Citations

Left atrial (LA) myopathy plays a critical role in the pathophysiology of heart failure with preserved ejection fraction (HFpEF). This study aimed to evaluate LA function in a murine model of hypertension-induced HFpEF, with and without obesity, by analyzing left ventricular (LV) pressure signals to quantify the contribution of LA systole to total LV volume. HFpEF was induced in C57BL/6J mice using the SAUNA model, which involves uninephrectomy, chronic aldosterone infusion, and high-salt drinking water for four weeks. To model obesity-associated HFpEF, a high-fat diet (HFD) was added to the SAUNA protocol. Invasive hemodynamic measurements were obtained, and a novel method was developed to assess atrial systole using the second-order derivative of the LV pressure signal during diastolic filling. SAUNA HFpEF mice exhibited preserved LVEF, moderate hypertension, pulmonary congestion, and diastolic dysfunction as evidenced by a prolonged time constant of relaxation (Tau). In sham control mice, atrial systole accounted for 23.0 ± 2.4% of LV volume, whereas this contribution was significantly reduced to 13.9 ± 2.3% in SAUNA HFpEF mice ( P < 0.01). In obese sham controls, atrial systole contributed 53.1 ± 2.2%, which was markedly decreased to 36.8 ± 3.6% in obese-SAUNA HFpEF mice ( P < 0.003). This novel method enables precise quantification of LA systolic contribution to LV filling in mice and highlights the pathophysiological effects of hypertension and obesity on LA function in HFpEF.

Ex vivo liver perfusion (EVLP) sustains human or large animal livers outside the body under near-physiological conditions, enabling functional monitoring for lactate clearance, bile production, and oxygen consumption and allowing targeted therapeutic interventions. Originally developed to optimize donor grafts for transplantation, EVLP has evolved into a powerful translational research platform bridging preclinical discovery and early clinical translation. This review discusses EVLP as a platform for gene therapy, immunotherapy, pharmacology, and personalized medicine, with particular emphasis on gene- and immune-based interventions as mechanistically mature exemplars. We consolidate advances in pharmacological testing and toxicity modeling, viral and non-viral gene delivery, genome engineering, and immunomodulation using perfused livers. We further describe emerging applications, including autologous EVLP pathways for organ-confined therapy, ex vivo liver surgery, and bioengineering strategies such as biliary organoid repair, RNA interference, and mitochondrial transfer. We highlight how these applications align with a paradigm shift in biomedical research, including the NIH’s recent initiative to prioritize human-based experimental models over animal-only studies. By leveraging transplant-declined or surgically resected organs that would otherwise be unused, ex vivo perfusion bridges the gap between pre-clinical testing and clinical practice, enabling real-time evaluation of interventions in functional human tissue. We discuss both the scientific opportunities afforded by EVLP and the technical, biosafety, and ethical challenges that must be addressed to enable responsible clinical translation.

Introduction Large-scale production of mammalian cells is pivotal for applications in biotechnology, regenerative medicine, and therapeutic manufacturing. However, current bioreactor technologies face significant technical and economic challenges, including excessive cell aggregation, shear stress-induced cell death, batch-to-batch inconsistencies, and limited scalability. We propose that engineering a cell-friendly microenvironment can enhance culture efficiency. Previously, we developed alginate hydrogel microtubes (AlgTubes) that significantly improved cell density and growth rates; however, AlgTubes lack adhesion sites essential for anchorage-dependent cells and frequently break, causing cell leakage and production inconsistencies. Methods To address these limitations, we reinforced AlgTubes with collagen nanofibers, creating collagen-alginate hybrid hydrogel microtubes (ColAlgTubes). Collagen was integrated to form a dense nanofiber network interwoven with the alginate mesh, with the dual aim of enhancing mechanical properties and providing cell adhesion sites. ColAlgTubes were fabricated to maintain cell mass within a 400 μm diameter to ensure efficient nutrient exchange and waste removal. Results ColAlgTubes successfully formed a reinforced hybrid hydrogel structure in which the collagen nanofiber network improved the mechanical integrity of the alginate matrix while supplying adhesion sites for anchorage-dependent cells. The optimized 400 μm diameter microenvironment supported high cell viability, rapid proliferation, and exceptional yields of 5 × 10 8 cells/mL. Discussion These results demonstrate that ColAlgTubes overcome the key limitations of AlgTubes by combining structural reinforcement with biological functionality. The improved mechanical properties reduce tube breakage and cell leakage, while collagen-derived adhesion sites broaden compatibility to anchorage-dependent cell types. With their scalability, cost-effectiveness, and high efficiency, ColAlgTubes represent a transformative solution for large-scale mammalian cell production across biotechnology, regenerative medicine, and therapeutic manufacturing.

Hepatic steatosis, characterized by the accumulation of triacylglycerol in hepatocytes, complicates liver transplantation by increasing the risk of ischemia-reperfusion injury (IRI), leading to suboptimal graft outcomes. This condition has become a major cause of liver graft discarding, exacerbating the ongoing organ shortage for transplantation. Recent advances in ex vivo normothermic perfusion machines (NMPs) have provided promising solutions for reconditioning and preserving steatotic liver. NMP supports cellular metabolism and mitigates IRI by maintaining the physiological conditions. In addition, pharmacological strategies to reduce hepatic steatosis have been explored to increase graft viability. In this review, we summarize current insights into hepatic lipid metabolism and the molecular mechanisms underlying steatosis, while discussing emerging pharmacological defatting approaches. These interventions have the potential to address the challenges posed by hepatic steatosis, improve graft quality, expand the availability of transplantable livers, and enhance clinical outcomes of transplant candidates. Finally, we highlight current limitations and outline directions for future research and clinical applications.

Abstract Anaplastic large cell lymphomas (ALCLs) are CD30+ T-cell lymphomas that share pathologic features but differ in presentation, outcome, and genetics. Current classification incorporates clinical presentation and anaplastic lymphoma kinase (ALK) status but inadequately addresses molecular heterogeneity and therapeutic vulnerabilities. We studied 689 patients with ALCL in the LLMPP (Lymphoma/Leukemia Molecular Profiling Project) and performed expert consensus review, genetic subtyping (ALK, DUSP22, TP63, and triple negative), and immunohistochemistry for phosphorylated STAT3Tyr705. RNA sequencing with unsupervised gene expression profiling in 393 patients identified 2 main molecular types of ALCL that could be predicted with 91% accuracy based on the presence (type I) or absence (type II) of phosphorylated STAT3Y705 expression. Type I ALCLs included ALK+ ALCL and a subset of triple-negative ALCLs (TN-I); type II ALCLs included tumors with DUSP22 and/or TP63 rearrangements and the remaining triple-negative ALCLs (TN-II). Type I ALCLs were enriched for JAK-STAT3, whereas type II ALCLs were enriched for non–tyrosine kinase pathways, particularly epigenetic regulators such as EZH2. Immunohistochemistry showed overexpression of EZH2 and its trimethylated substrate H3K27. Prognosis in systemic ALCL was favorable for DUSP22-rearranged ALCL (5-year overall survival, 95%) and ALK+ ALCL (88%), intermediate for triple-negative ALCL (TN-I, 52% and TN-II, 37%), and poor for TP63-rearranged ALCL (0%). We introduce an integrated molecular classification that preserves currently diagnosed ALCL entities but identifies 4 molecularly distinct ALK− ALCL subtypes (DUSP22-rearranged, TP63-rearranged, TN-I, and TN-II). This classification can be easily implemented on paraffin tissue in routine practice or clinical trials, and stratifies ALCL into diagnostically, prognostically, biologically, and potentially therapeutically relevant subtypes.

Abstract A major barrier in understanding adult diffuse gliomas lies in the inability to track tumor evolution in real time. To address this, we employed a novel method known as continuous lineage tracing, which integrates CRISPR/Cas9-based expressed DNA barcoding with single-cell RNA sequencing, enabling a phylogenetic approach to studying tumor development. Patient-derived glioma-initiating cell lines were engineered with expressed barcodes targeted by CRISPR/Cas9 and implanted into mice to create intracranial xenografts. Tumors underwent single-cell RNA sequencing; expressed barcodes were used to infer clonal relationships, and transcriptomic profiles enabled cell state classification. Phylogenetic lineage trees were reconstructed using lineage inference algorithms to characterize cell fitness, expansion, and plasticity. Our analysis uncovered extensive intra-clonal cell state heterogeneity, indicating active phenotype switching prior to therapy. We identified consistent transcriptional programs associated with tumor engraftment and in vivo clonal advantage. Lineage tracing revealed gene expression signatures linked to key phenotypes: fitness, enriched for neural-mesenchymal and injury-response pathways; expansion, associated with RNA splicing; and plasticity, correlated with cell cycle and DNA repair programs. Glioma stem cells appeared to span a transcriptional continuum from undifferentiated, high-fitness states to more differentiated, low-fitness states, with high-fitness cells potentially representing transitional phenotypes. We then validated these findings in a cohort of 185 surgically resected gliomas with matched bulk RNA and proteomic data. Phylogenetic gene signatures differed markedly between IDH-mutant and IDH-wildtype tumors. When stratified by tumor type, both fitness and expansion signatures were significantly associated with overall survival in GBM (median 12.6 months, p=0.041) and oligodendrogliomas (median 66 months, p=0.027). This study demonstrates the utility of continuous lineage tracing to reconstruct tumor evolution and identify transcriptional programs linked to tumor growth and prognosis. Our approach provides a powerful framework for dissecting glioma biology and identifying potential therapeutic vulnerabilities.

Objectives: This study evaluates the efficacy of existing and new aerosol mitigation methods during nebulization (Neb) in combination with high-flow nasal cannula (HFNC) oxygen supplementation and non-invasive ventilation (NIV). Methods: We recorded fugitive aerosol particle concentrations over time and assessed the peak (P) and area (A) efficacy of active and passive mitigation methods, comparing them to a no-mitigation condition. Peak efficacy was measured by the reduction in maximum aerosol concentration, while area efficacy was quantified by the reduction of the area under the aerosol concentration–time curve. Results: For HFNC with Neb, we found that active mitigation using a mask with a biofilter and a fan (referred to as the aerosol barrier mask) significantly outperformed passive mitigation with a face mask. The peak and area efficacy for aerosol reduction were 99.0% and 96.4% for active mitigation and 35.9% and 7.6% for passive mitigation, respectively. For NIV with Neb, the active mitigation method, using a box with a biofilter and fan, also outperformed passive mitigation using only the box. The peak and area efficacy for aerosol reduction were 92.1% and 85.5% for active mitigation and 53.7.0% and 25.4% for passive mitigation, respectively. Conclusion: We concluded that active mitigation set up systems advantageous for effective reduction of airborne aerosols during aerosol generated procedures.

Abstract Objective Rare large B-cell lymphomas (LBCLs) present with concurrent or subsequent lymphomatous effusion (solid-effusion LBCL, SE-LBCL), which may have an inferior prognosis compared with their noneffusion counterpart. In addition, the relationship between SE-LBCL and human herpesvirus 8–negative effusion-based LBCL (EB-LBCL) remains unclear. Methods We collected 141 cases of SE-LBCL and a control cohort of 101 cases of stage IV solid-only LBCL. The clinicopathologic features were analyzed and compared between SE-LBCL and solid-only LBCL. Results Patients with SE-LBCL had a median age of 67 years with a male-to-female ratio of 1.3:1. Eighty-six patients had concurrent solid lymphoma and lymphomatous effusion, whereas 55 developed lymphomatous effusion subsequently. Most cases involved the pleural cavities (79%, 112/141), followed by the peritoneal (21%, 29/141) and pericardial (11%, 16/141) cavities. BCL6, CD10, and MUM1 were expressed in 77% (90/117), 46% (60/130), and 61% (58/95) of cases, respectively, and 58% (71/123) were subclassified into the germinal center B-cell (GCB) subtype. Rearrangements of BCL2, BCL6, and MYC were detected in 42% (31/73), 35% (22/63), and 40% (35/88), respectively, and 22% (19/87) had both MYC and BCL2 rearrangements. The patients with SE-LBCL had a dismal prognosis, with a median survival of 5.7 months, which was significantly worse than solid-only LBCL (147.5 months; P < .0001). Conclusions The pathologic features of SE-LBCL were similar to those of solid-only LBCL but distinct from those of EB-LBCL; in particular, lymphomatous effusion was an independently adverse prognostic factor in LBCL. Our study underscores the need for surveillance of lymphomatous effusion during LBCL staging and development of effective therapeutic regimens for SE-LBCL.

Background: Anaplastic large cell lymphomas represent a group of T-cell lymphomas that have long been broadly classified by the presence or absence of ALK fusion proteins (ALK+ or ALK−, respectively). However, recent discovery of extensive additional molecular heterogeneity has both promoted refinement of disease classification and uncovered new opportunities for biomarker-guided targeted therapy. In addition to recognition of a new genetic subtype, ALK− ALCL with DUSP22 rearrangement (R), a new molecular classification has been proposed based on the recent discovery of 2 overarching molecular types of ALCL, defined by the presence (Type I) or absence (Type II) of a gene expression signature highly enriched for JAK-STAT3 activation. BCL2 is a mitochondrial membrane protein that functions to inhibit the actions of pro-apoptotic proteins such as BAX and BAK and can be targeted clinically by BCL2 small molecule inhibitors. BCL2 has been reported to be expressed more frequently in ALK− ALCL than in ALK+ ALCL, but its expression in the context of current molecular findings is unknown. Methods: ALCLs were centrally reviewed and classified by current WHO/ICC criteria. BCL2 was performed using IHC and scored as positive if ≥30% of tumor cells showed staining. DUSP22-R was assessed by fluorescence in situ hybridization (FISH) using a breakapart probe set and a dual-color, dual-fusion (D) probe set for the most common DUSP22 translocation, t(6;7)(p25.3;q32.3). pSTAT3Y705 IHC, which is >90% accurate for Type I/II assignment based on gene expression, was performed and scored based on a previously validated cut-off of ≥30%. Results: We evaluated 217 ALCLs (148M/69F; age: mean, 57 y; range, 5-93 y), including 55 systemic ALK+ ALCLs, 127 systemic ALK− ALCLs, and 35 primary cutaneous (pc) ALCLs. Overall, 86/217 cases were positive for BCL2 (40%). BCL2 was positive in 67/127 ALK− ALCLs (53%) and 15/35 pcALCLs (43%; P=NS), but in only 4/51 ALK+ (7%; P<0.0001). DUSP22-R was present in 90 cases (63 ALK− ALCL and 27 pcALCL). BCL2 was positive in 57/90 cases with DUSP22-R (63%) compared to 29/127 without DUSP22-R (23%; P<0.0001). Among 85 cases with DUSP22-R and informative D-FISH testing, BCL2 was positive in 24/36 cases with t(6;7)(p25.3;q32.3) (67%) and 31/49 cases without t(6;7)(p25.3;q32.3) (63%; P=NS). Among 213 cases with informative pSTAT3Y705 staining, BCL2 was positive in 13/88 pSTAT3+ cases (Type I, 15%) and 70/125 pSTAT3− cases (Type II, 56%; P<0.0001). Conclusions: We identified a complex pattern of BCL2 expression across ALCLs that strongly correlates with a recently introduced molecular classification based on gene expression profiling. While our findings are in keeping with the previously reported low expression in ALK+ ALCL, we found that the higher expression in both systemic ALK− ALCL and pcALCL is particularly associated with DUSP22-R and the absence of pSTAT3Y705 expression (Type II ALCLs). The discovery of 2 overarching types of ALCL associated with the presence or absence JAK-STAT3 pathway activation represents a new conceptual framework for classifying ALCL and understanding its therapeutic vulnerabilities. Of note, previous data demonstrated that Type II ALCLs are enriched for epigenetic alterations and particularly overexpress the histone methyltransferase EZH2. Correlation between BCL2 expression and EZH2 expression also is observed in other lymphomas such as diffuse large B-cell lymphoma, where dual targeting with EZH2 and BCL2 inhibitors has been proposed. Thus, our findings provide further rationale for stratifying Type I and Type II ALCLs and identify BCL2 as a differentially expressed oncoprotein that might represent a target for precision therapy approaches.