Aluminium ammonium sulphate (AAS) is a food additive used in some countries, but its effects on intestinal epithelial cells (IECs) remain poorly understood. We investigated whether AAS promotes IEC death and eosinophilic infiltration under dysbiotic conditions and whether heparin suppresses these effects. Mice pretreated with antibiotics or control water were orally administered AAS. IEC death was assessed by RNA sequencing, western blotting, and flow cytometry. Microbiota composition was analysed by 16S rRNA sequencing, and eosinophilic infiltration was evaluated by histology and flow cytometry. Immortalised murine IECs were stimulated with AAS in the presence or absence of bacterial components, and the effects of heparin were examined in vitro and in vivo. Antibiotic-induced dysbiosis altered IEC metabolic programmes and reduced polysaccharide-degrading bacteria. Under these conditions, AAS further reduced Actinobacteria, including Bifidobacterium, increased mitochondrial reactive oxygen species (mtROS), and promoted cleavage of caspase-1, caspase-6, caspase-8, caspase-11, interleukin-33, gasdermin D (GSDMD), and gasdermin E (GSDME) in IECs. These changes were associated with epithelial pyroptosis and eosinophilic infiltration in the small intestine. AAS induced similar cleavage events in IECs primed with lipopolysaccharide and lipoteichoic acid in vitro. Antioxidant studies showed that caspase-1/11-GSDMD activation was ROS-dependent, whereas caspase-6/8-GSDME activation involved ROS-independent mechanisms. Heparin suppressed these cleavage events, reduced IEC death both in vitro and in vivo, and attenuated eosinophilic inflammation. These findings identify a previously unrecognised pro-inflammatory effect of AAS in the dysbiotic gut and suggest low-dose heparin as a potential protective strategy for intestinal epithelial homoeostasis.

Transbronchial lung cryobiopsy for interstitial lung disease is described in guidelines as an "acceptable alternative" to surgical lung biopsy. However, the pathological diagnosis of interstitial lung disease by transbronchial lung cryobiopsy is difficult compared with surgical lung biopsy because of the small sample size, leading to diagnostic disagreement not only in pathological diagnosis but also in multidisciplinary discussion diagnosis. Therefore, a standardized approach for the pathological diagnosis of ILD using transbronchial cryobiopsy is necessary. We developed a diagnostic flowchart for the pathological diagnosis of ILD using transbronchial cryobiopsy based on the consensus reached by the Pathology and Cryobiopsy Subcommittee within the Study Group on Diffuse Pulmonary Disorders, Scientific Research/Research on Intractable Diseases of the Ministry of Health, Labour, and Welfare of Japan. This study reports the diagnostic flowchart.

BACKGROUND:The global increase in endometrial cancer, including in Japan, and a shortage of pathologists and cytotechnologists have increased the diagnostic burden, emphasizing the need for an AI-based diagnostic support model that uses deep learning. We evaluated the clinical application of an improved AI-supported endometrial cytology model. METHODS:Using YOLOv5x and YOLOv7 models evaluated by mean average precision (mAP), we compared two datasets-one annotated for both benign and malignant cell clusters, and one for malignant only. In addition, using the Two One-Sided Tests (TOST) procedure, we assessed the correlation between AI diagnostic accuracy and the level of difficulty perceived by human diagnosticians. Finally, we used Gradient-weighted Class Activation Mapping (Grad-CAM) to visualize and enhance the interpretability of the AI model's decision-making process. RESULTS:The YOLOv5x model with both benign and malignant annotations had the highest malignant mAP, 0.798, as compared with YOLOv7. The TOST analysis showed no significant difference in perceived diagnostic difficulty between cases that were correctly and incorrectly diagnosed by the AI model, indicating consistent AI accuracy regardless of case difficulty. Grad-CAM visualizations clarified the AI model's decision-making basis; in some cases, the model appeared to focus on regions that differed from those typically attended to by human diagnosticians. CONCLUSION:The AI support model showed high and consistent accuracy in endometrial cytological analysis, regardless of diagnostic difficulty as perceived by human diagnosticians. Grad-CAM visualizations revealed diagnostic patterns, and the AI occasionally focused on regions different from those emphasized by human diagnosticians. This study advanced a real-time microscope-integrated AI system toward clinical application.

Actinin-4 (gene name: ACTN4) is an actin-bundling protein implicated in cancer invasion and metastasis. This study evaluated whether ACTN4 amplification and actinin-4 protein expression were associated with osimertinib efficacy in epidermal growth factor receptor-mutant non-small cell lung cancer. We retrospectively analyzed 63 patients with epidermal growth factor receptor-mutant non-small cell lung cancer treated with osimertinib as first-line treatment. Immunohistochemistry was performed for pretreatment tumor tissues. Actinin-4 immunohistochemistry positivity was defined as positive staining of ≥ 30% tumor cells. In positive cases, ACTN4 amplification was assessed via fluorescence in situ hybridization. Progression-free survival and overall survival were compared across groups. Among 63 patients (median age: 73 years, 52 with Eastern Cooperative Oncology Group performance status 0-1, 63 with adenocarcinoma; epidermal growth factor receptor mutations: 19del/L858R/uncommon = 32/24/7), there were 33 and 30 actinin-4 immunohistochemistry-positive and actinin-4 immunohistochemistry-negative cases, respectively. The propensity score-weighted overall survival and progression-free survival were significantly shorter for actinin-4 immunohistochemistry-positive patients than for actinin-4 immunohistochemistry-negative patients (overall survival: hazard ratio, 2.76; 95% confidence interval, 1.02-7.45; progression-free survival: hazard ratio, 1.91; 95% confidence interval, 1.03-3.54). Among the 33 actinin-4 immunohistochemistry-positive cases, four showed positivity in ACTN4 fluorescence in situ hybridization. Overall survival and progression-free survival were numerically shorter for patients with ACTN4 positivity than for those with ACTN4 negativity in fluorescence in situ hybridization. The findings suggest that ACTN4 amplification and actinin-4 protein expression are prognostic markers for poor osimertinib efficacy in epidermal growth factor receptor-mutant non-small cell lung cancer.

Endometrial cytology, which is minimally invasive and available as an outpatient procedure, is widely used in Japan for early detection of endometrial cancer, but its diagnostic process is time-consuming and requires expert diagnosticians. We developed a real-time artificial intelligence (AI)-assisted system using a standard microscope, a charge-coupled device (CCD) camera, and the You-Only-Look-Once version 5x (YOLOv5x) (a well-established object detection model) to support endometrial cytology screening in resource-limited settings. A total of 146 pre-operative cytology cases were collected, and the model was trained to detect abnormal cell clusters. The system was evaluated in real-time using a CCD camera, and its diagnostic performance was compared with that of three pathologists and four medical students. In an independent test of 20 cases, the AI model achieved an accuracy of 85%, showing promising performance comparable to the average accuracy of 75% among human evaluators. Furthermore, the median diagnostic time was reduced by approximately 45% with AI assistance. The impact of AI support varied by user expertise, with notable improvements among non-specialists. This proof-of-concept study demonstrates the feasibility and potential of affordable, real-time AI support for endometrial cytology using widely available equipment. Further validation with larger, multicenter datasets is warranted to confirm the generalizability and clinical utility of this approach.

BACKGROUND:The interobserver agreement regarding airway-centered fibrosis (ACF), the key diagnostic feature of fibrotic hypersensitivity pneumonitis (fHP) has not been sufficiently addressed to date. We applied digital image analysis to investigate this issue and extracted histological features of ACF to correlate with fHP diagnosis. METHODS:A total of 111 selected glass slides from 17 fHP and 30 idiopathic pulmonary fibrosis (IPF) were scanned and seven expert pulmonary pathologists were tasked with digital annotation of ACF. Interobserver agreement on annotated ACF was assessed using Fleiss' kappa value. ACF recognized by majority of pathologists (4 or more) were considered as consensus ACF (cACF), and their frequencies were compared between fHP and IPF cases. RESULTS:Fleiss' kappa agreement in ACF recognition was 0.32 among seven pathologists. A significant difference between cryobiopsy and VATS specimens regarding an average ACF count per slide (p = 0.012) was found. The number of cACFs in a single case ranged from 0 to 20 (mean 5.71) for fHP cases and 0 to 13 (mean 1.80) for IPF cases (p = 0.011). When limited to surgical biopsies, the average number of cACF was 10.3 for fHP vs. 1.68 for IPF (p < 0.001). The common characteristic features of cACF in fHP were their confinement to the vicinity of respiratory bronchioles, frequent association with peribronchiolar metaplasia, and mild to moderate lymphocytic infiltration. CONCLUSIONS:The recognition of ACF varies widely among pathologists. We identified common histologic features of ACF in fHP cases, proposing criteria for ACF recognition in fHP.

ABSTRACT Introduction Anti‐neutrophil cytoplasmic antibody (ANCA) seropositivity strongly correlates to ANCA‐associated vasculitis. Patients with idiopathic interstitial pneumonias (IIPs) without systemic vasculitis are sometimes ANCA‐positive. Radiological and pathological differences between patients with myeloperoxidase (MPO)‐ANCA‐positive and those with proteinase 3 (PR3)‐ANCA‐positive IIPs remain unclear. To determine whether high‐resolution computed tomography (HRCT) features and pathology findings differ by ANCA subtype in ANCA‐positive IIP patients in a national database. Clinical, radiological, and pathological data were examined along with a web‐based multidisciplinary discussion. Methods We reviewed records of 10 MPO‐ANCA‐positive and 9 PR3‐ANCA‐positive IIP patients who underwent HRCT and surgical lung biopsy between April 2009 and March 2014. Pulmonologists, chest radiologists, and pathologists evaluated HRCT scans and pathological findings independently. Patterns were classified using ATS/ERS/JRS/ALAT 2011 guidelines for idiopathic pulmonary fibrosis. Results HRCT patterns were definite usual interstitial pneumonia (UIP) (n = 8; 42.1%), possible UIP (n = 6; 31.6%), and inconsistent with UIP (n = 5; 26.3%). Pathological patterns were definite UIP (n = 5; 26.3%), probable UIP (n = 8; 42.1%), possible UIP (n = 4; 21.1%), and not UIP (n = 2; 10.5%). HRCT and pathological patterns did not differ between MPO‐ANCA‐positive and PR3‐ANCA‐positive IIPs. Radiological features were reticulation (n = 13; 68.4%), nodules (n = 12; 63.1%), honeycombing (n = 10; 52.6%), and increased attenuation around honeycombing (n = 7; 36.8%). Pathological findings were cysts (n = 12; 63.1%), lymphoid follicles with germinal centers (n = 11; 57.9%), and peribronchiolar wall lymphocytic infiltration (n = 11; 57.9%). Conclusion HRCT and pathological patterns did not differ between MPO‐ANCA‐positive and PR3‐ANCA‐positive IIPs. This absence of significant differences suggests a similar mechanism underlying both types of interstitial pneumonia.

Immune checkpoint inhibitors (ICIs) can provide survival benefits to cancer patients; however, they sometimes result in the development of renal immune-related adverse events (irAEs). Tubulointerstitial nephritis (TIN) is the most representative pathological feature of renal irAEs. However, the clinicopathological entity and underlying pathogenesis of ICI-induced TIN are unclear. Therefore, we compared the clinical and histological features of this condition with those of non-ICI drug-induced TIN. Age and C-reactive protein levels were significantly higher in ICI-induced TIN, but there were no significant differences in renal function. Immunophenotyping of ICI-induced TIN showed massive T cell and macrophage infiltration with fewer B cells, plasma cells, neutrophils, and eosinophils. Compared with those in non-ICI drug-induced TIN, CD4+ cell numbers were significantly lower in ICI-induced TIN but CD8+ cell numbers were not significantly different. However, CD8/CD3 and CD8/CD4 ratios were higher in ICI-induced TIN. Moreover, CD25+ and FOXP3+ cells, namely regulatory T cells, were less abundant in ICI-induced TIN. In conclusion, T cell, B cell, plasma cell, neutrophil, and eosinophil numbers proved useful for differentiating ICI-induced and non-ICI drug-induced TIN. Furthermore, the predominant distribution of CD8+ cells and low accumulation of regulatory T cells might be associated with ICI-induced TIN development.

BACKGROUND Ventricular tachycardia (VT) associated with primary cardiac tumors (PCTs) originating from the ventricles is rare, but lethal, in young patients. OBJECTIVES This study aimed to clarify the mechanisms underlying primary cardiac tumor-related ventricular tachycardia (PCT-VT) and establish a therapeutic strategy for this form of VT. METHODS Among 67 patients who underwent surgery for VT at our institute between 1981 and 2020, 4 patients aged 1 to 34 years, including 3 males, showed PCT-VT (fibroma, 2; lipoma, 1; and hamartoma, 1), which was investigated using a combination of intraoperative electroanatomical mapping and histopathological studies. RESULTS All 4 patients developed electrical storms of sustained VTs refractory to multiple drugs and repetitive endocardial ablations. The VT mechanism was re-entry, and intraoperative electroanatomical mapping showed a centrifugal activation pattern originating from the border between the tumor and healthy myocardium, where fractionated potentials were detected during sinus rhythm. Histopathological studies of serial sections of specimens acquired from these areas revealed tumor infiltration into the surrounding myocardium with cell disorganization, exhibiting myocardial disarray. Several myocardia entrapped in the tumor edges contributed to the development and sustainment of re-entrant VT activation. In the 2 patients in whom complete resection was unfeasible, encircling cryoablation to entirely isolate the unresectable tumor was effective in suppressing VT occurrence. CONCLUSIONS The mechanism underlying PCT-VT involves re-entry localized at the tumor edges. Myocardial disarray associated with tumor infiltration is a substrate for this form of VT. Cryoablation along the border between the tumor and myocardium is a promising therapeutic option for unresectable PCT-VT. (J Am Coll Cardiol EP 2024;10:43-55) (c) 2024 Published by Elsevier on behalf of the American College of Cardiology Foundation.

BackgroundThe tumor microenvironment (TME) impacts the therapeutic efficacy of immune checkpoint inhibitors (ICIs). No liquid biomarkers are available to evaluate TME heterogeneity. Here, we investigated the clinical significance of PD-1-binding soluble PD-L1 (bsPD-L1) in gastric cancer (GC) patients and non-small cell lung cancer (NSCLC) patients treated with PD-1/PD-L1 blockade.MethodsWe examined bsPD-L1, matrix metalloproteinases (MMPs), and IFN-γ levels in plasma samples from GC patients (n = 117) prior to surgery and NSCLC patients (n = 72) prior to and 2 months after ICI treatment. We also examined extracellular matrix (ECM) integrity, PD-L1 expression, and T cell infiltration in tumor tissues from 25 GC patients by Elastica Masson-Goldner staining and immunohistochemical staining for PD-L1 and CD3, respectively.ResultsbsPD-L1 was detected in 17/117 GC patients and 16/72 NSCLC patients. bsPD-L1 showed strong or moderate correlations with plasma MMP13 or MMP3 levels, respectively, in both GC and NSCLC patients. bsPD-L1 expression in GC was associated with IFN-γ levels and intra-tumoral T cell infiltration, whereas MMP13 levels were associated with loss of ECM integrity, allowing tumor cells to access blood vessels. Plasma MMP3 and MMP13 levels were altered during ICI treatment. Combined bsPD-L1 and MMP status had higher predictive accuracy to identify two patient groups with favorable and poor prognosis than tumor PD-L1 expression: bsPD-L1+MMP13high in GC and bsPD-L1+(MMP3 and MMP13)increased in NSCLC were associated with poor prognosis, whereas bsPD-L1+MMP13low in GC and bsPD-L1+(MMP3 or MMP13)decreased in NSCLC were associated with favorable prognosis.ConclusionPlasma bsPD-L1 and MMP13 levels indicate T cell response and loss of ECM integrity, respectively, in the TME. The combination of bsPD-L1 and MMPs may represent a non-invasive tool to predict recurrence in GC and the efficacy of ICIs in NSCLC.

Antibody responses, involving B cells, CD4 + T cells, and macrophages, are implicated in autoimmune diseases and organ transplant rejection. We have previously shown that inhibiting FROUNT with disulfiram (DSF) suppresses macrophage activation and migration, effectively treating inflammatory diseases. In this study, we investigated the effectiveness of DSF in antibody-producing reactions. Using a heart transplantation mouse model with antibody-mediated rejection, we administered anti-CD8 antibody to exclude cellular rejection. DSF directly inhibited B cell responses in vitro and significantly reduced plasma donor-specific antibodies and graft antibody deposition in vivo, resulting in prolonged survival of the heart graft. DSF also mediated various effects, including decreased macrophage infiltration and increased Foxp3+ regulatory T-cells in the grafts. Additionally, DSF inhibited pyrimidine metabolism-related gene expression induced by B-cell stimulation. These findings demonstrate that DSF modulates antibody production in the immune response complexity by regulating B-cell and macrophage responses. The FDA-approved drug disulfiram can inhibit B cells and infiltration of macrophages in a mouse model of heart transplantation.

Libman-Sacks endocarditis is an important cause of embolic stroke in systemic lupus erythematosus, although the detailed pathogenesis of stroke remains unclear. We herein report two cases of stroke with Libman-Sacks endocarditis in which the emboli were retrieved by mechanical thrombectomy. The embolus consisted of eosinophilic homogeneous acellular structures, whereas fibrin-platelet thrombi were hardly observed in the embolus. Immunohistochemistry showed immunoglobulin deposits in the embolus, suggesting that immunological mechanisms were involved in the growth of the embolus. A pathological analysis of the embolus retrieved by mechanical thrombectomy provided useful information on the etiology, leading to optimal treatment.

Objective To explore the integration of artificial intelligence (AI)-assisted diagnostics into a cytology workflow, focusing on real-time detection of abnormal cell clusters in endometrial cytology without relying on whole-slide imaging (WSI), utilizing a YOLOv5x-based model. Methods We employed the YOLOv5x object detection model pretrained on the COCO dataset because of its high-speed and accurate detection capabilities. This study involved real-time direct detection of abnormal cell clusters using a CCD camera attached to a microscope, with the aim of enhancing diagnostic efficiency and accuracy in endometrial cytology. The model was further refined through transfer learning using actual cytology case images, emphasizing the need for a delicate balance between technological advancement and clinical integration. Results The integration of our AI model into the diagnostic workflow significantly reduced the time required for diagnosis compared to traditional methods, as demonstrated by the performance metrics that matched or exceeded those of pathologists. This breakthrough underscores the potential of AI to improve diagnostic workflows, particularly in settings where resources or pathology services are limited. Conclusion This study presents the first instance of an AI-assisted system for endometrial cytology that operates in real time under a microscope, negating the need for WSI. Our findings highlight the feasibility of embedding AI directly into existing clinical practices, offering significant time savings and potentially matching the diagnostic accuracy of specialists. The successful integration of this technology is a critical step forward in the application of AI in the medical field, paving the way for broader adoption and further research into user-friendly AI applications in pathology diagnostics.

Introduction: Osimertinib is a standard treatment for patients with EGFR -mutant NSCLC. Although some osimertinib resistance mechanisms have been identi fied, nearly 50% of the mechanisms remain to be elucidated. This study was aimed at identifying non -genetic mechanisms underlying osimertinib resistance. Methods: We established two osimertinib-resistant cell lines from EGFR mutation -positive PC -9 and HCC827 NSCLC cell lines (PC-9OR and HCC827OR, respectively) using a stepwise method. We compared the phosphoproteomic pro files of the osimertinib-resistant and parental cells using mass spectrometry. Upstream kinases were identi fied using the application Kinase Enrichment Analysis version 3. Results: Phosphoproteomic analysis revealed 80 phosphorylation sites that were mutually up -regulated in PC9OR and HCC827OR cells. The Kinase Enrichment Analysis version 3 analysis identi fied focal adhesion kinase (FAK) and proto-oncogene tyrosine -protein kinase Src (Src) as upstream kinases of these up -regulated phosphoproteins. The small -interfering RNA -mediated knockdown of FAK reduced Src phosphorylation and that of Src reduced FAK phosphorylation in both cell lines. Furthermore, FAK- or Src-speci fic small -interfering RNA treatments restored EGFR phosphorylation in PC-9OR and HCC827OR cells. The combination of FAK and Src inhibitors inhibited PC-9OR and HCC827OR cell proliferation in vitro and suppressed tumor growth in a xenograft mouse model. Immunohistochemistry of tumors from patients with EGFR -mutant NSCLC suggested that phosphorylated FAK and Src are involved in initial and acquired resistance to osimertinib. Conclusions: Phosphoproteomic analysis may help elucidate the mechanisms of resistance to molecular -targeted therapies in lung cancer. Mutual phosphorylation of FAK and Src is involved in osimertinib resistance. Thus, FAK and Src inhibition may be novel treatment strategies for osimertinib-resistant NSCLC. Copyright (c) 2024 by the International Association for the Study of Lung Cancer. This is an open access article under the CC BY -NC -ND license (http://creativecommons.org/ licenses/by-nc-nd/4.0/).

Introduction:Antineutrophil cytoplasmic antibody (ANCA)-associated glomerulonephritis (GN) is characterized by pauci-immune crescentic GN. Myeloperoxidase ANCA-associated GN (MPO-ANCA GN) with membranous nephropathy (MN), where bright granular capillary MPO and IgG staining along the glomerular basement membrane (GBM) is present, has been reported; however, its clinicopathological features remain unclear. Methods:We investigated 7 MPO-ANCA GN with MN and 11 control cases (6 MPO-ANCA GN and 5 primary MN cases). Proteomics of laser microdissected glomeruli followed by immunohistochemical analysis was performed to identify causal antigens in MPO-ANCA GN with MN. We described the clinicopathological features of MPO-associated MN compared with those of MPO-ANCA GN and primary MN. Results:We detected proteomic MPO and granular capillary MPO deposits in all MPO-ANCA GN with MN cases. Confocal microscopy revealed MPO and IgG colocalization along the GBM. MPO-associated MN clinicopathological features include greater proteinuria, a higher fibrous crescent rate, and a lower MPO-ANCA titer than MPO-ANCA GN. The estimated glomerular filtration rate (eGFR) and urinary protein excretion were lower in MPO-associated MN than in primary MN. Conclusion:MPO-associated MN, a unique type of secondary MN where MPO serves as the causal antigen, is a subset of MPO-ANCA GN with MN. Prolonged periods of MPO-ANCA GN and a low MPO-ANCA titer might be related to MPO-associated MN development.