Human Blood Research Articles

The Influence of Nanoparticles of Graphene Oxide‑PEG on Cytokine Profile of Monocytes from Human Blood In Vitro

The Influence of Nanoparticles of Graphene Oxide‑PEG on Cytokine Profile of Monocytes from Human Blood In Vitro

High-resolution, noninvasive single-cell lineage tracing in mice and humans based on DNA methylation epimutations.

In vivo lineage tracing holds great potential to reveal fundamental principles of tissue development and homeostasis. However, current lineage tracing in humans relies on extremely rare somatic mutations, which has limited temporal resolution and lineage accuracy. Here, we developed a generic lineage-tracing tool based on frequent epimutations on DNA methylation, enabled by our computational method MethylTree. Using single-cell genome-wide DNA methylation datasets with known lineage and phenotypic labels, MethylTree reconstructed lineage histories at nearly 100% accuracy across different cell types, developmental stages, and species. We demonstrated the epimutation-based single-cell multi-omic lineage tracing in mouse and human blood, where MethylTree recapitulated the differentiation hierarchy in hematopoiesis. Applying MethylTree to human embryos, we revealed early fate commitment at the four-cell stage. In native mouse blood, we identified ~250 clones of hematopoietic stem cells. MethylTree opens the door for high-resolution, noninvasive and multi-omic lineage tracing in humans and beyond.

LC-MS-Based Global Metabolic Profiles of Alternative Blood Specimens Collected by Microsampling

Blood microsampling (BμS) has recently emerged as an interesting approach in the analysis of endogenous metabolites but also in metabolomics applications. Their non-invasive way of use and the simplified logistics that they offer renders these technologies highly attractive in large-scale studies, especially the novel quantitative microsampling approaches such as VAMs or qDBS. Objectives: Herein, we investigate the potential of BµS devices compared to the conventional plasma samples used in global untargeted mass spectrometry-based metabolomics of blood. Methods: Two novel quantitative devices, namely, Mitra, Capitainer, and the widely used Whatman cards, were selected for comparison with plasma. Venous blood was collected from 10 healthy, overnight-fasted individuals and loaded on the devices; plasma was also collected from the same venous blood. An extraction solvent optimization study was first performed on the three devices before the main study, which compared the global metabolic profiles of the four extracts (three BµS devices and plasma). Analysis was conducted using reverse phase LC-TOF MS in positive mode. Results: BµS devices, especially Mitra and Capitainer, provided equal or even superior information on the metabolic profiling of human blood based on the number and intensity of features and the precision and stability of some annotated metabolites compared to plasma. Despite their rich metabolic profiles, BµS did not capture metabolites associated with biological differentiation of sexes. Conclusions: Overall, our results suggest that a more in-depth investigation of the acquired information is needed for each specific application, as a metabolite-dependent trend was obvious.

Acute Severe Hypoxia Decreases Mitochondrial Chain Complex II Respiration in Human Peripheral Blood Mononuclear Cells

Peripheral blood mononuclear cells’ (PBMCs) mitochondrial respiration is impaired and likely involved in myocardial injury and heart failure pathophysiology, but its response to acute and severe hypoxia, often associated with such diseases, is largely unknown in humans. We therefore determined the effects of acute hypoxia on PBMC mitochondrial respiration and ROS production in healthy volunteers exposed to controlled oxygen reduction, achieving an inspired oxygen fraction of 10.5%. We also investigated potential relationships with gene expression of key biomarkers of hypoxia, succinate and inflammation, as hypoxia and inflammation share common mechanisms involved in cardiovascular disease. Unlike global mitochondrial respiration, hypoxemia with a spO2 ≤ 80% significantly reduced PBMC complex II respiration (from 6.5 ± 1.2 to 3.1 ± 0.5 pmol/s/106 cell, p = 0.04). Complex II activity correlated positively with spO2 (r = 0.63, p = 0.02) and inversely correlated with the succinate receptor SUCNR1 (r = −0.68), the alpha-subunit of the hypoxia-inducible factor (HIF-1α, r = −0.61), the chemokine ligand-9 (r = −0.68) and interferon-stimulated gene 15 (r = −0.75). In conclusion, severe hypoxia specifically impairs complex II respiration in association with succinate, inflammation and HIF-1α pathway interactions in human PBMCs. These results support further studies investigating whether modulation of complex II activity might modify the inflammatory and metabolic alterations observed in heart failure.

Human PBMC-based humanized mice exhibit myositis features and serve as a drug evaluation model

Idiopathic inflammatory myopathies (IIMs) are a group of autoimmune disorders characterized by immune cell infiltration of muscle tissue accompanied by inflammation. Treatment of IIMs is challenging, with few effective therapeutic options due to the lack of appropriate models that successfully recapitulate the features of IIMs observed in humans. In the present study, we demonstrate that immunodeficient mice transplanted with human peripheral blood mononuclear cells (hPBMCs) exhibit the key pathologic features of myositis observed in humans and develop graft-versus-host disease. The hPBMC mice exhibit elevated serum levels of creatine kinase and aspartate transaminase, markers of myositis, and increased expression levels of myositis-related genes, such as vascular cell adhesion molecule 1, intercellular adhesion molecule 1, and serum amyloid A1, in muscle tissues. Histopathologic and flow cytometric analyses reveal the infiltration of CD8+ T cells in the muscle tissue of hPBMC mice, similar to that observed in patients with myositis, particularly in those with polymyositis. Transplantation of CD8+ T cell-depleted hPBMC leads to a significant reduction in polymyositis-like symptoms, in agreement with previous studies demonstrating CD8+ T cells as the main pathologic drivers of polymyositis. Furthermore, the transcriptome analysis of muscle tissue from hPBMC mice reveal extensive upregulation of characteristic genes of polymyositis, providing further support that hPBMC mice accurately reflect the pathophysiology of myositis in humans. Finally, administration of prednisolone or tacrolimus, which are commonly used for IIM treatment, leads to significant alleviation of myositis findings. Therefore, we propose that hPBMC mice should be considered as a model that accurately reflects the pathophysiology of myositis in human patients.

A Novel Approach for In Vitro Testing and Hazard Evaluation of Nanoformulated RyR2-Targeting siRNA Drugs Using Human PBMCs

Nucleic acid (NA)-based drugs are promising therapeutics agents. Beyond efficacy, addressing safety concerns—particularly those specific to this class of drugs—is crucial. Here, we propose an in vitro approach to screen for potential adverse off-target effects of NA-based drugs. Human peripheral blood mononuclear cells (PBMCs), purified from buffy coats of healthy donors, were used to investigate the ability of NA-drugs to trigger toxicity pathways and inappropriate immune stimulation. PBMCs were selected for their ability to represent potential human responses, given their likelihood of interacting with administered drugs. As proof of concept, a small interfering RNA (siRNA) targeting Ryanodine Receptor mRNA (RyR2) identified by the Italian National Center for Gene Therapy and Drugs based on RNA Technology as a potential therapeutic target for dominant catecholaminergic polymorphic ventricular tachycardia, was selected. This compound and its scramble were formulated within a calcium phosphate nanoparticle-based delivery system. Positive controls for four toxicity pathways were identified through literature review, each associated with a specific type of cellular stress: oxidative stress (tert-butyl hydroperoxide), mitochondrial stress (rotenone), endoplasmic reticulum stress (thapsigargin), and autophagy (rapamycin). These controls were used to define specific mRNA signatures triggered in PBMCs, which were subsequently used as indicators of off-target effects. To assess immune activation, the release of pro-inflammatory cytokines (interleukin-6, interleukin-8, tumor necrosis factor-α, and interferon-γ) was measured 24 h after exposure. The proposed approach provides a rapid and effective screening method for identifying potential unintended effects in a relevant human model, which also allows to address gender effects and variability in responses.

In vitro functional validation of anti-CD19 chimeric antigen receptor T cells expressing lysine-specific demethylase 1 short hairpin RNA for the treatment of diffuse large B cell lymphoma

BackgroundChimeric antigen receptor T (CAR-T) cell therapy is more effective in relapsed or refractory diffuse large B cell lymphoma (DLBCL) than other therapies, but a high proportion of patients relapse after CAR-T cell therapy owing to antigen escape, limited persistence of CAR-T cells, and immunosuppression in the tumor microenvironment. CAR-T cell exhaustion is a major cause of relapse. Epigenetic modifications can regulate T cell activation, maturation and depletion; they can be applied to reduce T cell depletion, improve infiltration, and promote memory phenotype formation to reduce relapse after CAR-T cell therapy.PurposeWe propose to develop and validate in vitro the function of novel CAR-T cells for the treatment of DLBCL, which simultaneously express an anti-CD19 CAR with lysine-specific demethylase 1 (LSD1) short hairpin (sh)RNA to prevent depletion and prolong the survival of CAR-T cells.MethodsWe designed an shRNA sequence targeting LSD1 mRNA, and created a vector with the following elements: the U6 promoter driving expression of the LSD1 shRNA sequence, the EF1a promoter driving a second-generation anti-CD19 CAR sequence encoding an anti-CD19 single-chain variable fragment (FMC63), the CD8 hinge and transmembrane structural domains, the CD28 co-stimulatory structural domain, and the CD3ζ-activating structural domain. The MFG-LSD1 shRNA anti-CD19 CAR plasmid was first constructed, then packaged in retroviral vectors and transduced into human primary peripheral blood mononuclear cell-derived T cells to generate the corresponding CAR-T cells. We examined by flow cytometry the efficiency of two CAR-T cells in killing U-2932 cells (a human DLBCL line) upon co-culture with RNAU6 anti-CD19 CAR-T cells or LSD1 shRNA anti-CD19 CAR-T cells. We analyzed Ki-67 staining of the CAR-T cells by flow cytometry on days 0, 5, and 10, and counted the cells to assess expansion. We also used flow cytometry to detect the central memory T cell (TCM) proportion.ResultsWe detected the expression of the CAR in the CAR-T cells by flow cytometry, and observed transduction rates of 31.5% for RNAU6 anti-CD19 CAR-T cells and 60.7% for LSD1 shRNA anti-CD19 CAR-T cells. The killing efficiency of LSD1 shRNA anti-CD19 CAR-T cells was significantly higher than that of RNAU6 anti-CD19 CAR-T cells at the low effector target ratio. We further found that LSD1 shRNA anti-CD19 CAR-T cells secreted more IFN-γ and granzyme B than RNAU6 anti-CD19 CAR-T cells. CAR-T cells proliferated after U-2932 cell stimulation and were able to sustain proliferation. After stimulation via U-2932 cell co-culture, both RNAU6 anti-CD19 CAR-T and LSD1 shRNA anti-CD19 CAR-T populations had increased proportions of cells with the TCM phenotype, with a higher percentage among LSD1 shRNA anti-CD19 CAR-T cells.ConclusionWe developed a novel, feasible CD19-LSD1 shRNA CAR-T cell strategy for the treatment of DLBCL. Our in vitro assay results showed that LSD1 shRNA anti-CD19 CAR-T cells more effectively killed target cells than RNAU6 anti-CD19 CAR-T cells, and developed a higher proportion of TCM phenotype cells. LSD1 shRNA anti-CD19 CAR-T cells may represent a potential treatment for DLBCL.

Anti-Tissue-Transglutaminase IgA Antibodies Presence Determination Using Electrochemical Square Wave Voltammetry and Modified Electrodes Based on Polypyrrole and Quantum Dots

A novel electrochemical detection method utilizing a cost-effective hybrid-modified electrode has been established. A glassy carbon (GC) modified electrode was tested for its ability to measure electrochemical tTG antibody levels, which are essential for diagnosing and monitoring Celiac disease (CD). Tissue transglutaminase protein biomolecules are immobilized on a quantum dots-polypyrrole nanocomposite in the improved electrode. Initial, quantum dots (QDs) were obtained from Bombyx mori silk fibroin and embedded in polypyrrole film. Using carbodiimide coupling, a polyamidoamine (PAMAM) dendrimer was linked with GQDs-polypyrrole film to improve sensor sensitivity. The tissue transglutaminase (tTG) antigen was cross-linked onto PAMAM using N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC)-N-hydroxy succinimide (NHS) chemistry to develop a nanoprobe that can detect human serum anti-tTG antibodies. The physicochemical characteristics of the synthesized nanocomposite were examined by FTIR, UV-visible, FE-SEM, EDX, and electrochemical studies. The novel electrode measures anti-tissue antibody levels in real time using human blood serum samples. The modified electrode has great repeatability and an 8.7 U/mL detection limit. Serum samples from healthy people and CD patients were compared to standard ELISA kit assays. SPSS and Excel were used for statistical analysis. The improved electrode and detection system can identify anti-tissue antibodies up to 80 U/mL.

Aptamer proximal enzyme cascade reactions for ultrafast detection of glucose in human blood serum.

Aninnovative colorimetric sensing strategy was developedfor the detection of glucose by the integration of glucose aptamer, glucose oxidase (GOx), and horseradish peroxidase (HRP), termed aptamer proximal enzyme cascade reactions (APECR). In the presence of glucose, aptamer binding enables GOx to catalyze glucose oxidation into H2O2 efficiently. Subsequently, the adjacent HRP catalyzes the oxidation of the peroxidase substrate, 2,2'-biazobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS2-), utilizing the generated H2O2, resulting in a distinct color change. In comparison to the free enzymes and the HRP-GOx system, APECR exhibited higher colorimetric signal. This approach achieved glucose detection within three minutes, which was significantly faster than previous methods. This method showed good sensitivity and selectivity with a limit of detection of 0.013mM. Moreover, the practical utility of this strategy was verified by achieving rapid detection of glucose in clinical serum samples. Hence, the developed strategy has the advantages of simple operation and rapid analysis time for the detection of glucose in human serum.

The associations between prenatal plastic phthalate exposure and lipid acylcarnitine levels in humans and mice.

Phthalates are ubiquitous environmental pollutants known for their endocrine-disrupting properties, particularly during critical periods such as pregnancy and early childhood. Phthalates alter lipid metabolism, but the role of prenatal exposure on the offspring lipidome is less understood. In particular, we focused on long chain acylcarnitines - intermediates of fatty acid oxidation that serve as potential biomarkers of mitochondrial function and energy metabolism. This study aimed (i) to investigate the association between prenatal phthalate exposure and the child's blood acylcarnitine concentrations and, (ii) to evaluate the impact of prenatal administration of di-(2-ethylhexyl) phthalate (DEHP) on acylcarnitine levels in mouse offspring blood, brain and liver. We conducted analyses of both a prospective birth cohort study and an experimental study in mice. From the Barwon Infant Study cohort (1074 mother-child pairs), prenatal phthalate exposure was assessed at 36 weeks' gestation and its association with acylcarnitine levels was examined in cord blood, and child's blood at 6 months, 12 months and 4 years. In mice, pregnant C57BL/6J mouse dams were exposed to 20μg/kg DEHP for 5 days mid-gestation, and offspring tissues were analyzed at 1 month of age postnatally for acylcarnitine profiles. Our findings demonstrate that prenatal phthalate levels (specifically butyl benzyl phthalate (BBzP) and diisobutyl phthalate (DiBP)) are inversely associated with total long chain acylcarnitine levels in human cord blood at birth. In contrast, BBzP was positively associated with the long chain acylcarnitines at 12 months of age. In mice, prenatal DEHP exposure for only 5 days led to decreased palmitoylcarnitine (AC16:0) levels in the brain and liver, but not in blood. Taken together, our findings highlight that prenatal phthalate exposure can alter acylcarnitine profiles, indicating disruptions in fatty acid metabolism that may have long-term effects on metabolic health.

Comparing acute versus AIDS ART initiation on HIV-1 integration sites and clonal expansion

Early antiretroviral therapy (ART) initiation is known to limit the establishment of the HIV reservoir, with studies suggesting benefits such as a reduced number of infected cells and a smaller latent reservoir. However, the long-term impact of early ART initiation on the dynamics of the infected cell pool remains unclear, and clinical evidence directly comparing proviral integration site counts between early and late ART initiation is limited. In this study, we used Linear Target Amplification-PCR (LTA-PCR) and Next Generation Sequencing to compare unique integration site (UIS) clonal counts between individuals who initiated ART during acute HIV infection stage (Acute-ART group) and those in the AIDS stage (AIDS-ART group). Our analysis revealed distinct clonal distribution patterns, with greater UIS heterogeneity in Acute-ART group and more homogeneity in AIDS-ART group. Monoclonal UIS accumulation, predominantly in-gene regions, was influenced by ART timing and duration, with early treatment delaying this process. Host cell genes integrated by HIV provirus as monoclonal types were enriched in cell cycle and lymphocyte activation pathways. Tumor suppressor genes (TSGs) were more frequently integrated as monoclonal types in AIDS-ART group, suggesting potential risk factors. Overall, we introduced a sequencing method to assess provirus size in human peripheral blood and identified the widespread presence of monoclonal distribution of UIS in AIDS-ART group after long-term treatment. The early intervention helps slow the progress of clonal expansion of infected cells, reducing the formation of stable and persistent reservoirs, and ultimately posing fewer barriers to achieving a functional cure.

High sensitivity tapered fiber refractive index biosensor using hollow gold nanoparticles

A localized surface plasmon resonance (LSPR) sensor based on tapered optical fiber (TOF) using hollow gold nanoparticles (HAuNPs) for measuring the refractive index (RI) is presented. This optical fiber sensor is a good candidate for a label-free RI biosensor. In practical biosensors, bioreceptors are immobilized on nanoparticles (NPs) that only absorb specific biomolecules. The binding of these biomolecules to the receptors changes the local RI around the sensor and this change is detected by the transmittance spectrum of the fiber. Fast, accurate, easy and low-cost disease diagnosis are the advantages of optical fiber biosensors. In this paper, the structure theory is reviewed and the sensor is simulated by the finite difference time domain (FDTD) method and the finite element method (FEM) and the effect of the thickness and diameter of the HAuNPs and the waist diameter of the TOF is investigated. For the structure with HAuNPs thickness (2.5 nm), diameter (50 nm), and the fiber waist diameter of 10 μm, the wavelength sensitivity of 489.8 nm/RIU and full width at half maximum (FWHM) of 50 nm are obtained, which are better than those specifications in some other LSPR fiber sensors. In addition, the sensitivity of the sensor increases about 2–3 times compared to those of sensors with the same structure. Although there are many parameters in human blood that can change its RI, in practical work, the special bioreceptors on the sensor can deactivate other markers except the specific cancer markers, which changes the effective RI. Therefore, this optical fiber sensor is used for label-free detecting the RI of cancer cells and can be used as a biosensor for the detection of early stages of cancers in a non-invasive way, just using human blood samples.

Identification of Drug-Targetable Genes for Eczema and Dermatitis Using Integrated Genomic and Proteomic Approaches.

Background: Eczema and dermatitis are common inflammatory skin conditions with significant morbidity. Identifying drug-targetable genes can facilitate the development of effective treatments. Methods: This study analyzed data obtained by meta-analysis of 2 genome-wide association studies on eczema/dermatitis (57,311 cases and 896,779 controls, European ancestry). We identified drug-targetable genes from the Drug-Gene Interaction Database and Finan et al's findings. Cis-expression quantitative trait loci (eQTL) data from human blood and skin tissues were used for Mendelian randomization (MR) analysis. Bayesian colocalization, proteomic MR, and meta-analysis validated the causal relationships. Finally, protein-protein interactions (PPIs) and correlation analysis of potential drug targets and cytokines were performed. Results: We identified 2532 drug-targetable genes; 3378 Single Nucleotide Polymorphism (SNPs) were associated with 1531 genes in blood cis-eQTLs, 664 SNPs with 667 genes in sun-exposed skin eQTLs, and 572 SNPs with 574 genes in nonsun-exposed skin eQTLs. Five genes (SLC22A5, NOTCH4, AGER, HLA-DRB5, and EHMT2) showed causal relationships with eczema/dermatitis across multiple datasets. Single-variable and multi-variable Mendelian randomization (SMR) and multi-SNP SMR analysis identified 8 genes (PIK3R4, DHODH, CXCR2, Interleukin (IL)18, LGALS9, RPS6KB2, SLC22A5, and AGER) across all tissues. Functional Summary Information for Variants in the Online Network (FUSION) analysis confirmed associations for SLC22A5 and AGER. Bayesian colocalization indicated AGER (PPH4: 0.95) as a shared causal variant. Proteomic MR and meta-analysis showed that increased AGER protein levels were associated with a lower risk of eczema or dermatitis (odds ratio: 0.995, 95% confidence interval: 0.997-0.993, P = 0.0002). A PPI network revealed interactions of AGER with NOTCH4 and multiple cytokines, whereas SLC22A5 showed no cytokine interactions. Conclusions: This study identified potential drug-targetable genes, with AGER showing strong potential as a target for reducing eczema/dermatitis risk. These findings provide a basis for developing targeted therapies.

Structure-Based Design of Novel TLR7/8 Agonist Payloads Enabling an Immunomodulatory Conjugate Approach.

Dual activation of the TLR7 and TLR8 pathways leads to the production of type I interferon and proinflammatory cytokines, resulting in efficient antigen presentation by dendritic cells to promote T-cell priming and antitumor immunity. We developed a novel series of TLR7/8 dual agonists with varying ratios of TLR7 and TLR8 activity for use as payloads for an antibody-drug conjugate approach. The agonist-induced production of several cytokines in human whole blood confirmed their functional activity. Structure-activity relationship studies guided by structure-based drug design are described.

Development of Hollow Fiber Membranes Suitable for Outside-In Filtration of Human Blood Plasma

Hemodialysis (HD) is a critical treatment for patients with end-stage kidney disease (ESKD). The effectiveness of conventional dialyzers used there could be compromised during extended use due to limited blood compatibility of synthetic polymeric membranes and sub-optimal dialyzer design. In fact, blood flow in the hollow fiber (HF) membrane could trigger inflammatory responses and thrombus formation, leading to reduced filtration efficiency and limiting therapy duration, a consequence of flowing the patients’ blood through the lumen of each fiber while the dialysate passes along the inter-fiber space (IOF, inside-out filtration). This study investigates the development of HF membranes for “outside-in filtration” (OIF) in HD. In OIF, blood flows through the inter-fiber space while dialysate flows within the fiber lumens, reducing the risk of fiber clogging and potentially extending treatment duration. For the OIF mode, the membrane should have a blood-compatible outer selective layer in contact with the patient’s blood. We develop HFs for OIF via liquid-induced phase separation using PES/PVP (polyethersulphone/polyvinylpyrrolidone) blends. The fibers’ surface morphology (SEM, scanning electron microscopy), chemistry (ATR-FTIR—attenuated total reflection-Fourier transform infrared spectroscopy, XPS—X-ray photoelectron spectroscopy), transport properties, and uremic toxin removal from human plasma are evaluated and compared to commercial HFs. These membranes feature a smooth, hydrophilic outer layer, porous lumen, ultrafiltration coefficient of 13–34 mL m2 h−1 mmHg−1, adequate mechanical properties, low albumin leakage, and toxin removal performance on par with commercial membranes in IOF and OIF. They offer potential for more efficient long-term HD by reducing clogging and systemic anticoagulation needs and enhancing treatment time and toxin clearance.

Predicting in vivo concentrations of dietary hop phytoestrogens by physiologically based kinetic modeling.

Hop extracts containing prenylated polyphenols such as 8-prenylnaringenin (8-PN) and its precursor isoxanthohumol (iXN) are popular among women seeking natural alternatives to hormone therapy for postmenopausal symptoms. Due to structural similarities with estrogens, these compounds act as estrogen receptor agonists. Especially 8-PN, described as the most potent phytoestrogen known to date, poses a potential risk for endocrine disruption. Therefore, its use as a hormone replacement raises concerns for human health. However, a significant challenge in assessing the potential endocrine-disruptive effects of hop polyphenols is the lack of data on their toxicokinetics. Particularly, information on in vivo concentrations in target tissues is lacking. To address this gap, we developed a physiologically based kinetic (PBK) model tailored to female physiology. The model was used to predict the levels of hop polyphenols in human blood and target tissues under realistic exposure scenarios. The predictions suggest that iXN and 8-PN concentrations in target tissues reach the low nanomolar range after dietary supplementation. This study enhances our understanding of internal concentrations of iXN and 8-PN after dietary consumption and is of direct applicability for respective risk assessment.

Tuning chitosan properties to enhance blood coagulation.

The search for new hemostatic materials remains a priority for researchers, as the problem of uncontrolled hemorrhage during surgical interventions or traumatic injuries represents a significant challenge. The objective of the study was to identify novel polysaccharide structures with enhanced hemostatic properties based on chitosan. The number of chitosan derivatives with two substituents was synthesized and characterized by 1H NMR, FTIR. One of these was a structural analogue of L-DOPA - N-3,4-dihydroxybenzyl, while the other comprised fragments of different nature, including hydrophobic (4-(tetradecyloxy)benzyl), negatively charged groups (4-carboxybenzyl) and aminocaproic acid residue. The hemostatic potential of the novel compounds was evaluated in vitro/in vivo on human blood and in mouse model of tail bleeding. Solutions of chitosan derivatives showed the ability to aggregate with blood about 3-5 times higher than chitosan in a neutral saline medium (0.9 % NaCl) and slightly acidic conditions both when (Ca2+) was added and in the case of citrated blood. Chitosan derivatives demonstrated low toxicity (3 T3, HepG2 and Huh7) and did not induce plasma coagulation or platelet aggregation at low concentrations. The novel compounds can be used to modify the surface of biomaterials in order to improve their hemostatic properties.

Enhanced Killing of Methicillin-Resistant Staphylococcus aureus with Ceftaroline or Vancomycin in Combination with Carbapenems.

Methicillin-resistant Staphylococcus aureus (MRSA) bacteremia is associated with high rates of treatment failure, even when antibiotics showing in vitro susceptibility are used. Early optimization of therapy is crucial to reduce morbidity and mortality. Building on our previous research on carbapenem therapy for methicillin-susceptible S. aureus (MSSA) bacteremia, we examined the utility of adjunctive carbapenems (ertapenem or meropenem) to enhance the efficacy of ceftaroline or vancomycin for treatment of MRSA. The effectiveness of combination therapy versus monotherapy against MRSA was assessed using checkerboard, time-kill, and human whole blood killing assays, as well as a murine bacteremia model. Additionally, we performed transcriptomic analysis and conducted human platelet and antimicrobial peptide killing assays on MRSA pretreated with subtherapeutic concentrations of ceftaroline and carbapenems. The supernatants from these MRSA were used to treat platelets, and cytotoxicity was assessed via LDH release assays. Although not typically used for MRSA, we identified striking in vitro and in vivo synergy between carbapenems and ceftaroline or vancomycin. MRSA pretreated with subtherapeutic ceftaroline-carbapenem therapy revealed transcriptional shifts indicative of reduced antibiotic resistance, virulence, and host immune evasion. Supernatants from these MRSA also caused less platelet injury compared to monotherapy. Furthermore, MRSA pretreated with ceftaroline and carbapenems demonstrated increased susceptibility to killing by human platelets and the antimicrobial peptide LL-37. The therapeutic success of adjunctive carbapenems appears to be driven by multiple mechanisms, including direct drug-drug synergy with first-line anti-MRSA agents, attenuation of resistance and virulence factors, and enhancement of immune-mediated killing, each warranting further investigation.

Endogenous CD4 T cells that recognize ALK and the NPM1::ALK fusion protein can be expanded from human peripheral blood.

Anaplastic lymphoma kinase (ALK)-fusion proteins resulting from chromosomal rearrangements are promising targets for cancer immunotherapy. While ALK-specific CD8+ T cells and epitopes presented on MHC class I have been identified in patients with ALK-positive malignancies, little is known about ALK-specific CD4+ T cells. We screened peripheral blood of ten ALK-positive anaplastic large cell lymphoma (ALK+ALCL) patients in remission and six healthy donors for CD4+ T-cell responses to the whole ALK-fusion protein, nucleophosmin (NPM1)::ALK. ALK-specific CD4+ T cells were detected in 15 individuals after stimulation with autologous dendritic cells pulsed with long-overlapping ALK peptide pools. CD4+ T-cell epitopes were predominantly located within three specific regions (p102-188, p257-356, p593-680) in the ALK portion of the fusion protein. We detected CD4+ T cells in one patient that recognized the NPM1::ALK fusion neoepitope and identified a corresponding T-cell receptor (TCR) by TCR single-cell sequencing. The NPM1::ALK fusion-specific TCR was HLA-DR13 restricted and conferred antigen specificity when expressed in a TCR- reporter cell line (58--). Together, our data provide evidence of ALK-specific CD4+ T cells in human peripheral blood, describe target epitopes in patients and support the consideration of CD4+ T cells in the development of ALK-specific immunotherapies.

Plasmodium falciparum histidine-rich protein 2 exhibits cell penetration and cytotoxicity with autophagy dysfunction.

Plasmodium falciparum is a major cause of severe malaria. This protozoan infects human red blood cells and secretes large quantities of histidine-rich protein 2 (PfHRP2) into the bloodstream, making it a well-known diagnostic marker. Here, however, we identified PfHRP2 as a pathogenic factor produced by P. falciparum. PfHRP2 showed cell penetration and cytotoxicity against various human cells. PfHRP2 also exhibited significant cytotoxicity at concentrations found in P. falciparum-infected patients' blood (90-100nM). We also showed that PfHRP2 binds to Ca2+ ions, localizes to intracellular lysosomes, increases lysosomal Ca2+ levels, and inhibits the basal level of autophagy by preventing autolysosome formation. Furthermore, the Ca2+-dependent cytotoxicity of PfHRP2 was suppressed by the metal ion chelator ethylenediaminetetraacetic acid. In summary, our findings suggest PfHRP2 as a crucial pathogenic factor produced by P. falciparum and its mode of action. Overall, this study provides preliminary insights into P. falciparum malaria pathogenesis.