Predominant CD8+ cell infiltration and low accumulation of regulatory T cells in immune checkpoint inhibitor-induced tubulointerstitial nephritis.

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.

Development of angiogenic periglomerular microvessels after acute glomerular lesions in IgA nephropathy.

AIM: To clarify the clinicopathological characteristics and role of periglomerular angiogenesis in IgA nephropathy. METHODS AND RESULTS: The renal biopsy specimens of 114 patients with IgA nephropathy were examined. Among them, 46 (40%) showed periglomerular angiogenesis around the glomeruli. CD34 and α-smooth muscle actin (α-SMA) staining in serial sections revealed that these vessels contained CD34+ α-SMA+ microarterioles along with CD34+ α-SMA- capillaries. We termed these "periglomerular microvessels (PGMVs)". Patients with PGMVs (PGMV group) had clinically and histologically more severe disease than those without PGMVs (non-PGMV group) at the time of biopsy. Even after adjusting for age, there were significant differences in the degree of proteinuria and estimated glomerular filtration rate reduction between the PGMV and non-PGMV groups. The PGMV group showed a higher incidence of segmental and global glomerulosclerosis and crescentic lesions than the non-PGMV group (P < 0.01). Here, PGMVs were undetectable in the acute and active inflammation phase, but were observed in the acute to chronic or chronic glomerular remodelling phase. PGMVs mainly developed around glomerular adherent lesions to the Bowman's capsule with small or minimal glomerular sclerotic lesions. Conversely, they were rarely observed in segmental sclerosis areas. CONCLUSION: The PGMV group is clinically and pathologically more severe than the non-PGMV group; however, they were undetectable in segmental sclerosis with mesangial matrix accumulation. PGMVs might occur after acute/active glomerular lesions, suggesting that PGMVs may inhibit segmental glomerulosclerosis progression and could be a marker for good repair response after acute/active glomerular injury in severe IgA nephropathy cases.

The reduced number of nephrons with shortening renal tubules in mouse postnatal adverse environment.

BACKGROUND: The intrauterine adverse environment during nephrogenesis reduces the nephron number, probably associates with impaired ureteric bud (UB) branching. METHODS: The kidneys in C57/BL6 mice were irradiated with a single dose of 10 gray (10 Gy) as adverse environment on postnatal day 3 (irradiated PND3 kidneys) after UB branching ceased. The renal functions and pathological findings of irradiated PND3 kidneys were compared with those of non-irradiated control and 10 Gy irradiation on PND14 (irradiated PND14 kidney) from 1 to 18 months. RESULTS: The number and density of glomeruli in irradiated PND3 kidneys were reduced by 1 month with renal dysfunction at 6 months. The morphologically incomplete glomeruli with insufficient capillaries were involuted by 1 month in the superficial cortex. Reduced tubular numbers and developmental disability with shortening renal tubules occurred in irradiated PND3 kidneys with impaired urine concentration at 6 months. Hypertrophy of glomeruli developed, and occasional sclerotic glomeruli appeared in the juxtamedullary cortex with hypertension and albuminuria at 12 to 18 months. CONCLUSIONS: The reduced number of nephrons with shortening renal tubules occurred with impaired renal functions in a postnatal adverse environment after cessation of UB branching, and glomerular hypertrophy with occasional glomerulosclerosis developed accompanied with hypertension and albuminuria in the adulthood. IMPACT: The reduced number of nephrons with shortening renal tubules occurred with impaired renal functions in a postnatal adverse environment after cessation of ureteric bud branching. The reduced number of glomeruli were associated with not only the impaired formation of glomeruli but also involution of morphologically small incomplete glomeruli after an adverse environment. The insufficiently developed nephrons were characterized by the shortening renal tubules with impaired urine concentration. In addition, glomerular hypertrophy and occasional glomerulosclerosis developed with hypertension and albuminuria in adulthood. The present study can help to understand the risk of alternations of premature nephrons in preterm neonates.

Immune checkpoint inhibitors associated granulomatous small vessel vasculitis accompanied with tubulointerstitial nephritis: a case report.

BACKGROUND: Immune checkpoint inhibitors (ICIs) have provided significant benefits in cancer treatment, but they could develop immune-related adverse events (irAE). ICI-associated renal adverse effects are rare and tubulointerstitial nephritis (TIN) is the most common in the renal irAE. However, only a few case reports of renal vasculitis associated with ICI have been reported. In addition, the characteristics of infiltrating inflammatory cells of ICI-associated TIN and renal vasculitis have been uncertain. CASE PRESENTATION: A 65-year-old man received immune checkpoint inhibitors (ICIs), anti-CTLA-4 (cytotoxic T-lymphocyte-associated protein 4) and anti-PD-1 (programmed cell death 1) antibodies for aggravated metastatic malignant melanoma. About 1 week after the second administration of nivolumab and ipilimumab, acute kidney injury developed. A renal biopsy was performed that showed TIN and non-necrotizing granulomatous vasculitis in interlobular arteries. Massive CD3+ T cells and CD163+ macrophages infiltrated both tubulointerstitium and interlobular arteries. Many infiltrating cells tested positive for Ki-67 and PD-1 ligand (PD-L1), but negative for PD-1. In CD3+ T cells, CD8+ T cells were predominantly infiltrated, and these cells were positive for Granzyme B (GrB) and cytotoxic granule TIA-1, but negative for CD25, indicating antigen-independent activated CD8+ T cells. Infiltration of CD4+ T cells was noted without obvious CD4+ CD25+ regulatory T (Treg) cells. His renal dysfunction recovered within 2 months of treatment with prednisolone in addition to discontinuation of nivolumab and ipilimumab. CONCLUSIONS: We herein reported a case of ICI-related TIN and renal granulomatous vasculitis with infiltration of massive antigen-independent activated CD8+ T cells and CD163+ macrophages, and none or few CD4+ CD25+ Treg cells. These infiltrating cells might be a characteristic of the development of renal irAE.

Alpha-actinin-4 (ACTN4) gene amplification is a predictive biomarker for adjuvant chemotherapy with tegafur/uracil in stage I lung adenocarcinomas.

Although adjuvant tegafur/uracil (UFT) is recommended for patients with completely resected stage I non-small-cell lung cancer (NSCLC) in Japan, only one-third of cases has received adjuvant chemotherapy (ADJ) according to real-world data. Therefore, robust predictive biomarkers for selecting ADJ or observation (OBS) without ADJ are needed. Patients who underwent complete resection of stage I lung adenocarcinoma with or without adjuvant UFT were enrolled. The status of ACTN4 gene amplification was analyzed by FISH. Statistical analyses to determine whether the status of ACTN4 gene amplification affected recurrence-free survival (RFS) were carried out. Formalin-fixed, paraffin-embedded samples from 1136 lung adenocarcinomas were submitted for analysis of ACTN4 gene amplification. Ninety-nine (8.9%) of 1114 cases were positive for ACTN4 gene amplification. In the subgroup analysis of patients aged 65 years or older, the ADJ group had better RFS than the OBS group in the ACTN4-positive cohort (hazard ratio [HR], 0.084, 95% confidence interval [CI], 0.009-0.806; P = .032). The difference in RFS between the ADJ group and the OBS group was not significant in ACTN4-negative cases (all ages: HR, 1.214; 95% CI, 0.848-1.738; P = .289). Analyses of ACTN4 gene amplification contributed to the decision regarding postoperative ADJ for stage I lung adenocarcinomas, preventing recurrence, improving the quality of medical care, preventing the unnecessary side-effects of ADJ, and saving medical costs.

Inhibition of the chemokine signal regulator FROUNT by disulfiram ameliorates crescentic glomerulonephritis.

Activated monocytes/macrophages promote glomerular injury, including crescent formation, in anti-glomerular basement membrane (GBM) glomerulonephritis. Disulfiram, an alcohol-aversion drug, inhibits monocyte/macrophage migration by inhibiting FROUNT, a cytosolic protein that enhances chemokine receptor signaling. Our study found that disulfiram at a human equivalent dose successfully blocked albuminuria and crescent formation with podocyte loss, and later stage kidney fibrotic lesions, in a rat model of anti-GBM glomerulonephritis. A disulfiram derivative, DSF-41, with more potent FROUNT inhibition activity, inhibited glomerulonephritis at a lower dose than disulfiram. Disulfiram markedly reduced the number of monocytes or macrophages at the early stage of glomerulonephritis and that of CD3+ and CD8+ lymphocytes at the established stage. Impaired pseudopodia formation was observed in the glomerular monocytes/macrophages of the disulfiram group; consistent with the in vitro observation that disulfiram blocked chemokine-dependent pseudopodia formation and chemotaxis of bone marrow-derived monocytes/macrophages. Furthermore, disulfiram suppressed macrophage activation as revealed by reduced expression of inflammatory cytokines and chemokines (TNF-α, CCL2, and CXCL9) and reduced CD86 and MHC class II expressions in monocytes/macrophages during glomerulonephritis. The dramatic reduction in monocyte/macrophage number might have resulted from disulfiram suppression of both the chemotactic response of monocytes/macrophages and their subsequent activation to produce cytokines and chemokines, which further recruit monocytes. Additionally, FROUNT was expressed in CD68+ monocytes/macrophages infiltrating the crescentic glomeruli in human anti-GBM glomerulonephritis. Thus, disulfiram can be a highly effective and safe drug for the treatment of glomerulonephritis by blocking the chemotactic responses of monocytes/macrophages and their activation status in the glomerulus.

Cholesterol Crystal in Thrombus Removed by Mechanical Thrombectomy Should be a Strong Marker for Aortogenic Embolic Stroke.

Aortogenic embolic stroke (AES) is an important stroke mechanism. However, as many stroke patients have aortic atheromatous lesions, it is unclear whether these lesions are the cause of these strokes. Cholesterol crystals are the solid, crystalline form of cholesterol that is found in atherosclerosis, but not in cardiac diseases such as atrial fibrillation, valvular diseases, and cardiomyopathy. Therefore, if a cholesterol crystal is found in a thrombus removed by mechanical thrombectomy (MT), this makes it possible to diagnose a patient as having an atheromatous lesion. Here, we report an AES case with a cholesterol crystal found in a thrombus removed by MT. A 67-year-old man was admitted due to consciousness disturbance, aphasia, and right hemiplegia. Diffusion-weighted imaging (DWI) showed a hyperintense area in the left frontal lobe, and magnetic resonance angiography demonstrated a branch occlusion of the left middle cerebral artery (MCA). MT was performed 1.5 h after stroke onset, with the thrombus removed and a left occluded MCA completely recanalized. Carotid duplex ultrasonography did not reveal any plaque in the carotid artery. Echocardiography did not show any abnormal function or findings, including thrombus. Transesophageal echocardiography showed a 4.9 mm atheromatous lesion at the aortic arch. Therefore, we suspected this patient as having an AES due to the embolic source of atheromatous lesion at the aortic arch. Pathological examination of the embolus revealed a cholesterol crystal cleft in the thrombus. Therefore, we diagnosed this patient as having AES caused by an atheromatous lesion at the aortic arch.

Effect of H2 treatment in a mouse model of rheumatoid arthritis-associated interstitial lung disease.

Rheumatoid arthritis (RA)-associated interstitial lung disease (ILD), a primary cause of mortality in patients with RA, has limited treatment options. A previously established RA model in D1CC transgenic mice aberrantly expressed major histocompatibility complex class II genes in joints, developing collagen II-induced polyarthritis and anti-cyclic citrullinated peptide antibodies and interstitial pneumonitis, similar to those in humans. Molecular hydrogen (H2 ) is an efficient antioxidant that permeates cell membranes and alleviates the reactive oxygen species-induced injury implicated in RA pathogenesis. We used D1CC mice to analyse chronic lung fibrosis development and evaluate H2 treatment effects. We injected D1CC mice with type II collagen and supplied them with H2 -rich or control water until analysis. Increased serum surfactant protein D values and lung densities images were observed 10 months after injection. Inflammation was patchy within the perilymphatic stromal area, with increased 8-hydroxy-2'-deoxyguanosine-positive cell numbers and tumour necrosis factor-α, BAX, transforming growth factor-ß, interleukin-6 and soluble collagen levels in the lungs. Inflammatory and fibrotic changes developed diffusely within the perilymphatic stromal area, as observed in humans. H2 treatment decreased these effects in the lungs. Thus, this model is valuable for studying the effects of H2 treatment and chronic interstitial pneumonia pathophysiology in humans. H2 appears to protect against RA-ILD by alleviating oxidative stress.

Naftopidil reduced the proliferation of lung fibroblasts and bleomycin-induced lung fibrosis in mice.

Naftopidil, an α-1 adrenoceptor antagonist with few adverse effects, is prescribed for prostate hyperplasia. Naftopidil inhibits prostate fibroblast proliferation; however, its effects on lung fibroblasts and fibrosis remain largely unknown. Two normal and one idiopathic pulmonary fibrosis human lung fibroblast lines were cultured with various naftopidil concentrations with or without phenoxybenzamine, an irreversible α-1 adrenoceptor inhibitor. We examined the incorporation of 5-bromo-2'-deoxyuridine into DNA and lactic acid dehydrogenase release by enzyme-linked immunosorbent assay, cell cycle analysis by flow cytometry, scratch wound-healing assay, and mRNA expressions of type IV collagen and α-smooth muscle actin by polymerase chain reaction. Effects of naftopidil on bleomycin-induced lung fibrosis in mice were evaluated using histology, micro-computed tomography, and surfactant protein-D levels in serum. Naftopidil, dose-dependently but independently of phenoxybenzamine, inhibited 5-bromo-2'-deoxyuridine incorporation in lung fibroblasts. Naftopidil induced G1 cell cycle arrest, but lactic acid dehydrogenase release and migration ability of lung fibroblasts were unaffected. Naftopidil decreased mRNA expressions of type IV collagen and α-smooth muscle actin in one normal lung fibroblast line. Histological and micro-computed tomography examination revealed that naftopidil attenuated lung fibrosis and decreased serum surfactant protein-D levels in bleomycin-induced lung fibrosis in mice. In conclusion, naftopidil may have therapeutic effects on lung fibrosis.

Analyses of alveolar epithelial injury via lipid-related stress in mammalian target of rapamycin inhibitor-induced lung disease.

Although mammalian target of rapamycin inhibitors (mTORi) are used to treat various malignancies, they frequently induce active alveolitis and dyslipidemia. Abnormal lipid metabolism affects alveolar surfactant function and results in pulmonary disorders; however, the pathophysiology of lung injury and its relationship with lipid metabolism remain unknown. We investigated the relationship between lipid metabolism and alveolar epithelial injury, focusing on peroxisome proliferator-activated receptor-γ (PPAR-γ) as a lipid stress-related factor in mTORi-induced lung injury. We clinicopathologically examined three patients with mTORi-induced lung injury. We constructed an mTORi injury mouse model using temsirolimus in mice (30 mg/kg/day), with the vehicle control and bleomycin injury groups. We also constructed a cultured alveolar epithelial cell injury model using temsirolimus (0-40 µM) in the mouse lung epithelial cell line MLE-12 and performed analysis with or without pioglitazone (PPAR-γ agonist) treatment. All three patients had dyslipidemia and lung lesions of hyperplastic pneumocytes with foamy and enlarged changes. In the mouse model, temsirolimus induced significantly higher levels of total cholesterol and free fatty acids in serum and higher levels of surfactant protein D in serum and BAL fluid with an increase in inflammatory cytokines in the lung compared to control. Temsirolimus also induced hyperplastic foamy pneumocytes with increased lipid-associated spots and larger round electron-lucent bodies compared to the control or bleomycin groups in microscopic analyses. Multiple lipid-associated spots within the cytoplasm were also induced by temsirolimus administration in MLE-12 cells. Temsirolimus downregulated PPAR-γ expression in mouse lung and MLE-12 cells but upregulated cleaved caspase-3 in MLE-12 cells. Pioglitazone blocked the upregulated cleaved caspase-3 expression in MLE-12 cells. The pathogenesis of mTORi-induced lung disease may be involved in alveolar epithelial injury, via lipid metabolic stress associated with downregulated PPAR-γ expression. Focusing on the relationship between lipid metabolic stress and alveolar epithelial injury represents a potentially novel approach to the study of pulmonary damage.

Nationwide cloud-based integrated database of idiopathic interstitial pneumonias for multidisciplinary discussion.

Multidisciplinary discussion (MDD) requiring close communication between specialists (clinicians, radiologists and pathologists) is the gold standard for the diagnosis of idiopathic interstitial pneumonias (IIPs). However, MDD by specialists is not always feasible because they are often separated by time and location. An online database would facilitate data sharing and MDD. Our aims were to develop a nationwide cloud-based integrated database containing clinical, radiological and pathological data of patients with IIPs along with a web-based MDD system, and to validate the diagnostic utility of web-based MDD in IIPs.Clinical data, high-resolution computed tomography images and lung biopsy slides from patients with IIPs were digitised and uploaded to separate servers to develop a cloud-based integrated database. Web-based MDD was performed using the database and video-conferencing to reach a diagnosis.Clinical, radiological and pathological data of 524 patients in 39 institutions were collected, uploaded and incorporated into the cloud-based integrated database. Subsequently, web-based MDDs with a pulmonologist, radiologist and pathologist using the database and video-conferencing were successfully performed for the 465 cases with adequate data. Overall, the web-based MDD changed the institutional diagnosis in 219 cases (47%). Notably, the MDD diagnosis yielded better prognostic separation among the IIPs than did the institutional diagnosis.This is the first study of developing a nationwide cloud-based integrated database containing clinical, radiological and pathological data for web-based MDD in patients with IIPs. The database and the web-based MDD system that we built made MDD more feasible in practice, potentially increasing accurate diagnosis of IIPs.

Pathologic glomerular characteristics and glomerular basement membrane alterations in biopsy-proven thin basement membrane nephropathy.

BACKGROUND: Thin basement membrane nephropathy (TBMN) is diagnosed by diffuse thinning of the glomerular basement membrane (GBM) without any clinical and pathologic findings of Alport syndrome and the other renal diseases. TBMN is characterized clinically by benign familial hematuria but rarely develops into end-stage renal disease. METHODS: In 27 cases of biopsy-proven TBMN, we evaluated the pathologic characteristics of TBMN, and examined the correlation between these pathologic characterizations and renal dysfunction. RESULTS: All patients had hematuria, and 21 patients (77.8%) had proteinuria. In six patients (28.6%) who were more than 50 years of age, the estimated glomerular filtration rate (eGFR) decreased from G3a to G4 in the chronic kidney disease stage. Pathologically, an irregular decrease in intensity of type IV collagen α5(IV) chain was seen in GBM, and irregular thinning with diffuse rough etched images was observed on the GBM surface with several sizes of holes by low-vacuum scanning electron microscopy. The glomerular morphology of TBMN was characterized by an increased number of small glomerular capillaries with an increased extracellular matrix (ECM). These characteristic morphologic alterations were evident from a young age in patients with TBMN, but were not correlated directly with the decrease of eGFR, the degree of hematuria, and proteinuria. The decrease of eGFR in patients with TBMN who were more than 50 years of age might be primarily mediated by arteriolosclerosis-associated glomerulosclerosis and interstitial fibrosis. CONCLUSION: Characteristic pathological glomerular findings and GBM alterations occurred from a young age but were not associated directly with renal impairment in biopsy-proven TBMN.

Optimal conditions and the advantages of using laser microdissection and liquid chromatography tandem mass spectrometry for diagnosing renal amyloidosis.

BACKGROUND: Liquid chromatography-tandem mass spectrometry (LC-MS/MS) has recently been utilized to accurately detect the amyloid proteins of renal amyloidosis. The present study investigated the optimal procedures for analyzing samples by LCMS/MS, and the advantage of using this technique to diagnosis renal amyloidosis. METHODS: To detect amyloid proteins, laser microdissected glomeruli from AL (n = 13) or AA (n = 10) renal amyloidosis patients were digested and analyzed by LCMS/MS. To determine the best procedures for analyzing samples by LCMS/MS, we examined the suitability of tissue samples, frozen or formalin-fixed paraffin-embedded (FFPE), the number of dissected glomeruli required for analysis (2, 10, or 50 glomeruli), and the amount of trypsin with or without dithiothreitol (DTT). We additionally compared the detection of amyloid proteins between immunostaining and LCMS/MS. RESULTS: Examining 10 dissected glomeruli from FFPE sections digested with trypsin 3 µL (0.1 mg/mL) without DDT made it possible to detect amyloid protein in all 10 AA and in 10 out of 12 AL amyloidosis cases. All AA amyloidosis cases were diagnosed using immunohistochemistry for amyloid A. With immunostaining, however, there were several inconclusive immunoglobulin and/or their light chain staining noted in the AA or AL amyloidosis cases. Even so, LCMS/MS was able to accurately detect amyloid protein in renal amyloidosis. CONCLUSION: The use of 10 laser microdissected glomeruli (170,000-220,000 µm2) with amyloid deposition from FFPE sections digested with trypsin 3 µL (0.1 mg/mL) allowed the accurate detection of amyloid protein in AA and AL amyloidosis.

Role of α1 and α2 chains of type IV collagen in early fibrotic lesions of idiopathic interstitial pneumonias and migration of lung fibroblasts.

Early fibrotic lesions are thought to be the initial findings of fibrogenesis in idiopathic interstitial pneumonias, but little is known about their properties. Type IV collagen comprises six gene products, α1-α6, and although it is known as a major basement membrane component, its abnormal deposition is seen in fibrotic lesions of certain organs. We studied the expression of type I and III collagen and all α chains of type IV collagen in lung specimens from patients with usual interstitial pneumonia (UIP) or organizing pneumonia (OP) via immunohistochemistry. With cultured lung fibroblasts, we analyzed the expression and function of all α chains of type IV collagen via immunohistochemistry, western blotting, real-time quantitative PCR, and a Boyden chamber migration assay after the knockdown of α1 and α2 chains. Although we observed type I and III collagens in early fibrotic lesions of both UIP and OP, we found type IV collagen, especially α1 and α2 chains, in early fibrotic lesions of UIP but not OP. Fibroblasts enhanced the expression of α1 and α2 chains of type IV collagen after transforming growth factor-ß1 stimulation. Small interfering RNA against α1 and α2 chains increased fibroblast migration, with upregulated phosphorylation of focal adhesion kinase (FAK), and adding medium containing fibroblast-produced α1 and α2 chains reduced the increased levels of fibroblast migration and phosphorylation of FAK. Fibroblasts in OP were positive for phosphorylated FAK but fibroblasts in UIP were not. These results suggest that fibroblasts in UIP with type IV collagen deposition, especially α1 and α2 chains, have less ability to migrate from early fibrotic lesions than fibroblasts in OP without type IV collagen deposition. Thus, type IV collagen deposition in early fibrotic lesions of UIP may be implicated in refractory pathophysiology including migration of lesion fibroblasts via a FAK pathway.

Pathological Findings of Embolus Retrieved by Mechanical Thrombectomy in Cerebral Embolism with Libman-Sacks Endocarditis: A Report of Two Cases.

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.

Myeloperoxidase-Associated Membranous Nephropathy in Antineutrophil Cytoplasmic Antibody-Associated Glomerulonephritis.

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.

Phosphoproteomic Analysis Identified Mutual Phosphorylation of FAK and Src as a Mechanism of Osimertinib Resistance in EGFR-Mutant Lung Cancer.

Introduction: Osimertinib is a standard treatment for patients with EGFR-mutant NSCLC. Although some osimertinib resistance mechanisms have been identified, 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 profiles of the osimertinib-resistant and parental cells using mass spectrometry. Upstream kinases were identified using the application Kinase Enrichment Analysis version 3. Results: Phosphoproteomic analysis revealed 80 phosphorylation sites that were mutually up-regulated in PC-9OR and HCC827OR cells. The Kinase Enrichment Analysis version 3 analysis identified 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-specific 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.

Disulfiram treatment suppresses antibody-producing reactions by inhibiting macrophage activation and B cell pyrimidine metabolism.

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.

Combination of plasma MMPs and PD-1-binding soluble PD-L1 predicts recurrence in gastric cancer and the efficacy of immune checkpoint inhibitors in non-small cell lung cancer.

Background: The 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. Methods: We 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. Results: bsPD-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. Conclusion: Plasma 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.

Electrophysiological and Histopathological Characteristics of Ventricular Tachycardia Associated With Primary Cardiac Tumors.

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.