Cell Production Research Articles

A singular plasmonic-thermoelectric hollow nanostructure inducing apoptosis and cuproptosis for catalytic cancer therapy

Thermoelectric technology has recently emerged as a distinct therapeutic modality. However, its therapeutic effectiveness is significantly limited by the restricted temperature gradient within living organisms. In this study, we introduce a high-performance plasmonic-thermoelectric catalytic therapy utilizing urchin-like Cu2−xSe hollow nanospheres (HNSs) with a cascade of plasmonic photothermal and thermoelectric conversion processes. Under irradiation by a 1064 nm laser, the plasmonic absorption of Cu2−xSe HNSs, featuring rich copper vacancies (VCu), leads to a rapid localized temperature gradient due to their exceptionally high photothermal conversion efficiency (67.0%). This temperature gradient activates thermoelectric catalysis, generating toxic reactive oxygen species (ROS) targeted at cancer cells. Density functional theory calculations reveal that this vacancy-enhanced thermoelectric catalytic effect arises from a much more carrier concentration and higher electrical conductivity. Furthermore, the exceptional photothermal performance of Cu2−xSe HNSs enhances their peroxidase-like and catalase-like activities, resulting in increased ROS production and apoptosis induction in cancer cells. Here we show that the accumulation of copper ions within cancer cells triggers cuproptosis through toxic mitochondrial protein aggregation, creating a synergistic therapeutic effect. Tumor-bearing female BALB/c mice are used to evaluate the high anti-cancer efficiency. This innovative approach represents the promising instance of plasmonic-thermoelectric catalytic therapy, employing dual pathways (membrane potential reduction and thioctylated protein aggregation) of mitochondrial dysfunction, all achieved within a singular nanostructure. These findings hold significant promise for inspiring the development of energy-converting nanomedicines.

A Non-Canonical p75HER2 Signaling Pathway Underlying Trastuzumab Action and Resistance in Breast Cancer.

Overexpression of HER2 occurs in 25% of breast cancer. Targeting HER2 has proven to be an effective therapeutic strategy for HER2-positive breast cancer. While trastuzumab is the most commonly used HER2 targeting agent, which has significantly improved outcomes, the overall response rate is low. To develop novel therapies to boost trastuzumab efficacy, it is critical to identify the mechanisms underlying trastuzumab action and resistance. We recently showed that the inhibition of breast cancer cell growth by trastuzumab is not through the inhibition of HER2 canonical signaling. Here we report the identification of a novel non-canonical HER2 signaling pathway and its interference by trastuzumab. We showed that HER2 signaled through a non-canonical pathway, regulated intramembrane proteolysis (RIP). In this pathway, HER2 is first cleaved by metalloprotease ADAM10 to produce an extracellular domain (ECD) that is released and the p95HER2 that contains the transmembrane domain (TM) and intracellular domain (ICD). p95HER2, if further cleaved by an intramembrane protease, γ-secretase, produced a soluble ICD p75HER2 with nuclear localization signal (NLS). p75HER2 is phosphorylated and translocated to the nucleus. Nuclear p75HER2 promotes cell proliferation. Trastuzumab targets this non-canonical HER2 pathway via inhibition of the proteolytic cleavage of HER2 by both ADAM10 and γ-secretase. However, p75HER2 pathway also confers resistance to trastuzumab once aberrantly activated. Combination of trastuzumab with ADAM10 and γ-secretase inhibitors completely blocks p75HER2 production in both BT474 and SKBR3 cells. We concluded that HER2 signals through the RIP signaling pathway that promotes cell proliferation and is targeted by trastuzumab. The aberrant HER2 RIP signaling confers resistance to trastuzumab that could be overcome by the application of inhibitors to ADAM10 and γ-secretase.

CAIP-Induced ROS Production Contributes to Sustaining Atherosclerotic Process Associated with Helicobacter cinaedi Infection through Macrophages and Endothelial Cells Activation.

Several lines of evidence have linked the intestinal bacterium Helicobacter cinaedi with the pathogenesis of atherosclerosis, identifying the Cinaedi Antigen Inflammatory Protein (CAIP) as a key virulence factor. Oxidative stress and inflammation are crucial in sustaining the atherosclerotic process and oxidized LDL (oxLDL) uptake. Primary human macrophages and endothelial cells were pre-incubated with 10 µM diphenyl iodonium salt (DPI) and stimulated with 20 µg/mL CAIP. Lectin-like oxLDL receptor (LOX-1) expression was evaluated by FACS analysis, reactive oxygen species (ROS) production was measured using the fluorescent probe H2DCF-DA, and cytokine release was quantified by ELISA assay. Foam cells formation was assessed by Oil Red-O staining, and phosphorylation of p38 and ERK1/2 MAP kinases and NF-κB pathway activation were determined by Western blot. This study demonstrated that CAIP triggered LOX-1 over-expression and increased ROS production in both macrophages and endothelial cells. Blocking ROS abrogated LOX-1 expression and reduced LDL uptake and foam cells formation. Additionally, CAIP-mediated pro-inflammatory cytokine release was significantly affected by ROS inhibition. The signaling pathway induced by CAIP-induced oxidative stress led to p38 MAP kinase phosphorylation and NF-κB activation. These findings elucidate the mechanism of action of CAIP, which heightens oxidative stress and contributes to the atherosclerotic process in H. cinaedi-infected patients.

Multicomponent double Povarov reaction for julolidine: Synthesis and mechanistic insights

Julolidines constitute a class of N-heterocyclic molecules that behave as molecular rotors. Due to their fluorescent properties, they are applied in a wide range of technological fields, such as the production of solar cells, fluorescent biomarkers or bioimaging, and photoconductive materials. In the present work, we developed a methodology for the synthesis of julolidines through the one-pot tandem reaction-based application of multicomponent double Povarov reaction (MCPR) using BF3.OEt2 as the catalyst (25 mol%), star anise oil rich in trans-anethole as the substrate, and water as the solvent in reactions at room temperature. The julolidines were obtained with total yields ranging from 55 to 95 %. A mechanistic study allowed for the conclusion that these MCPRs take place through an ionic mechanism and also that released protons by the reaction between BF3.Et2O and water are involved in the catalytic process. After extracting the products from the reaction medium, the remaining aqueous phase containing the catalyst can be reused for up to six catalytic cycles with a minimal loss of catalytic efficiency. DFT calculations were conducted to investigate the catalytic cycle using BF3.Et2O and ZnCl2 as catalysts. The results indicate that the formation of iminium ions is a prerequisite for the progress of the reaction (MCPR). When BF3.Et2O and water are involved, the reaction proceeds via Brønsted catalysis, and the formation of iminium ions occurs in an exothermic and spontaneous process. However, when the ZnCl2 is used, the process is Lewis catalyzed to form the first iminium ion and is endergonic, shifting the equilibrium towards the reactants and inhibiting the cyclization.

Apheresis product total CD34+ cell count prediction at peripheral stem cell collection via a formula: A multicenter study

IntroductionEffective mobilization of Stem Cells(SCs) to peripheral blood (PB) is crucial for obtaining sufficient CD34+ cell numbers via apheresis. The ratio of pre-apheresis PB CD34+ cells is the best parameter for predicting the product CD34+ cell count. However, quantitating CD34+ PB cells requires flow cytometry, which usually takes two or more hours to obtain the results. We hypothesized that the product CD34+ cell count could be predicted using the counts of white blood cells (WBCs), mononuclear cells (MNCs), and pre-apheresis CD34+ cells. A formula that achieves this would substantially affect the efficiency and effectiveness of apheresis. We, therefore, aimed to estimate the number of CD34+ cells in the product using a formula that incorporates pre-apheresis PB WBC, MNC, and CD34+ cell counts and product WBC and MNC counts. MethodsWe examined the results of 373 leukapheresis procedures for SC mobilization. Effective separation of CD34+ PBSCs (count/μL) via apheresis was estimated using the following formula: [Product WBC (count/μL) × MNC (count/μL) × pre-apheresis CD34+ cell (percentage/μL)] ÷ [PB WBC count/μL × PB MNC (count/μL)]. ResultsA strong correlation was observed between the CD34+ cell count calculated using our formula and the post-apheresis CD34+ cell count measured via flow cytometry (R = 0.939, based on linear regression analysis). In the subgroup analysis, this correlation was observed for all the disease subgroups and healthy donors. ConclusionWe developed a formula that predicts the product CD34+ cell count and is useful for determining whether a second apheresis procedure will be required.

PELO regulates erythroid differentiation through interaction with MYC to upregulate KLF10.

Erythropoiesis is a multistep process of erythroid cell production that is controlled by multiple regulatory factors. Ribosome rescue factor PELO plays a crucial role in cell meiotic division and mice embryonic development. However, the function of PELO in erythroid differentiation remains unclear. Here, we showed that knockdown of PELO increased hemin-induced erythroid differentiation of K562 and HEL cells, exhibiting a higher number of benzidine-positive cells and increased mRNA levels of erythroid genes. PELO knockdown inhibited the proliferation and cell cycle progression and promoted apoptosis of K562 cells. Mechanistically, PELO could regulate the expression of KLF10 through interaction with MYC. Moreover, KLF10 knockdown also enhanced erythroid differentiation of K562 and HEL cells induced by hemin. Collectively, our results demonstrated that PELO regulates erythroid differentiation and increases KLF10 expression levels by interacting with MYC.

A New Immunogenic Structure of Polyepitopic Fusion against Leishmania major: In Silico Study

Background: The lack of complete protection against leishmaniasis and the challenges of anti-leishmaniasis drug treatment have made the treatment process more difficult. This study aimed to develop a new strategy for preparing a vaccine against cutaneous leishmaniasis using some of the antigenic proteins of the Leishmania parasite. Methods: This study was carried out in 2022 at Shahid Chamran University of Ahvaz, Ahvaz, Iran. After preparing suitable epitopes of the Leishmania parasite and examining their antiparasitic properties, the process of making a fusion vaccine was performed and with the help of various bioinformatics tools, physicochemical and structural properties as well as immunological and simulation properties were studied and finally optimized. Construction and cloning were performed in the E.coli K12 system and finally, the docking process was performed with Toll-like receptors (TLRs), major histocompatibility complex I (MHC-I), and MHC-II receptors. With the help of selected epitopes of the Leishmania parasite, which had a high percentage of population coverage, a stable, antigenic, and non-allergenic chimeric vaccine was predicted. Results: The results of the structural analysis of the TLR5\vaccine complex and simulation of its molecular dynamics showed a sufficiently stable binding. It also showed good potential for stimulation and production of active B cells and memory, as well as the potential for CD8+ T, CD4+ T cell production and development of Th2 and Th1-induced immune responses. Conclusion: Computational results showed that the designed immunogenic structure has the potential to adequately stimulate cellular and humoral immune responses against Leishmania parasitic disease. As a result of evaluating the effectiveness of the candidate vaccine through in vivo and in vitro immunological tests, it can be suggested as a vaccine against Leishmania major.

Effects of selection for litter size variability on ovarian folliculogenesis, ovarian cell proliferation, apoptosis, and production of regulatory peptides in rabbits

The present study aimed to identify the novel mechanisms regulating rabbit fecundity. For this purpose, the association between litter size variability, fecundity, ovarian morphology, and markers of proliferation, apoptosis, steroidogenesis, and the presence of regulatory proteins were examined in ovarian cells from two rabbit lines divergently selected for low (LL) and high (HL) variability in litter size throughout sixteen generations. Ovaries and uteri were isolated from multiparous non-lactating female rabbits from each line at sixteen generations of selection. One part of the ovary was subjected to histomorphometric analysis of folliculogenesis. From the rest of ovary, ovarian granulosa cells (OGCs) were isolated, cultured, and cell viability, proliferation (accumulation of PCNA and of cyclin B1, and BrdU-positive cells), and apoptosis (accumulation of caspase 3, bax and DNA fragmentation) were evaluated by the Trypan blue exclusion test and BrdU, quantitative immunocytochemistry, and cell death detection assays. Furthermore, OGCs were subjected to proteomic analysis by using the nano HPLC-Chip-MS/MS method. The release of progesterone and oestradiol was measured by ELISA. The LL had more than one kit per litter than the HL (7.6 kits vs. 6.5 kits, p ≤ 0.05). No differences were found in the diameter of primordial and primary ovarian follicles, theca, and granulosa thickness, but the diameter of oocytes in the primary and secondary follicles was higher in the LL than in the HL (88.49 µm in the LL vs. 77.86 µm in the HL for oocytes of primary follicles, p ≤ 0.05; 122.10 µm in the LL vs. 109.87 µm in the HL for oocytes of secondary follicles, p ≤ 0.05). Preovulatory follicles were presented only in the ovaries of the LL. The LL had higher incorporation of BrdU and reduced accumulation of bax within OGCs (0.92% in the LL vs. 0.44% in the HL for incorporation of BrdU, p ≤ 0.05; 41% in the LL vs. 48% in the HL for accumulation of bax, p ≤ 0.05). Ovarian fragments from the LL produced less progesterone and oestradiol than those of the HL (12 ng/mg tissue/day in the LL vs, 45 ng/mg tissue/day in the HL for progesterone, p ≤ 0.05; 11 ng/mg tissue/day in the LL vs. 30 ng/mg tissue/day in the HL for oestradiol, p ≤ 0.05). Besides, the OGCs from the LL produced a higher number of specific regulatory proteins involved in cell differentiation, proliferation, and adhesion than the HL (50 vs 38, p ≤ 0.05). In conclusion, higher prolificacy in the LL line would be caused by: (1) the selection of growing primordial ovarian follicles; (2) better transformation to preovulatory follicles; (3) increased cytoplasmic maturation of oocytes; (4) increased DNA synthesis and decreased cytoplasmic apoptosis in OGCs; (5) changes in ovarian steroidogenesis; and (6) changes in the number of peptides involved in cell differentiation, proliferation, and adhesion. HIGHLIGHTS Assessing fecundity in two divergently rabbit lines for variability in litter size. Fecundity and its variability are related to the diameter and quality of oocytes. Selection and better growth of preovulatory follicles have been related to fecundity.

Effects of the oxoaporphine alkaloid hernandonine on dengue virus. Evidence for its mechanisms of action

BackgroundDengue, caused by the dengue virus (Orthoflavivirus dengue, encompassing DENV types 1-4), is a member of the Flaviviridae family. The symptoms of dengue range from subclinical or mild manifestations to potentially fatal complications. The management of severe dengue is exceptionally challenging due to the absence of effective antiviral medications. In this context, natural products, whether in the form of pure compounds or standardized plant extracts, have emerged as a promising source for the development of novel antiviral therapeutics. Hernandonine, an oxoaporphine alkaloid found in Hernandia nymphaeifolia (C. Presl) Kubitzki. serves both as a metabolite and an inhibitor of human immunodeficiency virus type 1 (HIV-1) integrase. PurposeThis study investigated the ability of hernandonine to inhibit DENV infection and explored its potential mechanisms. Study designTo assess the in vitro anti-DENV activity, cells or induced pluripotent stem cell (iPSC)-derived cerebral organoids were exposed to hernandonine before or after infection with DENV. Along with hernandonine, the endocytosis modulators, genistein, wortmannin, Methyl-β-cyclodextrin (MβCD) and lovastatin, were used in the assays. MethodsThe DENV infectivity and virion production in cells or cerebral organoids treated with compounds were determined. Various methods, including cell and cerebral organoids imaging, protein and gene detection were conducted to explore their antiviral mechanisms. ResultsThe results revealed notable antiviral properties of hernandonine, particularly in inhibiting DENV during the early stages of infection. Mechanistic analysis demonstrated that, akin to genistein, wortmannin, methyl-β-cyclodextrin (MβCD), and lovastatin, hernandonine exerted an influence on cholesterol-rich lipid rafts. It also restrained the pseudopodial movement ability of cells, potentially through the downregulation of cytoskeleton and endocytosis regulatory genes or protein expression. Moreover, hernandonine's virucidal activity was demonstrated. Hernandonine's inhibition of DENV infection was further validated in a disease-relevant iPSC-derived cerebral organoids model, a novel DENV-2 infection system worthy of further application. ConclusionThis study evidenced the potential of hernandonine as a novel candidate in the fight against DENV infection.

A mechanistic insight on the biomimetic activity of surface engineered NH2-MIL-125 with α-Asarone for enhanced ROS production in lung cancer cells

The α-Asarone-loaded on NH2-MIL-125 (AS@AMIL) was demonstrated to be exceptional potential as a chemo dynamic therapeutic agent (CDT) for lung cancer cells (A549), showing superior efficiency as compared to α-Asarone as well as pristine NH2-MIL-125. The loading of α-Asarone was achieved through cation-π interactions between –NH3+ groups of NH2-MIL-125 and the aromatic ring of α-Asarone. This interaction enriches the framework of AS@AMIL with electrons enhancing the basicity of the framework (O-atoms), which substantially accelerates the decomposition of endogenous H2O2 into reactive oxygen species (ROS) to exhibit enhanced CDT activity. The AS@AMIL exhibited ∼a 1.36-fold increase in ROS production (CDT activity) compared to pristine NH2-MIL-125. The study suggests that the loading of aromatic compounds in amine-functionalized MOFs through cation-π interactions (with –NH3+ groups) can enhance the basicity of MOFs for improved chemodynamic therapeutic activity, and such materials can also be promising as catalysts and CO2 adsorbents.

The Novel Anti-Cancer Agent, SpiD3, Is Cytotoxic in CLL Cells Resistant to Ibrutinib or Venetoclax

Background: B-cell receptor (BCR) signaling is a central driver in chronic lymphocytic leukemia (CLL), along with the activation of pro-survival pathways (e.g., NF-κB) and aberrant anti-apoptotic mechanisms (e.g., BCL2) culminating to CLL cell survival and drug resistance. Front-line targeted therapies such as ibrutinib (BTK inhibitor) and venetoclax (BCL2 inhibitor) have radically improved CLL management. Yet, persisting CLL cells lead to relapse in ~20% of patients, signifying the unmet need of inhibitor-resistant refractory CLL. SpiD3 is a novel spirocyclic dimer of analog 19 that displays NF-κB inhibitory activity and preclinical anti-cancer properties. Recently, we have shown that SpiD3 inhibits CLL cell proliferation and induces cytotoxicity by promoting futile activation of the unfolded protein response (UPR) pathway and generation of reactive oxygen species (ROS), resulting in the inhibition of protein synthesis in CLL cells. Methods: We performed RNA-sequencing using CLL cells rendered resistant to ibrutinib and venetoclax to explore potential vulnerabilities in inhibitor-resistant and SpiD3-treated CLL cells. Results: The transcriptomic analysis of ibrutinib- or venetoclax-resistant CLL cell lines revealed ferroptosis, UPR signaling, and oxidative stress to be among the top pathways modulated by SpiD3 treatment. By examining SpiD3-induced protein aggregation, ROS production, and ferroptosis in inhibitor-resistant CLL cells, our findings demonstrate cytotoxicity following SpiD3 treatment in cell lines resistant to current front-line CLL therapeutics. Conclusions: Our results substantiate the development of SpiD3 as a novel therapeutic agent for relapsed/refractory CLL disease.

The Development of Rotation in Simulated Dust-Devil-Like Vortices

Abstract The analysis uses a numerical simulation of an initially resting, dry, atmosphere, in which uniform surface heating leads to the development of a growing convective boundary layer (CBL). As soon as convective mixing sets in, regions of weak vertical vorticity develop at the lowest model level. Using forward trajectories, this vorticity is shown to originate from horizontal baroclinic production and subsequent reorientation into the vertical within the descending branches of the convective cells. The requirement for vertical vorticity production in the downdraft cells is shown to be a non-axisymmetric horizontal footprint of the downdraft regions. The resulting vertical vorticity is not initially associated with rotation. However, as the CBL matures, like-signed vortex patches merge, the vertical vorticity magnitude increases due to stretching, and deformation in the vortex core decreases, leading to the development of vortices. The ultimate origin of the vortices is thus initially horizontal vorticity that has been produced baroclinically, and that has subsequently been reoriented into the vertical in sinking air.

Microcystin-LR Regulates Interaction between Tumor Cells and Macrophages via the IRE1α/XBP1 Signaling Pathway to Promote the Progression of Colorectal Cancer.

Microcystin-LR (MC-LR), a cyanobacterial toxin, is a potent carcinogen implicated in colorectal cancer (CRC) progression. However, its impact on the tumor microenvironment (TME) during CRC development remains poorly understood. This study investigates the interaction between tumor cells and macrophages mediated by MC-LR within the TME and its influence on CRC progression. CRC mice exposed to MC-LR demonstrated a significant transformation from adenoma to adenocarcinoma. The infiltration of macrophages increased, and the IRE1α/XBP1 pathway was activated in CRC cells after MC-LR exposure, influencing macrophage M2 polarization under co-culture conditions. Additionally, hexokinase 2 (HK2), a downstream target of the IRE1α/XBP1 pathway, was identified, regulating glycolysis and lactate production. The MC-LR-induced IRE1α/XBP1/HK2 axis enhanced lactate production in CRC cells, promoting M2 macrophage polarization. Furthermore, co-culturing MC-LR-exposed CRC cells with macrophages, along with the IRE1α/XBP1 pathway inhibitor 4μ8C and the hexokinase inhibitor 2-DG, suppressed M2 macrophage-induced CRC cell migration, clonogenicity, and M2 macrophage polarization. This study elucidates the mechanism by which MC-LR-mediated interactions through the IRE1α/XBP1 pathway promote CRC progression, highlighting potential therapeutic targets.

HBHA induces IL-10 from CD4+ T cells in patients with active tuberculosis but IFN-γ and IL-17 from individuals with Mycobacterium tuberculosis infection.

To effectively control tuberculosis (TB), it is crucial to distinguish between active TB disease and latent TB infection (LTBI) to provide appropriate treatment. However, no such tests are currently available. Immune responses associated with active TB and LTBI are dynamic and exhibit distinct patterns. Comparing these differences is crucial for developing new diagnostic methods and understanding the etiology of TB. This study aimed to investigate the relationship between pro- and anti-inflammatory CD4+ cytokine production following stimulation with two types of latency-associated Mycobacterium tuberculosis (M.tb) antigens to allow differentiation between active TB and LTBI. Cryopreserved PBMCs from patients with active TB disease or LTBI were stimulated overnight with replication-related antigen [ESAT-6/CFP-10 (E/C)] or two latency-associated antigens [heparin-binding hemagglutinin (HBHA) and alpha-crystallin-like protein (Acr)]. Responses were analyzed using multiparameter flow cytometry: active TB disease (n=15), LTBI (n=15) and ELISA: active TB disease (n=26) or LTBI (n=27). CD4+ central memory T cells (Tcm) specific to E/C and CD4+ effector memory T cells specific to Acr and HBHA were higher in LTBI than in TB patients. IFN-γ+Tcm and IL-17+ Tem cells was higher in the LTBI group (p= 0.012 and p=0.029 respectively), but IL-10+ Tcm was higher in the active TB group (p= 0.029) following HBHA stimulation. Additionally, following stimulation with HBHA, IL-10 production from CD4+ T cells was significantly elevated in patients with active TB compared to those with LTBI (p= 0.0038), while CD4+ T cell production of IL-17 and IFN-γ was significantly elevated in LTBI compared to active TB (p= 0.0076, p< 0.0001, respectively). HBHA also induced more CCR6+IL-17+CD4Tcells and IL-17+FoxP3+CD25+CD4Tcells in LTBI than in TB patients (P=0.026 and P=0.04, respectively). HBHA also induced higher levels of IFN-γ+IL-10+CD4+ T cells in patients with active TB (Pp=0.03) and higher levels of IFN-γ+IL-17+ CD4+ T cells in those with LTBI (p=0.04). HBHA-specific cytokine production measured using ELISA showed higher levels of IFN-γ in participants with LTBI (P=0.004) and higher levels of IL-10 in those with active TB (P=0.04). Stimulation with HBHA and measurement of CD4+ T cell production of IFN-γ, IL-10, and IL-17 could potentially differentiate active TB from LTBI. The characteristics of cytokine-expressing cells induced by HBHA also differed between participants with active TB and LTBI.

Next-generation BCMA-targeted chimeric antigen receptor CARTemis-1: the impact of manufacturing procedure on CAR T-cell features.

CAR therapy targeting BCMA is under investigation as treatment for multiple myeloma. However, given the lack of plateau in most studies, pursuing more effective alternatives is imperative. We present the preclinical and clinical validation of a new optimized anti-BCMA CAR (CARTemis-1). In addition, we explored how the manufacturing process could impact CAR-T cell product quality and fitness. CARTemis-1 optimizations were evaluated at the preclinical level both, in vitro and in vivo. CARTemis-1 generation was validated under GMP conditions, studying the dynamics of the immunophenotype from leukapheresis to final product. Here, we studied the impact of the manufacturing process on CAR-T cells to define optimal cell culture protocol and expansion time to increase product fitness. Two different versions of CARTemis-1 with different spacers were compared. The longer version showed increased cytotoxicity. The incorporation of the safety-gene EGFRt into the CARTemis-1 structure can be used as a monitoring marker. CARTemis-1 showed no inhibition by soluble BCMA and presents potent antitumor effects both in vitro and in vivo. Expansion with IL-2 or IL-7/IL-15 was compared, revealing greater proliferation, less differentiation, and less exhaustion with IL-7/IL-15. Three consecutive batches of CARTemis-1 were produced under GMP guidelines meeting all the required specifications. CARTemis-1 cells manufactured under GMP conditions showed increased memory subpopulations, reduced exhaustion markers and selective antitumor efficacy against MM cell lines and primary myeloma cells. The optimal release time points for obtaining the best fit product were > 6 and < 10 days (days 8-10). CARTemis-1 has been rationally designed to increase antitumor efficacy, overcome sBCMA inhibition, and incorporate the expression of a safety-gene. The generation of CARTemis-1 was successfully validated under GMP standards. A phase I/II clinical trial for patients with multiple myeloma will be conducted (EuCT number 2022-503063-15-00).

Ghost cells as a two-phase blood analog fluid-high-volume and high-concentration production.

Hemolysis in mechanical circulatory support systems is currently determined quantitatively. To also locally resolve hemolysis, we are developing a fluorescent hemolysis detection method. This requires a translucent two-phase blood analog fluid combined with particle image velocimetry, an optical flow field measurement. The blood analog fluid is composed of red blood cell surrogates. However, producing surrogates in sufficient volume is a challenge. We therefore present a high-volume and high-concentration production for our surrogates: ghost cells, hemoglobin-depleted erythrocytes. In the ghost cell production, the hemoglobin is removed by a repeated controlled osmolar lysis. We have varied the solution mixture, centrifugation time, and centrifugation force in order to increase production efficiency. The production is characterized by measurements of output volume, hematocrit, transparency, and rheology of the blood analog fluid. The volume of produced ghost cells was significantly increased, and reproducibility was improved. An average production of 389 mL of ghost cells were achieved per day. Those ghost cells diluted in plasma have a rheology similar to blood while being permeable to light. The volume of ghost cells produced is sufficient for optical measurements as particle image velocimetry in mechanical circulatory support systems. This makes further work on experimental measurements for a locally resolved hemolysis detection possible.

Biofunctionalization of Cellulose Microcarriers Using a Carbohydrate Binding Module Linked with Fibroblast Growth Factor for the Expansion of Human Umbilical Mesenchymal Stromal Cells in Stirred Suspension Bioreactors.

Mesenchymal stromal cells (MSCs) have the potential to be used as autologous or allogenic cell therapy in several diseases due to their beneficial secretome and capacity for immunomodulation and differentiation. However, clinical trials using MSCs require a large number of cells. As an alternative to traditional culture flasks, suspension bioreactors provide a scalable platform to produce clinically relevant quantities of cells. When cultured in bioreactors, anchorage-dependent cells like MSCs require the addition of microcarriers, which provide a surface for cell attachment while in suspension. The best performing microcarriers are typically coated in animal derived proteins, which increases cellular attachment and proliferation but present issues from a regulatory perspective. To overcome this issue, a recombinant fusion protein was generated linking basic fibroblast growth factor (bFGF) to a cellulose-specific carbohydrate binding module (CBM) and used to functionalize the surface of cellulose microcarriers for the expansion of human umbilical MSCs in suspension bioreactors. The fusion protein was shown to support the growth of MSCs when used as a soluble growth factor in the absence of cellulose, readily bound to cellulose microcarriers in a dose-dependent manner, and ultimately improved the expansion of MSCs when grown in bioreactors using cellulose microcarriers. The use of CBM fusion proteins offers a simple method for the surface immobilization of growth factors to animal component-free substrates such as cellulose, which can be used alongside bioreactors to increase growth factor lifespan, decrease culture medium cost, and increase cell production in the manufacturing of therapeutic cells.

Fatty acid metabolism constrains Th9 cell differentiation and antitumor immunity via the modulation of retinoic acid receptor signaling.

T helper 9 (Th9) cells are interleukin 9 (IL-9)-producing cells that have diverse functions ranging from antitumor immune responses to allergic inflammation. Th9 cells differentiate from naïve CD4+ T cells in the presence of IL-4 and transforming growth factor-beta (TGF-β); however, our understanding of the molecular basis of their differentiation remains incomplete. Previously, we reported that the differentiation of another subset of TGF-β-driven T helper cells, Th17 cells, is highly dependent on de novo lipid biosynthesis. On the basis of these findings, we hypothesized that lipid metabolism may also be important for Th9 cell differentiation. We therefore investigated the differentiation and function of mouse and human Th9 cells in vitro under conditions of pharmacologically or genetically induced deficiency of the intracellular fatty acid content and in vivo in mice genetically deficient in acetyl-CoA carboxylase 1 (ACC1), an important enzyme for fatty acid biosynthesis. Both the inhibition of de novo fatty acid biosynthesis and the deprivation of environmental lipids augmented differentiation and IL-9 production in mouse and human Th9 cells. Mechanistic studies revealed that the increase in Th9 cell differentiation was mediated by the retinoic acid receptor and the TGF-β-SMAD signaling pathways. Upon adoptive transfer, ACC1-inhibited Th9 cells suppressed tumor growth in murine models of melanoma and adenocarcinoma. Together, our findings highlight a novel role of fatty acid metabolism in controlling the differentiation and in vivo functions of Th9 cells.

Effect of docosahexaenoic acid as an anti-inflammatory for Caco-2 cells and modulating agent for gut microbiota in children with obesity (the DAMOCLE study).

Docosahexaenoic acid (DHA) is a long-chain omega-3 polyunsaturated fatty acid. We investigated the dual health ability of DHA to modulate gut microbiota in children with obesity and to exert anti-inflammatory activity on human intestinal Caco-2 cells. In a pilot study involving 18 obese children (8-14years), participants received a daily DHA supplement (500mg/day) and dietary intervention from baseline (T0) to 4months (T1), followed by dietary intervention alone from 4months (T1) to 8months (T2). Fecal samples, anthropometry, biochemicals and dietary assessment were collected at each timepoint. At preclinical level, we evaluated DHA's antioxidant and anti-inflammatory effects on Caco-2 cells stimulated with Hydrogen peroxide (H2O2) and Lipopolysaccharides (LPS), by measuring also Inducible nitric oxide synthase(iNOS) levels and cytokines, respectively. Ten children were included in final analysis. No major changes were observed for anthropometric and biochemical parameters, and participants showed a low dietary compliance at T1 and T2. DHA supplementation restored the Firmicutes/Bacteroidetes ratio that was conserved also after the DHA discontinuation at T2. DHA supplementation drove a depletion in Ruminococcaceae and Dialisteraceae, and enrichment in Bacteroidaceae, Oscillospiraceae, and Akkermansiaceae. At genus level, Allisonella was the most decreased by DHA supplementation. In Caco-2 cells, DHA decreased H2O2-induced reactive oxygen species (ROS) and nitric oxide (NO) production via iNOS pathway modulation. Additionally, DHA modulated proinflammatory (IL-1β, IL-6, IFN-γ, TNF-α) and anti-inflammatory (IL-10) cytokine production in LPS-stimulated Caco-2 cells. An improvement in gut dysbiosis of children with obesity seems to be triggered by DHA and to continue after discontinuation. The ability to modulate gut microbiota, matches also with an anti-inflammatory effect of DHA on Caco-2 cells.

Fast Screening of Tyrosinase Inhibitors in Coreopsis tinctoria Nutt. by Ligand Fishing Based on Paper-Immobilized Tyrosinase.

Coreopsis tinctoria Nutt. is an important medicinal plant in traditional Uyghur medicine. The skin-lightening potential of the flower has been recognized recently; however, the active compounds responsible for that are not clear. In this work, tyrosinase, a target protein for regulating melanin synthesis, was immobilized on the Whatman paper for the first time to screen skin-lightening compounds present in the flower. Quercetagetin-7-O-glucoside (1), marein (2), and okanin (3) were found to be the enzyme inhibitors. The IC50 values of quercetagetin-7-O-glucoside (1) and okanin (3) were 79.06 ± 1.08 μM and 30.25 ± 1.11 μM, respectively, which is smaller than 100.21 ± 0.11 μM of the positive control kojic acid. Enzyme kinetic analysis and molecular docking were carried out to investigate their inhibition mechanism. Although marein (2) showed a weak inhibition effect in vitro, it inhibited the intracellular tyrosinase activity and diminished melanin production in melanoma B16 cells as did the other two inhibitors. The paper-based ligand fishing method developed in this work makes it effective to quickly screen tyrosinase inhibitors from natural products. This is the first report on the tyrosinase inhibitory effect of those three compounds, showing the promising potential of Coreopsis tinctoria for the development of herbal skin-lightening products.