Healthy Human Blood Research Articles

New insights into the oxidative and cytogenotoxic effects of Tetraglyme on human peripheral blood cells

The present study investigated the oxidative and cytogenotoxic potential of Tetraethylene glycol dimethyl ether (known as Tetraglyme) on healthy human peripheral blood lymphocytes, widely used as an in vitro model for assessing the human health risk posed by different chemical compounds. In a first step, Nuclear Magnetic Resonance (1H NMR) spectroscopy, and Ultra-High Performance Liquid Chromatography-Mass Spectrometry (UHPLC-MS) were employed to estimate Tetraglyme's stability under a wide range of pH values (4–12), and thus to identify potential by-products. Thereafter, isolated lymphocytes were treated with different concentrations of Tetraglyme (0.02–20 mg L−1) for assessing its oxidative (using the DCFH-DA staining), and cytogenotoxic potential (using the trypan blue exclusion test for estimating cell viability, Comet assay, as well as the cytokinesis-block micronucleus assay, with or without the addition of S9 metabolic activation system). According to the results, Tetraglyme remains stable at pH 4, but two additional derivatives (i.e. 1-[2-(2-ethoxyethoxy)ethoxy]-2-methoxyethane [C9H20O4] and 1-ethoxy-2-(2-ethoxyethoxy)ethane (Diethylene glycol diethyl ether) [C8H18O3]) were found in traces, under alkaline conditions (pH ≥7). Moreover, although Tetraglyme (and/or its derivatives) showed negligible alterations of cell viability (>92 %) in all cases, the pronounced ROS formation, DNA damage, cell proliferation arrest, and MN frequencies in challenged cells are indicative of its oxidative and cytogenotoxic potential. The significant alterations of Cytokinesis-Block Proliferation Index (CBPI) and Micronucleus (MN) frequencies in S9 challenged cells give further evidence for the potential involvement of Tetraglyme's metabolites in the observed cytogenotoxic mode of action. Although not conclusive, the present findings give rise to further research, utilizing different cell types and biological models, for elucidating Tetraglyme's toxic mode of action, as well as its environmental and human risk.

Generation of the CTRL EiPS J9 mR6F-8 iPSC line derived from healthy human outgrowth blood endothelial cells (BOECs) using mRNA reprogramming methodology

mRNA reprogramming is a technology for generating iPSCs with high efficiency and safety. However, it is not suitable for reprogramming non-adherent cells, the primary cell type in blood. An alternative is to obtain adherent cells from blood for this technology. To validate this approach, we generated a human iPSC line from blood outgrowth endothelial cells (BOECs) using mRNA-based reprogramming. The resulting line, CTRL EiPS J9 mR6F-8, meets iPSC criteria, including an undifferentiated state, pluripotency, genome integrity. This line is available upon request.

Dose-related effect of acetylcholine on human gingival blood flow

Abstract Background This study investigates the dose-response relationship of acetylcholine (ACh) on healthy human gingival blood flow (GBF). Understanding this dose-response relationship contributes to studying vasodilatory mechanisms in various pathological conditions. Methods The study involved 22 young healthy men (21 - 32 years) to investigate the dose-response relationship of ACh on GBF. Semi-circular wells were created on the labial surface of the upper right second incisor (FDI #12) and upper left first incisor (FDI #21), including the gingival sulcus, for the application of drugs. ACh-chloride solutions at 0.1, 1, and 10 mg/mL were administered to the gingival sulcus of tooth FDI #12 with a Hamilton syringe. Physiological saline was applied on the contralateral side to FDI #21 as a control. The GBF was measured non-invasively by the laser speckle contrast imaging method in four 1mm high adjacent regions: coronal, midway1, midway2, and apical, and was expressed in a laser speckle perfusion unit (LSPU). After the baseline blood flow recording, ACh doses were applied sequentially, with washout periods in between. Data were statistically analyzed using a linear mixed model. Results The GBF did not change on the saline site throughout the experiment. The GBF was significantly higher at the coronal region after all ACh doses (baseline: 218±31 LSPU, and 227±38 LSPU p < 0.05, 239±40 LSPU p < 0.001, 291±54 LSPU p < 0.001, respectively) compared to the saline. It was also elevated following 1 and 10 mg/mL at the midway1 (245±48 LSPU, p < 0.05, 293±65 LSPU p < 0.001). At midway2 and apical, only the 10 mg/mL dose was effective (285±71 LSPU, p < 0.001; 302±82 LSPU, p < 0.001). Conclusions Our findings suggest a dose-dependent vasodilation to ACh, emphasizing its role in human gingival microcirculation. Only the 10 mg/mL ACh could evoke remote vasodilation 3 mm from the application. The described method could facilitate the investigation of endothelium-dependent vasodilation in disorders affecting microcirculation, such as periodontitis or diabetes.

Human decidua basalis mesenchymal stem/stromal cells enhance anticancer properties of human natural killer cells, in vitro.

Mesenchymal stem cells/stromal cells from the Decidua Basalis of the human placenta (DBMSCs) express wide range of effector molecules that modulate the functions of their target cells. These properties make them potential candidate for use in cellular therapy. In this study, we have investigated the consequences of interaction between DBMSCs and natural killer (NK) cells for both cell types. DBMSCs were cultured with IL-2-activated and resting non-activated NK cells isolated from healthy human peripheral blood and various functional assays were performed including, NK cell proliferation and cytolytic activities. Flow cytometry and microscopic studies were performed to examine the expression of NK cell receptors that mediate these cytolytic activities against DBMSCs. Moreover, the mechanism underlying these effects was also investigated. Our findings revealed that, co-culture of DBMSCs and NK cells resulted in inhibition of proliferation of resting NK cells, while proliferation of IL-2 activated NK cells was increased. Contrarily, treatment of DBMSC's with comparatively high numbers of IL-2 activated NK cells, resulted in their lysis, whereas treatment with low numbers resulted in reduction in their proliferation. Cytolytic activity of NK cells against DBMSCs was mediated by several activating NK cell receptors. In spite of the expression of HLA class I molecules by DBMSCs, they were still lysed by NK cells, excluding their involvement in cytolytic activity. In addition, preconditioning NK cells by DBMSCs, enhanced their ability to suppress tumor cell proliferation and in severe cases resulted in their partial lysis. Lysis and decrease of tumor cell proliferation is associated with increased expression of important molecules involved in anticancer activities. We conclude that DBMSCs exhibit dualfunctions on NK cells that enhance their anticancer therapeutic potential.

The β2-adrenergic biased agonist nebivolol inhibits the development of Th17 and the response of memory Th17 cells in an NF-κB-dependent manner.

Adrenergic receptors regulate metabolic, cardiovascular, and immunological functions in response to the sympathetic nervous system. The effect of β2-adrenergic receptor (AR) as a high expression receptor on different subpopulations of T cells is complex and varies depending on the type of ligand and context. While traditional β2-AR agonists generally suppress T cells, they potentially enhance IL-17A production by Th17 cells. The effects of pharmacological drugs that count as biased agonists of AR like nebivolol are not completely understood. We investigated the impact of nebivolol on human memory CD4+ T (Th1, Th2, Th17) cells and polarized naive Th17 cells, highlighting its potential for IL-17A suppression via a non-canonical β2-AR cell signaling pathway. The effects of nebivolol were tested on healthy human peripheral blood mononuclear cells, purified memory Th cells, and polarized naive Th17 cells activated with anti-CD3/anti-CD28/anti-CD2 ImmunoCult reagent. IFN-γ, IL-4, and IL-17A, which are primarily derived from Th1, Th2, and Th17 cells, respectively, were quantified by ELISA and flow cytometry. IL-10 was measured by ELISA. Gene expression of RORC, ADRB1, ADRB2, and ADRB3 was evaluated by qPCR. The ADRB2 gene was knocked out in memory Th cells using CRISPR/Cas9. Protein expression of phosphorylated serine133-CREB and phosphorylated NF-κB p65 was assessed by Western blot. Proliferation was assessed by fluorescent dye loading and flow cytometry. Nebivolol treatment decreased IL-17A and IFN-γ secretion by activated memory Th cells and elevated IL-4 levels. Nebivolol reduced the proportion of IL-17A+ Th cells and downregulated RORC expression. Unlike the β2-AR agonist terbutaline, nebivolol inhibited the shift of naive CD4+ T cells toward the Th17 phenotype. IL-10 and the proliferation index remained unchanged. Nebivolol-treated β2-knockout memory Th cells showed significant inhibition of β2-AR-mediated signaling, evidenced by the absence of IL-17A suppression compared to controls. Phosphorylation of the NF-κB p65 subunit was inhibited by nebivolol, but CREB phosphorylation was not changed, suggesting a selective transcriptional control. The findings demonstrate that nebivolol acts through a β2-AR-mediated signaling pathway, as a distinctive anti-inflammatory agent capable of selectively shifting Th17 cells and suppressing the phosphorylation of NF-κB. This highlights nebivolol's potential for therapeutic interventions in chronic autoimmune conditions with elevated IL-17A levels.

Control of Hepatitis B Virus with Imdusiran, a Small Interfering RNA Therapeutic.

Chronic hepatitis B is a global health concern with a high risk of end-stage liver disease. Current standard-of-care agents have low cure rates, and new therapies are needed. Small interfering RNAs (siRNAs) that target viral RNAs fulfill a gap not addressed by standard-of-care agents and may contribute to a functional cure. Here, we describe the preclinical characterization of imdusiran (AB-729), a novel, pan-genotypic siRNA therapeutic that effectively reduces HBsAg, viral antigens, and viral replication in chronic hepatitis B patients and is currently in Phase 2 clinical studies. In hepatitis B virus (HBV) cell-based systems, imdusiran possessed pan-genotypic nanomolar potency and retained activity against HBV target site polymorphisms. Imdusiran was active against nucleos(t)ide analogue- and capsid assembly modulator-resistant HBV isolates, and combination with standard-of-care agents was additive. In an HBV adeno-associated virus mouse model, HBsAg was reduced up to 3.7 log10 after a single imdusiran dose, with sustained suppression for 10 weeks. Imdusiran did not intrinsically stimulate cytokine release in healthy donor human whole blood, supportive of its mechanism of action as a direct acting RNA interference antiviral. Taken together, these data support imdusiran in combination treatment approaches toward chronic hepatitis B functional cure.

Establishment of a humanized mouse model of HIV-1 infection and quantification of integrated HIV proviral DNA in vivo

In recent years, virological, pathological, and immunological studies need to be carried out for the emerging anti-human immunodeficiency virus (HIV) therapies such as gene therapy, broadly neutralizing antibodies, and the derived chimeric antigen receptor (CAR)-T immunotherapy, which necessitates suitable, simple, and inexpensive small-animal models and methods for accurate quantification of the viral genome in the HIV-1 infected. In our research, the HIV-∆ENV-Jurkat-EGFP-mCherry cell line was engineered through the infection with a dual-labelled HIV pseudovirus. A nested quantitative PCR (nested-qPCR) method with the cellular genome as the integrated standard was established for the quantification of HIV proviral copies. We administered intravenous injections of healthy human peripheral blood mononuclear cell (PBMC) into NOD/Prkdcscid/IL2rgnull (NPG) mice. To verify engraftment kinetics, we analyzed the percentages of hCD45+, hCD3+, hCD4+, and hCD8+ cells in the peripheral blood of hu-PBMC-NPG mice. To evaluate HIV-1 infection in hu-PBMC-NPG mice, we inoculated these mice with HIV NL4-3-NanoLuc by intraperitoneal (IP) injection. We then monitored the luciferase expression by the small animal imaging system and measured the viral load in the spleen by qPCR. The infiltration of human PBMCs in mice was detected 3-5 weeks after intravenous injection, and the percentage of hCD45 in humanized mouse PBMCs were more than 25% five weeks after IP inoculation. The expression of the virus-associated luciferase protein was detected by luciferase imaging 27 days post infection. Moreover, the viral total DNA, RNA, and proviral DNA copies reached 18 000 copies/106 cells, 15 000 copies/μg RNA, and 15 000 copies/106 cells, respectively, in the mouse spleen. Taken together, we reported a convenient method for building a simple humanized mouse model of HuPBMC-NPG/severe combined immunodeficiency (SCID) by intravenous injection with hu-PBMCs without advanced surgical skills and irradiation. Furthermore, we established a convenient method for the efficient determination of proviral DNA to assess HIV replication in vivo, viral reservoir sizes, and efficacy of novel anti-HIV therapies including CAR-T immunotherapy and gene therapy.

High-dimensional single-cell analysis of human natural killer cell heterogeneity

Natural killer (NK) cells are innate lymphoid cells (ILCs) contributing to immune responses to microbes and tumors. Historically, their classification hinged on a limited array of surface protein markers. Here, we used single-cell RNA sequencing (scRNA-seq) and cellular indexing of transcriptomes and epitopes by sequencing (CITE-seq) to dissect the heterogeneity of NK cells. We identified three prominent NK cell subsets in healthy human blood: NK1, NK2 and NK3, further differentiated into six distinct subgroups. Our findings delineate the molecular characteristics, key transcription factors, biological functions, metabolic traits and cytokine responses of each subgroup. These data also suggest two separate ontogenetic origins for NK cells, leading to divergent transcriptional trajectories. Furthermore, we analyzed the distribution of NK cell subsets in the lung, tonsils and intraepithelial lymphocytes isolated from healthy individuals and in 22 tumor types. This standardized terminology aims at fostering clarity and consistency in future research, thereby improving cross-study comparisons.

Pan-cancer profiling of tumor-infiltrating natural killer cells through transcriptional reference mapping

The functional diversity of natural killer (NK) cell repertoires stems from differentiation, homeostatic, receptor–ligand interactions and adaptive-like responses to viral infections. In the present study, we generated a single-cell transcriptional reference map of healthy human blood- and tissue-derived NK cells, with temporal resolution and fate-specific expression of gene-regulatory networks defining NK cell differentiation. Transfer learning facilitated incorporation of tumor-infiltrating NK cell transcriptomes (39 datasets, 7 solid tumors, 427 patients) into the reference map to analyze tumor microenvironment (TME)-induced perturbations. Of the six functionally distinct NK cell states identified, a dysfunctional stressed CD56bright state susceptible to TME-induced immunosuppression and a cytotoxic TME-resistant effector CD56dim state were commonly enriched across tumor types, the ratio of which was predictive of patient outcome in malignant melanoma and osteosarcoma. This resource may inform the design of new NK cell therapies and can be extended through transfer learning to interrogate new datasets from experimental perturbations or disease conditions.

Platelet and red cell responses to three North American pit vipers

We investigated the hemotoxic effects of three North American pit vipers in healthy human donor blood. Using experiments focusing on platelet and red blood cell activity, we found differential effects of these venoms on these cellular components. Platelet aggregation was most induced by C. adamanteus. Platelet activation was highest with C. atrox. Red blood cells had calcium expression and erythrocyte formation most induced by C. adamanteus and A. piscivorus. These results demonstrate the complex interplay of individual cellular effects with clinical presentations seen in envenomings from these species.

Non-enzymatic sensor based on doped polyindole/multi-walled carbon nanotube for detecting neurotransmitter acetylcholine

A facile non-enzymatic acetylcholine sensor composed of doped-polyindole/multi-walled carbon nanotube (dPIn-MWCNT) composite was designed, fabricated, and presented here. The negative current response toward acetylcholine concentration was first found and reported here. The confirmation of acetylcholine detection mechanism was elucidated by XPS, where the acetylcholine interaction resulted in a decrease in the % (dPIn doping level and an increase of the dPIn structural defect. The response optimizations with dPIn concentrations, applied potentials, and incubation times were systematically investigated. The 45dPIn-15MWCNT operated at + 0.6 V vs. Ag/AgCl with 5 min of incubation time was the optimum condition to obtain the highest acetylcholine current response. Chronoamperometric acetylcholine detection revealed two distinct current regimes from the acetylcholine surface interaction and the de-doping of dPIn. Regime I was related to the diffusion-controlled process as observed in the low concentration range of 10−8–10−5 M. Regime II corresponded to the mixed diffusion-controlled and adsorption-controlled process as observed in the concentration range of 10−5–10−2 M. For regime I, the calculated LOD was as low as 1.27 nM with a very high acetylcholine response sensitivity. Regime I is suggested for the point-of-care utility, where the detectable concentration range covered the acetylcholine level normally found in the healthy human blood. Regime II can be applicable and useful in the treatment monitoring of neurological diseases.

In vitro study on device-induced damage to blood cellular components and degradation of von Willebrand factor in a CentriMag pump-assisted circulation.

High mechanical shear stress (HMSS) generated by blood pumps during mechanical circulatory support induces blood damage (or function alteration) not only of blood cell components but also of plasma proteins. In the present study, fresh, healthy human blood was used to prime a blood circuit assisted by a CentriMag centrifugal pump at a flow rate of 4.5 L/min under three pump pressure heads (75, 150, and 350 mm Hg) for 4 h. Blood samples were collected for analyses of plasma-free hemoglobin (PFH), von Willebrand factor (VWF) degradation and platelet glycoprotein (GP) IIb/IIIa receptor shedding. The extent of all investigated aspects of blood damage increased with increasing cross-pump pressure and duration. Loss of high-molecular-weight multimers (HMWM)-VWF in Loop 2 and Loop 3 significantly increased after 2 h. PFH, loss of HMWM-VWF, and platelet GPIIb/IIIa receptor shedding showed a good linear correlation with mean shear stress corresponding to the three pump pressure heads. HMSS could damage red blood cells, cause pathological VWF degradation, and induce platelet activation and platelet receptor shedding. Different blood components can be damaged to different degrees by HMSS; VWF and VWF-enhanced platelet activation may be more susceptible to HMSS.

Single Cell Profiling Distinguishes Leukemia-Selective Chemotypes.

A central challenge in chemical biology is to distinguish molecular families in which small structural changes trigger large changes in cell biology. Such families might be ideal scaffolds for developing cell-selective chemical effectors - for example, molecules that activate DNA damage responses in malignant cells while sparing healthy cells. Across closely related structural variants, subtle structural changes have the potential to result in contrasting bioactivity patterns across different cell types. Here, we tested a 600-compound Diversity Set of screening molecules from the Boston University Center for Molecular Discovery (BU-CMD) in a novel phospho-flow assay that tracked fundamental cell biological processes, including DNA damage response, apoptosis, M-phase cell cycle, and protein synthesis in MV411 leukemia cells. Among the chemotypes screened, synthetic congeners of the rocaglate family were especially bioactive. In follow-up studies, 37 rocaglates were selected and deeply characterized using 12 million additional cellular measurements across MV411 leukemia cells and healthy peripheral blood mononuclear cells. Of the selected rocaglates, 92% displayed significant bioactivity in human cells, and 65% selectively induced DNA damage responses in leukemia and not healthy human blood cells. Furthermore, the signaling and cell-type selectivity were connected to structural features of rocaglate subfamilies. In particular, three rocaglates from the rocaglate pyrimidinone (RP) structural subclass were the only molecules that activated exceptional DNA damage responses in leukemia cells without activating a detectable DNA damage response in healthy cells. These results indicate that the RP subset should be extensively characterized for anticancer therapeutic potential as it relates to the DNA damage response. This single cell profiling approach advances a chemical biology platform to dissect how systematic variations in chemical structure can profoundly and differentially impact basic functions of healthy and diseased cells.

Abstract 4042: Cbl inhibition increases the response of cytotoxic T cells to sub-optimal stimulation and drives enhanced anti-tumor responses alone or in combination with PD-1 blockade

Abstract INTRODUCTION: Herein, we describe a series of experiments to define the unique biology of dual Cbl-b/c-Cbl (Cbl) inhibition in amplifying T cell activation and anti-tumor responses. Through their E3 ubiquitin ligase activity, Cbl-b and c-Cbl have been shown to negatively regulate multiple signaling proteins downstream of the T cell receptor (TCR), suggesting that inhibition may yield enhanced anti-tumor T cell activity. Indeed, using a dual Cbl-b/c-Cbl small molecule inhibitor, we demonstrate that Cbl blockade enhances T cell activity under standard and sub-optimal stimulation conditions. Moreover, Cbl inhibition increases mouse syngeneic tumor control alone or in combination with α-PD-1, as described below. METHODS: Human CD8+ T cells were isolated from healthy human blood and activated using α-CD3 or α-CD3/CD28 stimulation. Mouse OT-I splenocytes were stimulated with SIINFEKL or lower affinity ovalbumin peptides and cytokine production was assessed. For tumor studies, mice were inoculated with syngeneic cancer cells and were treated with Cbl inhibitor (30 mg/kg, QD) +/- α-PD-1 (10 mg/kg, Q5D) starting at a tumor volume of 75 mm3. RESULTS: Human CD8+ T cells, stimulated in the presence of a Cbl inhibitor, demonstrated a concentration-dependent increase in activation hallmarks including cytokine secretion (IL-2, IFN-γ and granzyme B), proliferation, and the presence of highly polyfunctional cells. Furthermore, using serially stimulated CD8+ T cells with reduced overall activity, Cbl inhibition showed a marked enhancement of IFN-γ production and cytotoxicity compared to controls upon re-stimulation. We then used an antigen-specific T cell assay where OT-I splenocytes were stimulated by peptides with optimal or reduced TCR affinities, Cbl inhibition produced significantly higher IFN-γ levels with all peptides relative to controls demonstrating that Cbl inhibition can increase the response of T cells to sub-optimal stimulation. Using mouse CT26 and MC38 tumor models, Cbl inhibition was able to significantly enhance tumor control in combination with α-PD-1 and as a monotherapy in CT26 tumors. Notably, a greater number of tumor antigen-specific cytotoxic T cells were found in Cbl inhibitor-treated tumors. Higher expression levels of the co-stimulatory molecule CD226 and the TCR in these anti-tumor cells were also observed, demonstrating the unique biology of Cbl inhibition in enhancing anti-tumor responses. Finally, Cbl inhibition had no effect on the growth or survival of cancer cells in vitro consistent with the notion that enhanced tumor control was driven by immune activity. CONCLUSIONS: Altogether, these data demonstrate that pharmacologic Cbl inhibition robustly enhances T cell activity in vitro and in vivo and provide a mechanistic rationale for targeting Cbl-b/c-Cbl to amplify anti-tumor immune responses. Citation Format: Livia Yamashiro, Sachie Marubayashi, Rameshwari Rayaji, Anudari Battsooj, Karim Mrouj, Hema Singh, Yue Tong Lee, Ada Chen, Lunda Shen, Artur Mailyan, Kai Yu, Masha Podunavac, Julie Yu, Dongdong Liu, Clayton Hardman, Johnny Yin, Srikanth Gangam, Ken Lawson, Manmohan Leleti, Matthew Walters, Daniel DiRenzo. Cbl inhibition increases the response of cytotoxic T cells to sub-optimal stimulation and drives enhanced anti-tumor responses alone or in combination with PD-1 blockade [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4042.

Lactate metabolism promotes in vivo fitness during Acinetobacter baumannii infection.

Acinetobacter baumannii is one of the most prevalent causes of nosocomial infections worldwide. However, a paucity of information exists regarding the connection between metabolic capacity and in vivo bacterial fitness. Elevated lactate is a key marker of severe sepsis. We have previously shown that the putative A. baumannii lactate permease gene, lldP, is upregulated during in vivo infection. Here, we confirm that lldP expression is upregulated in three A. baumannii strains during a mammalian systemic infection. Utilising a transposon mutant disrupted for lldP in the contemporary clinical strain AB5075-UW, and a complemented strain, we confirmed its role in the in vitro utilisation of l-(+)-lactate. Furthermore, disruption of the lactate metabolism pathway resulted in reduced bacterial fitness during an in vivo systemic murine competition assay. The disruption of lldP had no impact on the susceptibility of this strain to complement mediated killing by healthy human serum. However, growth in biologically relevant concentrations of lactate observed during severe sepsis, led to bacterial tolerance to killing by healthy human blood, a phenotype that was abolished in the lldP mutant. This study highlights the importance of the lactate metabolism pathway for survival and growth of A. baumannii during infection.

Influence of a Passive Tilt Test on the Proteomic Composition of the Blood of Healthy Humans.

In order to identify changes in the blood proteome of healthy volunteers after passive tilt test carried out on day 19 of head-down bed rest, a chromato-mass-spectrometric analysis of samples of dried blood spots was carried out. It was revealed that the body's response to the tilt test was characterized by a decrease in the level of HDL and kininogen-1. After the tilt test, we observed an increase in the level of vimentin, vitamin K-dependent protein C, Wnt signaling pathway proteins, proteins involved in autophagy and adaptive immune response, focal adhesion proteins, vascular damage marker S100A8, PEDF regulator, and some proteins of the heart: cardiac actin ACTC1 and transcription factor GATA4. The obtained results lay the foundation for future research in the framework of identifying the risks of developing cardiovascular changes in astronauts after space flights.

Evaluating of aqueous and Alcoholic extracts of (Urtica pilulifera L.) in the hypersensitivity

This study was devoted to the biological efficacy of the U.Pilulifera aqueous and Alcoholic extract at concentration of 100 mg/kg on hypersensitivity in adult Wiser albino rats induced by Bisphenol compound orally at a concentration of 1 mg/kg, for the period of experiment was 14 days. U.Pilulifera plants leafs were cleaned, the leaves were separated and dried, then well extracted with distilled water (D.W), a powder dissolved in distilled water and orally administered to the animals. Cytotoxicity of all plant extracts of U. Pilulifera used the human healthy blood hemolysis methods were show no hemolytic effect was recorded in all treatments with all extract concentration used. white laboratory rats weighing (168 –180 g) were used in this study were divided to 6 groups for each experiment, three replicates per group with a total of 18 rats for any experiment, and given oral treatment for a straight 14 days. The result showed that animals treated with Bisphenol had a significant (p <0.05) increase in IgE U/ml and Histamine U/ml content. From all the above results, the use of aqueous and Alcoholic extracts of U.Pilulifera plants may lead to the potential development of new treatments and a significant advance in pharmacology.

Investigation of the role of von Willebrand factor in shear-induced platelet activation and functional alteration under high non-physiological shear stress.

von Willebrand factor (vWF) plays a crucial role in physiological hemostasis through platelet and subendothelial collagen adhesion. However, its role in shear-induced platelet activation and functional alteration under non-physiological conditions common to blood-contacting medical devices (BCMDs) is not well investigated. Fresh healthy human blood was treated with an anti-vWF antibody to block vWF-GPIbα interaction. Untreated blood was used as a control. They were exposed to three levels of non-physiological shear stress (NPSS) (75, 125, and 175 Pa) through a shearing device with an exposure time of 0.5 s to mimic typical shear conditions in BCMDs. Flow cytometric assays were used to measure the expression levels of PAC-1 and P-Selectin and platelet aggregates for platelet activation and the expression levels of GPIbα, GPIIb/IIIa, and GPVI for receptor shedding. Collagen/ristocetin-induced platelet aggregation capacity was characterized by aggregometry. The levels of platelet activation and aggregates increased with increasing NPSS in the untreated blood. More receptors were lost with increasing NPSS, resulting in a decreased capacity of collagen/ristocetin-induced platelet aggregation. In contrast, the increase in platelet activation and aggregates after exposure to NPSS, even at the highest level of NPSS, was significantly lower in treated blood. Nevertheless, there was no notable difference in receptor shedding, especially for GPIIb/IIIa and GPVI, between the two blood groups at the same level of NPSS. The block of vWF exacerbated the decreased capacity of collagen/ristocetin-induced platelet aggregation. High NPSS activates platelets mainly by enhancing the vWF-GPIbα interaction. Platelet activation and receptor shedding induced by high NPSS likely occur through different pathways.

Adoptively Infused Memory-like Natural Killer Cells Impact Adaptive Immune Responses in Patients with Acute Myeloid Leukemia

Natural killer (NK) therapies have been challenging owing to antigen escape, restricted trafficking, and limited tumor infiltration, translating into modest clinical benefit. We have shown that memory-like (ML)-NK cells acquire an in vivo differentiated phenotype distinct from conventional NK cells and are well-tolerated without cytokine release syndrome, GVHD, or neurotoxicity. WU-NK-101 (W-NK) is an allogeneic cytokine-reprogrammed, expanded, cryopreserved ML-NK cell product derived from healthy human peripheral blood mononuclear cells, with scalable manufacturability, that is currently in clinical development in relapsed/refractory (R/R) AML (NCT# 05470140). We conducted a multi-modal analysis of the immune tumor microenvironment (TME) in 13 patients with R/R AML receiving ML-NK cells (NCT#01898793) with the aim to determine whether TME modifications induced by ML-NK cells correlate with clinical benefit. NK cells were purified from leukapheresate, and activated with IL-12, IL-15, and IL-18 for 12-16 hours. Patients were lymphodepleted with fludarabine and cyclophosphamide. NK cells were infused in escalating doses: 0.5 × 10 6/kg, 1.0 × 10 6/kg, and all NK cells from a single leukapheresis, capped at 10 × 10 6/kg. Response was defined as <5% BM blasts on d+28. BM trephines for immune gene expression profiling (nCounter IO360®) and spatial proteomics (GeoMx™ DSP; n=53 immuno-oncology [IO] targets; NanoString Technologies, Seattle) were collected at baseline (BL) and on treatment (OT). Immune infiltration, NK and T cell number from BM samples was quantified manually on immunofluorescent imaging. Oncomine™ TCR Pan-Clonality assay (Thermo Fisher) was used to profile TCRb and g repertoires by NGS. The Shannon index was used to describe TCR repertoire diversity, and clonal evenness was used as a measure of the similarity of clone sizes. BM infiltration with CD8 + effectors, enhanced glycolysis and cell cycling was higher in BM samples from responders (CR) to ML-NK cells. By contrast, OXPHOS RNA scores and gene signatures of immune senescence and terminal T-cell exhaustion were higher in non-responders (NR). We employed machine learning to construct an 8-gene predictor of response which encompassed T/NK markers, such as CD7, and IFN regulatory factors and TLRs. This score was significantly higher at baseline in CR and positively correlated with response duration. Principal component analysis (PCA) of spatially resolved IO proteins separated CR from NR, particularly in OT specimens, where T-cell markers CD3/CD8 and inhibitory receptors CTLA4/LAG3 were among the top contributing features. CD34, type I IFN signaling molecule STING1 and PARP1 were significantly higher at baseline compared with OT, with OT showing higher expression of cytotoxicity markers and inhibitory receptors (e.g., Tim-3 and CD276). This observation strongly suggests modulation of the immune TME after the infusion of ML-NK cells. Response to ML-NK cells was associated with coordinated up-regulation of T cell, NK cell and B cell protein markers. To further substantiate the co-localization of T cells and NK cells in the BM FFPE sections (Fig. A), and its correlation with clinical efficacy we annotated the 93 ROIs in our spatial proteomics dataset based on a median split of CD3 and CD56 protein barcodes. ROIs from CR were largely CD3 highCD56 high, both at BL and OT. Conversely, ROIs from BM sections of NR to ML-NK cells had a predominantly CD3 lowCD56 low phenotype. Quantitative analysis of T-cell infiltration post ML-MK dosing showed a significant association with response, 0.9 (PD) vs. 5.3 (CR)-fold-increase ( P=0.01). Interestingly, data demonstrate selected intra-BM clonal expansion as evidenced by decreased TCR diversity (mean ± SEM, 4.6±0.3 vs. 3.3±0.4, pre vs. post; P=0.013)and increased clonality (Fig. B). Thus, pre-existing T-cell responses appear to be co-opted in the AML TME of CR, suggesting the potential for durable effectiveness of ML-NK cells. Multi-modal analyses of WU-NK-101 highlight unique transcriptional and functional states, which could underpin in vivo potency and persistence. WU-NK-101 are currently being evaluated in a phase 1 study of R/R AML (#NCT05470140).

WU-NK-101 (W-NK), a Memory-like (ML) NK Cell, Intrinsically Overcomes Factors Restricting Adoptive Cell Therapy (ACT) in Acute Myeloid Leukemia (AML)

ML-NK cells are generated from conventional (c) NK cells activated with cytokines IL-12/15/18 which results in enhanced fitness, in vivo persistence, and function against tumor targets. While effective in early phase clinical trials, a 1 donor to 1 recipient cell product hinders commercial development as a cellular therapeutic. Here, W-NK is a cytokine-reprogrammed, expanded, cryopreserved ML-NK product derived from healthy human peripheral blood (PBMC) cNK cells, with scalable manufacturability. W-NKs harness the benefits of ML-NKs, and further overcome challenges previously reported for adoptive cell therapy (ACT) in AML: antigen escape, limited tumor trafficking/infiltration, and restricted function in tumor microenvironment (TME). W-NK characterization was performed by scRNA-seq (10X Genomics) and multidimensional flow and mass cytometry.Functionality was evaluated by in vitro cytotoxicity assays against AML cell lines (THP-1, and HL-60) and bioenergetics assays (Seahorse XF), under different conditions: conventional (N) media (pH 7, glucose 11 mM), or two models for TME (TME-aligned media - pH 5.9, glucose 6 mM, immune suppressive agents, e.g., PGE2, TGFb1, and malignant ascites), and in hypoxia (0.1% O 2; AVATAR). In vivo efficacy was evaluated in NSG mice bearing THP-1 AML tumors. Proteomic data was acquired by tandem-mass spectrometry (Sciex Zeno TOF 7600). Cell trafficking was measured by fluorescent-labeled W-NK in NSG mice. W-NK cells are transcriptionally distinct from cNKs. Sc-RNA-seq analysis revealed important transcriptional and compositional differences compared to cNK cells, including novel NK clusters expressing metabolic ( GLUL), proliferation-associated ( Ki67) and IL-15-inducible genes ( TXNIP) known to enhance NK responses. Additionally, W-NKs express higher levels of activating receptors ( i.e. 2B4, DNAM-1, NKG2D, NKp30, etc.), cytotoxic effector proteins ( i.e. granzyme B), and lower levels of inhibitory receptors leading to increased potency in vitro against HL-60 cells (mEC50 1.7 and 5.2; p= 0.032; W-NK vs. cNK respectively) and demonstrated a dose dependent reduction of tumor burden in THP-1 xenografts ( p=0.015). W-NK also express high levels of CXCR4 (81.6% positive, MFI 5552), contributing to improved bone marrow (BM) homing (16.5 ± 2.4%) compared to data previously reported for IL-2-expanded NK cells (3%; Sato et al.CCR. 2020). Once in the TME, unlike cNK and T-cells, W-NK cytotoxic function is preserved (TME-media vs. N-media-; W-NK: 96.5% vs. 88.2%, p=0.45; cNK: 44.1% vs.13.5%, p=0.009; T-cell: 53.2 % vs. 25.2%, p=0.041, respectively). This is potentially due to W-NK resistance to hypoxia, and improved metabolic fitness/flexibility. W-NK cytotoxic activity was also preserved in hypoxic conditions (90% vs. 89.5%, p=0.2) compared to cNK (24% vs. 6.9%, p=0.02; normoxia vs. hypoxia). Metabolically, W-NK express higher levels of cell surface nutrient transporters, implying better adaptation to adverse TMEs ( i.e. upregulation of amino acids, lactate, and pyruvate). This might enable utilization of diverse macronutrients, activation of metabolic pathways, and optimal ATP manufacturing, and is supported by upregulation of REACTOME pathways capturing amino-acid and lipid metabolism, as well as mitochondrial function. Lastly, W-NK did not attack cells from healthy human tissues including PBMCs, indicating that W-NK are not indiscriminate killers. This observation would predict low myelosuppression and a favorable safety profile. In summary, W-NK are effective against AML cells, robustly home to the BM, overcome hypoxia, immunosuppression, and nutritional scarcity to survive/function within the leukemia TME. Moreover, they are not restricted to a single antigen target thereby mitigating antigen escape. These features, which overcome challenges for ACT, herald the promise of NK cell therapy and augur positively for patients being treated with W-NK in a Phase 1 study in R/R AML (NCT# 05470140).