Increased erythropoiesis in mice injected with submicrogram quantities of pseudouridine-containing mRNA encoding erythropoietin

Abstract: Advances in the optimization of in vitro-transcribed mRNA are bringing mRNA-mediated therapy closer to reality. In cultured cells, we recently achieved high levels of translation with high-performance liquid chromatography (HPLC)-purified, in vitro-transcribed mRNAs containing the modified nucleoside pseudouridine. Importantly, pseudouridine rendered the mRNA non-immunogenic. Here, using erythropoietin (EPO)-encoding mRNA complexed with TransIT-mRNA, we evaluated this new generation of mRNA in vivo. A single injection of 100 ng (0.005 mg/kg) mRNA elevated serum EPO levels in mice significantly by 6 hours and levels were maintained for 4 days. In comparison, mRNA containing uridine produced 10-100-fold lower levels of EPO lasting only 1 day. EPO translated from pseudouridine-mRNA was functional and caused a significant increase of both reticulocyte counts and hematocrits. As little as 10 ng mRNA doubled reticulocyte numbers. Weekly injection of 100 ng of EPO mRNA was sufficient to increase the hematocrit from 43 to 57%, which was maintained with continued treatment. Even when a large amount of pseudouridine-mRNA was injected, no inflammatory cytokines were detectable in plasma. Using macaques, we could also detect significantly-increased serum EPO levels following intraperitoneal injection of rhesus EPO mRNA. These results demonstrate that HPLC-purified, pseudouridine-containing mRNAs encoding therapeutic proteins have great potential for clinical applications.

Karikó K
Muramatsu H
Keller JM
Weissman D

Animals
Cell Count
Cell Proliferation
Chromatography, High Pressure Liquid
Drug Delivery Systems
Erythropoiesis
Erythropoietin
Female
Genetic Vectors
Hematocrit
Humans
Injections, Intraperitoneal
Macaca mulatta
Mice
Mice, Inbred BALB C
Pseudouridine
RNA, Messenger
Reticulocytes


Nucleofection induces transient eIF2α phosphorylation by GCN2 and PERK

Abstract: Nucleofection permits efficient transfection even with difficult cell types such as primary and non-dividing cells, and is used to deliver various nucleic acids, including DNA, mRNA, and small interfering RNA. Unlike DNA and small interfering RNA, mRNA is subject to rapid degradation, which necessitates instant early translation following mRNA delivery. We examined the factors that are important in translation following nucleofection and observed rapid phosphorylation of eukaryotic initiation factor 2 alpha (eIF2α) following nucleofection, which occurred in the absence of the delivered nucleic acid. We studied the involvement of three ubiquitous kinases capable of phosphorylating eIF2α in mammalian cells and identified that nucleofection-mediated phosphorylation of eIF2α was dependent on general control non-derepressible 2 (GCN2) and RNA-dependent protein kinase (PKR)-like endoplasmic reticulum kinase (PERK) but not PKR. A reduction in translation due to eIF2α phosphorylation was observed post nucleofection, demonstrating functional significance. Understanding the impact of nucleofection on translational machinery has important implications for therapeutics currently under development based on the delivery of mRNA, DNA, and small interfering RNA. Strategies to circumvent eIF2α phosphorylation and other downstream effects of activating GCN2 and PERK will facilitate further advancement of nucleic acid-based therapies.

Animals
Cell Line
Eukaryotic Initiation Factor-2
Humans
Mice
Phosphorylation
Protein Serine-Threonine Kinases
Transfection
eIF-2 Kinase

Journal Article
Research Support, N.I.H., Extramural


Toll-like receptor 4 contributes to poor outcome after intracerebral hemorrhage

Abstract: Intracerebral hemorrhage (ICH) is a devastating stroke subtype in which perihematomal inflammation contributes to neuronal injury and functional disability. Histologically, the region becomes infiltrated with neutrophils and activated microglia followed by neuronal loss, but little is known about the immune signals that coordinate these events. This study aimed to determine the role of Toll-like receptor 4 (TLR4) in the innate immune response after ICH and its impact on neurobehavioral outcome. Transgenic mice incapable of TLR4 signaling and wild-type controls were subjected to striatal blood injection to model ICH. The perihematomal inflammatory response was then quantified by immunohistochemistry, whole brain flow cytometry, and polymerase chain reaction. The critical location of TLR4 signaling was determined by blood transfer experiments between genotypes. Functional outcomes were quantified in all cohorts using the cylinder and open field tests. TLR4-deficient mice had markedly decreased perihematomal inflammation, associated with reduced recruitment of neutrophils and monocytes, fewer microglia, and improved functional outcome by day 3 after ICH. Moreover, blood transfer experiments revealed that TLR4 on leukocytes or platelets within the hemorrhage contributes to perihematomal leukocyte infiltration and the neurological deficit. Together, these data identify a critical role for TLR4 signaling in perihematomal inflammation and injury and indicate this pathway may be a target for therapeutic intervention.

Sansing LH
Harris TH
Welsh FA
Kasner SE
Hunter CA
Kariko K

Animals
Brain Injuries
Cerebral Hemorrhage
Inflammation
Male
Mice
Mice, Inbred C3H
Mice, Knockout
Severity of Illness Index
Signal Transduction
Toll-Like Receptor 4

Journal Article
Research Support, N.I.H., Extramural
Research Support, Non-U.S. Gov't


Generating the optimal mRNA for therapy: HPLC purification eliminates immune activation and improves translation of nucleoside-modified, protein-encoding mRNA

Abstract: In vitro-transcribed mRNA has great therapeutic potential to transiently express the encoded protein without the adverse effects of viral and DNA-based constructs. Mammalian cells, however, contain RNA sensors of the innate immune system that must be considered in the generation of therapeutic RNA. Incorporation of modified nucleosides both reduces innate immune activation and increases translation of mRNA, but residual induction of type I interferons (IFNs) and proinflammatory cytokines remains. We identify that contaminants, including double-stranded RNA, in nucleoside-modified in vitro-transcribed RNA are responsible for innate immune activation and their removal by high performance liquid chromatography (HPLC) results in mRNA that does not induce IFNs and inflammatory cytokines and is translated at 10- to 1000-fold greater levels in primary cells. Although unmodified mRNAs were translated significantly better following purification, they still induced high levels of cytokine secretion. HPLC purified nucleoside-modified mRNA is a powerful vector for applications ranging from ex vivo stem cell generation to in vivo gene therapy.

Karikó K
Muramatsu H
Ludwig J
Weissman D

Animals
Cells, Cultured
Chromatography, High Pressure Liquid
Cytidine
Genetic Therapy
HEK293 Cells
Humans
Mice
Protein Biosynthesis
Pseudouridine
RNA, Double-Stranded
RNA, Messenger
Transcription, Genetic
Transfection


Autologous blood injection to model spontaneous intracerebral hemorrhage in mice

Abstract: Investigation of the pathophysiology of injury after intracerebral hemorrhage (ICH) requires a reproducible animal model. While ICH accounts for 10-15% of all strokes, there remains no specific effective therapy. The autologous blood injection model in mice involves the stereotaxic injection of arterial blood into the basal ganglia mimicking a spontaneous hypertensive hemorrhage in man. The response to hemorrhage can then be studied in vivo and the neurobehavioral deficits quantified, allowing for description of the ensuing pathology and the testing of potential therapeutic agents. The procedure described in this protocol uses the double injection technique to minimize risk of blood reflux up the needle track, no anticoagulants in the pumping system, and eliminates all dead space and expandable tubing in the system.

Journal of visualized experiments : JoVE

Sansing LH
Kasner SE
McCullough L
Agarwal P
Welsh FA
Kariko K

Animals
Basal Ganglia
Blood
Cerebral Hemorrhage
Disease Models, Animal
Mice
Wounds and Injuries

Journal Article
Research Support, N.I.H., Extramural
Research Support, Non-U.S. Gov't
Video-Audio Media


Nucleoside modifications in RNA limit activation of 2'-5'-oligoadenylate synthetase and increase resistance to cleavage by RNase L

Abstract: The interferon-induced enzymes 2'-5'-oligoadenylate synthetase (OAS) and RNase L are key components of innate immunity involved in sensory and effector functions following viral infections. Upon binding target RNA, OAS is activated to produce 2'-5'-linked oligoadenylates (2-5A) that activate RNase L, which then cleaves single-stranded self and non-self RNA. Modified nucleosides that are present in cellular transcripts have been shown to suppress activation of several RNA sensors. Here, we demonstrate that in vitro transcribed, unmodified RNA activates OAS, induces RNase L-mediated ribosomal RNA (rRNA) cleavage and is rapidly cleaved by RNase L. In contrast, RNA containing modified nucleosides activates OAS less efficiently and induces limited rRNA cleavage. Nucleoside modifications also make RNA resistant to cleavage by RNase L. Examining translation in RNase L(-/-) cells and mice confirmed that RNase L activity reduces translation of unmodified mRNA, which is not observed with modified mRNA. Additionally, mRNA containing the nucleoside modification pseudouridine is translated longer and has an extended half-life. The observation that modified nucleosides in RNA reduce 2-5A pathway activation joins OAS and RNase L to the list of RNA sensors and effectors whose functions are limited when RNA is modified, confirming the role of nucleoside modifications in suppressing immune recognition of RNA.

Anderson BR
Muramatsu H
Jha BK
Silverman RH
Weissman D
Karikó K

2',5'-Oligoadenylate Synthetase
Animals
Cells, Cultured
Endoribonucleases
Half-Life
Humans
Mice
Nucleosides
Protein Biosynthesis
Pseudouridine
RNA
RNA, Ribosomal
Uridine


Neutrophil depletion diminishes monocyte infiltration and improves functional outcome after experimental intracerebral hemorrhage

Abstract: Inflammation contributes to secondary injury and neuronal loss after intracerebral hemorrhage, but the role of individual immune populations in these processes is unclear. In a mouse model, the injection of autologous blood into the striatum was associated with an intense inflammatory cell infiltrate composed of neutrophils, monocytes, and dendritic cells. Selective depletion of neutrophils resulted in decreased infiltration of monocytes and improved functional outcomes at day 3 post-hemorrhage. These findings indicate that neutrophil infiltration into the site of hemorrhage contributes to brain injury either by direct cellular damage or the recruitment of monocytes.

Sansing LH
Harris TH
Kasner SE
Hunter CA
Kariko K

Analysis of Variance
Animals
Antibodies, Monoclonal
Antigens, Ly
CD11b Antigen
CD11c Antigen
Cerebral Hemorrhage
Dendritic Cells
Disease Models, Animal
Functional Laterality
Inflammation
Lymphocyte Count
Male
Mice
Mice, Inbred C3H
Monocytes
Neutrophil Infiltration
Neutrophils


DICER1 deficit induces Alu RNA toxicity in age-related macular degeneration

Abstract: Geographic atrophy (GA), an untreatable advanced form of age-related macular degeneration, results from retinal pigmented epithelium (RPE) cell degeneration. Here we show that the microRNA (miRNA)-processing enzyme DICER1 is reduced in the RPE of humans with GA, and that conditional ablation of Dicer1, but not seven other miRNA-processing enzymes, induces RPE degeneration in mice. DICER1 knockdown induces accumulation of Alu RNA in human RPE cells and Alu-like B1 and B2 RNAs in mouse RPE. Alu RNA is increased in the RPE of humans with GA, and this pathogenic RNA induces human RPE cytotoxicity and RPE degeneration in mice. Antisense oligonucleotides targeting Alu/B1/B2 RNAs prevent DICER1 depletion-induced RPE degeneration despite global miRNA downregulation. DICER1 degrades Alu RNA, and this digested Alu RNA cannot induce RPE degeneration in mice. These findings reveal a miRNA-independent cell survival function for DICER1 involving retrotransposon transcript degradation, show that Alu RNA can directly cause human pathology, and identify new targets for a major cause of blindness.

Kaneko H
Dridi S
Tarallo V
Gelfand BD
Fowler BJ
Cho WG
Kleinman ME
Ponicsan SL
Hauswirth WW
Chiodo VA
Karikó K
Yoo JW
Lee DK
Hadziahmetovic M
Song Y
Misra S
Chaudhuri G
Buaas FW
Braun RE
Hinton DR
Zhang Q
Grossniklaus HE
Provis JM
Madigan MC
Milam AH
Justice NL
Albuquerque RJ
Blandford AD
Bogdanovich S
Hirano Y
Witta J
Fuchs E
Littman DR
Ambati BK
Rudin CM
Chong MM
Provost P
Kugel JF
Goodrich JA
Dunaief JL
Baffi JZ
Ambati J

Alu Elements
Animals
Cell Death
Cell Survival
Cells, Cultured
DEAD-box RNA Helicases
Gene Knockdown Techniques
Humans
Macular Degeneration
Mice
MicroRNAs
Molecular Sequence Data
Oligonucleotides, Antisense
Phenotype
RNA
Retinal Pigment Epithelium
Ribonuclease III


Single-Atom-Enhanced Fully Inkjet-Printed Electrochemical Sensor for Dopamine Detection

Abstract: Single-atom (SA) engineering offers atomic-level control over interfacial reactivity, yet the lack of printable SA-based inks hinders its practical translation into electrochemical sensing. Here, a fully water-based inkjet-printable ink is introduced based on nitrogen-doped graphene acid (NGA) hosting atomically dispersed Cu centers (NGA-Cu-ink). The ink enables digitally controlled, spatially defined deposition and fabrication of low-cost ($0.04 per sensor), fully inkjet-printed sustainable electrodes on paper. A comparison of NGA functionalized with different SA dopants (Cu, Mn, Fe, Ce) identifies a strong dopant-dependent electrochemical response, with Cu uniquely enhancing the analyte signal while other dopants suppress it, demonstrating that SA identity is a decisive design parameter in printed sensing interfaces profiling sensitivity and selectivity. The functional role of the NGA support is to provide dense anchoring sites (nitrogen and carboxylate groups) that stabilize atomically dispersed metal centers and create adsorption- and electron-transfer-active microenvironments. The NGA-Cu-ink yields enhanced dopamine oxidation, enabling quantitative detection on fully printed devices (limit of detection 9.7 µM; linear range 50-400 µM) and printed-on-electrode platforms (10.6 µM), while maintaining <10% signal variation over 11 weeks. The approach establishes a general route to single-atom graphene-based inks for scalable, reproducible, and low-material-consumption manufacturing of advanced electrochemical sensors.

Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Nalepa MA
Panáček D
Hrubý V
Jendrišák M
Langer R
Langer M
Jakubec P
Dědek I
Kupka V
Mazur M
Zbořil R
Otyepka M


Industrialization of sandwich composite panels for portable outdoor tabletops

Abstract: Sandwich composite panels offer a promising pathway for developing lightweight yet mechanically robust portable outdoor tabletops, but their industrialization outside traditional aerospace and transportation sectors remains challenging. This study investigates how sandwich composites can be adapted for mass‑market outdoor tabletops by linking market‑driven requirements to engineering design decisions. Five representative panel configurations were developed, varying in core materials, face‑sheet systems, surface finishing, edge‑sealing strategies, and fastening solutions. A structured evaluation matrix was established to assess both material‑level and product‑level performance, aligned with customer‑relevant requirements, applicable standards, and company‑defined acceptance criteria. Results show that industrial feasibility is governed not by any single metric but by the coupled interactions among material selection, surface durability, moisture‑resistant edge sealing, fastening reliability, and manufacturability-cost trade‑offs. Blind rivets emerged as a practical fastening solution, while surface durability and thermal stability were strongly influenced by the combined behavior of finishing chemistry and substrate. The findings establish practical architecture-performance relationships and outline viable industrialization pathways for mass‑market portable outdoor tabletops based on sandwich composite panels.


Invasive Paget's Disease of the Breast: A Staging Pitfall Mimicking pT4 Disease

Abstract: Invasive Paget's disease of the breast is a rare entity with therapeutic challenges. Herein, we report three patients with Paget's disease of the breast with dermal invasion, of which two cases had coexistent invasive breast carcinoma, while the third had an extensive in situ component. Case 1 was a 49-year-old lady presenting with nipple ulceration and discharge along with concomitant multicentric right breast lesions detected on mammography. Breast conservation surgery was performed which revealed extensive high-grade ductal carcinoma in situ on histopathology, associated with invasive Paget's disease. The patient completed adjuvant radiotherapy and shows no evidence of disease at present, four months following the completion of therapy. Case 2 was a middle-aged woman of 45 years, who was detected with multiple hypoechoic space-occupying lesions in the right breast on ultrasonography. Modified radical mastectomy was performed which showed a tumor-infiltrating lymphocyte-rich breast carcinoma along with invasive Paget's disease. Following surgery, she is currently on adjuvant chemotherapy with no evidence of disease. Case 3 was a 47-year-old lady presenting with a lump in the right breast with nipple ulceration. Microscopic examination of the modified radical mastectomy specimen showed a high-grade invasive breast carcinoma along with invasive mammary Paget's disease. Follow-up imaging showed generalized lymphadenopathy. The patient was advised adjuvant chemotherapy; however, she was lost to follow-up after receiving the first cycle of treatment. We present three cases of invasive Paget's disease of the breast highlighting the importance of recognition of this rare entity with distinct therapeutic implications.

Sharma A
Langer R
Chadha P
Gupta G
Singh S


A deep learning framework for efficient pathology image analysis

Abstract: Artificial intelligence has transformed digital pathology by enabling biomarker prediction from high-resolution whole-slide images. However, current methods are computationally inefficient, processing thousands of redundant tiles per slide and requiring complex aggregation models. We introduce EAGLE (Efficient Approach for Guided Local Examination), a deep learning framework that emulates pathologists by selectively analyzing informative regions. EAGLE combines task-agnostic tile selection with detailed feature extraction and is benchmarked against leading slide- and tile-level foundation models across 43 tasks from nine cancer types spanning morphology, biomarker prediction, treatment response and prognosis. EAGLE outperforms patch aggregation methods by up to 23% and achieves the highest overall classification performance. It processes one slide in 2.27 s, reducing computational time by more than 99% compared with existing models. This efficiency supports rapid and auditable workflows by enabling review of the exact tiles used for each prediction and reducing dependence on high-performance computing. By reliably identifying informative regions and minimizing artifacts, EAGLE provides robust and auditable outputs, supported by systematic negative controls and attention concentration analyses. Its unified embedding enables rapid slide search, integration into multi-omics pipelines and emerging clinical foundation models.

Neidlinger P
Lenz T
Foersch S
Loeffler CML
Clusmann J
Gustav M
Shaktah LA
Langer R
Dislich B
Boardman LA
French AJ
Goode EL
Gsur A
Brezina S
Gunter MJ
Steinfelder R
Behrens HM
Röcken C
Harrison T
Peters U
Phipps AI
Curigliano G
Fusco N
Marra A
Hoffmeister M
Brenner H
Kather JN

Deep Learning
Humans
Image Processing, Computer-Assisted
Neoplasms

Journal Article
Research Support, Non-U.S. Gov't
Research Support, N.I.H., Extramural


A modular hydrogel system with independent control of bioadhesion, fibrosis, and stiffness

Abstract: Hydrogels that adhere to biological tissues and resist fibrosis are required to provide both optimal functionality and appropriate stiffness on diverse soft tissues to achieve therapeutic efficacy and biocompatibility. However, their performance is often limited by an intrinsic trade-off between functionality and stiffness. Through the incorporation of polymer brush coatings, we develop a modular hydrogel system to enable independent control of functionality and stiffness. By tailoring coating chemistry, coating thickness, and hydrogel network topology, we obtain consistent bioadhesion (~100 joules per square meter) and fibrosis suppression across the full stiffness range of soft tissues (1 kilopascal to 1 megapascal). Using this approach, we design a hydrogel that can maintain stable adhesion in vivo on a beating mouse heart and a hydrogel with no fibrotic capsule in immunocompetent mice over 40 days. This modular system offers a customizable approach for designing functional implants with tailored mechanical properties.

Yang J
Zhao Y
Jeang WJ
Pabel S
Wong BM
Manan R
Nahrendorf M
Langer R
Anderson DG

Animals
Mice
Hydrogels
Fibrosis
Biocompatible Materials
Polymers


Effects of short-course preoperative endocrine therapy on tumour morphology and immunohistochemical profile in oestrogen receptor-positive, HER2-negative breast cancer

Abstract: Short-term preoperative endocrine therapy (ET) is increasingly used in oestrogen receptor (ER)-positive, HER2-negative breast cancer as a functional test of endocrine sensitivity. We aimed to characterise histomorphological and immunophenotypic changes following preoperative ET and to identify predictors of endocrine response, defined as post-treatment Ki67 ≤ 10%. In this retrospective single-centre study, 180 patients treated with short-course preoperative ET (median duration 29 days) were compared with 151 patients undergoing primary surgery without ET. Paired biopsy and resection specimens were assessed for histological features, stromal proportion, stromal tumour-infiltrating lymphocytes (strTILs) and expression of ER, progesterone receptor (PR), HER2 and Ki67. Genomic risk was determined using the MammaPrint assay. Preoperative ET was associated with a significant reduction in tumour proliferation, with 73.9% of cases showing post-treatment Ki67 ≤ 10% compared with none in controls (P < 0.001). Histological grade decreased in 36.7% of ET-treated tumours versus 7.9% of controls (P < 0.001), predominantly reflecting reduced mitotic activity. ER expression remained stable, whereas PR expression decreased more frequently following ET (P < 0.001) and was independently associated with Ki67-defined response. HER2-low status was more frequently observed after ET (P < 0.001), but HER2 expression and microenvironmental parameters, including strTILs, were not associated with response. High genomic risk was independently associated with a lower likelihood of achieving post-treatment Ki67 ≤ 10% (P < 0.001). Short-course preoperative ET induces rapid and reproducible morphological and immunophenotypic changes in ER-positive, HER2-negative breast cancer. Ki67-defined response is associated with genomic risk and PR expression, whereas microenvironmental features appear to have limited predictive value.

Grosse C
Grosse A
Noack P
Preuss CI
Schwarz HK
Schneeweiss B
Gitter T
Schrenk P
Langer R


The Use of Deep Learning in RNA Therapeutic Development

Abstract: Ribonucleic acid (RNA)-based therapeutics have emerged as promising methods of disease treatment due to their ability to target the human genome and influence protein production, their versatility, and their relative lack of toxicity compared to other gene therapies. However, the RNA therapeutic design space is extremely large, encompassing multiple variables, including codon identities, secondary structure, and design of specific regions. RNA therapeutic optimization is difficult due to the impracticality of exploring such a vast design space experimentally. To address this limitation, deep learning methods have been employed to optimize RNA therapeutic development. In this review, we examine the application of deep learning models across three key aspects of RNA therapeutic development (RNA structure prediction, CRISPR activity, and RNA delivery), highlighting major contributions in these fields and analyzing how deep learning model architectures could affect model performance. We then discuss challenges associated with using deep learning for RNA therapeutics, such as computational and data limitations. Finally, we offer perspectives on areas for future exploration, such as emerging model architectures and methods of integration with more advanced high-throughput screening techniques. Ultimately, this review provides an overview of how deep learning is used in RNA therapeutic development and how it can evolve in the future.

Subramanian DA
Yao SL
Chan A
Witten J
Reker D
Anderson DG
Langer R
Traverso G

Deep Learning
Humans
RNA
Nucleic Acid Conformation
Genetic Therapy


Am80-lipid nanoparticles serve as an enteric mucosal adjuvant following parenteral immunization with inactivated polio vaccine

Abstract: Enteric pathogens are a major contributor to the global disease burden, necessitating vaccines capable of inducing robust gastrointestinal mucosal immunity. Achieving this response is especially challenging with inactivated or subunit vaccines, which lack the ability of live-attenuated formulations to mimic the natural infection process needed to induce strong intestinal mucosal immunity. Specifically, the inactivated polio vaccine (IPV), administered parenterally, elicits strong systemic immunity but fails to induce the mucosal IgA responses required to fully block poliovirus transmission, a critical step toward complete eradication. To address this limitation, we developed a clinically translatable and scalable nanoparticle-based intestinal mucosal adjuvant by encapsulating Am80, a small hydrophobic molecule known to promote intestinal mucosal immunity, within nanoparticles (NPs) designed for lymph node delivery, without requiring antigen modification, encapsulation, or adsorption. Encapsulation in NPs eliminated the need for the use of organic solvents, reduced toxicity, and prevented degradation, while lipid nanoparticles (Am80-LNPs) were engineered for sustained release over 3 to 5 days with high encapsulation efficiency (78 ± 9%). Cy5-labeled Am80-LNPs localized to draining lymph nodes within 6 hours and were retained for up to 72 hours. Coadministration of Am80-LNPs with the licensed IPV-2 in a Wistar rat model significantly boosted IPV-2-specific fecal IgA by 20-fold compared to IPV-2 alone and threefold over free Am80. These findings demonstrate that Am80-LNPs significantly enhance IPV-2-specific mucosal immunity in vivo and suggest their potential as a potent mucosal adjuvant against other enteric pathogens.

Eshaghi B
Wang EY
Mursalova S
Forster TA
Lasrado N
Tian J
Artzi D
Lin SQ
Wu Z
Yang X
Chung W
Sadeghi I
Garcia J
Chen Z
Sheridan O
Das R
Lau A
Gurram A
Mainou BA
Jones KAV
Liu Y
Barouch DH
von Andrian U
Langer R
Jaklenec A

Animals
Female
Rats
Adjuvants, Immunologic
Immunity, Mucosal
Immunization
Immunoglobulin A
Intestinal Mucosa
Lipids
Lymph Nodes
Nanoparticles
Nanovaccines
Poliomyelitis
Poliovirus Vaccine, Inactivated
Liposomes
Humans


Author Correction: Extracellular vesicles incorporating retrovirus-like capsids for the enhanced packaging and systemic delivery of mRNA into neurons

Gu W
Luozhong S
Cai S
Londhe K
Elkasri N
Hawkins R
Yuan Z
Su-Greene K
Yin Y
Cruz M
Chang YW
McMullen P
Wu C
Seo C
Guru A
Gao W
Sarmiento T
Schaffer C
Nishimura N
Cerione R
Yu Q
Warden M
Langer R
Jiang S


Wireless battery-free oxygenation devices enable extended immunosuppression-free islet transplantation in minimally invasive sites

Abstract: Here, we develop a next-generation wireless, battery-free oxygen generating O2-Macrodevice and wearable power transfer platform that can enable long-term immune protection and subcutaneous function of therapeutic cells. We demonstrate this device supports xenogeneic islet transplantation in C57BL/6J mice evidenced by 90-day diabetes reversal and glucose responsiveness in vivo. We also show partial glycemic control via high-density (>8,000 islets/cm2) human stem-cell derived islets (SC-islets) without immune-suppression in subcutaneous sites for 90 days. Additionally, we confirmed the device supports allogenic islet cell survival and 90-day diabetic reversal in rats. Finally, we demonstrate 1-month islet survival in a nonhuman primate without the need for immune suppression in the subcutaneous space. Collectively, these results indicate the device supports cell survival and function across multiple transplant models in three species without the need for any immunosuppression or external user intervention. These results represent an important set of advances towards immunosuppression free, minimally invasive islet transplantation.

Krishnan SR
Bochenek MA
Pan J
Pendyala S
Chan SN
Liu C
Prokop E
Hogrebe N
Rios PD
Lopez D
Potdar S
Gumustop D
Her BS
O'Keeffe L
Millman JR
Oberholzer J
Langer R
Anderson DG

pmc-id: PMC13196877;manuscript-id: NIHMS2161415;embargo-date: 2027/05/15;


Expansion Revealing of Pathology Resolves Nanostructures Associated with Inflammatory Phenotypes in COVID-19 Decedent Human Brain Tissue

Abstract: Expansion revealing (ExR) elucidates cellular organization by separating proteins within dense nanostructures by 20x linear expansion, but requires fixation procedures incompatible with human pathology specimens. Here, we report ExR of pathology (ExRPath), which attains ~20 nm resolution and decrowding of such tissues, through iterative 20x expansion, adapted to human brain pathology specimens. We also report a single-shot 15x expansion protocol for such tissues (15ExMPath), achieved through one-shot 15x expansion. Applying ExRPath and 15ExMPath to COVID-19-decedent brain tissue reveals periodic amyloid nanoclusters that co-localize with SARS-CoV-2 in a rare minority of patient specimens, pointing to a potential neuroinflammatory phenotype associated with COVID-19, and highlighting the power of high-throughput nanoimaging, empowered by expansion microscopy, for discovering potential novel disease mechanisms.

bioRxiv : the preprint server for biology

Stanton AE
Kang J
Blanchard JW
Boix CA
Schroeder ME
Lee Y
Su H
Wang S
Yu E
Emenari A
Peng Z
Agbas E
Cerit O
Park D
Zhang R
Bennett DA
Yin P
Kellis M
Langer R
Boyden E
Tsai LH


Immune-remodeling mRNAs expressing IRF8 or NIK generate durable antitumor immunity in multiple cancer models

Abstract: Although immunotherapy has benefited a subset of persons with cancer, its broader efficacy remains limited, primarily because of an immunosuppressive tumor microenvironment characterized by insufficient numbers of functional tumor-specific T cells, antigen-presenting cells (APCs) and tumor-infiltrating lymphocytes. Here we engineer immune cells in the tumor microenvironment using lipid nanoparticles (LNPs) to deliver immune-remodeling mRNAs (IR-mRNAs) encoding NF-κB-inducing kinase or interferon regulatory factor 8. These IR-mRNAs activate APCs in tumors, significantly increasing activated type 1 conventional dendritic cells, immunostimulatory cytokines and priming antitumor CD8+ T cells. IR-mRNAs encapsulated in LNPs elicited durable antitumor responses in multiple syngeneic mouse tumor models through both intratumoral and intravenous delivery. Coadministration of IR-mRNA and ovalbumin mRNA elicited a ~10-fold increase in antigen-specific CD8+ T cell responses, sustained long-term memory and effectively prevented tumor growth in vaccinated mice. Additionally, coadministration of IR-mRNA and hemagglutinin mRNA enhanced the humoral response ~5-fold and the cellular response ~15-fold, underscoring their potential as adjuvants for boosting adaptive immunity.

Gupta A
Das R
Reed K
Jeon T
Nguyen QTC
Rudra A
Ge X
Trongjit S
Vanrobaeys YS
Langer R
Weissleder R
Garris C
Anderson DG


A platform for near real-time and multiplexed monitoring of cerebrospinal fluid biomarkers and flow in neurocritical care

Abstract: Real-time monitoring of cerebrospinal fluid (CSF) is critical in intensive care units for the timely management of complications such as infection and mechanical malfunction in patients with external ventricular drainage systems. Current practice relies on intermittent CSF sampling for laboratory-based biomarker analysis and manual inspection, resulting in delayed reporting and intervention. To address these limitations, we developed NeuroSense, a multiplexed sensing platform that integrates with standard external ventricular drainage systems to enable near real-time monitoring of key CSF (bio)markers, including glucose, lactate, pH, and flow rate, that are essential for detecting infection and drain dysfunction. NeuroSense incorporates glucose and lactate aptamer-based electrochemical biosensors, a polydopamine pH sensor, and an impedance-based flow sensor. Validation in simulated conditions demonstrated sensor specificity, stability in human CSF for several days, and ethylene-oxide sterilization compatibility. Evaluation in patients hospitalized in intensive care unit demonstrated strong correlation with clinical reference standards. By providing near real-time bedside assessment, NeuroSense has the potential to improve temporal resolution for detection of biomarker trends and drain malfunction indicators.

Keyvani F
Muller LM
Fudge N
MacLean B
Saini A
Khatri J
Kriesen T
Wittstock M
Gessler FA
Langer R
Bernstock JD
Srinivasan S
Poudineh M

Humans
Biomarkers
Critical Care
Biosensing Techniques
Hydrogen-Ion Concentration
Monitoring, Physiologic
Lactic Acid
Time Factors


Microneedle array platforms for drug delivery and biomarker sensing: From skin mechanics guided design to scalable manufacture for clinical utility

Abstract: Microneedle (MN) devices provide a platform for development of products that can facilitate intra-and trans-dermal delivery and sampling of active pharmaceutical ingredient (APIs) and biomarkers that would otherwise be restricted by the skin barrier. Despite significant progress in the development of laboratory-based prototypes, no Microneedle Array Patch (MAP) drug delivery products have been approved for commercial use by the relevant national regulatory authorities. This discrepancy reflects the technical, commercial, and regulatory challenges to their clinical translation. This review presents some of the pertinent challenges facing MN development, discusses potential approaches to them, and provides a future outlook. MN design is discussed, including their intrinsically conflicting requirement of being slender enough for skin penetration and voluminous enough to accommodate clinically relevant doses of APIs. The anatomy and constitutive behavior of skin tissue, and the related insertion mechanics of MNs with distinct morphologies are also considered, enabling understanding of how MNs achieve the mechanical strength to withstand handling and use. The fabrication methods for these materials are compared, including their amenability to large-scale manufacturing based on factors such as high throughput, cost efficiency, and process capability. Biocompatibility and dissolution behaviors in the dermal compartment are also considered. MN applicator designs are summarized, with a focus on their different mechanisms of application and impact energies. Key elements of the regulatory science of MAPs are also discussed. This review highlights challenges in the development of MN-based drug delivery systems compatible with applicators for on-demand, self-applied patient-specific intra- and trans-dermal therapy.

Journal of controlled release : official journal of the Controlled Release Society

Zhou J
Wu L
Wyttenbach M
Lennon R
Liu Y
Christian M
Scherr S
Charara M
Kommalapati R
Blum J
Chatzilakou E
Schlueter A
Theobald F
Valenti L
Kosuda K
Jacobson GB
Kwon M
Ding S
Quinn C
Rosen C
Springall IC
Tran KTM
Tian J
Yetisen AK
Jaklenec A
Shalabi N
Li J
Kang Z
Farra R
Kendall M
Park JH
DeSimone J
Donnelly RF
Wang J
Mistilis JJ
Prausnitz MR
Birchall JC
Coulman SA
Hart AJ
Portela CM
Langer R
Francese G
Traverso G

Animals
Humans
Administration, Cutaneous
Biomarkers
Equipment Design
Microneedle Drug Delivery
Needles
Skin
Skin Absorption

Journal Article
Review
Research Support, Non-U.S. Gov't


Cryopreservation of stem cell-derived aggregates for type 1 diabetes cell therapy: Considerations and challenges

Abstract: Type 1 Diabetes (T1D) is a devastating disease in which the immune system attacks insulin- producing beta cells in the pancreas, disrupting the normal blood glucose regulation mechanism and resulting in the significant burden of ongoing blood glucose monitoring and management and longer-term damage to organs and tissues. An emerging therapy for T1D includes transplanting insulin-producing stem cell (SC)-derived aggregates into patients, restoring normal regulation of blood glucose and eliminating the need for insulin injections. To enable stable storage, distribution, and clinical administration of this therapeutic, reliable cryopreservation methods are required. However, current cryopreservation protocols result in low cell viability post thaw and have challenges in scalability. This review provides background on stem cell therapy for T1D and the production and storage pipeline of these SC-derived aggregates, with a focus on the challenges of cryopreservation. We review the fundamental physics involved in cryopreservation, including cryoprotective agents (CPAs), CPA loading and unloading, the importance of cooling and rewarming rate selection, and why the cell aggregate microstructure of islets presents a particularly difficult challenge for cryopreservation. Finally, we highlight important developments in SC-derived aggregate cryopreservation and the state of the art.

Barsukova AD
Weng L
Szymanska-Vandendriessche K
Scott M
Langer R
Traverso G

Humans
Cryopreservation
Diabetes Mellitus, Type 1
Animals
Cryoprotective Agents
Stem Cells
Stem Cell Transplantation
Insulin-Secreting Cells


Correction: Cell docking inside microwells within reversibly sealed microfluidic channels for fabricating multiphenotype cell arrays

Abstract: Correction for 'Cell docking inside microwells within reversibly sealed microfluidic channels for fabricating multiphenotype cell arrays' by Ali Khademhosseini et al., Lab Chip, 2005, 5, 1380-1386, https://doi.org/10.1039/B508096G.

Khademhosseini A
Yeh J
Eng G
Karp J
Kaji H
Borenstein J
Farokhzad OC
Langer R


Towards intelligent and miniaturized drug delivery devices

Abstract: Advances at the intersection of biotechnology, artificial intelligence, electronics and materials science are reshaping how drugs can be delivered inside the body. Intelligent and miniaturized drug delivery devices (IMDDDs) leverage these technologies to achieve precise pharmacokinetics, targeted distribution and programmable release while minimizing toxicity and improving patient adherence. Unlike conventional approaches, IMDDDs can incorporate real-time sensing and adaptive control, enabling drug administration that is more precise and more responsive to dynamic physiological conditions. In this Review, we outline key categories and design principles, highlight artificial intelligence technologies for augmenting performance, discuss potential clinical applications across cancer, diabetes, cardiovascular disease, vaccination and beyond, and examine translation challenges and opportunities. By uniting engineering innovation with medical need, IMDDDs exemplify the next generation of drug delivery technologies.

Wei X
Buse JB
Chen H
Desai TA
Stevens MM
Traverso G
Langer R
Gu Z

Humans
Drug Delivery Systems
Miniaturization
Artificial Intelligence
Animals
Neoplasms
Cardiovascular Diseases


Laser-Processed 2D Germanane on Graphene for Organohydrogel-Based Zinc-Ion Hybrid Capacitors

Abstract: Group 14 monoelemental two-dimensional (2D) materials beyond graphene, such as silicene and germanene, have gained significant attention in the scientific community. Covalent functionalization of germanene with hydrogen and methyl leads to germanane (hydrogen/methyl-terminated germanene; HGe/MGe). While the optical and electronic properties of HGe and MGe were explored previously, there is no report on their zinc ion storage electrochemistry. Though the layered HGe/MGe sheets have tunable interlayer spacing, which cushions the volume expansion during ion storage, their inferior electrical conductivity limits the charge transfer kinetics. Herein, we demonstrate a single-step, facile approach for in situ decoration of 2D HGe/MGe sheets over laser-induced graphene (LIG) using a pulsed laser and examine their morphological, chemical, and electrochemical (EC) characteristics. The HGe/MGe-decorated LIG is tested as a cathode for a zinc ion hybrid capacitor (ZHC) in an aqueous electrolyte and polyacrylamide organohydrogel to unveil the selective sites for zinc ion electrochemistry by experimental and theoretical aspects. This ZHC design enables a notable Zn2+ storage capacity (104 F g-1 @ 0.25 A g-1 in aqueous electrolyte) for HGe-decorated LIG, whereas MGe-decorated LIG records impressive cyclic stability (capacity retention 83% after 12000 cycles). Density functional theory calculations elucidate favorable adsorption of Zn at MGe and HGe networks. These findings summarize the applicability of 2D functionalized germanane, which has the potential to expand by numerous alkyl chains and terminal groups for targeted energy storage applications.

Deshmukh S
Sonigara KK
Langer R
Otyepka M
Pumera M


Chronic Pelvic Pain in Medical Education: A Needs Assessment of British Columbian Health Care Providers in Training

Abstract: Chronic pelvic pain (CPP) affects ∼15% of women and gender-diverse people, yet it is often dismissed by health care providers, partly due to a lack of related medical education. This brief communication describes the findings of a needs assessment survey for health care trainees. The findings suggest that current health care trainees felt inadequately prepared to manage CPP. Resources tailored to the diagnostic workup and treatment of CPP are needed in medical education.

Journal of obstetrics and gynaecology Canada : JOGC = Journal d'obstetrique et gynecologie du Canada : JOGC

Orr NL
Yang L
Howard AF
Langer R
Bruce B
Lisonek M
Phulka N
Hayward A
Currie L
Qayumi K
Carlyle M
Daudt H
Yong PJ

Humans
Pelvic Pain
Needs Assessment
Chronic Pain
Female
British Columbia
Health Personnel
Education, Medical
Clinical Competence


Lipid Nanoparticle Database towards structure-function modeling and data-driven design for nucleic acid delivery

Abstract: Lipid nanoparticles (LNPs) are the leading nonviral nucleic acid delivery technology, but LNP structure-function data remains fragmented and nonstandardized. Unlike protein engineering which is anchored by the centralized Protein Data Bank, the LNP field lacks a unified repository for systematic analysis. To address this, we develop Lipid Nanoparticle Database (LNPDB) ( https://lnpdb.molcube.com ), an integrated database and web tool that consolidates structural and functional data for 19,528 LNPs. LNPDB standardizes LNP featurization by encoding lipid composition, experimental methods, and functional results, and generates CHARMM force field files for constituent lipids to enable molecular dynamics simulations. LNPDB also supports future data contributions for continued growth. We examine the utility of LNPDB through two applications: advancing our deep learning model for predicting LNP delivery performance, and simulating bilayer dynamics to identify structural features - bilayer stability and critical packing parameter - that correlate with LNP delivery performance. Altogether, LNPDB provides the digital framework for LNP modeling and data-driven rational design.

Collins E
Ji J
Kim SG
Witten J
Kim S
Zhu R
Park P
Jung M
Park A
Manan RS
Rudra A
Keum G
Bang EK
Jin JO
Jeang WJ
Langer R
Anderson DG
Im W

Nanoparticles
Lipids
Nucleic Acids
Molecular Dynamics Simulation
Structure-Activity Relationship
Lipid Bilayers
Liposomes


Crystallized colony-stimulating factor-1 receptor inhibitor protects immunoisolated allo but not xeno transplants in primates

Abstract: Immunoisolation devices containing therapeutic protein-secreting cells offer potential for long-term therapy without immune suppression. However, scar tissue formation driven by the foreign body response (FBR) hinders nutritional exchange and ultimately leads to graft failure. We previously showed that inhibiting the colony-stimulating factor-1 receptor (CSF1R) pathway in monocytes and macrophages can block the FBR to implanted materials. Here, we demonstrate that coencapsulation of slow-releasing CSF1R inhibitor (GW2580) crystals with human stem cell-derived β cells (SC-β) in alginate spheres enables stable glycemic control for 1 year in immune-competent diabetic C57BL/6 mice. In nonhuman primates (NHPs), GW2580 crystals similarly protected viable, glucose-responsive allogeneic β cells for 1 month without systemic immune suppression. In contrast, the same xenogeneic human SC-β cell formulation that functioned long-term in mice elicited extensive sphere overgrowth and graft failure in NHPs. Serum cytokine profiling and transcriptomic analysis of omental biopsies at day 30 revealed pronounced adaptive immune activation in xenogeneic recipients, including enrichment of CD4+ T cells, CD19+ B cells, and antigen-presenting cell programs marked by elevated MHC class II expression. Chemokines CCL17, CCL22, and CXCL13 were among the most highly up-regulated transcripts, mirroring responses observed previously with profibrotic alginate formulations without cells. These findings underscore the issues associated with xenogeneic cell sources in higher-order species yet indicate that targeting innate immune pathways with localized CSF1R inhibition may be sufficient to enable function of encapsulated allogeneic cell therapies.

Bochenek MA
Farah S
Doloff JC
Han HJ
Sadraei A
Jeang WJ
Shaheen-Mualim M
Kutner N
Odeh E
Facklam A
DiNardo A
Engquist EN
Rios P
Isa D
Ghani S
Joshi I
Xing Y
Wang Y
Pop R
Greiner DL
Oberholzer J
Langer R
Anderson DG

Receptor, Macrophage Colony-Stimulating Factor

Receptors, Granulocyte-Macrophage Colony-Stimulating Factor


Hybrid microneedle patch for administration of measles and rubella vaccine with an on-patient medical record

Abstract: Decentralized on-patient medical record (OPMR) storage would enable medical staff to access patients' medical information on demand, especially in low-resource settings lacking centralized recordkeeping infrastructure. We incorporated near-infrared (NIR) microparticles into microneedle patches (MNPs) to invisibly encode medical information in the skin for OPMR using a specialized reader. NIR particles were co-encapsulated with inactivated polio vaccine in the same microneedles without loss of antigenicity. However, measles and rubella vaccines combined with NIR particles lost almost all potency. We therefore developed hybrid MNPs that spatially separated NIR particles in patterned microneedles in the central part of the patch and vaccines in separate microneedles around the periphery. This approach preserved vaccine potency. Ex vivo testing in pig skin demonstrated effective microneedle penetration and reliable OPMR signal readability. In vivo studies in rats and guinea pigs demonstrated OPMR signal retention for up to 21 months. Immunogenicity studies in cotton rats confirmed that neutralizing antibody titers were similar after measles vaccination by hybrid MNPs (containing NIR particles), conventional MNPs (without NIR particles) and subcutaneous injection. These findings suggest that hybrid MNPs enable co-administration of vaccines and NIR particles for OPMR or co-delivery of other materials requiring sequestration.

Sadeqi A
Lee JW
Gomaa Y
Nguyen TC
Azizoglu E
Hasin T
Ryu YJ
Dixon JB
Kanelli M
Han J
Langer R
Jaklenec A
Prausnitz MR

Animals
Microneedle Drug Delivery
Measles Vaccine
Guinea Pigs
Rubella Vaccine
Female
Antibodies, Neutralizing
Needles
Humans
Rats
Antibodies, Viral
Administration, Cutaneous

Journal Article
Research Support, Non-U.S. Gov't
Research Support, N.I.H., Extramural
Research Support, U.S. Gov't, P.H.S.


Endoscopic Submucosal Dissection (ESD) of Upper Gastrointestinal Carcinomas: An Integrated Clinical and Pathological Perspective

Abstract: Endoscopic submucosal dissection (ESD) has revolutionized the management of early upper gastrointestinal (GI) carcinomas. While technically demanding, it offers, in experienced hands, definitive local therapy for early GI neoplasia by allowing complete En bloc resection of mucosal and superficially invasive neoplasms, thus enabling precise histopathological risk stratification and organ preservation. Appropriate patient selection relies on meticulous endoscopic assessment using high-definition and image-enhanced endoscopy to define lesion boundaries and predict invasion depth. The principal indications include high-grade intraepithelial neoplasia and early carcinomas without endoscopic evidence of deep submucosal invasion or lymph node metastasis risk factors. Pathological analysis of the resection specimens includes histological typing and grading per WHO classification and precise assessment of invasion depth-in case of submucosal invasion measurement in micrometers-and evaluation of margin status and lymphovascular invasion. The presence of risk factors such as deep invasion in the submucosa, poor differentiation, or lymphovascular invasion may require additional surgery, guided by validated risk scores such as the eCura system. This narrative review summarizes current clinical and pathological practices for ESD in upper GI lesions. This includes the discussion of technical and biological challenges and the need of accurate assessment of risk factors for systemic metastatic spread and local recurrence as a limitation for this sophisticated but highly effective therapeutic method.

Ziachehabi A
Worm M
Liu DHW
Pimingstorfer P
Langer R


[Biomarker testing for carcinomas of the upper gastrointestinal tract]

Abstract: Predictive biomarker testing is nowadays an integral part of modern diagnostics and treatment planning for esophageal and gastric cancer. It currently consists mainly of immunohistochemically determined markers, namely the PD-L1 status (stated by the Tumor Proportion Score [TPS], Combined Positivity Score [CPS] and Tumor Area Proportion [TAP] Score) in esophageal squamous cell carcinomas, as well as the HER2 status (supplemented by in situ hybridization in case of equivocal results), the mismatch repair status (optionally supplemented by molecular pathological determination of microsatellite status), and, since mid-2024, the expression of claudin 18.2 in gastroesophageal adenocarcinomas. An expansion of the panel by FGFR2b is expected in the near future. In addition to the quality guideline-compliant technical performance of the tests, the biological heterogeneity of many of these biomarkers is also a diagnostic and clinical challenge. The current article provides an overview of current biomarker testing in the context of current treatment options for cancer of the upper gastrointestinal tract.

Humans
Biomarkers, Tumor
Esophageal Neoplasms
Stomach Neoplasms
Adenocarcinoma
B7-H1 Antigen
Erb-b2 Receptor Tyrosine Kinases
DNA Mismatch Repair
Carcinoma, Squamous Cell
Gastrointestinal Neoplasms
Immunohistochemistry


Diversity of axon initial segment geometry in the mouse hippocampus and its predicted influence on neuronal excitability

Abstract: Action potentials, the primary information units of the nervous system, are usually generated at the axon initial segment. Changes in the length and position of the axon initial segment are associated with alterations in neuronal excitability, but there is only limited information about the baseline structural variability of this compartment. This work provides a comprehensive analysis of the diversity of proximal cell geometries across all anatomical axes of the murine hippocampus, encompassing dorsal-ventral, superficial-deep, and proximal-distal regions. We analyzed the morphology of 3,681 hippocampal pyramidal neurons in 12 animals of both sexes, focusing on axon initial segment length, position, and association with proximal cellular features such as the soma and apical dendrite. Notably, neurons with axon-carrying dendrites were significantly more common in ventral compared to dorsal hippocampal areas, which we also found in two of three human samples. We employed NEURON simulations to assess the functional implications of this variability. Here, variation in proximal geometry contributed only minimally to neuronal homeostasis, but instead increased heterogeneity of response patterns across neurons.

Stevens NA
Achilles M
Monath J
Langer R
Engelhardt M
Both M
Thome C

Animals
Male
Hippocampus
Axon Initial Segment
Female
Pyramidal Cells
Mice
Mice, Inbred C57BL
Action Potentials
Axons
Dendrites
Humans


Design of CellProfiler-Based Pipelines Enabling the Attribution of Molecular Stress Markers to Specific Tissue and Subcellular Compartments of the Colonic Mucosa

Abstract: Stress granules (SGs) represent membrane-free cytoplasmic structures rapidly aggregating during cellular stress responses arguably useful as markers of molecular inflammation. To provide an automated, reproducible, and unbiased analytic workflow, we used the open-source software CellProfiler to quantify SGs in distinct cell types in inflammatory bowel disease. The EpiCellProfiler (ECP) and PropiCellProfiler (PCP) pipelines enable segmentation within intestinal epithelial cells and lamina propria cells, respectively. The SG marker Ras GTPase-activating protein-binding protein 1 (G3BP1) was quantified for fluorescence intensity, granule size, and morphology on tissue sections of patients with ulcerative colitis (UC) and Crohn's disease (CD) in deep remission. Both pipelines detected elevated G3BP1 fluorescence intensities in inactive UC and CD. Additionally, SGs spot counts and spot sizes were increased in CD and UC compared with controls. The distribution of G3BP1 was homogenous in intestinal epithelial cells, without SG typical aggregations. In UC, PCP analysis revealed nuclear morphology alterations in terms of size, regularity, and compactness. Herein, we provide a powerful, reproducible, versatile and open-source software tool to quantify remnant molecular inflammation in patients with CD and UC, enabling research to openly share, reproduce and compare results within the field of quantitative image analysis. Our pipeline separates and distinguishes between epithelial and lamina propria events and provides insights into the spatial distribution and dynamics of SGs, revealing their homogeneous distribution and persistent accumulation in patients with CD and UC, notably in such without clinical, endoscopic, biochemical and histological disease activity. The sensitivity of the pipelines allows detection of subtle morphologic alterations that warrant further investigation, as does the usage of G3BP1 as an inflammatory bowel disease stress marker.

Cellular and molecular gastroenterology and hepatology

Hödlmayr H
Watschinger C
Wallner GK
Knipp S
Rohwedder A
Prommer R
Langer R
Moschen AR

Humans
Intestinal Mucosa
RNA Recognition Motif Proteins
Colitis, Ulcerative
Biomarkers
RNA Helicases
Colon
Software
Stress Granules
Crohn Disease
DNA Helicases
Male
Epithelial Cells
Female
Stress, Physiological
Adult
Poly-ADP-Ribose Binding Proteins


The critical role of food fortification in combatting malnutrition and disease susceptibility in Africa

Abstract: Malnutrition can severely compromise immune function, rendering populations more vulnerable to both communicable and non-communicable diseases. Here, we discuss food fortification and micronutrient supplementation programs that have been instrumental in mitigating malnutrition across Africa and in controlling disease spread, marking considerable advances in public health throughout the continent.

Yang X
Zhang L
MacDonald S
Langer R
Jaklenec A


Characterisation and impact of intratumoural stroma in melanoma and carcinoma brain metastases

Abstract: Research on the tumour microenvironment in brain metastases (BM) has predominantly focused on the immune response, while the presence, morphological patterns, and potential clinical relevance of intratumoural stroma remain less intensively investigated. We retrospectively analysed 604 BM (529 carcinomas, 75 melanomas) from 556 patients. Intratumoural stroma was histomorphologically classified into absent/unclear (Group 0), present without desmoplasia (Group 1) or with desmoplasia (Group 2). Associations with histological features, clinical parameters, and survival were evaluated. Intratumoural stroma was absent in 63.2% of tumours (n = 382), was present without desmoplasia in 14.9% (n = 90), and was desmoplastic in 21.9% (n = 132). Desmoplasia was most frequent in metastases from breast carcinomas and pulmonary squamous cell carcinomas. No significant associations were found between stroma groups and age, sex, brain location, PD-L1 expression, oncogenic mutations in lung carcinoma, or breast-cancer molecular subtype. Independent predictors of poorer survival were increasing age (HR = 1.029, 95% CI: 1.018-1.039, p < 0.001), male sex (HR = 1.492, 95% CI: 1.204-1.849, p < 0.001), and infratentorial location (HR = 1.402, 95% CI: 1.125-1.748, p = 0.003). Stroma groups showed no independent prognostic value in the overall cohort. However, subgroup analysis of non-small cell lung cancer revealed a U-shaped relationship, with Group 1 stroma linked to better survival (p = 0.020). In summary, intratumoural stroma in BM is a carcinoma-specific phenomenon, absent in melanoma. Patient outcomes, however, were primarily determined by demographic and anatomical factors rather than stromal morphology in the overall cohort but may have clinical relevance in particular tumour subgroups.

The journal of pathology. Clinical research

Bandke D
Weis S
Gruber A
Noack P
Kalev O
Ornig K
Langer R

Humans
Female
Male
Brain Neoplasms
Middle Aged
Melanoma
Retrospective Studies
Aged
Stromal Cells
Tumor Microenvironment
Adult
Aged, 80 and over
Skin Neoplasms
Lung Neoplasms
Carcinoma

Journal Article
Research Support, Non-U.S. Gov't


Rare non-neoplastic gastrointestinal diseases-drugs and bugs

Abstract: Rare diseases of the gastrointestinal (GI) tract present a significant diagnostic challenge. This paper focusses on two particular groups of conditions: drug-induced injuries and parasitic infections. With aging populations and increased use of complex pharmacologic regimens-including immunosuppressants and biologics-drug-related GI pathology is nowadays more frequently encountered. However, the histologic changes are often non-specific and overlap with more common conditions such as inflammatory bowel disease (IBD), ischemia, autoimmune diseases, or infections. Key patterns include lymphocytic or neutrophilic inflammation, crypt apoptosis, crypt abscesses, and architectural distortion. Certain agents, such as mycophenolate, olmesartan, immune checkpoint inhibitors, or ion exchange resins (e.g. sodium polystyrene sulfonate/kayexalate, sevelamer), have distinct but subtle histopathologic signatures. Parasitic infections, although less frequent in high-income countries, remain relevant due to global travel and migration. Organisms such as Schistosoma, Strongyloides, or Giardia can mimic IBD, neoplasia, or cause unexpected eosinophilic or granulomatous inflammation. Parasite ova may require special stains and careful morphologic assessment to be identified. Importantly, some helminths have been associated with chronic complications including cancer or fibrosis, thus underscoring the need for accurate recognition. For the practicing pathologist, these rare but impactful conditions demand a high index of suspicion, especially in cases with atypical histology or poor clinical correlation. Using some illustrative cases, this paper highlights diagnostic strategies and key morphologic features to improve recognition and avoid misdiagnosis of these underappreciated entities.

Humans
Gastrointestinal Diseases
Rare Diseases
Animals
Diagnosis, Differential
Parasitic Diseases


Delivery of macromolecular drugs: An update

Abstract: In 2019, in this journal, I discussed approaches for controlling the movement of molecules, in particular macromolecules, with an emphasis on how this enabled advances in the field of drug delivery - a field that has impacted billions of people worldwide. Since 2019, there have been advances in our work and this field including a striking demonstration in which drug delivery nanoparticles were crucial to the success of mRNA therapies and the Covid-19 vaccine. In this paper, I provide updates in such areas as i) developing new methods for oral drug delivery systems, ii) delivery of molecules to specific sites of the body, iii) new types of delivery systems, and iv) examples of machine learning/artificial intelligence in these areas. I also discuss advances in mRNA technology as it relates to drug delivery and the development of nanoparticles to protect and deliver vaccines, which saved and improved the lives of hundreds of millions of people throughout the world.

Humans
Drug Delivery Systems
Macromolecular Substances
Nanoparticles
COVID-19 Vaccines
COVID-19
Animals
SARS-CoV-2
Machine Learning

Journal Article
Review
Research Support, N.I.H., Extramural
Research Support, Non-U.S. Gov't


Wafer-Scale Laser-Writing of Nanoporous Membranes with Monodisperse Pores for Robust Immunoisolation

Abstract: Membranes that prevent cellular infiltration are essential components of immune-isolation devices. Stringent pore size control is critical to robustly exclude immune cells while preserving the efficient transport of nutrients and therapeutic molecules. Conventional membrane fabrication methods, however, struggle to balance these two properties. For instance, phase separation and fiber spinning generate highly tortuous pores that restrict diffusion, while random pore overlaps due to ion track etching result in oversized defects that compromise immune isolation. Here, we leverage scalable laser-writing tools to achieve spatially controlled open-through and dense pores with uniformly distributed submicrometer sizes for rapid transport and robust immune cell exclusion. Given the optical limits of laser aligners when fabricating submicrometer features, we tuned substrate reflectivity and exposure parameters to overcome these resolution limits. Self-standing and flexible membranes were achieved via thickness-tunable grid layers supporting membrane mechanical integrity. We report pore sizes as small as ca. 600 nm (below the resolution limits of the laser exposure systems) and over 20% open area at the exposure times of ∼3 min/cm2. Compared with conventional methods, this approach minimizes pore tortuosity, narrows the pore distribution, and improves the open pore density. Enhanced pore control is highlighted by macrophage infiltration studies, which indicate that these membranes reliably exclude macrophages at nominal pore sizes greater than those previously achievable. This approach thus provides a scalable pathway toward next-generation immunoisolation membranes for enhanced implantable therapeutic devices.

Montazerian H
Jeang WJ
Zhao Y
Manan RS
Bochenek MA
Krishnan SR
Langer R
Anderson DG

Lasers
Nanopores
Porosity
Particle Size
Animals
Mice
Membranes, Artificial
Surface Properties
Macrophages
Humans


Degradable cyclic amino alcohol ionizable lipids as vectors for potent influenza mRNA vaccines

Abstract: The next generation of mRNA vaccines must address several limitations, including enhancing vaccine potency and reducing toxicity. Here we develop a class of degradable, cyclic amino ionizable lipids via sequential combinatorial chemistry and rational design. Lipid nanoparticles (LNPs) formulated with the top-performing ionizable lipid, AMG1541, elicited similar protective neutralization antibody titres against an H3 influenza antigen when compared with the FDA-approved ionizable lipid SM-102 at a 100-fold lower dose, with enhanced clearance in vivo. AMG1541 mRNA LNPs substantially reduced expression in the liver following intramuscular injection, mitigating the associated toxicity. We also observed improved mRNA delivery to antigen-presenting cells at the injection site and the draining lymph node, leading to stronger germinal centre reactions. Structure-activity relationship studies suggest that cyclic headgroups and β-amino alcohols facilitate interactions with the mRNA backbone and enhance endosomal escape. The formulations developed here significantly enhance the potency of mRNA vaccines, and our structural insights may guide the development of next-generation vaccine delivery systems.

Rudra A
Gupta A
Reed K
Deik A
Min J
Mansour HMA
Nguyen QTC
Danko A
Hu Y
Berger A
Prado M
Beck A
Clish CB
Klauda JB
Langer R
Anderson DG

Influenza Vaccines
Lipids
Animals
RNA, Messenger
Nanoparticles
Mice
Amino Alcohols
mRNA Vaccines
Female
Humans
Mice, Inbred BALB C
Liposomes


Regioselective Multiboration and Hydroboration of Alkenes and Alkynes Enabled by a Platinum Single-Atom Catalyst

Abstract: Selective multiboration including di- and triboration and hydroboration of alkynes and alkenes face significant challenges in organic synthesis, including achieving high regioselectivity, functional group tolerance, and catalyst stability while requiring mild conditions to maintain reactivity. These transformations have been predominantly explored by using homogeneous catalysts. In this study, we report the scalable synthesis of heterogeneous platinum single-atom catalyst (Pt-SAC) supported on ultrathin nanosheets of graphitic carbon nitride via a rapid microwave-assisted method. The Pt-SAC enables 1,2-diboration of sterically hindered alkenes and 1,2,2-triboration of alkynes with B2pin2 under mild conditions. For the diboration of styrene, the catalyst achieves 99% yield with 95% selectivity, a turnover number (TON) of 3711, and a turnover frequency (TOF) of 247 h-1. The catalyst also promotes the regioselective hydroboration of alkenes and alkynes, yielding anti-Markovnikov alkylboranes and vinylboranes, respectively. Computational calculations reveal that the enhanced reactivity on the Pt-SAC catalyst arises from adsorption-induced weakening of key bonds (C=C and B-H), thereby significantly lowering the activation energy barriers. The Pt-SAC exhibits stability and recyclability, maintaining performance over at least eight consecutive runs without detectable Pt leaching. This study highlights the potential of Pt-SAC as a robust and versatile platform for organoboron transformations under mild conditions, with relevance to applications in pharmaceutical, agrochemical, and polymer synthesis.

Huninik P
Sharma P
Saptal VB
Slaby M
Langer R
Kumar P
Shayesteh Zeraati A
Wang X
Petr M
Otyepka M
Gawande MB
Zbořil R
Kment S
Walkowiak J


Enhanced macromolecule bioavailability in rats and pigs using an in situ forming synthetic epithelial lining

Abstract: Oral delivery of macromolecules is hindered by enzymatic degradation, poor epithelial permeability, and rapid gastric transit, leading to low bioavailability. Existing permeation enhancers (PEs), such as salcaprozate sodium and sodium caprate, improve absorption but do not fully address proteolytic degradation and require high doses due in part to short gastrointestinal residence times. We developed the Peroral Mucosal Epithelium Absorption Enhancer (PERMEATE) system, an orally administered polymer film designed to adhere to the small intestinal mucosa, maximizing contact between therapeutics, PEs, and the absorptive tissue. Utilizing Synthetic Tissue-Lining (SYNT™) technology, PERMEATE triggers endogenous catalase-dependent dopamine polymerization to form an in situ polydopamine coating, creating a temporary depot that enhances co-localization and prolongs exposure to the absorptive mucosa. We assessed PERMEATE's potential to enhance the oral bioavailability of semaglutide (SEMA). High-throughput screening using the GI tissue robotic interface system (GI-ORIS) identified glycocholic acid (GCA) and ammonium carbonate (NHCO) as effective PEs when combined with SYNT. Ex vivo studies (n = 8-24) and in vivo tests in Sprague-Dawley rats (n = 5-11/group) demonstrated a 200-fold increase in bioavailability compared to SEMA alone (P = 0.0001) and a 6-fold increase relative to SEMA+PE without SYNT (P = 0.0011). In Yorkshire pigs (n = 3-4), PERMEATE achieved a 2.4 % absolute bioavailability, a 6-fold improvement over SEMA+PE controls (P = 0.0316). These results suggest PERMEATE as a promising platform for improving oral macromolecule delivery through enhanced mucosal adhesion and prolonged therapeutic contact, supporting further development for clinical application.

Kanelli M
Yu AC
Susilo D
Petropulos O
Kadasia K
Sandoval L
Hudson MA
Sim D
Dial C
Mena MB
Liang J
Rivera-Delgado E
Sepich L
Hollfelder K
Wright J
von Erlach T
Hayward A
Gaspie KA
Barron SM
Pombo M
Basani RR
Lanchantin M
Lopes A
Pizzo S
Sethuraman V
Dhanda RK
Langer R
Traverso G

Animals
Biological Availability
Rats, Sprague-Dawley
Male
Swine
Intestinal Mucosa
Administration, Oral
Rats
Polymers
Drug Delivery Systems
Intestinal Absorption
Indoles


Engineered 3D immuno-glial-neurovascular human miBrain model

Abstract: Patient-specific, human-based cellular models integrating a biomimetic blood-brain barrier, immune, and myelinated neuron components are critically needed to enable accelerated, translationally relevant discovery of neurological disease mechanisms and interventions. To construct a human cell-based model that includes these features and all six major brain cell types needed to mimic disease and dissect pathological mechanisms, we have constructed, characterized, and utilized a multicellular integrated brain (miBrain) immuno-glial-neurovascular model by engineering a brain-inspired 3D hydrogel and identifying conditions to coculture these six brain cell types, all differentiated from patient induced pluripotent stem cells. miBrains recapitulate in vivo-like hallmarks inclusive of neuronal activity, functional connectivity, barrier function, myelin-producing oligodendrocyte engagement with neurons, multicellular interactions, and transcriptomic profiles. We implemented the model to study Alzheimer's Disease pathologies associated with APOE4 genetic risk. APOE4 miBrains differentially exhibit amyloid aggregation, tau phosphorylation, and astrocytic glial fibrillary acidic protein. Unlike the coemergent fate specification of glia and neurons in other organoid approaches, miBrains integrate independently differentiated cell types, a feature we harnessed to identify that APOE4 in astrocytes promotes neuronal tau pathogenesis and dysregulation through crosstalk with microglia.

Proceedings of the National Academy of Sciences of the United States of America

Stanton AE
Bubnys A
Agbas E
James B
Park DS
Jiang A
Pinals RL
Liu L
Truong N
Loon A
Staab C
Cerit O
Wen HL
Mankus D
Bisher ME
Lytton-Jean AKR
Kellis M
Blanchard JW
Langer R
Tsai LH

Humans
Neuroglia
Induced Pluripotent Stem Cells
Neurons
Coculture Techniques
Brain
Blood-Brain Barrier
Alzheimer Disease
Cell Differentiation
Astrocytes
Models, Biological
Oligodendroglia


[Combined gastric and respiratory heterotopia in the rectum suggesting malignancy]

Abstract: Heterotopia refers to the presence of normal cells or tissue located in anatomically unusual sites of the body. In the gastrointestinal tract, most heterotopic lesions do not cause any symptoms, but rarely, they can be mistaken for neoplasms during endoscopic examinations. A 45-year-old man presented with lower abdominal pain and blood in the stool. Macroscopically, a polypoid lesion was found and resected by endoscopic submucosal dissection. Histologically, ectopic gastric mucosa, salivary gland-like structures compatible with bronchial glands, and respiratory epithelium were found. This combination of a gastric and respiratory ectopia has so far been rarely documented in the literature.

Humans
Male
Choristoma
Middle Aged
Gastric Mucosa
Rectal Diseases
Diagnosis, Differential
Respiratory Mucosa
Salivary Glands
Rectal Neoplasms
Rectum

Journal Article
Case Reports
English Abstract


Inflammatory reprogramming of human brain endothelial cells compromises blood-brain barrier integrity in Alzheimer's disease

Abstract: Blood-brain barrier (BBB) dysfunction is an early feature of Alzheimer's disease (AD), yet the endothelial gene-regulatory programs involved remain incompletely understood. We integrate postmortem human single-nucleus transcriptomics with iPSC-based BBB models to define a conserved, inflammation-driven pathway that compromises barrier integrity. We identify an NF-κB-associated endothelial gene module endoM2 that is elevated in AD, inversely correlated with cognition, and enriched for inflammation and endothelial-to-mesenchymal transition signatures. Cytokine stimulation of iPSC-derived brain endothelial cells induces morphological remodeling, lipid accumulation, junctional disruption, and transcriptomic shifts that mirror endoM2. A targeted drug screen identifies the NF-κB inhibitor BAY11-7082 as protective against cytokine-induced changes. In our perfusable iPSC-derived BBB-Chip that recapitulates human BBB signatures, single-cell profiling reveals inflammatory endothelial state-specific programs reflecting those in AD brains and demonstrates that BAY11-7082 suppresses cytokine-triggered dysfunction and reverses inflammation-associated gene activation. Together, these findings position cerebrovascular inflammation as a therapeutic target to preserve BBB integrity in AD.

Pinals RL
Islam MR
King O
Choi A
Kang E
Nakano M
Tuyéras A
Naomi MT
Ngo A
Jiang A
Truong N
Agbas E
Lozano Cruz CF
Staab C
Ko T
Bennett DA
Stanton AE
Langer R
Tsai LH


Benchmarking foundation models as feature extractors for weakly supervised computational pathology

Abstract: Numerous pathology foundation models have been developed to extract clinically relevant information. There is currently limited literature independently evaluating these foundation models on external cohorts and clinically relevant tasks to uncover adjustments for future improvements. Here we benchmark 19 histopathology foundation models on 13 patient cohorts with 6,818 patients and 9,528 slides from lung, colorectal, gastric and breast cancers. The models were evaluated on weakly supervised tasks related to biomarkers, morphological properties and prognostic outcomes. We show that a vision-language foundation model, CONCH, yielded the highest overall performance when compared with vision-only foundation models, with Virchow2 as close second, although its superior performance was less pronounced in low-data scenarios and low-prevalence tasks. The experiments reveal that foundation models trained on distinct cohorts learn complementary features to predict the same label, and can be fused to outperform the current state of the art. An ensemble combining CONCH and Virchow2 predictions outperformed individual models in 55% of tasks, leveraging their complementary strengths in classification scenarios. Moreover, our findings suggest that data diversity outweighs data volume for foundation models.

Neidlinger P
El Nahhas OSM
Muti HS
Lenz T
Hoffmeister M
Brenner H
van Treeck M
Langer R
Dislich B
Behrens HM
Röcken C
Foersch S
Truhn D
Marra A
Saldanha OL
Kather JN

Humans
Benchmarking
Neoplasms
Computational Biology
Algorithms
Female
Prognosis


Self-driving labs for biotechnology


Vascular-Perfusable Human 3D Brain-on-Chip

Abstract: Development and delivery of treatments for neurological diseases are limited by the tight and selective human blood-brain barrier (BBB). Although animal models have been important research and preclinical tools, the rodent BBB exhibits species differences and fails to capture the complexity of human genetics. Microphysiological systems incorporating human-derived cells hold great potential for modeling disease and therapeutic development, with advantages in screening throughput, real-time monitoring, and tunable genetic backgrounds when combined with induced pluripotent stem cell (iPSC) technology. Existing 3D BBB-on-chip systems have incorporated iPSC-derived endothelial cells but not the other major brain cell types from iPSCs, each of which contributes to brain physiology and disease. Here we developed a 3D Brain-Chip system incorporating endothelial cells, pericytes, astrocytes, neurons, microglia, and oligodendroglia from iPSCs. To enable this multicellular 3D co-culture in-chip, we designed a GelChip microfluidic platform using a 3D printing-based approach and dextran-based engineered hydrogel. Leveraging this platform, we co-cultured and characterized iPSC-derived brain-on-chips and modeled the brain microvasculature of APOE4 , the strongest known genetic risk factor for sporadic Alzheimer's disease. These 3D brain-on-chips provide a versatile system to assess BBB vascular morphology and function, investigate downstream neurological effects in disease, and screen therapeutics to optimize delivery to the brain. The blood-brain barrier (BBB) is both a contributing factor to neurological disease and a major obstacle to its treatment, yet human-relevant models remain limited. Most existing brain-on-chip systems incorporate only subsets of BBB cell types and cannot capture the full cellular complexity of the human neurovascular unit. Here, we establish a vascular-perfusable 3D Brain-Chip using human induced pluripotent stem cell-derived brain cells including endothelial cells, pericytes, astrocytes, neurons, microglia, and oligodendroglia. This system enables systematic analysis of human genetic risk factors, such as APOE4 in Alzheimer's disease, and provides a powerful platform to investigate BBB function and dysfunction and accelerate the development of more effective neurological therapies.

Stanton AE
Pinals RL
Choi A
Truong N
Kang E
Jiang A
Lozano Cruz CF
Hawkins S
Sarcar R
Volkova A
King O
Agbas E
Nakano M
Chiu CC
Bubnys A
Wright S
Staab C
Bikdash R
Forden E
Langer R
Tsai LH


Engineered prime editors with minimal genomic errors

Abstract: Prime editors make programmed genome modifications by writing new sequences into extensions of nicked DNA 3' ends1. These edited 3' new strands must displace competing 5' strands to install edits, yet a bias towards retaining the competing 5' strands hinders efficiency and can cause indel errors2. Here we discover that nicked end degradation, consistent with competing 5' strand destabilization, can be promoted by Cas9-nickase mutations that relax nick positioning. We exploit this mechanism to engineer efficient prime editors with strikingly low indel errors. Combining this error-suppressing strategy with the latest efficiency-boosting architecture, we design a next-generation prime editor (vPE). Compared with previous editors, vPE features comparable efficiency yet up to 60-fold lower indel errors, enabling edit:indel ratios as high as 543:1.

Gene Editing
INDEL Mutation
CRISPR-Associated Protein 9
CRISPR-Cas Systems
Deoxyribonuclease I
Humans
Genome
DNA
Genomics

Research Support, U.S. Gov't, Non-P.H.S.


Mining the CD4 antigen repertoire for next-generation tuberculosis vaccines

Abstract: Tuberculosis (TB) is the leading cause of death from infectious disease worldwide, and Bacillus Calmette-Guérin (BCG) remains the only clinically approved vaccine. An enduring challenge in TB vaccine development is systematic antigen selection from a large repertoire of potential candidates. We performed an efficacy screen in mice of antigens that are targets of CD4 T cells in humans. We found striking heterogeneity in protective efficacy, and most of the top protective antigens are not currently in clinical development. We observed immunologic cross-reactivity among phylogenetically clustered antigens, reflecting common CD4 epitopes. We developed a trivalent mRNA vaccine consisting of PPE20 (Rv1387), EsxG (Rv0287), and PE18 (Rv1788), which augmented and exceeded BCG protection in multiple mouse models. Finally, we observed cellular immune responses to these antigens in 84% of humans exposed to M. tuberculosis. These data advance our understanding of TB vaccine immunology and define a vaccine concept for clinical development.

Vidal SJ
Lasrado N
Tostanoski LH
Chaudhari J
Mbiwan ER
Neka GD
Strutton EA
Espinosa Perez AA
Sellers D
Barrett J
Lifton M
Wakabayashi S
Eshaghi B
Borducchi EN
Aid M
Li W
Scriba TJ
Jaklenec A
Langer R
Barouch DH

Tuberculosis Vaccines
Animals
Humans
Antigens, Bacterial
Mice
Mycobacterium tuberculosis
Tuberculosis
CD4-Positive T-Lymphocytes
Female
Mice, Inbred C57BL
Cross Reactions
BCG Vaccine
Vaccine Development


Corrigendum to "Poly(ethylene oxide)-modified poly(β-amino Ester) nanoparticles as a pH-sensitive biodegradable system for paclitaxel delivery" [Journal of Controlled Release, 86 (2003) 223-234]


Monomer-Dependent Selectivity in Sulfur-Containing Ring-Opening Copolymerisation: Bimetallic Catalysis for Predictive Design of Degradable Polymers

Abstract: Sulfur-containing polymers offer unique opportunities for advanced materials due to their inherent degradability, high refractive indices, and potential for chemical recyclability. Yet, synthetic strategies for their precise construction remain underdeveloped and monomer selection rules to achieve this are elusive. Herein, we achieve the first comprehensive evaluation of sulfurated ring-opening copolymerisations (ROCOP) performed under unified conditions using a single heterobimetallic Cr(III)/Rb(I) catalyst platform. Although such catalysts are largely unexplored in sulfur-based ROCOP, our study demonstrates their remarkable synergistic performance in controlling both activity and selectivity across diverse heteroallene/epoxide systems. We identify carbonyl sulfide (COS) as the most promising monomer, enabling perfectly alternating copolymers under mild conditions on kinetic grounds. As a close second, PhNCS emerges as a viable, easier to handle, alternative that offers selective access to sulfur-containing copolymers. In contrast, thioanhydrides and CS2 show progressively lower selectivity, with increasing O/S scrambling and small molecule byproduct formation. This study provides the first predictive framework linking sulfur monomer identity to selectivity and reactivity under unified conditions, enabling rational design of degradable sulfur-rich polymers.

Angewandte Chemie (International ed. in English)

Manjunatha BR
Sengoden M
Stühler MR
Langer R
Darensbourg DJ
Plajer AJ


Survival outcomes after systemic treatment of high-grade triple-negative metaplastic breast cancer versus triple-negative breast cancer of no special type

Abstract: This study aimed to evaluate the prognostic impact of adjuvant and neoadjuvant chemotherapy (ACT, NACT) in high-grade triple-negative metaplastic breast cancer (TNMBC) and to compare survival outcomes with those of triple-negative breast cancer of no special type (TNBC NST). A total of 73 patients with high-grade TNMBC and 369 patients with TNBC NST were included in the study. In the non-NACT and ACT subgroups, TNMBC patients exhibited significantly worse overall survival (OS), distant disease-free survival (DDFS), breast cancer-specific survival (BCSS), and disease-free survival (DFS) than TNBC NST patients (non-NACT: p < 0.001 for all endpoints; ACT: OS, p < 0.001; DDFS, p < 0.001; BCSS, p = 0.004; DFS, p < 0.001). In the NACT subgroup, TNMBC and TNBC NST patients had similar survival outcomes. Within the TNMBC cohort, patients treated with NACT without achieving a pathological complete response (pCR) demonstrated improved OS (p = 0.045) and a trend toward improved BCSS (p = 0.056) compared to TNMBC patients who did not receive CT. No significant survival difference was observed between TNMBC patients treated with ACT and those without CT, nor between NACT-treated TNMBC patients without a pCR and those treated with ACT. We conclude that survival outcomes of TNMBC vs. TNBC NST patients may be influenced by systemic treatment. NACT-treated TNMBC patients without a pCR demonstrated superior OS compared to TNMBC patients receiving no CT, while no survival difference was observed among TNMBC patients based on treatment sequencing (ACT vs. NACT).

Virchows Archiv : an international journal of pathology

Grosse C
Grosse A
Schwarz HK
Frauchiger-Heuer H
Rordorf T
Ring A
Langer R
Varga Z


Arthroscopic Transosseous Rotator Cuff Repair Using Bone Tunneling Device

Abstract: Rotator cuff injuries are a common cause of shoulder pain, primarily affecting individuals older than 40 years, with the incidence increasing progressively with age. Advancements in surgical techniques have led to the development of arthroscopic techniques. This evolution has transitioned through various arthroscopic suture anchor rotator cuff repair configurations, including single-row, double-row, and transosseous-equivalent constructs. Although these innovations have replicated the biomechanics of the original transosseous technique conducted using an open approach, they often require multiple anchors, significantly increasing surgical costs. Additionally, anchor-related complications persist. To enhance the feasibility and affordability of implant-free transosseous repair, a reusable instrumentation system, was developed by Drillbone (Brno, Czechia). This innovative device facilitates robust and reproducible transosseous rotator cuff repairs while substantially reducing procedural costs. We present a surgical technique using bone tunneling device, showing its application in arthroscopic rotator cuff repair.

Hudeček F
Apostolopoulos V
Kužma J
Liskay J
Langer R
Tomáš T


T-cell immunity in the experimental autoimmune vasculitis rat model

Abstract: ANCA-vasculitis (AAV) is a small-vessel vasculitis characterized by the presence of autoantibodies against proteinase-3 (PR3) or myeloperoxidase (MPO). The dynamics of the T-cell response within tissues is studied best in animal models. It was the aim to analyze the lesional T-cell dynamics in the experimental autoimmune vasculitis model. Female Wistar Kyoto-rats were immunized with human MPO emulsified in complete Freund's adjuvant. Control animals received complete Freund's adjuvant without MPO. Selected groups received anti-IL17A treatment. Lesional T-cells from kidneys were assessed by flow cytometry (FACS), realtime polymerase chain reaction (PCR) and EliSpot. All animals immunized with MPO developed signs of vasculitis. At week six, lung damage expressed as petechial bleeding score and renal damage quantified by albuminuria were highest. As analyzed by FACS, the fraction of renal Th17 cells peaked at week six in MPO rats equaling the proportion of Th1 cells. MPO-specific renal Th1 and Th17 cells were detectable by EliSpot at weeks four and six post-immunization in MPO-immunized rats being absent in control rats. Neutralization of IL-17A did not affect the development of humoral and cellular anti-MPO immunity. Likewise, pulmonary and renal vasculitis were not ameliorated. In summary, the dynamics of the lesional T-cell response in the EAV model shows a major participation of MPO-specific Th17 and Th1 cells in renal vasculitis. Simple cytokine neutralization was not efficacious in this disease model so that combined neutralization approaches should be studied further.

Zeng Y
Boschmann E
Kotte J
Sun M
Hess L
Dolff S
Hinkeldein T
Hagedorn J
Langer R
van Paassen P
Damoiseaux J
Cohen Tervaert JW
Witzke O
Kribben A
Wilde B


Gastrointestinal neuroprosthesis for motility and metabolic neuromodulation

Abstract: Gastrointestinal (GI) dysmotility and associated conditions affect over 20% of population, yet pharmacological, behavioural, and surgical interventions offer limited therapeutic efficacy. Targeted electrical stimulation addressing underlying neuromuscular pathology stands to transform our ability to treat dysmotility. Here, we developed a closed-loop GI neuroprosthesis which activates or relaxes GI tract musculature through electrochemical stimulation in response to sensed food stimuli. We additionally describe a tool supporting minimally invasive endoscopically guided implantation that can penetrate the mucosa, accurately localize the submucosa, and safely deploy this device to directly interface with the enteric nervous system. The neuroprosthesis enables generation of coordinated peristaltic waves, significantly increasing the motility rate in a swine model of oesophageal and stomach dysmotility (p < 0.05, student's t-test). Further, by directly modulating the myenteric plexus and thus mimicking meal ingestion, we induce peristalsis in a fasted state and achieve a metabolic response commensurate with a fed or satiated state. This neuroprosthesis and implantation platform expand opportunities in fundamental studies and treatments of metabolic and neuromuscular pathologies affecting the GI tract.

Srinivasan S
Antonini MJ
Alshareef A
Sahasrabudhe A
Jenkins J
Ishida K
Kuosmanen J
Hayward A
Min S
Langer R
Anikeeva P
Traverso G

Animals
Swine
Gastrointestinal Motility
Peristalsis
Electric Stimulation
Gastrointestinal Tract
Neural Prostheses
Enteric Nervous System
Myenteric Plexus
Electric Stimulation Therapy
Esophagus
Female


Clinical and pathological features of lymphomas in the breast: a comprehensive multicentric study

Abstract: Breast lymphomas, though rare, present a unique spectrum of clinical and pathological characteristics. This multicenter retrospective study evaluates 92 cases from four international institutions over a ten-year period to provide a comprehensive analysis of primary breast lymphomas (PBLs), secondary breast lymphomas (SBLs), and related subtypes, including breast implant-associated lymphomas (BIA-ALCL) and intra-mammary lymph node lymphomas. Primary breast lymphomas accounted for 46.7% of cases, with a predominance of diffuse large B-cell lymphoma (DLBCL). Secondary breast involvement, observed in 43.5% of cases, displayed a more diverse histological distribution, with follicular lymphoma being the most common subtype. Breast implant-associated lymphomas (BIA-ALCL) constituted 6.5% of cases, exclusively involving anaplastic large cell T-cell lymphoma, ALK-negative. The study highlights significant differences in patient demographics, clinical presentation, and survival outcomes. PBLs primarily affected older patients (mean age: 68.4 years) and were associated with longer lymphoma-specific survival compared to SBLs (76.12 vs. 59.45 months; p = 0.001). Notably, survival differences were evident within histological subtypes, emphasizing the impact of disease origin on prognosis. BIA-ALCL cases were distinct in clinical features and histology, with a younger mean age (47.5 years) and frequent association with unilateral effusion. This analysis underscores the necessity for precise diagnostic and therapeutic strategies tailored to the unique biology of breast lymphomas. Future research should aim to elucidate molecular mechanisms and optimize management protocols to improve patient outcomes.

Maccio U
Rets A
Grosse C
Ferguson NA
Langer R
Bühler MM
Gallo A
Witzel I
Beham-Schmid C
Wieler J
Orsaria M
Varga Z

Humans
Female
Aged
Middle Aged
Breast Neoplasms
Retrospective Studies
Adult
Aged, 80 and over
Lymphoma
Lymphoma, Large-Cell, Anaplastic
Breast Implants
Prognosis


Emergency delivery of particulate drugs by active ejection using in vivo wireless devices

Abstract: Rapidly administered emergency drug therapy represents life-saving treatment for a range of acute conditions including hypoglycaemia, anaphylaxis and cardiac arrest. Devices that automate emergency delivery, such as pumps and automated injectors, are limited by the low stability of liquid formulations. In contrast, dry particulate formulations of these drugs are stable but are incompatible with drug pumps and require reconstitution before administration. Here we develop a miniaturized (<3 cm3), lightweight (<2 g), minimally invasive, fully wireless emergency rescue device for the storage and active burst-release of indefinitely stable particulate forms of peptide and hormone drugs into subcutaneous sites for direct reconstitution in interstitial biofluids and rapid (<5 min) therapeutic effect. Importantly, the device delivers drug across fibrotic tissue, which commonly accumulates following in vivo implantation, thereby accelerating systemic delivery. Fully wireless delivery of dry particulate glucagon in vivo is demonstrated, providing emergency hypoglycaemic rescue in diabetic mice. In addition, triggered delivery of epinephrine is demonstrated in vivo. This work provides a platform for the long-term in vivo closed-loop delivery of emergency rescue drugs.

Krishnan SR
O'Keeffe L
Rudra A
Gumustop D
Khatib N
Liu C
Yang J
Wang A
Bochenek MA
Lu YC
Bose S
Reed K
Langer R
Anderson DG

Animals
Mice
Drug Delivery Systems
Wireless Technology
Epinephrine
Glucagon
Male
Mice, Inbred C57BL
Diabetes Mellitus, Experimental


mRNA-LNP hepatitis C vaccine encoding p7 elicits improved humoral immunogenicity

Abstract: Nucleoside-modified messenger RNA lipid nanoparticle (mRNA-LNP) vaccines have had a tremendous impact on vaccine development against infectious pathogens, particularly since the onset of the COVID-19 pandemic. The platform is an effective stimulator of humoral and cellular immune responses due to unique features of the mRNA and LNP components. We sought to optimize the performance of the mRNA-LNP platform against a specific viral target, developing a vaccine against Hepatitis C virus (HCV) that would improve HCV-like particle secretion and enhance the humoral immune response. We designed an mRNA-LNP vaccine targeting HCV and improved its efficacy by including the nonstructural HCV viroporin p7, and measured cellular and humoral immune responses in immunized mice. Both constructs induced antigen-specific functional CD4+ and CD8+ T cell responses, as well as T follicular helper cell responses associated with humoral immunity, consistent with other applications of the mRNA-LNP platform. Mice immunized with the optimized construct showed superior binding and neutralizing antibody responses compared to the non-optimized one. Notably, the optimized construct elicited stronger humoral responses against heterologous viral strains. Our findings underscore the potential of mRNA-LNP vaccines for HCV, yet translating these findings into humans requires further investigation. These results demonstrate the value of optimizing the mRNA-LNP platform through inclusion of elements with utility beyond presenting epitopes of the pathogen being targeted. This study pushes HCV vaccine development forward and extends the utility of the mRNA-LNP platform against infectious diseases.

Reagan EK
Wright DA
Clark KE
Mercado-Lopez X
Atochina-Vasserman EN
Ni H
Mui BL
Hu F
Bailey JR
Bar KJ
Weissman D


Lipid Nanoparticle Co-Delivery of mRNA and a Small Molecule Drug for Oral Cancer Chemoimmunotherapy

Abstract: Oral squamous cell carcinoma (OSCC) represents 90% of all head and neck cancers. Despite decades of research, the 5-year survival rate is 50%. Strikingly, the overall incidence rate is projected to increase by 30% in the next 10 years, which may result in a sharp increase in mortality. Two fundamental aspects of OSCC are that it progresses via the inactivation and mutation of tumor suppressor genes (TSGs) and has a "cold" tumor microenvironment (TME). A major barrier in the treatment of OSCC is the lack of novel therapies clinicians have at their disposal that are designed to disrupt tumor progression by reshaping the cold tumors into inflammatory "hot" tumors. To overcome these obstacles, we employed a lipid nanoparticle (LNP) that co-encapsulates p53 mRNA and the small molecule ciclopirox (CPX). We demonstrate that both drugs have innate chemotherapeutic properties by facilitating caspase activation. Moreover, these therapies can create a less immunosuppressive TME in part by repolarizing tumor-associated macrophages (TAMs) to M1-like phenotypes. When formulated together, our platform provides an all-in-one approach for OSCC, effective in both p53-therapy-susceptible and p53-therapy-resistant models. Additionally, this work offers a template for a delivery platform capable of tackling multiple mechanisms of OSCC progression and survival.

Advanced materials (Deerfield Beach, Fla.)

Padilla MS
Li JJ
Zhang Q
Patwari K
Shi S
Yamagata HM
Joseph RA
Hymms BN
Teerdhala SV
Fitzgerald E
Chalom OZ
Gupta K
Alameh MG
Weissman D
Le AD
Mitchell MJ


mRNA lipid nanoparticle cancer vaccine platform delivering multiple STING activators for enhanced antitumor activity

Abstract: mRNA-based cancer vaccines offer a modular and safe platform to elicit antitumor immunity, yet their efficacy is often limited by inefficient mRNA delivery and inadequate dendritic cell (DC) activation, both of which are essential for initiating robust cytotoxic T cell responses. Inadequate innate immune activation coupled with poor antigen presentation further diminishes their effectiveness, particularly in immunologically "cold" tumors. While stimulator of interferon genes (STING) agonists can enhance DC maturation and cross-presentation, their therapeutic utility is constrained by poor intracellular delivery and limited colocalization with tumor antigens. In this study, we developed a lipid nanoparticle (LNP) platform via high-throughput screening of ionizable lipids for potent mRNA delivery to DCs both in vitro and in vivo. To amplify immune activation, we coencapsulated the STING agonists c-di-AMP (AMP) and manganese (Mn2+) together with tumor antigen-encoding mRNA into the lead LNP formulation. This codelivery strategy synergistically activated type I interferon signaling, upregulated costimulatory molecules, enhanced antigen presentation, and elicited potent tumor-specific T cell responses and superior antitumor efficacy. Our results demonstrate that integrating innate immune stimulation with mRNA-LNP delivery provides a promising strategy to overcome current limitations in mRNA vaccine efficacy and to improve cancer immunotherapy outcomes.

Zeng Y
Xu J
Wang J
Xue L
Liu J
Geisler HC
Ma X
Melamed JR
Shi Q
Padilla MS
Luo Z
Zhu J
Thatte AS
Figueroa-Espada CG
Ang MJY
Murray AM
Yamagata HM
Kim D
Metzloff AE
Weissman D
Mitchell MJ

Cancer Vaccines
Animals
Membrane Proteins
Mice
Dendritic Cells
Nanoparticles
RNA, Messenger
STING Protein
Lipids
cGAS-STING Signaling Pathway
Nanovaccines
Female
Mice, Inbred C57BL
Humans
Antigens, Neoplasm
Cell Line, Tumor
Antigen Presentation
Interferon Type I
Liposomes