Citations

Abstract Background Lupus nephritis remains a major cause of chronic kidney disease and kidney failure in systemic lupus erythematosus. Glucocorticoids are central to treatment but are limited by systemic toxicity. We evaluated whether a previously characterized collagen IV α3-targeted liposomal nanoparticle formulation carrying low-dose prednisolone could attenuate established lupus nephritis in MRL/lpr mice. Methods Female MRL/lpr mice with disease present at treatment initiation and C57BL/6J control mice received saline or collagen IV α3-targeted prednisolone-loaded nanoparticles (Col4-α3-Pred-NPs). Renal outcomes were assessed by longitudinal proteinuria, glomerular filtration rate (GFR), survival, kidney histopathology, renal IgG and C3d deposition, dUTP/TUNEL-associated injury staining, and renal cytokine/chemokine profiling. Body weight, food and water intake, and blood glucose were monitored as measures of general condition and preliminary tolerability. Results Col4-α3-Pred-NPs improved survival in MRL/lpr mice, reduced cumulative proteinuria burden, and attenuated terminal GFR decline compared with saline-treated MRL/lpr controls. Treatment reduced glomerular and tubulointerstitial injury, lowered composite EGTI histopathology scores, decreased terminal kidney enlargement, reduced glomerular IgG deposition and renal dUTP-positive injury signals, and reduced renal signals for IL-28A/B, IL-7, PD-ECGF, IL-11, CCL6/C10, and IL-15. C3d deposition was not significantly altered. Nanoparticle treatment was not associated with sustained treatment-related increases in blood glucose or body-weight loss during the measured study period. Conclusions Collagen IV α3-targeted liposomal delivery of low-dose prednisolone attenuated established lupus nephritis in MRL/lpr mice and improved renal structural, functional, inflammatory, and survival outcomes. These findings support further evaluation of glomerulus-targeted nanotherapy as a potential strategy to improve the precision and therapeutic index of glucocorticoid treatment in lupus nephritis.

Abstract Lupus nephritis (LN) requires long-term immunosuppressive therapy, which is often associated with severe systemic side effects. Therefore, new therapeutic strategies that maintain high efficacy while minimizing adverse effects are essential. Although nanomedicine has advanced systemic and kidney-targeted drug delivery, a reliable method for glomerulus-specific delivery is lacking. Collagen IV (Col4)-alpha 3, located in the glomerular basement membrane at the blood–tissue interface through fenestrated capillary endothelium, represents an ideal target for glomeruli delivery. Herein, we developed a novel liposomal nanoparticle conjugated with a Col4-alpha 3-binding peptide (Col4-α3-NPs) for selective glomerular targeting. Prednisolone-loaded Col4-α3-NPs were administered to lupus-prone mice twice weekly for 8 weeks. Kidney injury and function were evaluated biweekly, and renal immune cell populations were analyzed by flow cytometry at study completion. The results show that rhodamine-labeled NPs predominantly accumulate in kidney glomeruli 48 h after intravenous injection. The Col4-NP system demonstrated stable and prolonged release of the encapsulated drug for over 48 h. Lupus-prone mice treated with prednisolone-loaded Col4-NPs showed significantly improved renal function and histology, including a 30% increase in glomerular filtration rate, a 56% reduction in proteinuria, and decreased IgG deposition and fibrosis. Notably, treatment also enhanced renal regulatory T cell populations. These findings suggest that glomerulus-targeted Col4-α3-NPs hold significant translational promise. This platform may offer an effective, site-specific treatment for LN while minimizing systemic side effects and could be adapted for other glomerular diseases requiring targeted therapy.

ABSTRACT Lupus nephritis requires long-term immunosuppressive therapy, which is often associated with severe systemic side effects. Therefore, new therapeutic strategies that maintain high efficacy while minimizing adverse effects is essential. Although nanomedicine has advanced systemic and kidney-targeted drug delivery, a reliable method for glomerulus-specific delivery is lacking. Collagen IV (Col4)-alpha 3, located in the glomerular basement membrane (GBM) at the blood–tissue interface through fenestrated capillary endothelium, represents an ideal target for glomeruli delivery. Herein, we developed a novel liposomal nanoparticle conjugated with a Col4-alpha 3 binding peptide (Col4-α3-NPs) for selective glomerular targeting. Prednisolone-loaded Col4-α3-NPs were administered to lupus-prone mice twice weekly for 8 weeks. Kidney injury and function were evaluated biweekly, and renal immune cell populations were analyzed by flow cytometry at study completion. The results show that rhodamine-labeled NPs predominantly accumulate in kidney glomeruli 48 hours after intravenous injection. The Col4-NP system demonstrated stable and prolonged release of the encapsulated drug for over 48 hours. Lupus-prone mice treated with prednisolone-loaded Col4-NPs showed significantly improved renal function and histology, including a 30% increase in glomerular filtration rate (GFR), a 56% reduction in proteinuria, and decreased IgG deposition and fibrosis. Notably, treatment also enhanced renal regulatory T cell (Treg) populations. These findings suggest that glomerulus-targeted Col4-α3-NPs hold significant translational promise. This platform may offer an effective, site-specific treatment for lupus nephritis while minimizing systemic side effects and could be adapted for other glomerular diseases requiring targeted therapy.

ABSTRACT Our study aimed to develop a novel, reproducible, minimally invasive turkey knee osteoarthritis (OA) model and evaluate whether intra‐articular hyaluronan (HA) alone or combined with purified exosome product (PEP) attenuates OA‐related functional and structural deterioration. OA was induced arthroscopically by transection of the anterior and posterior cruciate ligaments with resection of the anterior horn of the medial meniscus. Animals received intra‐articular injections of saline, HA, or PEP + HA at postoperative 2–5 weeks. Gait was assessed at 4, 8, and 12 weeks, and cartilage degeneration, subchondral bone sclerosis, osteophyte formation, synovial fibrosis, heterotopic ossification, and meniscal integrity were evaluated by quantitative CT and histology at week 12. Compared with saline, HA improved lameness, preserved cartilage glycosaminoglycan content, and reduced meniscal rupture, subchondral bone sclerosis, and periarticular heterotopic ossification. PEP + HA provided additional benefits, including prolonged stance phase, reduced synovial fibrosis, and greater chondroprotection than HA alone, while plantar pressure variables, subchondral bone remodeling, and patellar osteophytes did not differ between HA and PEP + HA. This turkey model reliably produces human‐like OA changes and enables longitudinal functional and structural assessment. Intra‐articular HA mitigated OA progression, and the addition of PEP conferred incremental joint protection.

INTRODUCTION: Nosocomial infections (NIs) in cirrhosis are associated with high mortality but could be preventable. Logistic regression (LR) models have failed to identify high-risk patients. We aimed to develop machine learning (ML) models to predict NI. METHODS: The CLEARED consortium consists of prospectively enrolled cirrhosis inpatients from >120 centers. Using day-of-admission clinical data, 3 ML approaches (random forest [RF], extreme gradient boosting, and neural networks [NNs]) were used to predict NI. Data were split 80:20 for training and testing stratified by the outcome. Models were compared using area under the receiver operating characteristic curve (AUC). RESULTS: In total, 8,263 patients (55.90 ± 13.34 years; 64.1% men) from 127 centers in 37 countries were included. NI developed in 869 (10.5%), a median of 6 (4–11) days of postadmission. Major NIs were respiratory (29.6%) and urinary tract infection (15.7%), and spontaneous bacterial peritonitis (13.5%). NIs occurred more frequently in patients from low/low-middle income countries and those with severe liver disease, alcohol etiology, and admission infections. NIs were associated with inpatient mortality (31.9% vs 8.1%, P < 0.001) and liver transplantation (4.8% vs 1.9%, P < 0.001). Although the RF model (AUC 0.69) showed good calibration (Brier score 0.09), outperforming extreme gradient boosting, neural network, and LR models (AUC 0.66 for all; LR comparison P = 0.043), no model achieved AUC ≥0.80 for clinical utility. At 10% predicted probability threshold, the RF model demonstrated only 75.4% sensitivity, 52.9% specificity, and 15.9% positive predictive value (PPV). DISCUSSION: NIs cannot be accurately predicted from day-of-admission data using ML models, even in a large, prospective, global cirrhosis cohort. Every hospitalized patient with cirrhosis should receive protocolized infection control measures.

Abstract Acute kidney injury (AKI) causes rapid loss of renal function with high morbidity and mortality. Our previous research showed that neuronal nitric oxide synthase (NOS1) mediates nitric oxide-dependent dilation of the afferent arteriole, inhibiting tubuloglomerular feedback (TGF), which critically regulates single nephron glomerular filtration rate (GFR). This mechanism could affect how vulnerable the organ is to ischemic injury, but this has not been directly tested. In the present study, we generated inducible macula densa (MD)-specific NOS1 knockout mice (NKCC2-Cre-NOS1 flox/flox) using a new inducible NKCC2-Cre line crossed with NOS1 flox mice. AKI was induced by 18-min bilateral renal pedicle clamping at 37°C, followed by 48-h reperfusion. Kidney injury was assessed by measuring GFR, plasma creatinine, histology, cytokines, apoptosis, fibrotic factors, and proteomics. Complete NOS1 deletion in MD cells was confirmed through NKCC2/NOS1 immunofluorescence. MD NOS1 deletion enhanced the TGF response, which was further amplified following AKI compared with control (Cre −/−). Following AKI, compared with controls (Cre −/−), NOS1 knockouts showed a significantly lower GFR (236 ± 66 to 24 ± 22 μl/min) and higher plasma creatinine levels, alongside more severe tubular damage evidenced by H&E staining. Cytokine array analysis showed chemokines such as MCP-1 and CXCL1 and macrophage marker CD68 were significantly increased; Western blot analysis showed cleaved caspase-3 levels were significantly increased, indicating enhanced apoptosis. Additionally, fibrosis markers TIMP-1, collagen-3, and α-SMA were significantly up-regulated at both mRNA and protein levels. We further observed increased hypoxia marker hypoxia-inducible factor-1α in MD-specific NOS1 knockout mice (Cre +/−). Global label-free proteomic profiling with targeted validation identified genotype-dependent responses involving haptoglobin, Tacstd2, and Cyp20a1, linking NOS1 deficiency to exaggerated inflammatory, fibrotic, and metabolic pathways. These findings highlight a novel role of MD NOS1 and TGF in AKI pathophysiology, suggesting NOS1 targeting as a therapeutic strategy. Haptoglobin was identified as a downstream plasma signal of NOS1-dependent AKI responses, indicating potential translational value.

ABSTRACT Tumorigenesis and metastasis are frequently attributed to the intricate interplay between cancer cells and the tumor microenvironment (TME). Comprehending the mechanisms and key regulators of cancer‐immune crosstalk in the TME is imperative for developing efficacious immunotherapy. Through a series of in vivo CRISPR screens, we identified tumor‐intrinsic ITGB1 as a critical regulator of triple‐negative breast cancer (TNBC) development and deciphered its underlying mechanisms. Tumoral ITGB1 facilitated the establishment of pro‐tumorigenic TME by orchestrating tumor‐associated myeloid populations. Suppressing ITGB1 favored the enrichment of anti‐tumorigenic myeloid cells and enhanced infiltration of CD4 and CD8 T cells, culminating in superior antitumor effects. CRISPR scanning pinpointed a previously unrecognized functional domain essential for ITGB1's pro‐tumorigenic activity. This domain is distinct from all known ligand‐binding sites in ITGB1. An antibody capable of sterically blocking this domain significantly impaired TNBC progression. These findings position tumoral ITGB1 as a promising therapeutic target for reprogramming the TME from a pro‐ to an anti‐tumorigenic state, thereby effectively inhibiting TNBC development. Our study uncovers a novel mechanism of TNBC development and provides a unique therapeutic strategy for targeting ITGB1 in TNBC treatment.

Background: Ischemia–reperfusion injury (IRI) is a major contributor to acute kidney injury (AKI). While the precise mechanisms of AKI are still incompletely defined, extensive evidence highlights tubular cell injury and death as key factors in its development. Necroptosis has recently emerged as a critical pathway in the pathogenesis of ischemia–reperfusion-induced AKI (IR-AKI). Although sex differences in susceptibility to IR-AKI have been reported, it remains unclear whether there are sex differences in necroptosis dynamics and whether these differences underlie the observed sexual dimorphism in kidney IRI. This study aimed to address these questions. Methods: male and female C57BL/6 J mice were subjected to AKI via ischemia induced by bilateral renal pedicle clamping for 18 min at 37 °C. Plasma, urine, and kidney samples were collected at 0 h, 3 h, 6 h, 12 h, 24 h, 48 h, and 72 h post-reperfusion. Kidney injury and function were assessed by measuring plasma creatinine (PCr), blood urea nitrogen (BUN) levels, and histological damage (PAS and cleaved caspase3 staining). Necroptosis activation was assessed by quantifying phosphorylated forms of key markers: p-RIPK1 and p-MLKL. To explore the role of sex hormones in regulating necroptosis dynamics, ovariectomized female mice were subjected to the same IR-AKI protocol, and their kidney injury and functional outcomes were compared with those of intact counterparts. Results: The PCr was 0.35 ± 0.04 and 0.32 ± 0.06 mg/dL for males and females, respectively, at 3 h of IR. The levels exponentially increased to 2.05 ± 0.18 at 48 h post-reperfusion in the males but only gradually to 0.94 ± 0.13 mg/dL in females. Necroptosis activation began as early as 3 h post-IR in males but was delayed until ~6 h in females. Males exhibited stronger and more sustained necroptosis activation than females, showing elevated phosphorylation levels of pRIPK1 and pMLKL in Western blot. Female sex hormone deficiency exacerbated the female response to IR-induced injury, which reduced the sex difference in the dynamic of the necroptotic activation and subsequent kidney injury. To our knowledge, this is the first study to characterize sex differences in the initiation and progression of necroptosis and subsequent injury in a mouse model of IR-AKI. Conclusions: Our findings reveal distinct temporal patterns of programmed cell death between sexes. Necroptosis-targeted therapies require early intervention in males, which can be delayed in females after IR-AKI, highlighting the need for sex-specific therapeutic windows.

340 Background: TIS, an anti-programmed cell death protein 1 (PD-1) antibody, + chemo as 1L therapy demonstrated statistically significant and clinically meaningful improvement in overall survival (OS) vs PBO + chemo in patients with advanced or metastatic ESCC (hazard ratio [HR] 0.66 [95% confidence interval (CI) 0.54, 0.80]; P< 0.0001), with a manageable safety profile, at interim analysis of the phase 3, double-blind RATIONALE-306 study (NCT03783442). Here, we report data from the non-Asia subgroup (Europe, Northern America, and Oceania). Methods: Adults with advanced or metastatic ESCC, with no prior systemic treatment for advanced disease were randomized 1:1, (stratified by region, prior definitive therapy, and investigator [INV]-chosen chemo) to receive TIS 200 mg intravenously (IV) once every 3 weeks (Q3W) (Arm A) or PBO IV Q3W (Arm B), with platinum + fluoropyrimidine, or platinum + paclitaxel until disease progression by INV per RECIST v1.1, intolerable toxicity, or withdrawal. The primary endpoint was OS in the intent-to-treat population. Secondary endpoints included: progression-free survival (PFS), objective response rate (ORR), and duration of response (DoR) by INV per RECIST v1.1; OS in the programmed death-ligand 1 score ≥10%; and safety. Results: Of 649 randomized patients, 163 (25.1%) were from the non-Asia subgroup (Arm A, n = 83; Arm B, n = 80). At data cutoff (Feb 28, 2022), the median study follow-up time in the non-Asia subgroup was 16.0 months (mo) in Arm A vs 8.4 mo in Arm B. OS (median 16.3 vs 9.0 mo; unstratified HR 0.66 [95% CI 0.45, 0.96]) and PFS (median 7.7 vs 5.5 mo; unstratified HR 0.59 [95% CI 0.41, 0.83]) were improved in Arm A vs Arm B, respectively. Arm A had higher ORR (61.4% vs 41.3%, odds ratio 2.27 [95% CI 1.21, 4.25]) and longer median DoR (7.1 mo [95% CI 5.6, 9.6] vs 5.7 mo [95% CI 3.8, 8.3]) than Arm B. More patients in Arm A vs Arm B experienced ≥1 treatment-related adverse event (TRAE; 94.0% vs 88.5%), serious TRAEs (25.3% vs 17.9%), and discontinuation due to treatment-emergent AEs (42.2% vs 35.9%, respectively). Similar proportions of patients in Arm A vs Arm B had ≥grade 3 TRAEs (56.6% vs 52.6%), and TRAEs leading to death (1.2% vs 1.3%), respectively. Conclusions: In the non-Asia subgroup, 1L TIS + chemo showed a clinically meaningful improvement in OS vs PBO + chemo in patients with advanced or metastatic ESCC, with a manageable safety profile, consistent with published results in the overall population. Clinical trial information: NCT03783442 .