Bronchus-associated lymphoid tissue-resident Foxp3+ T lymphocytes prevent antibody-mediated lung rejection.

Antibody-mediated rejection (AMR) is a principal cause of acute and chronic failure of lung allografts. However, mechanisms mediating this oftentimes fatal complication are poorly understood. Here, we show that Foxp3+ T cells formed aggregates in rejection-free human lung grafts and accumulated within induced bronchus-associated lymphoid tissue (BALT) of tolerant mouse lungs. Using a retransplantation model, we show that selective depletion of graft-resident Foxp3+ T lymphocytes resulted in the generation of donor-specific antibodies (DSA) and AMR, which was associated with complement deposition and destruction of airway epithelium. AMR was dependent on graft infiltration by B and T cells. Depletion of graft-resident Foxp3+ T lymphocytes resulted in prolonged interactions between B and CD4+ T cells within transplanted lungs, which was dependent on CXCR5-CXCL13. Blockade of CXCL13 as well as inhibition of the CD40 ligand and the ICOS ligand suppressed DSA production and prevented AMR. Thus, we have shown that regulatory Foxp3+ T cells residing within BALT of tolerant pulmonary allografts function to suppress B cell activation, a finding that challenges the prevailing view that regulation of humoral responses occurs peripherally. As pulmonary AMR is largely refractory to current immunosuppression, our findings provide a platform for developing therapies that target local immune responses.

Recurrent WNT pathway alterations are frequent in relapsed small cell lung cancer.

Nearly all patients with small cell lung cancer (SCLC) eventually relapse with chemoresistant disease. The molecular mechanisms driving chemoresistance in SCLC remain un-characterized. Here, we describe whole-exome sequencing of paired SCLC tumor samples procured at diagnosis and relapse from 12 patients, and unpaired relapse samples from 18 additional patients. Multiple somatic copy number alterations, including gains in ABCC1 and deletions in MYCL, MSH2, and MSH6, are identifiable in relapsed samples. Relapse samples also exhibit recurrent mutations and loss of heterozygosity in regulators of WNT signaling, including CHD8 and APC. Analysis of RNA-sequencing data shows enrichment for an ASCL1-low expression subtype and WNT activation in relapse samples. Activation of WNT signaling in chemosensitive human SCLC cell lines through APC knockdown induces chemoresistance. Additionally, in vitro-derived chemoresistant cell lines demonstrate increased WNT activity. Overall, our results suggest WNT signaling activation as a mechanism of chemoresistance in relapsed SCLC.

Expression of mucins, SIMA, villin, and CDX2 in small-intestinal adenocarcinoma.

Expression of gastrointestinal biomarkers MUC1, MUC2, MUC5AC, small-intestinal mucin antigen (SIMA), villin, and CDX2 has been studied in colorectal adenocarcinoma (CRC). Little is known, however, about their expression in small-intestinal adenocarcinoma (SIA). We immunohistochemically compared 30 SIAs with 48 CRCs for the expression of these biomarkers. The results showed that all 6 proteins were variably expressed in SIA, but the frequencies of expression were significantly lower than those for CRC with the exception of MUC1. Specifically, positive staining for MUC1, MUC2, MUC5AC, SIMA, villin, and CDX2 was observed in 16 (53%), 17 (57%), 12 (40%), 15 (50%), 20 (67%), and 18 (60%) of SIAs and 25 (52%), 43 (90%), 39 (81%), 45 (94%), 47 (98%), and 47 (98%) of CRCs, respectively. In addition, SIAs more frequently exhibited a focal staining pattern for MUC2, MUC5AC, SIMA, and villin, whereas more diffuse immunoreactivity was evident in CRCs. Focal staining for MUC1 and diffuse staining for CDX2 were common for SIAs and CRCs. Furthermore, poorly differentiated SIAs tended to express MUC1 more frequently when compared with well- and moderately differentiated SIAs. These observations further support the notion that SIA is immunophenotypically distinct from CRC despite their morphologic similarity.

Differential expression of alpha-methylacyl coenzyme A racemase in adenocarcinomas of the small and large intestines.

Alpha-methylacyl coenzyme A racemase (AMACR), a novel immunomarker for prostatic adenocarcinoma, has recently been shown to be expressed in a number of malignancies including colorectal adenocarcinoma. In the current study, 59 surgically resected primary small intestinal adenocarcinomas (34 ampullary and 25 non-ampullary) were immunohistochemically examined for AMACR expression and compared with 66 colorectal adenocarcinomas (including 24 secondary tumors involving the small intestine by direct extension or metastasis). The results show that no AMACR immunoreactivity was detected in normal-appearing small and large intestinal mucosa. While 41 of 66 (62%) colorectal adenocarcinomas exhibited a variable degree of cytoplasmic staining, only 1 of 25 (4%) non-ampullary and 2 of 34 (6%) ampullary small intestinal adenocarcinomas showed positive AMACR immunoreactivity (P < 0.0001). Interestingly, AMACR appeared to be less frequently expressed in mucinous or poorly differentiated colorectal adenocarcinomas when compared with non-mucinous or better-differentiated counterparts, suggesting an association with microsatellite instability status. These results extend our previous observations that small intestinal adenocarcinomas differ markedly from colorectal adenocarcinomas despite their morphologic similarity. The different AMACR expression patterns may not only provide an additional diagnostic tool in the distinction between adenocarcinomas of the small and large intestinal origins but may also shed light on further understanding of intestinal tumorigenesis.