IgG4 monoclonal anti-CD47 for cancer immunotherapy resulted in anemia necessitating red cell (RBC) transfusions. Although the antibody readily binds to CD47 on RBCs, the mechanism underlying the anemia associated with IgG4 is poorly understood. We investigated samples from patients receiving Hu5F9-G4 (magrolimab) with positive reactivity in compatibility tests using a monocyte-monolayer assay (MMA). For RBC opsonization, patient plasma was diluted to avoid direct agglutination and to give a 4+ indirect antiglobulin test (IAT) and >103 mean fluorescent intensity (MFI) by flow cytometry. Fcγ receptor blocking used F(ab')2 antibodies. Antibody-dependent cellular cytotoxicity (ADCC) used purified natural killer (NK) cells. Recombinant IgG4 anti-K and polyclonal anti-D were tested by MMA concurrent with blocking of CD47 with a deglycosylated antibody. Five of six samples showed significant erythrophagocytosis (phagocytosis index [PI] = 20-60; PI > 5 clinically significant) with the exception having 30-fold lower MFI. Phagocytosis was not increased in the presence of complement. Fcγ receptor blocking showed FcγRI and FcγRIIa were involved but not FcγRIIIa, supported by negative results when tested for NK ADCC. IgG4 anti-K also showed significant erythrophagocytosis (PI = 30). Blocking of CD47 increased phagocytosis, 1.7 to 3.9-fold, respectively, of RBCs opsonized with IgG4 anti-K or polyclonal anti-D. IgG4 RBC antibodies, generally not considered clinically significant for transfusion, can result in erythrophagocytosis providing an explanation for anemia related to anti-CD47 mediated by FcγRI and FcγRIIa. Enhanced phagocytosis of alloantibody-coated RBCs with concurrent blocking CD47 suggests potential for enhanced complications for patients who develop RBC alloantibodies while receiving cancer therapies targeting CD47.

Retrospective Study on Simultaneous Liver-Kidney Transplantation: Renal Outcome and Prevalence of Acute Rejection.

High concentration of human platelet antigen-1a (HPA-1a) antibodies is reported to be associated with severe foetal and neonatal alloimmune thrombocytopaenia (FNAIT). The gold standard for quantification of anti-HPA-1a antibodies is the monoclonal antibody immobilization of platelet antigen (MAIPA) assay, which is a laborious method performed in only a few reference laboratories. The aim of this study was to evaluate the performance of the commercially available bead-based Luminex assay PakLx (Immucor) for quantitative measurement of anti-HPA-1a antibodies. We analysed anti-HPA-1a antibody levels in plasma samples from 42 HPA-1a-negative women who had given birth to a child with thrombocytopaenia. Quantification of antibodies was performed with two different techniques: MAIPA analysed by spectrophotometry with results expressed in international units (IU)/mL, and PakLx analysed in the Luminex assay with results expressed as the mean fluorescence intensity (MFI). In the comparison of the two methods' ability to stratify a result as either positive or negative, PakLx demonstrated 97.6% agreement with the MAIPA assay, with positive and negative predictive values of 96.7% and 100%, respectively. The correlation of the MFI values from PakLx with IU/mL in MAIPA assay was high, with a correlation coefficient (R2) of 0.92. MFI values were converted into semi-quantitative results: high, intermediate and low levels of anti-HPA-1a. PakLx shows high agreement with the MAIPA assay and allows fast laboratory turnaround time for the determination of anti-HPA-1a antibody levels. The result may be of predictive value in clinical assessments.

Mechanistic Insights into Yunpi-Xiefei-Huatan decoction effects on autophagy and MUC5AC secretion in rat tracheal epithelial cells via the TGF-β3/Smad2/Smad3 pathway.

In partially matched Stem Cell Transplantation (SCT), the presence of Donor-Specific Anti-HLA antibodies (DSAs) is a critical factor contributing to graft rejection. This is particularly challenging for patients with restricted donor availability who need rapid access to transplantation. Managing DSAs before transplantation is essential to improve engraftment success and overall transplant outcomes in these high-risk individuals. The use of partially HLA-mismatched donors is increasingly favoured in transplantation, particularly in cases where fully matched donors are scarce or when time-sensitive procedures are required. However, the presence of DSAs has emerged as a major barrier to effective engraftment, posing a threat to transplant viability. This case describes the application of a desensitisation protocol in a highly sensitised patient with DSAs exceeding 5000 Mean Fluorescence Intensity (MFI). The approach included alternate-day Plasma Exchange (PLEX), rituximab and Intravenous Immunoglobulin (IVIg), all administered before proceeding with a partially matched stem cell transplant. Post-transplant, neutrophil engraftment was achieved on day 17 and platelet engraftment on day 23, both slightly delayed relative to expected norms. Other than mild to moderate gastrointestinal and febrile symptoms, which were managed medically, no acute complications such as primary graft failure or Graft-versusHost Disease (GvHD) were observed. At six-month follow-up, the patient demonstrated stable trilineage haematopoiesis with no evidence of relapse, graft failure, or chronic GvHD, highlighting the potential utility of desensitisation in overcoming DSA-mediated barriers to successful transplantation

Safety and Tolerability of Tocilizumab in a Case Series of Heart Transplant Recipients With Chronic Antibody-Mediated Rejection.

Lipid nanoparticles (LNPs) have emerged as a promising nonviral nucleic acid delivery platform for clinical use. To expand LNPs as a treatment option in nonhepatic-based diseases, LNPs surface coated with targeting moieties produce a precise modular delivery method that can bind to and be internalized by specific receptors expressed on target cells. This study showcases the tetrazine-trans-cyclooctene inverse electron-demand Diels-Alder click reaction and directly compares its performance with the widely employed thiol-maleimide conjugation. We also compare direct mixing and micelle mixing insertion methods under different conditions to determine the optimal formulation to produce targeted LNPs. For thiol-maleimide chemistry, monoclonal antibody cetuximab was modified with N-succinimidyl S-acetylthioacetate, followed by reaction with either 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[maleimide(polyethylene glycol)-2000] (DSPE-PEG2000-maleimide) directly or in preformed DSPE-PEG2000-maleimide: 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG2000) micelles. For clickable tetrazine chemistry, cetuximab was modified with 2,5-dioxo-1-pyrrolidinyl 5-[4-(1,2,4,5-tetrazin-3-yl)benzylamino]-5-oxopentanoate, followed by reaction with either 1,2-Distearoyl-sn-glycero-3-PE-polyethylene glycol-2000- trans-cyclooctene (DSPE-PEG2000-TCO) directly or preformed DSPE-PEG2000-TCO:DMG-PEG2000 micelles. LNPs were prepared by mixing lipids in ethanol and mRNA (10 mM citrate buffer, pH 3.5) at a ratio of 3:1 (v/v). Insertion was carried out by combining either the direct conjugate solution or the micelle solution with the LNPs and mixing at 60 °C for 1 h. Only the micelle mixing method produced stable particles with mRNA encapsulation above 80%. The LNPs were found to be stable up to 3 weeks (stored at 4 °C) as indicated by the RiboGreen assay and particle sizes. Targeted LNPs displayed relatively weak and neutral zeta potential. In vitro studies revealed enhanced cellular uptake of Green Fluorescent Protein (GFP) mRNA by targeted LNPs with similar transfection rates with thiol-maleimide and tetrazine-TCO chemistries showing around 97% GFP+ cells, compared to less than 30% in control groups at 6 h. Both targeted LNP formulations showed a significant rise in mean fluorescence intensity, achieving at least a 3-fold increase over the controls.

Accurate B-lineage assignment in acute leukemia is critical for therapeutic decisions. While nuclear expression of PAX5 protein (nPAX5) is a highly reliable marker for B-cell differentiation, its conventional assessment by time-consuming immunohistochemistry (IHC) is not ideal for rapid diagnosis. This study aimed to validate the utility of flow cytometric (FCM) detection of nPAX5 for the diagnosis of B-lymphoblastic leukemia (B-ALL), potentially enhancing diagnostic efficiency. A retrospective study was conducted, comparing nPAX5 expression by FCM and IHC in 125 bone marrow biopsies (57B-ALL, 12T-ALL, 56 AML). Further diagnostic performance analysis, using ROC analysis of the geometric mean fluorescence intensity ratio (Blast/Normal T-cell), was performed for nPAX5, cCD22, and cCD79a across a larger cohort of 538 acute leukemia cases (123B-ALL, 29T-ALL, 386AML). FCM and IHC for PAX5 expression showed 100 % concordance across the 125 cases (57/57B-ALL positive; 0/12T-ALL negative). The single PAX5-positive AML case harbored the t(8;21) translocation. ROC analysis demonstrated excellent diagnostic performance for both cCD79a (AUC 0.980) and nPAX5 (AUC 0.955). At optimal criteria, cCD79a achieved 100.00 % specificity and 91.06 % sensitivity. nPAX5 showed strong utility, matching the 91.06 % sensitivity with 89.45 % specificity (criterion >6.30). cCD22 exhibited moderate discriminatory power (AUC 0.807). Flow cytometric nuclear PAX5 (nPAX5) demonstrated 100 % agreement with IHC and exhibited excellent diagnostic accuracy (AUC 0.955, 91.06 % sensitivity). These compelling results validate nPAX5 as a reliable and powerful FCM marker, supporting its immediate adoption for rapid and efficient B-lineage assignment in acute leukemia diagnostics.

Determination of unacceptable human leukocyte antigen (HLA) mismatches (UAM) before kidney transplantation (KT) aims at minimizing immunological risk and routinely involves Luminex single antigen bead (SAB) testing. SAB-UAM criteria, however, often lack standardization. We implemented standardized mean fluorescence intensity (MFI)-based SAB-UAM criteria in four German transplant centers and prospectively studied the consequences on waitlist composition as well as waiting time, early antibody-mediated rejection (AMR) and graft loss in 267 patients. HLA were deemed unacceptable in case of CDC-reactivity or antibodies against known HLA from previous transplants irrespective of MFI. For all other antibodies, the MFI cut-off was 5.000 with the exception of 10.000 for anti-HLA DQ. We observed significant accumulation of highly sensitized patients (virtual panel-reactivity >95%) on the waiting list during the study period. Median time to KT was longer in patients with UAM, but differences were not statistically significant. Patients with preformed donor-specific anti-HLA antibodies (DSA) below the UAM cut-off criteria (39/267) experienced more AMR episodes compared to DSA-negative patients (10.3% vs. 1.3%, p < 0.001). Graft survival, however, was not statistically different over a median follow-up of four years. Standardized SAB-UAM criteria associated with good short-term outcomes but resulted in accumulation of highly sensitized patients on the waiting list.

BackgroundZika virus (ZIKV), dengue virus (DENV) and chikungunya virus (CHIKV) are arboviruses transmitted by Aedes mosquitoes. While DENV serotypes 1 and 2 (DENV1 and DENV2) are the most prevalent in Guatemala, serotypes 3 and 4 (DENV3 and DENV4) have caused recent epidemics. In 2024, a public health emergency was declared by the Ministry of Health due to the rise in dengue cases. CHIKV and ZIKV outbreaks were reported between 2014-2017.Evolution of flavivirus serostatus among the AGRI cohort (2020-2022)The graph shows the serologic characteristics of the AGRI cohort against flavivirus infections. A rise in the multytipic DENV serostatus is observed over the years. On the contrary, there is a decline in the flavivirus-naive population.Density distribution of the MFI values against arbovirus-specific antigensThe MFI of the antigen-antibody reaction was recorded for each arbovirus. The evolution of cohort-level mean fluorescence intensity (MFI) readings over 3 years is shown. Positivity cutoffs are indicated by the dashed red line.MethodsMean Fluorescence Intensity (MFI) from anti-CHIKV/ZIKV/DENV1-4 antibodies was obtained using an IgG-based multiplex bead assay. Flavivirus-specific beads were coupled with the EDIII protein of ZIKV/DENV1-4. CHIKV beads were coupled with CHIKV E2 protein. We tested 1136 samples from 478 individuals, collected in 2020 (n=478), 2021 (n=338) and 2022 (n=320). A set of 15 samples from children born between 2018-2022 were used as regionally appropriate samples to calculate the positivity cutoffs (mean MFI + 3SD). A previously described algorithm for analyzing flavivirus MFI signals was used to discriminate between naive samples and primary/multitypic infections.ResultsCHIKV seropositivity was 56.9%, 51.5% and 53.4% for 2020, 2021 and 2022. During the follow up, 12 individuals had a negative-to-positive seroconversion against CHIKV. For 2020, 2021, and 2022, we detected seropositivity for primary infections of DENV1 (16.9%, 9.17%, 10.6%), DENV2 (9.41%, 21.0%, 11.2%), DENV3 (1.88%, 0.30%, 0.00%) and ZIKV (11.9%, 6.51%, 4.38%). Naïve-to-primary seroconversions were found for DENV1 in 2022 (n=12), for DENV2 in 2021 (n=14) and 2022 (n=10), and for ZIKV in 2021 (n=1) and 2022 (n=3). Flavivirus-naive prevalence went from 15.7% in 2020 to 7.8% in 2022.ConclusionThough confirmatory testing is needed, these findings suggest that active DENV1/2, ZIKV and CHIKV transmission was ongoing in the region for this period.DisclosuresBlair Weikel, MPH, Merck: Grant/Research Support Molly Lamb, PhD, Merck: Grant/Research Support Daniel Olson, MD, Fundacion para la Salud Integral de los Guatemaltecos: Board Member|Merck: Grant/Research Support|Roche Diagnostics: Grant/Research Support

Background/Objectives: While standard Luminex single antigen bead (SAB) detects total IgG antibodies, qualitative differences among IgG subclasses may influence their immunologic risk. In particular, complement fixing ability, assessed via C1q binding, is linked to poor transplant outcomes. This study aimed to evaluate the relationship between IgG subclasses and C1q-binding activity in HLA antibodies and to define clinically relevant subclass-specific mean fluorescence intensity (MFI) thresholds for predicting complement binding. Methods: We analyzed 4189 HLA IgG bead reactions from sera of 37 kidney transplant recipients using SAB assays for total IgG, IgG1-4 subclasses, and C1q-binding. IgG subclasses were assessed using a modified SAB assay with subclass-specific monoclonal secondary antibodies. Results: IgG reactivity (MFI ≥ 1000) was observed in 15.3% of beads (639/4189), with 31.0% (198/639) also positive for C1q binding. IgG+C1q+ beads exhibited significantly higher MFIs compared with IgG+C1q− beads. IgG1 showed positive correlations with both total IgG (rs = 0.5439, p < 0.0001) and C1q MFIs (rs = 0.4042, p < 0.0001), with the strongest correlations at HLA-DQ. Among subclass-positive beads, IgG1 predominated and was strongly associated with C1q binding, whereas isolated IgG2 or IgG4 positivity was rarely C1q-binding. ROC analysis identified an IgG1 MFI threshold of >837 to predict C1q positivity with 73.2% sensitivity and 92.3% specificity, while the cutoff for total IgG MFI was >7881 with 85.4% sensitivity and 88.9% specificity. At the patient level, IgG1-positive immunodominant DSAs were more frequent in antibody-mediated rejection than in non-rejection biopsies Conclusions: IgG1 predominates among complement-fixing antibodies and correlates strongly with total IgG and C1q binding. Quantitative IgG subclass assessment, especially IgG1, may serve as a useful predictor of complement activation.

Objectives: Adenoviruses (Ads) are among the most used vectors for gene therapy; human Ad serotype 5-derived (HuAd5) vectors are the most frequently used for gene transfer applications. However, Ad5 infection is endemic in humans, and 20% of the Western population has neutralizing antibodies (nAbs). Pre-existing immunity against HuAd5 represents a major issue for many gene therapy applications. In our study, we evaluated several Ad serotypes derived from chimpanzees (ChAds) in vitro and in vivo to assess their transduction efficiency in various cell types and tissues. We aimed at identifying Ad serotypes able either to transduce "challenging" cell types or to represent a possible alternative to Ad5-derived vectors with comparable infectivity and tropism. Methods: We evaluated the efficacy of transduction of twelve ChAds vectors expressing enhanced green fluorescent protein (EGFP) in human embryonic kidney cells, as well as human leukemic and human mesenchymal stem cells, using flow cytometry to determine the percentage of EGFP-expressing cells and their mean fluorescent intensity (MFI). We observed the highest transduction efficiency in the serotype CV1 ChAd; therefore, we proceeded to evaluate toxicity and biodistribution in vivo. Results: After in vitro evaluation of twelve ChAds serotypes, we observed that the CV1 serotype was the most efficient in transducing both leukemia cell lines (HL-60 and NB-4) and human mesenchymal stem cells. Furthermore, in vivo analysis of the CV1 serotype induced an inflammatory reaction similar to what was observed after HuAd5 administration. Conclusions: ChAds vectors represent an effective alternative for the transduction of cells resistant to HuAd5 infection, such as mesenchymal stem cells and leukemic cells. In addition, we observed that the CV1 ChAd serotype presented a transduction profile similar to HuAd5 in vitro and induced a similar inflammatory response in vivo; therefore, CV1 ChAd-derived vectors represent an interesting alternative for gene therapy applications.

Optimizing stereotaxic injection strategy for AAV-mediated corticospinal tract tracing in mice.

Dual targeting of B cell compartments using belatacept and daratumumab in highly sensitized kidney transplant candidates: A mechanistic proof-of-concept desensitization trial.

Physiological response of Ammonia confertitesta (phylotype T6) to oil-amended sediments: Evidence from confocal microscopy.

Regenerative medicine increasingly relies on stem cell-based therapies, yet their clinical success is largely determined by immunological compatibility. Fetal liver-derived progenitor and stem cells represent a promising, yet insufficiently characterized, source for transplantation. This study aimed to evaluate the immunogenicity of fetal liver cells by analyzing the expression of HLA class I and II molecules and detecting the presence of anti-HLA antibodies after cryopreservation at early gestational ages. Cell suspensions were obtained from fetal livers at 5-12 weeks of gestation. Flow cytometry was performed using a CyFlow Space cytometer (Sysmex, Germany), and results were analyzed with Statistica 10. HLA class I (HLA-ABC) and class II (HLA-DR/DP/DQ) expression was quantified as the percentage of positive cells and their mean fluorescence intensity. Anti-HLA antibodies were assessed in the cell suspensions. Statistical analysis included descriptive statistics, group comparisons, and correlation analyses with gestational age. HLA class I expression was consistently detectable across all samples. In contrast, HLA class II expression increased progressively with gestational age, reflecting the developmental maturation of the fetal immune system. Importantly, these antigens primarily indicated differentiation toward myeloid lineages, which are unlikely to provoke graft-versus-host immune reactions. Anti-HLA antibodies were not detected in any of the analyzed suspensions. This study provides the first systematic assessment of HLA expression in fetal liver-derived progenitor and stem cells following cryopreservation. The results suggest that these cells retain a favorable immunological profile, supporting their potential application in regenerative medicine. The findings highlight the importance of considering gestational age in evaluating immunogenicity. Further studies with larger sample sizes and in vivo validation are required to confirm clinical safety.

IRF4 exacerbates pulmonary inflammation in bronchopulmonary dysplasia mice model by regulating macrophage polarization and phagocytosis.

Clear-positive cut off determination of flow cytometric MOG-antibody assay in Korea: Alignment with international MOGAD diagnostic standards.

To assess the effect of AAD-2004 on spinal cord injury (SCI) and to explore its mechanism, we employed an in vitro model using OGD/R-challenged astrocytes to investigate the effects of AAD-2004 against cell death (terminal deoxynucleotidyl transferase dUTP nick-end labeling, tunel), oxidative stress (H2O2 level), and the expression of the key neuroprotective factor MAP2.AAD-2004[2-hydroxy-5-[2-(4-trifluoromethylphenyl)-ethylaminobenzoic acid] is a hydrogen peroxide(H2O2) scavenger primarily used for the treatment of amyotrophic lateral sclerosis and Alzheimer disease that has demonstrated certain neuroprotective properties. In parallel, modified allen’s method was adopted, further exploring the potential molecular mechanism in vivo. Based on these conditions, histological and behavioral analysis were performed by Nissl staining, basso mouse scale and footprint analysis. The level of molecules associated with glial scar formation, nerve regeneration, axonal regeneration and H2O2 level were analyzed using western blot, immunofluorescence staining and H2O2 kit. AAD-2004 significantly improved the movement function after SCI and inhibited the proliferation of astrocytes, thus preventing the formation of glial scar by inhibiting of H2O2. At the same time, AAD-2004 promoted nerve regeneration, and the effect was due to neuronal regeneration and axonal regeneration pathways. The expression levels of GFAP and vimentin were significantly downregulated in AAD-2004-treated, and the expression level of Ki67 and PH3 were downregulated. The mean fluorescence intensity of neuronal regeneration (Neun+and MAP2+) and axonal regeneration-related (NF+ and GAP43+) were significantly upregulated after AAD-2004 treatment. Scavenging H2O2 level is a viable therapeutic strategy, and that AAD-2004 is prospective, and that scavenging H2O2 facilitated nerve regeneration and inhibited glial scar formation for SCI.Supplementary InformationThe online version contains supplementary material available at 10.1038/s41598-025-33322-x.

Study DesignPilot randomized controlled trial.ObjectivesIatrogenic nerve injury is a major complication in endoscopic spinal surgery, potentially causing serious neurological deficits. Near-infrared (NIR) fluorescence imaging with indocyanine green (ICG) has shown promise for intraoperative nerve root identification. This study assessed the feasibility, optimal dosing, safety, and mechanism of ICG fluorescence for nerve root visualization, transitioning from preclinical to clinical phases.MethodsIn the preclinical phase, 36 rabbits were assigned to ICG dose groups (1.4, 2.8, or 5.5mg/kg, corresponding to 0.5, 1, or 2mg/kg in humans) and observation times (3, 6, 12, or 24 hours). Fluorescence signals in lumbar nerve roots were quantified by signal-to-background ratio (SBR) and mean fluorescence intensity (MFI). Histological analyses explored ICG retention mechanisms. In the clinical phase, 40 patients undergoing unilateral biportal endoscopic surgery for lumbar disc herniation were randomized into different ICG dose groups (0, 0.5, 1, or 2mg/kg), administered 1.5 hours preoperatively. Intraoperative fluorescence parameters, nerve root identification time, and perioperative outcomes (VAS and ODI scores) were assessed.ResultsIn preclinical studies, the 2.8 and 5.5mg/kg groups showed peak SBR and MFI at 3 hours post-injection. Histology revealed ICG accumulation in nerve root microvascular regions. In the clinical study, the 2mg/kg group had the highest SBR and MFI, reducing nerve root identification time without significant adverse events.ConclusionICG fluorescence imaging is a feasible and safe technique for intraoperative nerve root visualization, with ICG accumulation attributed to the enhanced permeability and retention effect.