Pathological Phenotypes Research Articles

Meteorin-like regulates cardiac hypertrophy and associated metabolic plasticity during gestation and lactation

Abstract Funding Acknowledgements Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Ministerio de investigación y ciencia de España Introduction Pregnancy-induced cardiac hypertrophy is considered a physiological, reversible process (1, 2). However, cardiac remodeling after delivery and through lactation, as well as mechanisms driving hypertrophy reversion, remain undefined. In recent studies, we described the cardioprotective role of Meteorin-like (Metrnl) during pathological remodeling of the myocardium (3). Its role during gestation and lactation in the maternal heart is completely unknown. Purpose To characterize pregnancy-induced cardiac hypertrophy and its underlying mechanisms, and to determine the role of Metrnl in the cardiac remodeling associated to pregnancy and lactation. Methods C57BL/6J wild-type (WT) and Metrnl knockout (Metrnl-KO) female mice were studied during gestation, lactation and weaning. Echocardiographic measurements and cardiac samples were obtained from virgin control animals (V), pregnant mice at term (E18), lactating females at days 14 and 21 (L14 and L21), and mice 7 and 21 days after L21 weaning (W7 and W21). Results In WT females, pregnancy induces eccentric cardiac hypertrophy characterized by a major dilation of the left ventricle, a phenomenon that is maintained and further increased during lactation, reaching the highest degree of eccentric hypertrophy by L14. After weaning, hypertrophy is totally reversed (Figure 1A). By contrast, Metrnl-KO mice show a more pathological cardiac phenotype characterised by an exacerbated eccentric hypertrophy development accompanied by reduced ejection fraction. Post-weaning resolution is also delayed compared to WT. Next, we analysed cardiac metabolism since during the latest stages of gestation, the heart relies more on fatty acid utilization due to the increased glucose demands of the foetus (Figure 1B). We found that in WT mice, cardiac expression of genes linked to fatty acid oxidation (FAO; Acadm, Pdk4 and Cpt1b) is increased during gestation and repressed during lactation in parallel with cardiac Metrnl expression, recovering basal levels by W7. By contrast, in Metrnl-KO mice mitochondrial pyruvate transporter Mpc1 expression is increased during the whole process, while Pdk4 and Cpt1b protein levels are lower, suggesting a reliance of these hearts on glucose metabolism during pregnancy and post-partum. Conclusions The maternal heart develops an adaptive pregnancy-induced cardiac hypertrophy, a condition that initiates at gestation, peaks by day 14 of lactation and starts reverting upon weaning. Metrnl is necessary for the correct development of cardiac adaptations associated with pregnancy/lactation.Figure 1

Zebrafish dspb -/- tert +/- mutant as model for arrhythmogenic cardiomyopathy. Impact of aging in the development of the disease

Abstract Funding Acknowledgements Type of funding sources: Public Institution(s). Main funding source(s): Instituto de Salud Carlos III Background/Objectives Zebrafish has been reported as model to study the Arrythmogenic Cardiomyopathy, which is generally associated with desmosomal gene mutations. We have identified a founder non-sense human variant in patients from our region (DSP p.Q447*) associated with left ventricle Arrythmogenic Cardiomyopathy and we have selected it for modelling in zebra fish by CRISPR/Cas9 method, choosing exon 10 as the region of homology to be mutated and obtaining the p.T449fs* variant that determines a premature stop codon. On the other hand, tert gene enables telomerase activity and telomere maintenance and mutations in this gene have been used for studying aging effects. After multiple crossings of our dspb model with tert mutants we have obtained dspb-/-tert+/- model line. Purpose Our aim was the characterization of dspb-/-tert+/- mutants, studying the impact of aging in the development of the disease. Methods The model was obtained by crossing dspb-/- and tert-/- mutants, crossing the heterozygous mutants and genotyping for the selection of the dspb-/-tert+/- mutants. The characterization of the phenotype was performed through viability assays, cardiac function measurements and gene expression assays in larvae (3 days post fertilization) young adult (8 months old) and old adult (3 years old) individuals. Results Viability assays showed that dspb-/-tert+/- mutants suffer a premature death, with much lower survival rates than the dspb-/- group (survival dspb-/- 61.3% vs dspb-/-tert+/- 35.6%, p<0.01). Larvae heart rate analysis showed no significant differences. Echocardiography in young adults showed no significant differences between dspb-/-tert+/- and dspb-/- mutants. In old adults, echocardiography showed differences in heart rate (dspb-/- 142.5 ± 15.5 vs dspb-/-tert+/ -97.5 ± 3.5, p<0.01), respiratory frequency (dspb-/- 288.7 ± 36.1 vs dspb-/-tert+/- 95 ± 49.4, p<0.01) and atrial contraction velocity (dspb-/-155.7 ± 26.09 vs dspb-/-tert+/ -227.2 ± 17.6, p<0.05). Gene expression analysis in larvae showed that dspb expression drastically decreased (dspb-/- 0.3 ± 0.1 vs dspb-/-tert+/ -0.01 ± 0.008, p<0.01). In adults, dspb expression drastically decreased in heart tissue (dspb-/- 0.4 ± 0.01 vs dspb-/-tert+/- 0.06 ± 0.004, p<0.01) and skin (dspb-/ -0.34 ± 0.02 vs dspb-/-tert+/- 0.25 ± 0.01, p<0.01). Conclusion The dspb-/-tert+/- zebrafish presented worse survival rates, lower dspb gene expression levels and worse pathological cardiac phenotype at adult stages than dspb-/- mutants, proving that age is an important factor in the development of the disease and proving that dspb-/-tert+/- zebrafish is an appropriate model to study the impact of aging.

Arrhythmogenic cardiomyopathy related phenotypes are detected and pharmacologically modulated in Galectin-3 zebrafish models

Abstract Funding Acknowledgements Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Italian Ministry of University and Research Introduction Arrhythmogenic Cardiomyopathy (AC) is a rare inherited cardiac disorder characterized by fibro-fatty replacement and progressive loss of the ventricular myocardium, causing life-threatening arrhythmias, syncope and sudden cardiac death in young and athletes. About 50% of AC cases carry one or more mutations in genes encoding for desmosomal proteins. However, mutations in non-desmosomal genes have been identified in 1-3% of AC patients, such as Galectin-3 (Gal-3). Purpose For a better understanding of Gal-3 role in AC pathogenesis, we have generated a stable Gal-3 knock-out (KO) zebrafish line, able to reproduce cardiac abnormalities and cell signaling dysregulation characteristic of AC, on which environmental factors and candidate drugs can be tested. Methods The zebrafish Gal-3 mutant line was generated by CRISPR/Cas9 strategy and then phenotypically characterized. Phenotyping included heart activity characterization, by pyHeart4Fish software, and gene expression analysis using Real Time PCR and signaling pathway reporters. Immunofluorescence analysis was performed using an antibody against L-plastin, a leukocyte marker for the detection of inflammatory cells, and Acridine Orange/Ethidium Bromide (AO/EB) staining to check for apoptotic and necrotic events. Transmission Electron Microscopy (TEM) analysis was carried out to detect possible ultrastructural alterations. A pathway-directed pharmacological treatment was applied to try to rescue pathological phenotypes. Results The Gal-3 KO zebrafish line, analysed at larval stage, presented structural abnormalities, pericardial effusion and/or hemopericardium associated with reduced contractility, bradycardia and presence of arrhythmia events. In addition to Wnt/β-catenin signaling dysregulation, as seen in other AC forms, we also observed cell death and inflammation. Specifically, the analysis of the L-plastin inflammatory marker showed remarkable infiltration of inflammatory cells in the cardiac region of Gal-3 mutants, whereas AO/EB staining demonstrated elevated number of apoptotic and necrotic cells. Moreover, TEM analysis revealed "pale", disorganized and delocalized desmosomes in mutant adult hearts. Of note, pharmacological treatment at larval stage, using a Wnt/β-catenin signaling agonist, partially rescued a set of AC cardiac phenotypes (contractility, bradycardia, arrhythmia). Conclusions Overall, the Gal-3 KO zebrafish line confirms in zebrafish the strict connection between Gal-3 mutation and AC-like cardiac alterations. Moreover, cell death and inflammation, known to be involved in the remodelling of the myocardium, were observed. The pharmacological rescue of the Wnt/β-catenin signaling partially recovered a set of cardiac phenotypes, confirming this pathway as a key modulator of AC progression. In conclusion, our results strongly support the usefulness of the Gal-3 zebrafish mutant to clarify Gal-3 involvement in AC, offering future perspectives for AC treatment.

Zebrafish Congenital Heart Disease Models: Opportunities and Challenges.

Congenital heart defects (CHDs) are common human birth defects. Genetic mutations potentially cause the exhibition of various pathological phenotypes associated with CHDs, occurring alone or as part of certain syndromes. Zebrafish, a model organism with a strong molecular conservation similar to humans, is commonly used in studies on cardiovascular diseases owing to its advantageous features, such as a similarity to human electrophysiology, transparent embryos and larvae for observation, and suitability for forward and reverse genetics technology, to create various economical and easily controlled zebrafish CHD models. In this review, we outline the pros and cons of zebrafish CHD models created by genetic mutations associated with single defects and syndromes and the underlying pathogenic mechanism of CHDs discovered in these models. The challenges of zebrafish CHD models generated through gene editing are also discussed, since the cardiac phenotypes resulting from a single-candidate pathological gene mutation in zebrafish might not mirror the corresponding human phenotypes. The comprehensive review of these zebrafish CHD models will facilitate the understanding of the pathogenic mechanisms of CHDs and offer new opportunities for their treatments and intervention strategies.

Characterization of a new arrhythmogenic cardiomyopathy mouse model due to filamin c haploinsufficiency

Abstract Funding Acknowledgements Type of funding sources: Public grant(s) – EU funding. Main funding source(s): Spanish Ministry of Universities FPU fellowship (FPU19/04044), Spanish Ministry of Science and Innovation Digital Transition call (TED2021-129774B-C22), EU HORIZON EIC Pathfinder Challenges 2022 Cardiogenomics call (Project 101115416). Background Filamin C (FLNC) is widely expressed in heart and skeletal muscle. It participates in cell junction and sarcomere assembly and function. We have previously shown that heterozygous FLNC truncating variants (FLNCtv) cause arrhythmogenic/dilated cardiomyopathy (ACM/DCM) in patients. Diverse studies have suggested haploinsufficiency as the potential mechanism of this disease. However, animal models of FLNCtv have only been reported to develop ACM/DCM in homozygosity. Purpose To develop a new model of ACM/DCM caused by a FLNCtv and study the progression of the disease in heterozygosity. Methods Two gRNAs flanking exon 15 of the Flnc gene and SpCas9 protein were microinjected into mouse zygotes to generate a mouse line with a constitutive deletion of Flnc exon 15 (FlncWt/Ex15del). mRNA and protein stability were determined by qRT-PCR and western blot, respectively. Cardiac function was analysed by ECG and echocardiography every 2 months, starting at 8 weeks of age (Fig. 1B). The mean ECG values were calculated using 3 time intervals and standard ECG waves. Results FlncWt/Ex15del mice showed a reduction in Flnc mRNA and protein similar to that observed in a human ACM/DCM patient bearing a similar mutation, suggesting that this FLNCtv causes haploinsufficiency. Both male and female FlncWt/Ex15del mice developed cardiac electrophysiological defects, including reduced amplitude of the QRS complex and the P-wave, notched R and/or S waves, and increased QT interval times, and these defects worsened with time. At 40 weeks of age, FlncWt/Ex15del mice showed some initial signs of left ventricular dilatation. Conclusions Mice carrying FLNCtv in heterozygosity develop electrophysiological changes resembling pathological features of ACM/DCM patients due to haploinsufficiency. The development of this pathological phenotype in heterozygosity, as opposed to previous models, paves the way for the development of new gene therapies using this model.

#1447 Calcimimetics improve impaired cardiac function and contractility in chronic phosphate loading by activating the CaSR/cAMP signaling cascade

Abstract Background and Aims High phosphate loading stimulate the synthesis of PTH and FGF23 and is associated with increased cardiovascular (CV) mortality. In hemodialysis patients, the administration of calcimimetics led to reductions in PTH and FGF23 and the latter was associated with a lower rate of CV events. We have previously shown that mice with a high dietary phosphate load show elevated PTH and FGF23 levels and develop impaired systolic cardiac function, which could be improved by calcimimetics. Calcimimetics are allosteric activators of the calcium-sensing receptor (CaSR). Phosphate (Pi) was shown to inactivate the parathyroid CaSR at concentrations >1.4 mM Pi. Whether calcimimetics contribute to CV event reduction indirectly via lowering PTH and FGF23 or directly via activation of the cardiac CaSR is unknown. Method Wild-type mice were fed a high phosphate diet (HPD) ± etelcalcetide (Etl) or a normal phosphate diet (NPD), cardiac function was assessed by echocardiography and Millar catheterization, and cardiac tissue was harvested for histology and RNAseq analyses. In addition, isolated adult mouse cardiomyocytes (AMCM) were stimulated ex vivo with PTH, FGF23 or Pi, contractility and calcium handling were measured with the addition of Etl and activity analyses were performed. Results In addition to the positive effect on HPD-induced systolic cardiac dysfunction, Etl improved impaired LV contractility and reduced fibrosis. RNAseq analyses revealed reduced Ca, PKA and cAMP signaling by HPD. Ex vivo stimulation of AMCM with PTH and FGF23 had no functional effects. In contrast, Pi significantly worsened contractility and Ca handling, which was blunted by Etl co-stimulation. Downstream of cardiac CaSR, Pi reduced the formation of cAMP, which led to PKA-dependent dephosphorylation of PLN and subsequently to lower Serca2a activity, which was ultimately responsible for the reduced contractility. Etl led to improved cardiomyocyte contractility via increased cAMP synthesis and increased PLN phosphorylation and Serca2a activity. Conclusion Our data suggest that Etl ameliorates the Pi-induced pathologic cardiac phenotype associated with high phosphate load via direct activation of CaSR/cAMP signaling cascade, resulting in improvement of cardiomyocyte contractility.

Transcriptomic Analysis of Lipid Metabolism Genes in Alzheimer's Disease: Highlighting Pathological Outcomes and Compartmentalized Immune Status.

The dysregulation of lipid metabolism has been strongly associated with Alzheimer's disease (AD) and has intricate connections with various aspects of disease progression, such as amyloidogenesis, bioenergetic deficit, oxidative stress, neuroinflammation, and myelin degeneration. Here, a comprehensive bioinformatic assessment was conducted on lipid metabolism genes in the brains and peripheral blood of AD-derived transcriptome datasets, characterizing the correlation betweendifferentially expressed genes (DEGs) of lipid metabolism and disease pathologies, as well as immune cell preferences. Through the application of weighted geneco-expression network analysis (WGCNA),modules eigengenes related to lipid metabolism were pinpointed, and the examination oftheir molecular functions within biological processes, molecular pathways, andtheir associations with pathological phenotypes and molecular networkshas been characterized. Analysis of biological networks indicates notable discrepancies in the expression patterns of the DEGs between neuronal and immune cells, as well as variations in cell type enrichments within both brain tissue and peripheral blood. Additionally, drugs targeting the DEGs from central and peripheral and a diagnostic model for hub genes from the blood were retrieved and assessed, some of which were shown to be useful for therapeutic and diagnostic.These results revealed the distinctive pattern of transcriptionally abnormal lipid metabolism in central, peripheral, and immune cell activation, providing valuable insight into lipid metabolism for diagnosing and guiding more effectivetreatment forAD.

Targeting autophagy impairment improves the phenotype of a novel CLN8 zebrafish model

CLN8 is an endoplasmic reticulum cargo receptor and a regulator of lysosome biogenesis whose loss of function leads to neuronal ceroid lipofuscinosis. CLN8 has been linked to autophagy and lipid metabolism, but much remains to be learned, and there are no therapies acting on the molecular signatures in this disorder. The present study aims to characterize the molecular pathways involved in CLN8 disease and, by pinpointing altered ones, to identify potential therapies. To bridge the gap between cell and mammalian models, we generated a new zebrafish model of CLN8 deficiency, which recapitulates the pathological features of the disease. We observed, for the first time, that CLN8 dysfunction impairs autophagy. Using autophagy modulators, we showed that trehalose and SG2 are able to attenuate the pathological phenotype in mutant larvae, confirming autophagy impairment as a secondary event in disease progression. Overall, our successful modeling of CLN8 defects in zebrafish highlights this novel in vivo model's strong potential as an instrument for exploring the role of CLN8 dysfunction in cellular pathways, with a view to identifying small molecules to treat this rare disease.

Lysosomal Channels as New Molecular Targets in the Pharmacological Therapy of Neurodegenerative Diseases via Autophagy Regulation.

Besides controlling several organellar functions, lysosomal channels also guide the catabolic "self-eating" process named autophagy, which is mainly involved in protein and organelle quality control. Neuronal cells are particularly sensitive to the rate of autophagic flux either under physiological conditions or during the degenerative process. Accordingly, neurodegeneration occurring in Parkinson's (PD), Alzheimer's (AD), and Huntington's Diseases (HD), and Amyotrophic Lateral Sclerosis (ALS) as well as Lysosomal Storage Diseases (LSD) is partially due to defective autophagy and accumulation of toxic aggregates. In this regard, dysfunction of lysosomal ionic homeostasis has been identified as a putative cause of aberrant autophagy. From a therapeutic perspective, Transient Receptor Potential Channel Mucolipin 1 (TRPML1) and Two-Pore Channel isoform 2 (TPC2), regulating lysosomal homeostasis, are now considered promising druggable targets in neurodegenerative diseases. Compelling evidence suggests that pharmacological modulation of TRPML1 and TPC2 may rescue the pathological phenotype associated with autophagy dysfunction in AD, PD, HD, ALS, and LSD. Although pharmacological repurposing has identified several already used drugs with the ability to modulate TPC2, and several tools are already available for the modulation of TRPML1, many efforts are necessary to design and test new entities with much higher specificity in order to reduce dysfunctional autophagy during neurodegeneration.

Idiopathic dendriform pulmonary ossification as the phenotype of interstitial lung abnormalities: CT–pathologic correlation and prevalence

Background and purposeIdiopathic dendriform pulmonary ossification (DPO) is mostly asymptomatic, and detected incidentally in lung CT. There have been no reports on the precise CT–pathologic correlation and the prevalence of idiopathic DPO. This study aimed to clarify the histological background and prevalence of idiopathic DPO.Materials and methodsSixteen patients with histologically confirmed idiopathic DPO (12 men and 4 women; mean age, 38.8 years; range 22–56 years) were identified in a nationwide epidemiological survey. Local HRCT findings of pre-biopsy examinations, such as branching, round, linear structures with or without high attenuation were compared side by side with histological findings. The attenuation of branching, round, and linear structures was classified into three-point levels on bone window images (width, 2500 HU; level, 500 HU). Furthermore, we collected continuous pulmonary CT images of 8111 cases for checking up metastasis from extrathoracic malignancy at a single institution, and evaluated the prevalence of interstitial lung abnormalities (ILAs) and DPO.ResultsIn all 16 cases, branching (n = 15, 93%), round (n = 5, 31%), or linear (n = 5, 31%) structures were identified, histologically corresponding to dendriform ossification and cicatricial organizing pneumonia (OP)/fibrosis. Histologically, ossification was confirmed in all the 16 patients. However, in two cases, a highly attenuated structure could not be detected on the pre-biopsy CT of the same area. Regarding the prevalence of idiopathic DPO, 283 (3.5%) of 8111 patients had ILAs, of which a total of 26 (0.3% of all cases, 9.2% of ILAs cases) had DPO.ConclusionIdiopathic DPO showed linear or branching structures with or without high attenuation on CT, corresponded to ossification, cicatricial OP/fibrosis. DPO was seen in 9.2% of ILAs cases. Idiopathic DPO is one of pathologic phenotypes of ILAs.

Rediscovery of the implication of albuminuria in heart failure: emerging classic index for cardiorenal interaction.

The development of new drugs and device therapies has led to remarkable advancements in heart failure (HF) treatment in the past couple of decades. However, it becomes increasingly evident that guideline-directed medical therapy cannot be one-size-fits-all across a wide range of ejection fractions (EFs) and various aetiologies. Therefore, classifications solely relying on EF and natriuretic peptide make optimization of treatment challenging, and there is a growing exploration of new indicators that enable efficient risk stratification of HF patients. Particularly when considering HF as a multi-organ interaction syndrome, the cardiorenal interaction plays a central role in its pathophysiology, and albuminuria has gained great prominence as its biomarker, independent from glomerular filtration rate. Albuminuria has been shown to exhibit a linear correlation with cardiovascular disease and HF prognosis in multiple epidemiological studies, ranging from normal (<30mg/g) to high levels (>300mg/g). However, on the other hand, it is only recently that the details of the pathological mechanisms that give rise to albuminuria have begun to be elucidated, including the efficient compaction/tightening of the glomerular basement membrane by podocytes and mesangial cells. Interestingly, renal disease, diabetes, and HF damage these components associated with albuminuria, and experimental models have demonstrated that recently developed HF drugs reduce albuminuria by ameliorating these pathological phenotypes. In this review, facing the rapid expansion of horizons in HF treatment, we aim to clarify the current understanding of the pathophysiology of albuminuria and explore the comprehensive understanding of albuminuria by examining the clinically established evidence to date, the pathophysiological mechanisms leading to its occurrence, and the outcomes of clinical studies utilizing various drug classes committed to specific pathological mechanisms to put albuminuria as a novel axis to depict the pathophysiology of HF.

Ben-JNK signaling is required for host mortality during Periplaneta fuliginosa densovirus infection.

Cockroaches are widely acknowledged as significant vectors of pathogenic microorganisms. The Periplaneta fuliginosa densovirus (PfDNV) infects the smoky-brown cockroach P. fuliginosa and causes host mortality, which identifies the PfDNV as a species-specific and environmentally friendly biopesticide. However, although the biochemical characterization of PfDNV has been extensively studied, the immune response against PfDNV remains largely unclear. Here, we investigated the replication of PfDNV and its associated pathological phenotype in the foregut and hindgut. Consequently, we dissected and performed transcriptome sequencing on the foregut, midgut, and hindgut separately. We revealed the up-regulation of immune response signaling pathway c-Jun N-terminal kinase (JNK) and apoptosis in response to viral infection. Furthermore, knockdown of the JNK upstream gene Ben resulted in a decrease in virus titer and delayed host mortality. Taken together, our findings provide evidence that the Ben-JNK signaling plays a crucial role in PfDNV infection, leading to excessive apoptosis in intestinal tissues and ultimately resulting in the death of the host. Our results indicated that the host response to PfDNV fosters viral infection, thereby increasing host lethality. This underscores the potential of PfDNV as a viable, environmentally friendly biopesticide. © 2024 Society of Chemical Industry.

Berbamine ameliorates DSS-induced colitis by inhibiting peptidyl-arginine deiminase 4-dependent neutrophil extracellular traps formation

Ulcerative colitis (UC) is a chronic inflammatory bowel disease with immune dysregulation affecting colon inflammatory response. Recent studies have highlighted that neutrophil extracellular traps (NETs) play an important role in the pathogenesis of UC. Berbamine (BBM), one of the bioactive ingredients extracted from Chinese herbal medicine Berberis vulgaris L, has attracted intensive attentions due to its significant anti-inflammatory activity and a marketing drug for treating leukemia in China. However, the exact role and potential molecular mechanism of BBM against UC remains elusive. In the present study, our results showed that BBM could markedly improve the pathological phenotype and the colon inflammation in mice with dextran sulfate sodium (DSS)-induced colitis. Then, comprehensive approaches combining network pharmacology and molecular docking analyses were employed to predict the therapeutic potential of BBM in treating UC by peptidyl-arginine deiminase 4 (PAD4), a crucial molecule involved in NETs formation. The molecular docking results showed BBM had a high affinity for PAD4 with a binding energy of −9.3 kcal/mol Moreover, PAD4 expression and NETs productions, including citrullination of histone H3 (Cit-H3), neutrophil elastase (NE), myeloperoxidase (MPO) in both neutrophils and colonic tissue were reduced after BBM administration. However, in the mice with DSS-induced colitis pretreated with GSK484, a PAD4-specific inhibitor, BBM could not further reduce disease related indexes, expression of PAD4 and NETs productions. Above all, the identification of PAD4 as a potential target for BBM to inhibit NETs formation in colitis provides novel insights into the development of BBM-derived drugs for the clinical management of UC.

Abstract PO4-25-01: Selective elimination of CD169+ macrophages in lymph nodes invaded by breast cancers

Abstract Lymph node metastasis is a prognostically significant factor in breast cancer, though the colonization mechanism of cancer cells in this immune cell-rich organ remains elusive. This study aimed to elucidate the impact of breast cancer on the lymph node immune cell landscape. Multiscale transcriptomic analyses were performed on both metastatic and non-metastatic lymph node samples from breast cancer patients with lymph node metastasis. Twenty laser-micro-dissected sections were obtained from 17 lymph nodes across six breast cancer patients at stages II–III, with each patient contributing both types of lymph nodes for direct comparison. Comparing the transcriptomes of paired lymph nodes with and without metastasis from the same patients revealed selective downregulation of CD169+ macrophage-related genes in metastatic lymph nodes. The spatial transcriptome indicated a potential depletion of CD169+ macrophages, initiators of anticancer immunity, from their residence (sinuses) in metastatic lymph nodes, while other principal immune cell types were unaltered. Mass spectrometry imaging revealed that the numbers of CD169+ macrophages were smaller in the metastatic lymph nodes than in the non-metastatic lymph nodes, suggesting that cancer cells uprooted CD169+ macrophages from the lymph nodes. Conversely, the count of B, T, Treg, and CD11c+ cells remained comparable in both lymph node types, albeit an enrichment of Treg cells around metastasized cancer tissues was observed. Additional immunohistochemistry analysis of 315 non-metastatic lymph nodes and 159 metastatic lymph nodes from 58 patients with breast cancer showed that a reduced CD169+ macrophage population was prevalent in various breast cancer subtypes. A subset of metastatic lymph nodes (37 out of 159) displayed a complete absence of detectable CD169+ macrophages. The data also depicted a gradual decline in CD169+ macrophages correlating with the pN classification, while no correlation was identified with pathological tumor size classification (pT) or metastasized cancer volume. The data suggest precedence of CD169+ macrophage elimination over other reported immune cell abnormalities, such as cell number and metabolic irregularities. CD169+ macrophages are a unique type of resident macrophages in the lymphoid organs that present cancer-derived antigens to CD8+ cells. The antigen-presenting role of CD169+ macrophages to T cells, a pivotal step in adaptive immunity, signifies the catastrophic implications of their suppression. This study underscores CD169+ macrophage suppression as a pronounced pathological phenotype in lymph nodes with breast cancer metastasis, thereby establishing it as a critical future therapeutic target. Citation Format: Yurina Maeshima, Tatsuki R. Kataoka, Alexis Vandenbon, Masahiro Hirata, Yasuhide Takeuchi, Yutaka Suzuki, Yukiko Fukui, Yumiko Ibi, Hironori Haga, Satoshi Morita, Masakazu Toi, Shinpei Kawaoka, Kosuke Kawaguchi. Selective elimination of CD169+ macrophages in lymph nodes invaded by breast cancers [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO4-25-01.

Hydralazine use can be associated with IgM-dominated immune complex-mediated glomerulonephritis

ABSTRACT Context IgM-dominant immune complex-mediated glomerulonephritis (IgM-dominant ICMGN) is a rare renal entity, characterized by a membranoproliferative pattern by light microscopy, dominant IgM staining by immunofluorescent staining, and subendothelial deposits by electron microscopy. This study was to investigate if some of IgM-ICMGN were associated with autoimmune disorders induced by hydralazine. Design Seven IgM-dominant ICMGN cases were identified over 8 years. Their pathologic phenotypes and clinical scenarios were analyzed in detail. Results Patients’ ages ranged from 47 to 87 years old with 5 women and two men. Six of seven patients had drug-induced autoimmune phenomenon (hydralazine-induced positive ANCA and ANA). All of them had renal dysfunction and some proteinuria. Most pathologic features showed a membranoproliferative pattern of glomerulonephritis with dominant IgM deposits at subendothelial spaces. IgM nephropathy (a variant of focal segmental glomerulosclerosis), chronic thrombotic microangiopathy, and cryoglobulinemic glomerulopathy were ruled out in the cases. Conclusion The hydralazine-induced autoimmune phenomenon can be seen in IgM-dominant ICMGN, which should be classified as a subtype of membranoproliferative glomerulonephritis.

Simple modeling of familial Alzheimer’s disease using human pluripotent stem cell-derived cerebral organoid technology

BackgroundCerebral organoids (COs) are the most advanced in vitro models that resemble the human brain. The use of COs as a model for Alzheimer’s disease (AD), as well as other brain diseases, has recently gained attention. This study aimed to develop a human AD CO model using normal human pluripotent stem cells (hPSCs) that recapitulates the pathological phenotypes of AD and to determine the usefulness of this model for drug screening.MethodsWe established AD hPSC lines from normal hPSCs by introducing genes that harbor familial AD mutations, and the COs were generated using these hPSC lines. The pathological features of AD, including extensive amyloid-β (Aβ) accumulation, tauopathy, and neurodegeneration, were analyzed using enzyme-linked immunosorbent assay, Amylo-Glo staining, thioflavin-S staining, immunohistochemistry, Bielschowsky’s staining, and western blot analysis.ResultsThe AD COs exhibited extensive Aβ accumulation. The levels of paired helical filament tau and neurofibrillary tangle-like silver deposits were highly increased in the AD COs. The number of cells immunoreactive for cleaved caspase-3 was significantly increased in the AD COs. In addition, treatment of AD COs with BACE1 inhibitor IV, a β-secretase inhibitor, and compound E, a γ-secretase inhibitor, significantly attenuated the AD pathological features.ConclusionOur model effectively recapitulates AD pathology. Hence, it is a valuable platform for understanding the mechanisms underlying AD pathogenesis and can be used to test the efficacy of anti-AD drugs.

Endogenous mutant Huntingtin alters the corticogenesis via lowering Golgi recruiting ARF1 in cortical organoid

Pathogenic mutant huntingtin (mHTT) infiltrates the adult Huntington’s disease (HD) brain and impairs fetal corticogenesis. However, most HD animal models rarely recapitulate neuroanatomical alterations in adult HD and developing brains. Thus, the human cortical organoid (hCO) is an alternative approach to decode mHTT pathogenesis precisely during human corticogenesis. Here, we replicated the altered corticogenesis in the HD fetal brain using HD patient-derived hCOs. Our HD-hCOs had pathological phenotypes, including deficient junctional complexes in the neural tubes, delayed postmitotic neuronal maturation, dysregulated fate specification of cortical neuron subtypes, and abnormalities in early HD subcortical projections during corticogenesis, revealing a causal link between impaired progenitor cells and chaotic cortical neuronal layering in the HD brain. We identified novel long, oriented, and enriched polyQ assemblies of HTTs that hold large flat Golgi stacks and scaffold clathrin+ vesicles in the neural tubes of hCOs. Flat Golgi stacks conjugated polyQ assemblies by ADP-ribosylation factor 1 (ARF1). Inhibiting ARF1 activation with Brefeldin A (BFA) disassociated polyQ assemblies from Golgi. PolyQ assembles with mHTT scaffolded fewer ARF1 and formed shorter polyQ assembles with fewer and shorter Golgi and clathrin vesicles in neural tubes of HD-hCOs compared with those in hCOs. Inhibiting the activation of ARF1 by BFA in healthy hCOs replicated impaired junctional complexes in the neural tubes. Together, endogenous polyQ assemblies with mHTT reduced the Golgi recruiting ARF1 in the neuroepithelium, impaired the Golgi structure and activities, and altered the corticogenesis in HD-hCO.

RIP1 kinase inactivation protects against LPS-induced acute respiratory distress syndrome in mice

Acute respiratory distress syndrome (ARDS) is characterized by lung tissue oedema and inflammatory cell infiltration, with limited therapeutic interventions available. Receptor-interacting protein kinase 1 (RIPK1), a critical regulator of cell death and inflammation implicated in many diseases, is not fully understood in the context of ARDS. In this study, we employed RIP1 kinase-inactivated (Rip1K45A/K45A) mice and two distinct RIPK1 inhibitors to investigate the contributions of RIP1 kinase activity in lipopolysaccharide (LPS)-induced ARDS pathology. Our results indicated that RIPK1 kinase inactivation, achieved through both genetic and chemical approaches, significantly attenuated LPS-induced ARDS pathology, as demonstrated by reduced polymorphonuclear neutrophil percentage (PMN%) in alveolar lavage fluid, expression of inflammatory and fibrosis-related factors in lung tissues, as well as histological examination. Results by tunnel staining and qRT-PCR analysis indicated that RIPK1 kinase activity played a role in regulating cell apoptosis and inflammation induced by LPS administration in lung tissue. In summary, employing both pharmacological and genetic approaches, this study demonstrated that targeted RIPK1 kinase inactivation attenuates the pathological phenotype induced by LPS inhalation in an ARDS mouse model. This study enhances our understanding of the therapeutic potential of RIPK1 kinase modulation in ARDS, providing insights for the pathogenesis of ARDS.

Pan-neuronal expression of human mutant SOD1 in Drosophila impairs survival and motor performance, induces early neuroinflammation and chromosome aberrations

Several mutations in the SOD1 gene encoding for the antioxidant enzyme Superoxide Dismutase 1, are associated with amyotrophic lateral sclerosis, a rare and devastating disease characterized by motor neuron degeneration and patients' death within 2–5 years from diagnosis. Motor neuron loss and related symptomatology manifest mostly in adult life and, to date, there is still a gap of knowledge on the precise cellular and molecular events preceding neurodegeneration.To deepen our awareness of the early phases of the disease, we leveraged two Drosophila melanogaster models pan-neuronally expressing either the mutation A4V or G85R of the human gene SOD1 (hSOD1A4V or hSOD1G85R).We demonstrate that pan-neuronal expression of the hSOD1A4V or hSOD1G85R pathogenic construct impairs survival and motor performance in transgenic flies. Moreover, protein and transcript analysis on fly heads indicates that mutant hSOD1 induction stimulates the glial marker Repo, up-regulates the IMD/Toll immune pathways through antimicrobial peptides and interferes with oxidative metabolism. Finally, cytological analysis of larval brains demonstrates hSOD1-induced chromosome aberrations. Of note, these parameters are found modulated in a timeframe when neurodegeneration is not detected.The novelty of our work is twofold: we have expressed for the first time hSOD1 mutations in all neurons of Drosophila and confirmed some ALS-related pathological phenotypes in these flies, confirming the power of SOD1 mutations in generating ALS-like phenotypes. Moreover, we have related SOD1 pathogenesis to chromosome aberrations and antimicrobial peptides up-regulation. These findings were unexplored in the SOD1-ALS field.

Improving diagnosis and outcome prediction of gastric cancer via multimodal learning using whole slide pathological images and gene expression

For the diagnosis and outcome prediction of gastric cancer (GC), machine learning methods based on whole slide pathological images (WSIs) have shown promising performance and reduced the cost of manual analysis. Nevertheless, accurate prediction of GC outcome may rely on multiple modalities with complementary information, particularly gene expression data. Thus, there is a need to develop multimodal learning methods to enhance prediction performance. In this paper, we collect a dataset from Ruijin Hospital and propose a multimodal learning method for GC diagnosis and outcome prediction, called GaCaMML, which is featured by a cross-modal attention mechanism and Per-Slide training scheme. Additionally, we perform feature attribution analysis via integrated gradient (IG) to identify important input features. The proposed method improves prediction accuracy over the single-modal learning method on three tasks, i.e., survival prediction (by 4.9% on C-index), pathological stage classification (by 11.6% on accuracy), and lymph node classification (by 12.0% on accuracy). Especially, the Per-Slide strategy addresses the issue of a high WSI-to-patient ratio and leads to much better results compared with the Per-Person training scheme. For the interpretable analysis, we find that although WSIs dominate the prediction for most samples, there is still a substantial portion of samples whose prediction highly relies on gene expression information. This study demonstrates the great potential of multimodal learning in GC-related prediction tasks and investigates the contribution of WSIs and gene expression, respectively, which not only shows how the model makes a decision but also provides insights into the association between macroscopic pathological phenotypes and microscopic molecular features.