Distinguishing BK virus nephropathy from T -cell -mediated rejection remains a clinicopathological challenge in kidney transplants, because the 2 entities may share overlapping histological features while requiring opposite therapeutic approaches. We report a kidney transplant recipient with biopsy -proven BK virus nephropathy who later underwent repeat biopsy showing low -level BK virus nephropathy with borderline T -cell -mediated rejection. Despite histological concern for rejection, the broader clinical picture did not support clinically significant T -cell -mediated rejection; specifically, serum creatinine remained stable, BK viral load showed marked improvement, donor -specific antibodies were negative, and there were no vascular or microvascular features of active rejection. A peripheral blood next-generation RNA sequencing gene expression profile yielded a low -risk acute rejection score of 6. Antirejection therapy was deferred, immunosuppression was not escalated, and the patient remained clinically stable. This case highlights the potential adjunctive role of peripheral blood RNA profiling for resolution of histologically ambiguous cases of BK virus nephropathy and borderline T -cell-mediated rejection.
Donor-derived cell-free DNA (dd-cfDNA) is a noninvasive biomarker for allograft injury in kidney transplant recipients. This single-center study aimed to evaluate the clinical indications for dd-cfDNA testing, its association with biopsy findings, and the comparative utility of dd-cfDNA expressed as a percentage versus absolute copy number. A retrospective analysis was conducted of 571 dd-cfDNA measurements from 313 kidney transplant recipients at the Charité Berlin Transplant Center from May 2020 to May 2025. The clinical indications for testing, biopsy, and rejection predictors were assessed using multivariable logistic regression and linear regression. Elevated creatinine (29%), elevated urine albumin-to-creatinine ratio (UACR, 10%), donor-specific antibody positivity (10%), history of rejection (9%), immunosuppression reduction (5%), and combined reasons (38%) were the most frequent indications for dd-cfDNA testing. Elevated UACR was the only independent predictor of biopsy (odds ratio [OR], 3.02; 95% confidence interval [CI], 1.39-6.58; p = 0.005). In the biopsy cohort, the dd-cfDNA percentage (OR, 5.46; 95% CI, 2.89-13.70; p = 0.0035) and copies/mL (OR, 7.05; 95% CI, 1.01-21.09; p = 0.0062) independently predicted rejection. ROC analysis indicated good discrimination of rejection for both copies/mL (AUC, 0.781) and percentage (AUC, 0.820). DCA revealed that both dd-cfDNA formats offered greater net clinical benefits for rejection prediction than the conventional markers. Although biopsy decisions primarily rely on conventional clinical parameters, dd-cfDNA can independently identify rejection and improve decision-making when integrated into multimodal post-transplant monitoring strategies.
Subclinical rejection after liver transplantation precludes immunosuppression withdrawal despite normal clinical function. To better understand the immunobiology of early subclinical rejection, we performed longitudinal, multimodal immune profiling and single-cell RNA sequencing (scRNA-seq) in adult living-donor recipients (n = 13) participating in an interventional immunosuppression withdrawal trial. Samples from patients with subclinical rejection (termed "nonpermissive") at 12 months posttransplant exhibited distinct immune trajectories from those with quiescent ("permissive") allografts despite comparable baseline profiles. Central to these distinct dynamics was biphasic Janus kinase (JAK)-signal transducer and activator of transcription (STAT)-interferon signaling. Greater interferon-stimulated gene (ISG) expression and JAK-STAT activation occurred postreperfusion in permissive allograft recipients, which reversed at 12 months, when elevated ISG expression and JAK-STAT signaling were evident in nonpermissive recipients exhibiting subclinical rejection. We then leveraged our findings to ascertain whether a comparable signature existed in internal and external bulk RNA-seq and scRNA-seq liver transplant cohorts. Across cohorts, including a rodent model, there was a similar elevation in JAK-STAT signaling in instances of allograft rejection. We also detected distinct, portal-based phosphorylated STAT1 staining in a preliminary analysis of biopsies exhibiting histologic rejection compared with nonrejecting controls. Moreover, ruxolitinib-mediated JAK inhibition suppressed alloreactive CD8+ T cell proliferation and inflammatory-mediator production in vitro. Together, these exploratory findings suggest a temporal, biphasic role for JAK-STAT signaling in the regulation and occurrence of T cell-mediated rejection after liver transplant and highlight JAK inhibition as a potential therapeutic strategy.
Kidney transplantation remains the most effective treatment for end-stage renal disease. However, acute rejection after transplantation is a major contributor to mortality and morbidity. This study investigates the role of CXCL10 and its receptor CXCR3 as early, non-invasive predictors of rejection by quantifying their mRNA and protein levels in both blood and urine during the first week post-transplant, addressing the limitations of serum creatinine and biopsy. The study included 65 adult kidney transplant recipients, divided into a rejected group (n = 27) and a non-rejected group (n = 38). Blood and urine samples were collected during the first week after transplantation. mRNA and protein levels of CXCL10 and its receptor CXCR3 were quantified using SYBR Green real-time PCR and ELISA, respectively. Urinary CXCL10 transcript (AUC = 0.8, P = 0.0002), CXCL10 protein (AUC = 0.79, P = 0.007) and CXCR3 transcript (AUC = 0.83, P = 0.0001) showed relatively high accuracy for detecting inflammation in KTRs. When comparing rejected versus non-rejected groups, urinary CXCL10 transcript demonstrated reliable diagnostic accuracy (AUC = 0.74, P = 0.02). By contrast, urinary CXCL10 protein had lower diagnostic performance (AUC = 0.58, P = 0.53). CXCR3 transcript and protein levels were higher in rejected compared with non-rejected KTRs in both blood and urine, but these markers did not reliably distinguish between rejected and non-rejected recipients. Urinary CXCL10 and CXCR3 markers showed rising transcript and protein levels in kidney transplant recipients, correlating with inflammation and suggesting their potential as noninvasive, early biomarkers to predict rejection within the first week post-transplant. While findings support integrating these markers into graft monitoring, standard thresholds require validation before clinical adoption.
Lactate, once regarded merely as a metabolic waste product of glycolysis, has recently emerged as a potent signaling molecule and regulator of cellular function. The discovery of protein lactylation, a novel post-translational modification derived from lactate metabolism, has revealed a critical mechanism linking metabolic activity with epigenetic and functional reprogramming of cells. Both histone and non-histone lactylation serve as integrators of glycolytic flux, modulating gene expression, enzyme activity, and immune responses in diverse physiological and pathological contexts. The immune system is particularly sensitive to these metabolic cues. Accumulation of lactate in hypoxic or inflamed tissues reprograms macrophages, neutrophils, NK cells, and T cells through lactylation, shaping their activation, polarization, and effector functions. In the kidney, which is highly vulnerable to hypoxia and metabolic stress, lactate-driven immune reprogramming has profound consequences. Acute insults such as ischemia-reperfusion injury (IRI) and acute rejection (AR) in transplantation are characterized by glycolytic metabolic reprogramming and lactate accumulation, which in turn influence immune and parenchymal cell behavior. Thus, understanding the role of lactylation and lactic acid-induced immune cell malfunction in renal pathophysiology provides not only mechanistic insight but also potential therapeutic targets for acute kidney injury, chronic kidney disease, and transplant rejection.
Swine leukocyte antigens (SLA) may be a new type of xenoantigen. Previous studies on SLA have primarily focused on their cross-reactivity with HLA. However, the role of SLA in stimulating xenogeneic immune responses after xenotransplantation remains unclear. In our recent kidney xenotransplantation study in rhesus monkeys using GTKO/hCD55 or GTKO/β4GalNT2KO/hCD55/hTBM pigs as donors, 5 of 13 recipients experienced early AMR accompanied by a marked increase in anti-donor pig antibodies. Flow cytometry analysis showed that the terminal sera of these recipients contained significant de novo anti-SLA antibodies, as evidenced by reduced antibody binding to GTKO/SLA-I/II KO pig PBMCs. Using GTKO/β4GalNT2KO pAECs with or without pIFN-γ stimulation as target cells, we found that the binding levels of the terminal sera to pAECs were positively correlated with SLA expression. IP-MS analysis identified multiple SLA class I and class II epitopes targeted by recipient IgG antibodies, with SLA-2 emerging as a dominant immunogenic antigen. In addition, xenogeneic MLR assays revealed that deletion of SLA, particularly SLA class II, markedly attenuated human anti-pig T-cell proliferation. These results demonstrate that induced anti-SLA antibodies can be generated in the early period after pig-to-monkey kidney xenotransplantation and may play a significant role in the development of AMR.
In a heart transplant recipient, a new cutaneous finding can be difficult to attribute to a single cause, and the clinical context does not always point to localized skin disease. We describe the case of a 54-year-old man five years after orthotopic heart transplantation who presented with severe periumbilical pain, hypoxemia, and abdominal wall erythema one week after completing intravenous immunoglobulin and rituximab for biopsy-proven antibody-mediated rejection (AMR). Computed tomography suggested paraumbilical cellulitis and possible pulmonary infection, and empiric antibiotics were appropriately initiated. However, the erythema was non-purulent and non-necrotizing, blood cultures were negative, there was no clear bacterial skin source, the patient's atypical pretransplant ischemic pain phenotype had recurred, and the tacrolimus trough was subtherapeutic at 3.4 ng/mL, together raising concern for ongoing allograft immune injury. This case supports treating apparent cellulitis while urgently considering an extracardiac manifestation of graft rejection when support for a localized infectious source is weak and the transplant context is high risk. Preserved left ventricular ejection fraction does not exclude recurrent AMR or cardiac allograft vasculopathy, and early transplant-center coordination remains the critical management step.
Antibody-mediated rejection (ABMR) is a major cause of chronic allograft dysfunction and graft loss after kidney transplantation (KTx). Daratumumab, antibody targeting the transmembrane glycoprotein CD38 (cluster of differentiation) on immune cells, has shown promise in the treatment of refractory ABMR in adult solid organ transplantation; however, pediatric data are scarce. We report two pediatric cases of refractory ABMR after KTx treated with daratumumab with followed-up protocol biopsies over 10-12 months. Treatment consisted of an induction phase with five weekly doses followed by maintenance dosing every two to four weeks. Donor-specific antibodies (DSAs), kidney function, albuminuria, and histologic response were assessed. The first case, a 10-year-old girl with recurrent ABMR after KTx showed histologic improvement from active ABMR with C4d positivity, glomerulitis, and severe microvascular inflammation to predominantly chronic changes without signs of active rejection. Microvascular inflammation became mild and C4d staining turned negative. The second case, a 9-year-old boy with active and chronic antiglomerular basement membrane disease and severe transplant ABMR glomerulopathy initially showed progression of chronic injury, followed by complete histologic resolution of active disease, disappearance of C4d staining, minimal fibrosis (<1%), and marked reduction of albuminuria after 10 months. Both patients demonstrated reduced DSA levels, stabilization of graft function, and no serious adverse events. Daratumumab was associated with histologic improvement, reduction of microvascular inflammation, and stabilization of graft function in two children with refractory ABMR after KTx. Controlled studies to determine the safety, efficacy, and optimal dosage of daratumumab in children with ABMR are needed.
Post-harvest losses are the most economically damaging phase of quality deterioration in the Egyptian strawberry export corridor. Egypt is the world's leading exporter of individually quick-frozen strawberries and a top-three supplier of fresh strawberries globally, yet substantial losses arise from cold chain failures, suboptimal packaging, and logistical inefficiencies. The objectives of this study are (1) to develop the first ML-based post-harvest loss prediction framework calibrated to Egyptian strawberry export conditions; (2) to benchmark four model architectures (Random Forest, eXtreme gradient boosting [XGBoost], support vector regression/support vector classification [SVC], and long short-term memory [LSTM]) for simultaneous shelf-life estimation, export rejection classification, and loss severity categorization; and (3) to apply SHapley Additive exPlanations (SHAP) explainability analysis to identify the dominant controllable cold chain predictors and translate model outputs into actionable guidance for cold chain operators, packinghouse managers, and export logistics coordinators. The framework was trained on a physics-calibrated synthetic dataset of 800 Egyptian export lots (30 input features, 10 target variables). An ensemble stacking model achieved shelf-life prediction R2 = 0.934 (root mean square error = 0.41 days), export rejection area under the curve-receiver operating characteristic = 0.961 (F1 = 0.862), and loss category quadratic weighted kappa = 0.887. SHAP analysis identified pre-cooling delay, storage temperature, temperature deviation events, and transport duration as the four dominant predictors.
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Kidney transplant recipients (KTRs) disproportionately experience severe COVID-19 infections. We previously identified concern regarding vaccine-induced rejection as a barrier to vaccine uptake. This study assesses COVID-19 vaccine uptake, infection outcomes and allograft rejection in KTRs during the delta and omicron waves (BA.1/BA.2). This cohort study included all adult KTRs with a functioning allograft at 22 March 2021 at our centre. KTR and vaccination-related risk factors for the primary outcome of severe COVID-19 infection (requiring hospitalisation) and the secondary outcomes of death and all COVID-19 infections were assessed using Cox proportional hazards models. Rejection risk following infection/vaccination was also assessed. Of the 986 included KTRs, there were 333 (33.8%) COVID-19 infections, 79 (23.7%) severe infections and 13 deaths (n = 13/333, 3.9%). Vaccine number was the most significant modifiable predictor of severe infection (adjusted hazards ratio, per additional dose, 0.5; 95% confidence interval, 0.40-0.65, P < 0.001) and death. While older age also predicted severe infection and death, lower estimated glomerular filtration rate, history of diabetes and previous allograft rejection predicted severe infection. Male gender was the only predictor of all infections. Mycophenolate dose, prednisolone use and vaccine type did not predict infection or severe disease. Infections and vaccinations did not increase the risk of rejection. KTRs should be encouraged to be optimally vaccinated to prevent severe disease and can be reassured about the low risk of allograft rejection. Risk factors that compound the state of immunocompromise and severe COVID-19 infections should prompt vigilance and targeted interventions to mitigate these risks.
Spousal living donor kidney transplant is commonly performed; however, the optimal induction immunosuppression strategy in recipients with low immunological risk remains unclear. We compared the early clinical and immunological outcomes of low -dose rabbit anti -thymocyte globulin and basiliximab in low -risk spousal kidney transplant recipients. We retrospectively analyzed 43 recipients with low immunological risk who underwent spousal living donor kidney transplant and received induction therapy with either rabbit anti -thymocyte globulin (n = 20 ) or basiliximab (n = 23 ). Low immunological risk was defined as first -time transplant with negative panel reactive antibody and negative complement -dependent cytotoxicity crossmatch. Primary endpoints were early graft function, as assessed by serum creatinine trends, and biopsy -proven acute rejection within the first 2 postoperative months. Secondary endpoints included perioperative lymphocyte dynamics and documented infections, including cytomegalovirus and urinary tract infections. Peripheral blood lymphocyte counts were significantly lower in the group treated with rabbit anti -thymocyte globulin on postoperative day 1 and day 7 versus the basiliximab -treated group (P < .001 ). In contrast, no significant differences were observed between groups in early graft function trajectories (P = .71 ) or the incidence of biopsy-proven acute rejection (rabbit anti -thymocyte globulin, 3 of 20; vs basiliximab, 2 of 23; P > .05 ). All documented urinary tract infections (n =3 ) and the single cytomegalovirus infection occurred in the group treated with rabbit anti -thymocyte globulin; however, these differences were not statistically significant (P > .05 ). In spousal kidney transplant recipients with low immunological risk, low -dose rabbit anti -thymocyte globulin (4.5 mg /kg ) and basiliximab provide comparable early graft function and similar protection against acute rejection. Although rabbit anti -thymocyte globulin induces more pronounced lymphocyte depletion, this observation does not translate into superior early clinical outcomes in this patient population.
Penile injury induces rapid structural disruption of the corpus cavernosum, ultimately culminating in erectile dysfunction (ED). Owing to the inherently limited regenerative capacity of injured tissue, current therapies fail to restore both structure and function. To address this unmet clinical need, we employed 3D printing technology to fabricate a hydrogel-based biomimetic penile corpus cavernosum with a sinusoidal architecture. It can simulate the penile erection process under fluid pressure in vitro. This construct was functionally modified by seeding with porcine umbilical cord-derived mesenchymal stem cells (MSCs), and its therapeutic potential was systematically evaluated in a preclinical porcine model of corpus cavernosum defects. The treatment brought about the relative improvement in reproductive performance compared with defect control groups. Mechanistically, MSCs accelerate hydrogel degradation to support tissue integration and facilitate nearly complete structural regeneration of damaged cavernous sinuses. Single-cell RNA sequencing (scRNA-seq) revealed a multifaceted regulatory network in which MSCs enhance the terminal differentiation of endothelial cells (ECs) to rebuild functional vascular networks, mitigate cluster of differentiation 4-positive (CD4+) T-cell-induced endothelial-to-mesenchymal transition (EndMT) by reducing transforming growth factor-beta (TGF-β) secretion, and reprogram the immune microenvironment, specifically by activating anti-inflammatory interleukin-10 (IL-10) signaling in M1 macrophages and downregulating prorejection pathways in M2 macrophages. Collectively, these effects inhibit excessive inflammation and attenuate graft rejection. These findings not only deepen our mechanistic understanding of MSC-based interventions for ED secondary to penile injury but also establish a novel, more efficient therapeutic strategy for penile reconstruction to restore the reproductive capability of males.
The tumour microenvironment (TME) influences breast cancer progression and treatment response. We investigated whether TME composition predicts tamoxifen benefit in postmenopausal women with oestrogen receptor-positive, HER2-negative (ER+HER2-) breast cancer. This study included 513 patients from the Stockholm Tamoxifen (STO-3) trial, which randomised postmenopausal, lymph node-negative women to tamoxifen or no endocrine therapy. Bulk tumour transcriptomes were deconvoluted with the ConsensusTME algorithm to estimate the relative abundance of 18 immune and stromal cell types. A summary score of combined immune cells was created on a per patient basis and evaluated alongside fibroblast and endothelial stromal compartments. Patients were categorised into immune and stromal tertiles on the basis of these scores. Associations between TME composition and tumour characteristics were evaluated using Spearman correlations and Fisher's exact test. Tamoxifen benefit was analysed by univariable Kaplan-Meier (log-rank) and multivariable Cox proportional hazards adjusting for age, tumour size, grade, progesterone receptor, Ki-67, and radiotherapy. Differential expression was assessed with limma and pathway enrichment with fgsea using Hallmark gene sets from MSigDB. Low immune abundance was significantly associated with higher ER expression (Kruskal-Wallis test p < 0.001). Among tamoxifen-treated patients, those with low immune scores showed improved distant recurrence-free interval (DRFI) relative to untreated patients (log-rank p < 0.001). Similarly, intermediate endothelial (p < 0.001) and low/intermediate fibroblast abundances (p = 0.042, p = 0.009) were associated with favourable DRFI. In multivariable models, low immune (adjusted HR = 0.17, 95% CI 0.08-0.40), intermediate endothelial (aHR = 0.21, 95% CI 0.09-0.51), and low/intermediate fibroblast tertiles (aHR = 0.50, 95% CI 0.27-0.93; aHR = 0.36, 95% CI 0.17-0.77) retained significance. Transcriptomic analysis revealed enrichment of oestrogen-response, MYC-target, and oxidative-phosphorylation pathways in low-immune and low-fibroblast tumours, while interferon-γ response and allograft rejection pathways were downregulated. TME composition modulates tamoxifen benefit in postmenopausal ER+HER2- breast cancer. Low immune, intermediate endothelial, and low/intermediate fibroblast abundances are associated with improved benefit from tamoxifen, suggesting that both immune and stromal compartments influence endocrine treatment efficacy.
Tacrolimus trough concentrations are essential after kidney transplantation but do not describe the dose required to achieve them. The concentration-to-dose (C/D) ratio adds this denominator and provides a low-cost, time-dependent dose-requirement phenotype from routine data. Low C/D identifies recipients with high dose requirements and is associated with impaired kidney function, rejection, BK virus complications, graft failure, and mortality. Large registry data show graded risk as C/D decreases and more favorable outcomes when low C/D normalizes than when it remains low. C/D can therefore support risk stratification and selective AUC-based exposure assessment, but it is not a stand-alone treatment algorithm: interpretation requires valid sampling, assay, timing, formulation, and clinical context, and outcome benefit from C/D-guided intervention remains unproven.
Macrophage inflammatory plasticity is a central determinant of immune-mediated tissue injury, yet the intrinsic checkpoint mechanisms that restrain inflammatory macrophage programming remain incompletely defined. Here, using acute cardiac allograft rejection as an alloimmune tissue-injury model, we found that Vsir, encoding V-domain Ig suppressor of T-cell activation (VISTA), was preferentially expressed in graft macrophages but declined as macrophages acquired inflammatory and antigen-presenting states. Single-cell RNA sequencing and pseudotime analysis revealed that VISTA downregulation accompanied macrophage progression from reparative/resident-like states toward inflammatory programs. In vitro, VISTA overexpression intrinsically restrained pro-inflammatory macrophage polarization, reduced inflammatory cytokine expression, and limited the induction of antigen-presenting molecules including MHC-II and CD80, thereby attenuating macrophage-driven CD4+ T-cell proliferation. Integrative CUT&Tag and RNA-seq analyses further showed that VISTA overexpression was associated with reduced H3K4me3 -associated chromatin remodeling at inflammatory regulatory loci, including TRAF5 and CHDH, together with transcriptional repression of NF-κB-, TNF-, MAPK-, and IL-17-associated inflammatory programs. In vivo F4/80 promoter-directed Vsir restoration shifted intragraft macrophages away from inflammatory polarization, reduced T-cell accumulation, and attenuated early rejection-associated tissue injury. Together, these findings identify VISTA as an intrinsic regulator of macrophage inflammatory programming and point to H3K4me3-associated chromatin remodeling as a regulatory layer linked to VISTA-mediated inflammatory restraint.
Artificial intelligence (AI)-based computer-aided diagnosis (CADx) systems are increasingly used to classify colorectal polyps during colonoscopy, but their impact depends on how endoscopists interpret AI predictions. This study aimed to evaluate endoscopists' decision-making after reviewing CADx outputs, using histopathology diagnosis as ground truth. We performed a secondary analysis of a prospective study (NCT06822816) conducted at our centre between 2022 and 2025. All patients and polyps ≤10 mm with documented CADx output and endoscopist diagnosis were included. Endoscopists either accepted CADx classifications (neoplastic vs hyperplastic) or provided alternatives. Our primary outcome was the accuracy of accepted CADx diagnoses; secondary outcomes assessed reclassification patterns after CADx output rejection. Our cohort included 754 polyps <10 mm from 367 patients. Accepted CADx diagnoses were correct for 89.1% of neoplastic and 68.7% of hyperplastic CADx predictions. Endoscopists accepted incorrect CADx classifications more frequently when these were neoplastic compared to hyperplastic (64.9% vs 26.5%). Upon rejecting correct CADx neoplastic predictions, endoscopists misclassified 23.4% as sessile serrated lesions (SSLs). Similarly, 29.5% of rejected correct CADx hyperplastic predictions were incorrectly reassigned as SSLs. Endoscopists exhibit a diagnostic bias toward neoplasia, particularly misclassifying hyperplastic polyps as SSLs. While this may enhance safety, it may risk unnecessary surveillance colonoscopies and increase costs.
Child sexual abuse (CSA) is a major public health concern, yet rigorously evaluated and fully manualized primary prevention programs for preschool children remain scarce. Evidence on the feasibility and acceptability of such interventions among young children and their educators is limited. This study assessed the acceptability, safety, and feasibility of the CARE Program, a CSA primary prevention intervention for preschool children in Portugal and explored changes in CSA-related knowledge. Participants were 195 preschool children aged 3 to 6 years from five schools and five educators from three schools in northern and central Portugal. A pragmatic, parallel-arm, waitlist-controlled mixed-methods design was used. Quantitative pre-post assessments examined children's CSA-related knowledge using a vignette-based instrument and evaluated program acceptability. Qualitative interviews with educators explored feasibility, safety, and perceived impact through framework analysis. The CARE Program demonstrated high levels of acceptability and safety among both children and educators. Feasibility indicators were favorable, with strong adherence and retention, although recruitment at the school level proved challenging. Preliminary findings indicated significant improvements among children in the intervention group in emotional recognition and rejection of secrecy, whereas no comparable changes were observed in the control group. Findings related to disclosure were less consistent across analytic approaches and should be interpreted with caution. The CARE Program appears to be a safe, acceptable, and feasible primary prevention strategy for preschool children in Portugal. Preliminary evidence supports its potential to improve CSA-related knowledge and justifies further evaluation in a fully powered cluster randomized controlled trial.
Xenotransplantation offers a promising solution to the shortage of human organs for transplantation but requires overcoming numerous immune responses-particularly those mediated by the innate immune system through natural killer (NK) cells and macrophages. This review examines advances demonstrating that the expression of human leukocyte antigen E (HLA-E) on porcine cells contributes to reduce cellular xenograft rejection. HLA-E expression partially inhibits both direct NK cell cytotoxicity and antibody-dependent cellular cytotoxicity (ADCC), while also attenuating macrophage-mediated lysis. Furthermore, perfusion of transgenic porcine organs expressing HLA-E with human blood resulted in significantly less tissue damage compared to wild-type counterparts, thereby confirming the protective effect of HLA-E against innate immunity. Inhibition of NK cell activation can be further enhanced by co-expression of HLA-G and HLA-E. These findings confirm the potential of HLA-E and HLA-G expression as a complementary strategy in the design of immune-compatible porcine organs for clinical xenotransplantation.
Stem cell spheroids, as an advanced platform in cell therapy, have shown great potential for improving therapeutic efficacy through enhanced cell-cell and cell-microenvironment interactions. Meanwhile, zinc-based biomaterials are attracting increasing attention for their biological functions. However, their roles in modulating stem cell spheroids remain largely unclear. In this study, we successfully synthesized zinc-doped urchin-like hydroxyapatite (Zn-uHA) via a hydrothermal method and verified its long-term antibacterial activity. Utilizing microwell array technology, we conducted 3D cell culture of human dental pulp stem cells (hDPSCs) with Zn-uHA, thereby achieving high-throughput and precise fabrication of 3D hDPSCs/Zn-uHA hybrid stem cell spheroids. Compared with pure cell spheroids, hDPSCs/Zn3-uHA hybrid spheroids demonstrated significantly enhanced cell migration and osteogenic differentiation, with osteogenesis-related gene expression upregulated 2.7-fold. In vivo studies further confirmed that the hybrid cell spheroids exhibited favorable biocompatibility, with no evident inflammatory response or immune rejection, and enabled prolonged retention at the implantation site. Additionally, they significantly promoted neovascularization and new bone formation. These findings suggest that Zn-uHA-integrated stem cell spheroids represent a promising strategy for enhancing both antibacterial and osteogenic performance, offering potential applications in bone tissue engineering, particularly in infection-prone clinical scenarios such as orthopedics and dentistry.