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Cardiovascular health depends critically on the integrity of the vascular extracellular matrix (ECM) and the behavior of vascular smooth muscle cells (VSMCs), both of which can be adversely affected in vascular diseases. This study quantitatively evaluates the therapeutic potential of conditioned medium (CM) derived from bone marrow (BM-MSCs) and adipose-derived stem cells (ADSCs) in an elastase-injured human aortic smooth muscle cell (HASMC) model. We systematically varied seeding densities (2000, 5000, and 10,000 cells/cm2) and serum conditions to optimize the SC-SMC secretome for vascular repair. Our results indicate that neither BM-SMC nor AD-SMC CM significantly enhanced lysyl oxidase (LOX) activity. In fact, serum-supplemented BM-SMC CM significantly suppressed LOX activity at seeding densities of 2000 cells/cm2 (p&#x2009;=&#x2009;0.0378) and 10,000 cells/cm2 (p&#x2009;=&#x2009;0.0080) compared to injured untreated controls. High-density AD-SMC CM (10,000 cells/cm2) also resulted in a significant decrease in elastin levels (p&#x2009;<&#x2009;0.05). In addition, serum presence was critical for maintaining the reparative phenotype. Serum-free (SF) conditions for both cell sources led to widespread, statistically significant reductions (p&#x2009;<&#x2009;0.0001) in key repair and inflammatory biomarkers, including PDGF-AA, Leptin, Lipocalin-2, Osteopontin, RBP4, MMP-1, and IL-6. IL-11 emerged as a primary discriminatory biomarker, showing significant differences between BM and AD treatments at high seeding densities, with both sources causing a significant decrease (p&#x2009;<&#x2009;0.0001) compared to injured untreated controls. These findings demonstrate that seeding density and serum conditions are critical variables that quantitatively modulate the efficacy of SC-SMC-CM. The study highlights that BM-SMC-derived CM offers a more stable platform for elastin maintenance under serum-free conditions, providing a foundation for developing tailored, cell-free regenerative therapies for cardiovascular disease.

Cholangiocarcinoma (CCA) is a biliary cancer with a poor prognosis and marked chemoresistance. The transcription factor SOX17, which is essential for cholangiocyte differentiation, is frequently downregulated in CCA, as is the case with several tumor suppressor genes. This study provides a proof-of-concept for further development of cellular therapeutic strategies to restore SOX17 expression in CCA cells. For this aim, we used EGI-1 cells as the target cell model derived from extrahepatic CCA. Recombinant Tat-SOX17 protein was first produced in bacteria and purified using Ni-NTA affinity columns and asymmetric flow-field-flow fractionation. Tat-SOX17 entered EGI-1 cells and reached their nucleus. The addition of the &#x3b1;-fetoprotein signal peptide (AFPsp) to the chimeric protein enabled the efficient secretion of the fusion protein formed by AFPsp, SOX17, and reverse Tat (TatR) by donor cells. For the production of secretome enriched in AFPsp-SOX17-TatR protein, HEK293T cells, derived from embryonic kidney, were used. In EGI-1 cells cultured in the presence of this secretome, target gene expression, cell cycle progression, apoptosis, proliferation, colony formation, and cell migration were assessed. Protein expression and localization were analyzed by Western blotting and immunofluorescence. AFPsp-SOX17-TatR enters EGI-1 cells, reaches their nucleus, and modulates the expression of SOX17-dependent genes, such as ABCB1 and ABCG2. Moreover, a marked reduction in proliferation and colony-forming ability was found. In contrast, no significant effect on cell cycle progression, apoptosis, or cell migration was observed. Similar treatment of immortalized human cholangiocytes also increased their SOX17 content, resulting in upregulation of the cholangiolar marker cytokeratin 7 (CK7), but did not affect their proliferation rate. In conclusion, using chimeric proteins such as AFPsp-SOX17-TatR, which contain components for secretion from donor cells and entry into target cancer cells, can provide a promising approach for treating tumors such as CCA, which are characterized by reduced expression of tumor suppressor genes, including SOX17.

Among the immune mechanisms underlying the pathogenesis of antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV), B-lineage cells play crucial roles by producing ANCA, presenting antigens to T cells, and secreting proinflammatory cytokines. The upregulated signaling pathways of the B-cell activation factor of tumor necrosis factor family (BAFF) and a proliferation-inducing ligand (APRIL), which are broadly produced by several types of myeloid cells, contribute to the activation and survival of autoreactive B cells, leading to disease onset and relapse. Furthermore, elevated levels of soluble BAFF and APRIL persist even in patients who achieve clinical remission following conventional induction therapies such as cyclophosphamide and rituximab (RTX), resulting in the cause of relapse. Active AAV is characterized by expansions of specific phenotypes in circulating B-cell lineages, including plasmablasts and plasma cells. Conversely, reductions in transitional, memory, and regulatory B cells are observed. Significant expressions of affinity receptors binding to secreting BAFF/APRIL, including decreased BAFF receptor expression on memory B cells and transitional B cells or increased transmembrane activator and calcium-modulator and cyclophilin ligand interactor (TACI) expression on transitional B cells and plasmablasts/plasma cells, are also observed during acute and remission AAV. Enhanced BAFF and APRIL signaling through these receptors, especially TACI, activates the intracellular nuclear factor-&#x3ba;B pathway in B cells, promoting their proliferation and ANCA production. B-cell depletion therapy using RTX is effective; however, it requires repeated administration to maintain remission, which can occasionally lead to infection. Belimumab, a BAFF-inhibiting monoclonal antibody, alone has shown limited efficacy in preventing relapse of AAV. Targeting BAFF/APRIL signaling pathways and their binding receptors, along with the downstream molecular pathways in targeted B cells, is essential for developing novel therapeutic strategies aimed at achieving complete and sustained AAV remission.

Currently, no targeted therapy exists for idiopathic pulmonary fibrosis (IPF). The hallmark pathological feature of excessive extracellular matrix (ECM) deposition severely undermines the efficacy of mesenchymal stem cell (MSC)-based treatments. While existing MSC therapeutic strategies primarily focus on modulating inflammation in early stages, they have not yet established precise interventions addressing the core pathological mechanism-ECM dysregulation. Previous studies demonstrated the therapeutic potential of human embryonic stem cell (hESCs)-derived immunity-and-matrix-regulatory cells (IMRCs) in lung injury and fibrosis models. However, the critical biomarkers and underlying mechanisms mediating IMRCs' efficacy in IPF remain poorly understood. In this study, we generated MMP1 knockout IMRCs (IMRCs-MMP1 KO) using CRISPR-based gene editing. We then characterized whether MMP1 ablation affected key properties of IMRCs, including cell morphology, proliferation, migration, marker protein expression, transcriptomic profile, and cytokine secretion. Subsequently, the ability of IMRCs-MMP1 KO to degrade collagen was tested using in&#xa0;vivo and in&#xa0;vitro pulmonary fibrosis models. MMP1 knockout was successfully achieved and did not compromise typical IMRC characteristics or impair their immunomodulatory capacity. However, MMP1 deficiency significantly attenuated the ability of IMRCs to degrade TGF-&#x3b2;1-induced collagen I deposition in A549 cells. Importantly, wild-type IMRCs demonstrated superior therapeutic efficacy in ameliorating bleomycin-induced lung injury and fibrosis in mice compared with IMRCs-MMP1 KO. Furthermore, IMRCs exhibited significantly greater capability to directly degrade the pericellular collagen I and modulate fibroblasts' activation progression within fibrotic lung tissues in a MMP1-dependent manner. In summary, our data establish that MMP1 plays an essential functional role in IMRC-mediated attenuation of PF. MMP1 thus represents a key therapeutic biomarker for IMRC-based treatment. This work provides a foundation for developing stem cell therapies tailored to the pathological features of IPF, potentially enabling adaptive treatment strategies.

Macrophages and their fused counterpart, foreign body giant cells (FBGCs), have a determining influence on the long-term functionality of implanted biomaterials, coordinating biomaterial resorption and fibrous encapsulation. Modulating macrophage phenotype into favorable polarization states is a widely adopted strategy to attempt steering the foreign body response. However, the links between macrophage phenotype and FBGC formation and function are unknown. The goal of this study was to determine the fusion potential of well-defined macrophage polarization states, and to explore whether prior polarization state translates to FBGC phenotype in primary human macrophages. Peripheral blood-derived human macrophages were first stimulated toward M0/M1/M2a/M2c polarizations after which they were stimulated to fuse into FBGCs using interleukin-4 (IL-4). After fusion, these cells were re-stimulated to investigate the effect of the respective cytokine cocktails on FBGC phenotype. The results showed that M1 macrophages had increased IL-4 receptor expression, an altered cytoskeleton, and significantly increased expression of fusion-associated genes, when compared to unstimulated, M2a and M2c macrophages. FBGCs could form from all polarization states. Although M1 polarization led to a decreased fusion percentage, the FBGCs that did form were larger in terms of cell size and number of nuclei. FBGCs showed significant changes in their cytoskeletal structure by forming rings of actin around the periphery of the cell, which was consistent for the different macrophage polarizations. Expression of select genes and proteins associated with macrophage polarization states were either maintained during fusion, or reacquired by FBGCs upon restimulation with polarizing factors. These results provide evidence for the existence of FBGC polarization states, emphasizing the importance of the macrophage population and microenvironment in regulating FBGC formation. This suggests that FBGC function may be steerable, for example using biomaterial design parameters, in order to improve long-term biomaterial functionality and integration.

Hyperthermia is a potential therapy for non-small cell lung cancer (NSCLC); however, cell resistance to hyperthermia remains challenging. This study investigated the role of heat shock protein 70 (HSP70) in regulating small ubiquitin-like modifier modification (SUMOylation) of P-glycoprotein (P-gp) in the resistance of NSCLC cells to hyperthermia. Human NSCLC cells (A549) were exposed to hyperthermia and subjected to HSP70 overexpression/knockdown, P-gp silencing, or Erastin/Ferrostatin-1 treatment. Nude mice received inoculation with A549 cells infected with adenoviral vectors overexpressing HSP70 (Ad-HSP70) before hyperthermia. Verapamil was selected as an inhibitor of P-gp in vivo. A series of experiments were performed to analyze cellular and molecular changes, including viability, migration, invasion, ferroptosis-related indices, and mitochondrial dysfunction. HSP70-P-gp interaction, SUMOylation, and protein stability were examined using co-immunoprecipitation and cycloheximide assays. HSP70 overexpression accelerated the malignant properties of A549 cells while restricting ferroptosis, thus driving thermoresistance. Furthermore, activating ferroptosis partially annulled these effects of HSP70 overexpression, whereas inhibiting ferroptosis partially counteracted the suppressive effect of HSP70 knockdown on the thermoresistance of A549 cells. HSP70 interacted with P-gp to modulate its SUMOylation, thereby increasing the stability and levels of P-gp protein. P-gp knockdown partially blocked the effect of HSP70 and restored the sensitivity of A549 cells to hyperthermia. HSP70 suppressed oxidative stress and mitochondrial dysfunction by upregulating P-gp. In vivo, HSP70-mediated SUMOylation of P-gp inhibited ferroptosis, promoting tumor growth and thermoresistance. HSP70 suppresses oxidative stress and mitochondrial dysfunction through SUMOylation of P-gp, thereby inhibiting ferroptosis and promoting thermoresistance in A549 NSCLC cells.

Myeloid cells can express lymphoid markers, including components of the CD3-TCR complex. CD3+ macrophages have been identified in both infectious and non-infectious pathologies; however, their origin and signaling mechanisms remain poorly studied, primarily due to their low prevalence in human samples. Utilizing the RAW murine macrophage cell line as a model, this study aimed to ascertain whether nutrient deprivation induces CD3 expression and to identify the signaling molecules involved in the activation of a CD3-dependent proinflammatory profile. Firstly, the impact of environmental stress, specifically nutrient deprivation, on CD3 expression over a period was assessed. Subsequently, RAW cells were stimulated with anti-CD3 and IgG2a, and the profile of proinflammatory cytokines was evaluated alongside the expression of signaling proteins, including NFAT, c-Jun, and IKK. Furthermore, the transcriptional regulation of IRF-1, GATA-3, and MAFB was examined. Lastly, the functional capacity of CD3+ RAW cells was determined through a phagocytosis assay. RAW cells were found to express molecules classically associated with T cells, including CD3, TCR, and CD4. Notably, CD3 expression was significantly upregulated at both the protein and transcriptional levels under nutrient deprivation, although it did not reach the levels observed in T cells. Functionally, CD3+ RAW cells exhibited enhanced phagocytic activity toward latex beads. Furthermore, stimulation with anti-CD3 plus IgG2a induced a robust proinflammatory cytokine response, characterized by the secretion of IFN-&#x3b3;, TNF, and IL-6 as early as 5 hours post-stimulation. This response was associated with activation of signaling pathways involving NFAT, c-Jun, and IKK, along with increased IRF-1 expression and downregulation of MAFB, supporting the establishment of a CD3-dependent proinflammatory profile in RAW cells. Our findings establish RAW macrophages as a model to study CD3+ signaling machinery in myeloid cells. We demonstrate for the first time that nutrient deprivation induces CD3 expression and that the resulting CD3-driven proinflammatory program is associated, at least in part, with NFAT and IRF-1. These conclusions provide new insights into the origin and functional significance of CD3+ macrophages and offer a valuable platform for investigating the impact of this pathway on innate immune responses across various pathological contexts.

A significant proportion of gastric diffuse large B-cell lymphoma with mucosa-associated lymphoid tissue [DLBCL(MALT)] and without MALT ('pure' DLBCL) can be resolved by Helicobacter pylori eradication (HPE). Gastric MALT lymphoma is an indolent lymphoma derived from memory B cells in the marginal zone. In the present study, we aimed to explore the origin of large cells in HPE-responsive gastric DLBCLs (complete remission after HPE). We investigated gastric lymphoma biopsies from 31 patients with HPE-responsive DLBCLs [15 'pure' DLBCLs, 16 DLBCL(MALT)s]. We used the Hans algorithm (CD10, BCL-6, and MUM1) to define the origins of germinal center B cell (GCB) and non-GCB. To further ascertain the cellular origin, 11 'pure' DLBCLs were examined using an Agilent whole-human genome microarray. Eleven DLBCLs [eight with 'pure' DLBCL and three with DLBCL(MALT)] were also assessed using Lymph2Cx. Specific GCB markers, including BACH2, AID, and BCL2 rearrangement and enhancer of zeste 2 polycomb repressive complex 2 subunit (EZH2) codon 641 mutations, were evaluated in 31 patients with HPE-responsive gastric DLBCLs. According to the Hans algorithm, 53% (8/15) of gastric 'pure' DLBCLs and 50% (8/16) of DLBCL(MALT)s were of the GCB phenotype. Gene expression assays revealed that five of six patients with 'Hans' GCB had GCB genetic signatures, whereas four of five patients with 'Hans' non-GCB had activated B-cell genetic signatures. The Lymph2Cx assay revealed the GCB subtype in seven of eight patients with 'Hans' GCB. The expression patterns of BACH2 (p&#x2009;=&#x2009;0.005) and AID (p&#x2009;=&#x2009;0.038) closely correlated with the 'Hans' GCB phenotype. BCL2 rearrangements and EZH2 codon 641 mutations were detected in 44% (7/16) and 13% (2/16) of patients with 'Hans' GCB, respectively. In another cohort of 29 HPE-unresponsive gastric DLBCLs [19 'pure' DLBCLs and 10 DLBCL(MALT)s], we found a close association between the 'Hans' GCB subtype and the GCB subtype as determined by the Agilent whole-human genome microarray and Lymph2Cx in lymphoma cells of these patients. In conclusion, more than half of HPE-responsive large cell lymphoma cases in the stomach were of GCB origin. &#xa9; 2026 The Pathological Society of Great Britain and Ireland.

Breast cancer is one of the most common cancers among women in Nigeria. Human epidermal growth factor receptor 2 (HER2) is an important prognostic and predictive biomarker that guides targeted therapy. The tumour grade is an important prognostic factor and is also important in the treatment of patients. In a resource-limited setting, cell block cytology may serve as an alternative for initial biomarker assessment and also as an initial diagnostic tool for planning definitive management. The study aims to compare HER2 overexpression of breast carcinoma using cell blocks and corresponding paraffin wax-embedded (FFPE) tissue blocks and to evaluate the concordance between both methods. This was a one-year prospective study involving 83 cases of breast carcinoma patients with both cell block and corresponding FFPE tissue specimens. HER2 immunohistochemistry was performed using the ASCO/CAP 2018 guideline. Sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) were calculated with 95% confidence intervals (CI). Concordance was assessed using Cohen's kappa statistic. McNemar's test was used for paired comparisons. The mean age of the study participants was 43.1 &#xb1;13.1 years, with a peak age group of 40-49 years. IHC HER2 overexpression was done on both cell blocks and histological blocks. In cell blocks, HER2 expression showed 15 cases (18.1%), 65 cases (78.3%), 3 cases (3.6%) were positive, negative, and equivocal, respectively while from histologic tissues, 15 cases (18.1%), 63 cases (75.9%), 5 cases (6.0%) were also positive, negative and equivocal respectively. The overall concordance rate between the two methods was 93.5%, with concordance rates of 100% for HER2-positive cases, 96.9% for HER2-negative cases, and 60% for equivocal cases. Sensitivity and specificity of cell block HER2 assessment were 96.9% (95% CI: 82.9-99.9) and 100% (95% CI: 94.3-100.0), respectively. The PPV of HER2 assessment on cell block was 100.0% (95% CI: 78.2-100.0), and the NPV was 97.1% (95% CI: 89.9-99.6). The kappa coefficient for agreement was 0.935, indicating excellent agreement. McNemar's test showed no statistically significant difference (p = 0.480). Equivocal (2+) cases were included without FISH confirmation. Most of the cases were invasive ductal carcinoma (NST), accounting for 97.6% (81 cases). Cell block cytology demonstrates strong concordance with FFPE tissue for HER2 assessment and may serve as a reliable alternative for initial triaging in resource-limited settings, particularly where tissue is not readily feasible. However, confirmatory testing on tissue biopsy remains essential, particularly for equivocal cases.

Chronic cough, particularly prevalent in post-COVID condition (PCC, also known as long COVID), remains a significant medical challenge. Recently, extracellular vesicles (EVs) have gained significant attention for their therapeutic potential. In this study, we explore the therapeutic effects of EVs derived from mesenchymal stem cells (MSCs) cultured in a 3D system (3D-EVs) in treating chronic cough, with a focus on post-COVID-19 patients. Our in vitro experiments demonstrated that 3D-EVs promote angiogenesis and cell migration, crucial processes in tissue repair and regeneration. Notably, 3D-EVs exhibited a robust suppressive effect on lymphocyte proliferation in human PBMCs, indicating their potential immunomodulatory role. At day 6, nebulized 3D-EVs treatment group showed significantly higher rates of significant improvement (22.5% vs. 5.0%, P = 0.023) and total effectiveness (67.5% vs. 47.5%, P = 0.035), as well as a shorter mean time to cough resolution (13.83 vs. 19.90 days, P = 0.037). By day 14, total effective rates were comparable (85% vs. 80%, P > 0.05). No safety concerns were observed. RNA sequencing revealed altered B cell receptor signaling and downregulation of the mitotic cell cycle pathway, while exploratory immunophenotyping identified a significant reduction in plasma cells (P < 0.05) and directional trends in other immune subsets, providing preliminary evidence that 3D-EVs may modulate B cell differentiation and immune responses. These findings demonstrate the feasibility and preliminary efficacy of 3D-EVs for rapid relief of post-COVID-19 chronic cough, supporting larger controlled trials to validate their role in this and related respiratory diseases.

Pyroptosis, a lytic and inflammatory form of programmed cell death, has emerged as a regulator of tumor immunity through its capacity to trigger localized cytokine storms (operationally defined in Section 3). This review examines the dual mechanisms by which macrophage pyroptosis-driven cytokine storms influence tumor progression. Pyroptosis is executed through canonical (caspase-1/nucleotide-binding oligomerization domain-like receptor family pyrin domain-containing 3 (NLRP3)-dependent) and non-canonical (caspase-4/5/11-dependent) pathways, both converging on cleavage of gasdermin D (GSDMD) to form membrane pores that release pro-inflammatory cytokines (interleukin-1 beta, IL-1&#x3b2;; interleukin-18, IL-18) and damage-associated molecular patterns (DAMPs). These primary signals initiate cascade amplification through chemokine and cytokine networks, recruit diverse immune cell populations, and establish distinct inflammatory microenvironments. The effects of pyroptotic cytokine storms show striking temporal and intensity dependence. Acute, moderate inflammatory responses activate anti-tumor immunity through induction of immunogenic cell death (ICD), dendritic cell maturation, and cytotoxic lymphocyte priming. Chronic, low-grade cytokine storms, in contrast, promote tumorigenesis through six interconnected mechanisms: genomic instability and epigenetic reprogramming, cancer stem cell enrichment, pro-angiogenic remodeling, pre-metastatic niche formation, establishment of an immunosuppressive microenvironment, and induction of epithelial-mesenchymal transition. This "double-edged sword" phenomenon depends on inflammation intensity, duration, spatial distribution, and tumor microenvironment (TME) characteristics. Clinical investigations indicate that pyroptosis-related biomarkers, including GSDMD, gasdermin E (GSDME), and inflammatory cytokine profiles, may support patient stratification and treatment-response prediction across multiple cancer types in patients with tumor-associated macrophages (TAMs)-rich tumors. Preclinical evidence from bioorthogonal chemical systems in murine models, together with mathematical modeling, has suggested that pyroptosis affecting approximately 10-15% of tumor cells may serve as a tentative threshold for initiating anti-tumor immunity. However, this value has been derived from a limited number of preclinical systems (primarily 4T1 mammary tumor models) and has not yet been validated in human tumors; it should therefore be interpreted as a working hypothesis rather than an established parameter. Current therapeutic strategies targeting this pathway include NLRP3 inhibitors, IL-1&#x3b2;/IL-18 blockers, and combination approaches with immune checkpoint inhibitors (ICIs). Looking forward, future research should prioritize: (i) quantitative in vivo mapping of macrophage pyroptosis using spatial multi-omics and intravital imaging; (ii) development of tumor-targeted, spatiotemporally controlled pyroptosis inducers (e.g., nano-delivery and bioorthogonal activation systems); (iii) rational combination with immune checkpoint inhibitors and epigenetic modulators; and (iv) establishment of pyroptosis-based biomarker panels to guide patient stratification and toxicity prediction in clinical trials. Achieving "controllable cytokine storms" through precise macrophage pyroptosis modulation represents a therapeutic paradigm that balances anti-tumor efficacy against inflammatory toxicity, with the potential to advance cancer immunotherapy toward precision inflammation regulation.

Malignant peripheral nerve sheath tumors (MPNSTs) are aggressive soft tissue sarcomas and the most common cause of disease-associated death for neurofibromatosis type 1 (NF1) patients. In the context of NF1, MPNSTs develop from benign premalignant precursors and the transition to malignancy is typically accompanied by loss of the polycomb repressive complex 2 (PRC2), which results in aberrant upregulation of over 1200 genes due to global depletion of histone H3 lysine 27 trimethylation (H3K27me3). Previous studies suggest cells compensate for the loss of this repressive histone mark via hypermethylation of the genome. Here we analyzed genome-wide DNA methylation and the transcriptome in MPNST cell lines and isogenic PRC2-deficient and -proficient CRISPR-engineered immortalized human Schwann cells. In addition to effects of PRC2 status, we also measured the effects of two DNA methyltransferase inhibitors (DNMTi), decitabine and azacitidine. We found that PRC2 status does not affect global DNA methylation or average methylation levels across specific genomic features. Furthermore, decitabine and azacitidine have differential effects on MPNSTs. While both DNMTis hypomethylate the genome, they upregulate different targets. Azacitidine upregulates genes involved in RNA processing pathways and exhibits direct tumor cell cytotoxicity, while decitabine upregulates genes involved in the immune response, has no direct-cell killing effects, and likely suppresses tumor growth in&#xa0;vivo by altering the tumor microenvironment. We show that DNA hypomethylation alone is insufficient to kill MPNST cells, regardless of PRC2 status. Consequently, these findings suggest that DNMT inhibitors should be utilized in combination with other targeted therapies for MPNST patients.

Chimeric antigen receptor T-cell (CAR-T) therapy is an established treatment for several hematological malignancies, with peripheral blood mononuclear cells (PBMCs) serving as the starting material for manufacturing. Cryopreservation of PBMCs may offer logistical flexibility, although its influence on manufacturing outcomes remains incompletely defined. This review aimed to compare the effect of fresh versus cryopreserved PBMC starting material on CAR-T cell manufacturing outcomes, including viability, fold expansion, and transduction efficiency. A systematic review was conducted following PRISMA guidelines. PubMed and Google Scholar were searched from inception through March 2026 for original studies comparing fresh and cryopreserved PBMCs in human CAR-T cell manufacturing. Methodological quality was assessed using the design-appropriate quality-appraisal tools, and findings were synthesized narratively due to heterogeneity in study design and protocols. Five studies published between 2019 and 2025 met the inclusion criteria, comprising two clinical and three experimental analyses. Post-thaw viability and recovery of cryopreserved PBMCs ranged between 77% and 97%, slightly lower than fresh material. Fold expansion, transduction efficiency, and cytotoxic activity were generally comparable between groups, although some studies reported transient early differences including prolonged doubling times, mitochondrial dysfunction signals, and increased TIM-3 expression in cryopreserved-derived products. Cryopreservation can be considered a feasible approach in CAR-T manufacturing, with generally comparable outcomes despite early post-thaw cellular changes. These differences do not seem to consistently compromise the overall manufacturing performance. However, the current evidence remains limited and heterogeneous, and further studies are required to increase confidence in our initial findings.

Glaucoma is a leading cause of irreversible blindness worldwide, yet available treatments fail to prevent disease progression for all patients. It is characterized by a progressive dysfunction and loss of retinal ganglion cells. Neuroinflammation has been recognized as an underlying neurodegenerative mechanism of glaucoma in animal models and human post-mortem samples, and targeting neuroinflammation may provide additional means to neuroprotection. Galectin-3, a pro-inflammatory mediator encoded by the LGALS3 gene in humans, holds promise as a treatable target as its pharmacological and genetic inhibition is neuroprotective in multiple models of experimental glaucoma. However, the role of Galectin-3 in glaucoma remains unclear, particularly whether its emergence is a consequence of degeneration, or occurs at earlier time points. To address these knowledge gaps, we labeled IBA1, GFAP, and Galectin-3 in retina sections at early glaucoma stages in the rat bead glaucoma model, and in human retina from glaucoma donors. In the rat, IBA1 volume, but not GFAP, increased at an early, pre-degenerative timepoint. Accompanying this, we identified a significant increase of Galectin-3/IBA1 colocalization compared to control at the same timepoint, supporting the upregulation of Galectin-3 in early inflammation, preceding retinal ganglion cell degeneration in experimental glaucoma. However, a significant increase in Galectin-3/GFAP colocalization compared to control at the same timepoint in the rat additionally associates Galectin-3 production with astrocytes and M&#xfc;ller glia. This Galectin-3 to M&#xfc;ller glia relationship was significantly pronounced in human glaucomatous retina, predominating over microglia co-labelling. We further demonstarted that human MIO-M1 M&#xfc;ller glia in vitro express Galectin-3, but this is not altered in response to glaucoma relevant stimuli (TNF-&#x3b1; or mild-metabolic stress from rotenone). Instead, Galectin-3 expression was altered in phagocytosing states from exposure to E. coli particles, brain synaptosomes, or apoptotic neuronal debris. These findings provide further insight into Galectin-3 and gross inflammatory responses in glaucoma pathology.

To determine whether circulating soluble Flt-1 (sFlt-1) and soluble Tie-2 (sTie-2) are associated with intraplaque neovascularization (IPN) in human carotid atherosclerotic plaques. Forty-four patients with &#x2265;50% carotid stenosis underwent conventional carotid ultrasound and superb microvascular imaging (SMI); 29 had plasma collected and 11 provided carotid endarterectomy specimens for histology. IPN was quantified as neovessel counts in 2-minute SMI cine loops and as microvessel counts in excised plaques. Plasma VEGF-A/B/C/D, Ang-2, sFlt-1, and sTie-2 were measured by immunoassays. Public single-cell RNA-seq data from human carotid plaques were interrogated to map FLT1, TEK, VEGFA, ANGPT1, and ANGPT2 expression to specific plaque cell populations. IPN was detected in 33/44 (75%) patients, with 0-16 neovessels on SMI (median 4). Plasma sFlt-1 correlated with SMI neovessel counts (r=0.42, p=0.030), with a similar trend for sTie-2 (r=0.37, p=0.057), whereas VEGF-A/B/C/D and Ang-2 showed no significant relationships. In the surgical subgroup, histological neovessel counts correlated with sFlt-1 (r=0.65, p=0.030) and SMI neovessel count (r= 0.68, p =0.02) but not sTie-2. sFlt-1 and sTie-2 correlated with BMI (and sTie-2 also with age), yet log-sFlt-1 remained independently associated with SMI-derived neovessel counts after adjustment for age and BMI (B&#xa0;=&#xa0;13.24, 95% CI 0.17-26.30, p=0.047; R&#xb2;=0.18), while sTie-2 was not. No significant associations were observed between IPN or sFlt-1/sTie-2 and lipid profile, CRP, leukocyte counts, or other conventional risk factors. Single-cell transcriptomics showed FLT1 and ANGPT2 broadly expressed across endothelial, smooth-muscle, and myeloid clusters, with TEK largely confined to endothelial cells and ANGPT1 to smooth-muscle cells, supporting local activation of VEGF-FLT1 and Ang-TEK/Tie-2 signaling within plaques. In this pilot study, higher plasma sFlt-1, and, to a lesser degree sTie-2, correlated with carotid IPN on SMI and histology, independent of age and BMI. These findings suggest that soluble VEGF- and Ang/Tie-2-receptor pathways may serve as circulating, context-sensitive markers of intraplaque angiogenesis and plaque vulnerability, meriting evaluation in larger longitudinal cohorts.

Pathogenic immune-cardiac crosstalk underlies maladaptive remodeling in chronic heart failure, yet therapies directly targeting this axis are lacking. Glycoconjugates, which are crucial for signal transduction and extracellular matrix integrity, represent an underexploited therapeutic avenue. This study sought to define the role of glycoconjugate-metabolizing enzymes at the immune-cardiac interface and evaluate their translational potential. We performed integrative analyses of bulk and single-cell RNA sequencing data from failing human and mouse hearts. Employing mouse models of pressure overload (transverse aortic constriction) and ischemia-reperfusion, we used global and mast cell (MC)-specific gene deletion, bone-marrow chimeras, and pharmacological neutralization. Mechanistic insights were gained through multiomics profiling, including RNA-seq, ATAC-seq, CUT&Tag, and proteomics. The ganglioside GD3 synthase, St8sia1, was selectively induced in cardiac MCs during pathological remodeling in both mice and humans. MC-specific or hematopoietic deletion of St8sia1 preserved ventricular function, attenuated fibrosis, and markedly reduced neutrophil and Ly6C+ monocyte recruitment after transverse aortic constriction and ischemia-reperfusion. Therapeutic neutralization of GD3 with the clinical-grade monoclonal antibody R24 improved cardiac function and diminished scar formation after ischemia-reperfusion. Mechanistically, GD3 bound specific histone variants, such as H2A.Z and H3.3C, thereby reprogramming chromatin accessibility to activate proinflammatory and profibrotic transcriptional programs in MCs. Consequently, GD3 inhibition suppressed MC degranulation, disrupted pathogenic MC-cardiomyocyte/fibroblast crosstalk, and preserved reparative macrophage populations. The MC-restricted St8sia1-GD3 axis functions as a glyco-epigenetic checkpoint driving maladaptive cardiac remodeling. Targeting this axis represents a translatable immunomodulatory strategy to prevent the progression to chronic heart failure.

Immunocompromised hosts have reduced immune responses to COVID-19 vaccination, and more severe disease. Antibody responses correlate with protection but markers of immunity vary across a spectrum of immunocompromise. We compared serologic and cellular responses following Ancestral COVID-19 vaccines in healthy controls (HC), people with HIV (PWH) and lung transplant (LTx) recipients. Anti-spike receptor binding domain (RBD) IgG, neutralising antibodies (nAb) and T-cell responses were assessed one-month post-dose 2 and dose 3 of Ancestral COVID-19 vaccination in HC, PWH and LTx. NAb responses to Ancestral, Delta and Omicron BA.2 and BA.5 variants were assessed. Twenty-nine HC, 21 PWH and 12 LTx recipients were included. PWH demonstrated lower anti-RBD-IgG responses (median post-dose 3: 80.3&#x2009;&#x3bc;g mL-1 vs 43.3&#x2009;&#x3bc;g mL-1, P&#x2009;=&#x2009;0.03) to mRNA COVID-19 vaccination than HC, while LTx recipients displayed diminished responses following any vaccine (15.3&#x2009;&#x3bc;g mL-1 vs 74.0&#x2009;&#x3bc;g mL-1, P&#x2009;=&#x2009;0.01). Dose 3 increased anti-RBD-IgG concentrations and nAb responses in HC and PWH, though Omicron variant neutralisation was attenuated. LTx recipients mounted limited nAb responses. PWH and HC had no difference in nAb responses for Ancestral (median 1738 vs 486.2, P&#x2009;>&#x2009;0.99) or BA.5 variants (median 34.0 vs 67.9, P&#x2009;>&#x2009;0.99). Compared with HC, PWH and LTx demonstrated reduced frequencies of SARS-CoV-2-specific memory T&#xa0;cells and a reduced functional memory T-cell response in LTx. Although Dose 3 was beneficial, LTx recipients demonstrated lower serological responses than HC, while reductions were modest in PWH. Immunocompromised groups had reduced but detectable SARS-CoV-2-specific T-cell responses, demonstrating the utility of COVID-19 vaccination despite poorer serological responses.

Recurrent spontaneous abortion (RSA) is a major reproductive health challenge with limited clinical options. Curcumin, a natural polyphenol widely consumed as a dietary supplement, has shown promise in improving pregnancy outcomes; however, its specific molecular targets remain obscure due to its broad bioactivity. This study aimed to define curcumin's impact on RSA and identify its direct molecular target to provide a mechanistic basis for its nutritional application. We employed an integrated approach combining phenotypic analysis in the RSA mouse model and target discovery in human endometrial stromal cells. Target identification was performed using unbiased Limited Proteolysis-Mass Spectrometry (LiP-MS). The interaction between curcumin and the identified target, BRD4, was confirmed through molecular docking, dynamics simulations, functional genetics, and chromatin immunoprecipitation (ChIP-qPCR). Functional outcomes were assessed by measuring key ferroptosis markers and mitochondrial morphology using transmission electron microscopy. Decidualization was analyzed in both mouse tissues and cells subjected to a decidualization and ferroptosis induction protocol. Curcumin significantly reduced embryo resorption and restored decidual morphology and marker expression (PRL and IGFBP1) in RSA mice. LiP-MS analysis in human decidual stromal cells identified ferroptosis as the primary pathway targeted by curcumin. Both in&#xa0;vivo and in&#xa0;vitro validation showed that curcumin inhibits ferroptosis, decreasing lipid peroxidation, restoring glutathione balance, and preserving mitochondrial integrity, which in turn rescued decidual marker expression. Proteomic integration with ferroptosis databases identified BRD4 as a central hub. Mechanistically, curcumin binds BRD4, thereby suppressing BRD4-driven expression of TFRC and ACSL4, which blocks ferroptosis and rescues decidual markers. This research uncovers a BRD4-driven ferroptosis pathway as a key pathogenic mechanism in RSA. Our results demonstrate that curcumin acts as an epigenetic modulator by directly targeting BRD4, thereby suppressing this pro-ferroptotic transcriptional program. These findings provide critical mechanistic evidence supporting curcumin as an evidence-based nutritional intervention for RSA.

Oral cavity (OC) and oropharyngeal (OP) squamous cell carcinoma (SCC) represent a major global health concern, accounting for most of head and neck malignancies. These tumors arise from the epithelial lining of the OC and oropharynx and are characterized by aggressive local invasion, lymph node metastasis, and high morbidity and mortality. In addition to known risk factors such as tobacco and alcohol consumption, several oncogenic viruses, including human papillomavirus (HPV), Epstein-Barr virus (EBV), human herpesviruses (HHV) 1 and 2, cytomegalovirus (CMV) and Merkel cell polyomavirus (MCPyV), have been implicated in the development, progression, or prognosis. Detection of viral genomes within tumor tissues suggests a complex interplay between viral oncogenes, host immune responses, and environmental factors in carcinogenesis. This systematic review synthesizes current evidence on the contribution of oncogenic viruses to the etiology of OC and OP SCC. Following the PRISMA 2020 guidelines, observational studies between January 2015 and December 2025 were identified through searches of PubMed, Scopus and Web of Science. Eligible studies were limited to human research and reporting viral detections. The results demonstrate a strong, consistent, and clinically relevant association between HPV infection and OP SCC, while evidence supporting a causal or prognostic role for non-HPV oncogenic viruses in OC and OP SCC remains limited and heterogeneous. Substantial methodological variability in viral detection approaches and epidemiological reporting was observed, underscoring the need for standardized assessment frameworks. Overall, these results support site-specific interpretation of viral oncogenesis and inform the development of targeted prevention, diagnostic stratification, and therapeutic strategies.