Culture Expansion Research Articles

Manufacturing, Characterization and Anti-Tumor Activity of Allogeneic CD19-CAR Cytokine-Induced Killer (CIK) Cells Engineered with Non-Viral Sleeping Beauty Transposon System and Armored with IL-18

Rationale & Objective: Adoptive cell therapy by chimeric antigen receptor- engineered cytokine-induced killer (CAR-CIK) cells has demonstrated high therapeutic potential with minimal alloreactivity and little to no toxicity in B-cell acute lymphoblastic leukemia patients, but further development is required to improve anti-tumor activity and persistence in vivo. The expression of the proinflammatory cytokine interleukin-18 (IL-18) has been shown to greatly enhance CAR-T cells potency and persistence. The purpose of the work described here was to develop a workflow for the manufacture of functional CAR-CIK cells, using the non-viral Sleeping Beauty transposon system, that co-express a second generation CD19-targeted CAR and the proinflammatory cytokine IL-18, hereafter referred to as CARCIK-1918 cells. The CAR molecule expressed by these cells includes an attenuated CD28 intracellular domain for enhancement of anti-tumor activity and in vivo persistence. Characterization of the manufactured armored CARCIK-1918 cells and evaluation of their anti-tumor activity in vitro and in vivo were performed. Methods: PBMCs isolated from healthy donors were cultured with IFN-γ, stimulated with anti-CD3 and IL-2, electroporated for non-viral genetic modification with Sleepy Beauty transposase RNA (SB100X) and pT4-1928'z-IL18 bicistronic plasmid DNA, and cultured for 15 days (17-day process). In process and final product characterization included monitoring of cell culture composition, cell expansion and viability, and evaluation of the expression of CAR, CIK proteins CD56 and NKG2D, and memory phenotype markers using flow cytometry. The final product CARCIK-1918 cells were further characterized by in vitro stimulation with CD19 + REH tumor cells to assess tumor killing activity and IL-18 secretion. CARCIK-1918 in vivo efficacy and persistence were also investigated in an NSG/Daudi mouse model. Mice were grafted with CD19 + Daudi tumor cells and, after 2 days, received an intravenous injection of 5x10 6 or 10x10 6 CARCIK-1918 cells. Daudi-grafted untreated mice were used as controls. Survival was monitored, and tumor burden and CAR-CIK cell persistence evaluated by using flow cytometry to assess human CD19 + and human CD3 + cells respectively, in peripheral blood, bone marrow, spleen, and kidney. Results: We manufactured >15 lots of CARCIK-1918 cells. After electroporation on day 2, CAR expression was already detected at day 7 and increased during the expansion culture. After the 17-day process, the range of harvested cells was 4.4E+09 to 1.6E+10 viable cells, with a cell viability >94%, and a fold expansion that varied among lots (range 20.1 - 161.4). The final CARCIK-1918 cells were predominantly CD3 +, expressed both the CAR (range 13 - 81%) and CIK-specific markers CD56 (range 27 - 88%) and NKG2D (range 59 - 86%) at variable levels among batches, and showed mostly a central memory/effector memory phenotype. CARCIK-1918 cells demonstrated potent and specific in vitro cytotoxicity towards the CD19 + REH target cell line. Cells secreted IL-18 only in the presence of CD19 + target cells (range 62.17 - 560.58 pg/mL). In addition, CARCIK-1918 cells had a substantial anti-leukemic activity in vivo. CARCIK-1918 injection resulted in a significant dose-dependent reduction of tumor growth and improved survival, as compared with untreated NSG/Daudi mice. CARCIK-1918 cells persisted in vivo in mouse peripheral blood up to 90 days. Post-mortem analysis demonstrated that CARCIK-1918 cells from both dose levels persisted over time and limited the level of leukemic cell dissemination in the animals, with 10x10 6 regimen allowing an almost total control of the disease progression in all tissue examined. Conclusion: In conclusion, this study demonstrates that manufacturing of armored CAR-CIK cells using Sleepy Beauty non-viral transposon system and 2 nd generation CD19 CAR with an attenuated CD28 costimulatory domain and armored with IL-18 is feasible and allows rapid and efficient expansion of highly potent CARCIK-1918 cells. These cells demonstrated potent and specific anti-tumor activity in vitro and a robust anti-leukemic activity in vivo. These findings provide the first reported successful production of IL-18 armored CAR-CIK cells offering a promising strategy for the treatment of B-cell malignancies.

New Optimized Academic Anti-BCMA CAR (CARTemis-1): How Does the Manufacturing Process Impact CAR-T Cell Features?

Introduction CAR therapy targeting BCMA (B-cell maturation antigen) is under research as treatment for multiple myeloma (MM). However, given the lack of a plateau in most studies, it becomes imperative to pursue more effective alternatives. BCMA downmodulation, the presence of soluble BCMA or the immune dysfunction observed in MM are some limitations that may affect efficacy. Next-generation CAR-T cells are being designed exploring new approaches to overcome restrictions to CAR-T efficacy in MM. We present a novel optimized anti-BCMA CAR (CARTemis-1) and its translation to the clinic. We have specifically focused on the study of the dynamics of the immunophenotype of CARTemis-1 along the manufacturing process, analyzing how these variables might impact the quality of the final product and influence the release decision. Materials and methods CARTemis-1 was evaluated in preclinical in vitro and in vivo models. CARTemis-1 generation from three healthy donors was validated under GMP-conditions in CliniMACS Prodigy with IL-7/IL-15. Flow cytometry was performed to characterize the immunophenotype along the manufacturing process. Quality controls of the final CARTemis-1 product were performed according to the criteria of the Spanish Medicine Agency. Stability of the final cryopreserved CARTemis-1 cell product was also analyzed. Results CARTemis-1 is a second generation anti-BCMA CAR that co-expresses the truncated epidermal growth factor receptor (EGFRt) as a transduction marker and suicide gene. Two different versions of CARTemis-1 were synthesized with different spacer regions (short vs long) with the long version showing significantly greater anti-myeloma function in vitro compared to the short version (p<0.001) (Figure 1A). During the preclinical validation, CARTemis-1 demonstrated potent anti-tumor capacity in vitro and in vivo, showing no susceptibility to the presence of soluble BCMA. Expansion with IL-2 or IL-7/IL-15 was compared (n=8), obtaining increased proliferation (mean 1.59x10 7 vs 3.07x10 7, respectively, p=0.002), increased stem cell memory and naïve population (mean 37.48% vs 43.95%, respectively, p=0.0052), and less PD1+LAG3+ levels (1.38% vs 0.55%, respectively, p=0.0338) with IL-7/IL-15. The generation of CARTemis-1 was validated under GMP-conditions closely monitoring the immunophenotype dynamics from leukapheresis to the final product. Through comprehensive evaluations, the optimal time-point for product release to achieve the best-fitness product was >6 (specifically, day 8-10) with reduced levels of exhaustion markers (Figure 1B) maintaining hallmarks of T cell activation and anti-tumor potency. In vivo biodistribution of CARTemis-1 cells was studied in mice models without myeloma cells, finding preserved tissue structure without relevant morphological alterations in brain, cerebellum, intestine, lung, endometrium, ovary, testicle, spleen, and liver. We performed graft-versus-host disease (GVHD) in vivo mice models and found no increase in GVHD incidence following CARTemis-1 inoculation. Additionally, we assessed the cryopreserved product stability, and it met all specifications and retained its functionality for up to 12 months after cryopreservation. With this positive outcome and having obtained the approval of the Spanish Medicine Agency, we plan to proceed with a phase I/II clinical trial using CARTemis-1 for patients with multiple myeloma who have experienced relapse following allogeneic transplantation (EudraCT 2021-001955-15). Conclusions CARTemis-1 has been rationally designed to increase anti-tumor efficacy and overcomes sBCMA inhibition. To our knowledge, this is the first study analyzing the impact of the manufacturing process in the dynamics of the CAR-T immunophenotype highlighting the importance of the immunophenotypic characterization of CAR-T cells throughout the manufacturing process to define the optimal cell culture protocol and expansion time to increase CAR-T cell product fitness.

Characterization of Hematopoietic Stem Cell Diversity Using Spatiotemporal Quantitative Phase Imaging

Cells undergo continual transformations, never retaining their original state. Hematopoietic stem cells (HSCs), at the apex of the hematopoietic hierarchy, are not exempt from this dynamic process. In recent years, the advent of single-cell analysis techniques such as scRNAseq has progressively unveiled the heterogeneity within cell populations previously presumed to be homogeneous. However, these techniques are limited to evaluating the cellular state at a single temporal point based on snapshot analysis, thus failing to account for temporal changes and dynamic behavior of cells. Therefore, the true diversity inherent to the ever-changing HSCs remains uncertain. In this study, we comprehensively elucidate HSC diversity, including temporal changes, by integrating our recently established ex vivo HSC expansion techniques (Wilkinson et al., Nature, Becker et al., Cell Stem Cell), single-cell quantitative phase imaging (QPI), and post-transplantation dynamics analysis. For live imaging, HSCs were harvested from hepatic leukemia factor ( Hlf)-tdTomato reporter mice, which specifically marks long-term HSCs. Index sorted single Hlf-tdTomato + HSC were expanded with a single-HSC culture system and monitored by QPI fluorescence time-lapse imaging. Surprisingly, even though the cells were derived from the same HSC fraction, significant variations in mitotic rate, motility, morphological changes, and Hlf expression levels were observed at the single-cell level. Stemness did not decrease stepwise over time, but always exhibited continuous changes. Thus, analysis including the temporal dimension illuminated the infinitely branched nature of HSC diversity. Interestingly, Hlf expression level correlated with the dynamic behavior of HSCs in the expansion culture system. Hlf-high HSCs exhibited a slow division rate, small, rounded morphology and low motility, while Hlf-low HSCs actively divided and differentiated, displayed larger morphological size with low sphericity, and exhibited high motility. Furthermore, spatiotemporal dynamics analysis of Hlf-high/low HSCs after transplantation, employing the highly sensitive in vivo imaging system Akaluc bioluminescence imaging (AkaBLI), revealed that Hlf-high HSCs initiate systemic hematopoiesis slowly, while Hlf-low HSCs expanded quickly, characterized by short-term explosive hematopoiesis in the spleen. As expected, only Hlf-high HSCs had long-term bone marrow reconstitution ability. Thus, based on the expression level of Hlf, it is possible to predict the dynamic behavior of the cells in the expansion medium and in the post-transplant body. Next, we investigated whether Hlf expression level could be predicted based on the kinetic features of cells during HSC ex vivo expansion. Interestingly, multidimensional analysis based on kinetic features extracted from time-lapse imaging showed multiple clusters, each linked to Hlf expression levels. In human HSCs, the dynamic diversity of HSCs was also observed in our recently established ex vivo expansion system (Sakurai et al., Nature). Notably, the kinetic features of human HSCs differed from those of mouse HSCs. Transplantation of human HSCs exhibiting specific morphological characteristics showed long-term reconstitution ability. Thus, classification of HSCs based on kinetic features facilitated enrichment of functional HSCs. This study demonstrates the importance of characterizing cells, including the temporal dimension, and illustrates the existence of infinite dynamic individuality in mouse and human HSCs.

Extracellular Vesicle Trafficking Regulates Hematopoietic Stem and Progenitor Cell Fitness

Among regulatory pathways critical to hematopoietic stem and progenitor cell (HSPC) function, sphingolipid metabolism has recently been recognized for its role in safeguarding essential HSPC properties of self-renewal and lineage fate. In particular, de novo ceramide (Cer) synthesis appears to regulate a coordinated stress response that results in enhanced self-renewal of human HSPCs. However, the fundamental molecular mechanisms underlying these regenerative stress responses have yet to be uncovered and broadly validated. Moreover, alternative synthetic pathways regulating Cer and sphingomyelin (SM) levels may serve additional regulatory roles in HSPCs. Here we target neutral sphingomyelinase-2 (nSMase2/SMPD3)-dependent SM hydrolysis through pharmacologic (GW4869) and genetic (Cas9-mediated) interruption, and similarly confirm an adaptive ATF4-mediated integrated stress response (ISR). Functionally, transient inhibition led to durable improvements in HSPC reconstitution after primary (>8 fold at steady-state, p<0.001) and secondary (>20 fold at steady-state, p<0.001) transplantation. Surprisingly, lipidomic profiling of GW4869-treated HSPCs demonstrated neither global Cer depletion nor SM accumulation, suggesting an alternative mechanism underlying the observed gains in HSPC fitness. Sphingolipid metabolism also regulates the biogenesis of select subclasses of secreted extracellular vesicles (EVs). To segregate metabolic regulation of HSPCs by sphingolipids from their role in EV formation and secretion, we began to systematically modulate and compare several core EV biogenesis pathways by Cas9/ribonucleoprotein-mediated knockout (KO) of SMPD3, CD63 and TSG101 in HSPC ex vivo expansion cultures . Similar to pharmacologic nSMase2 inhibition, loss of all 3 genes suppressed EV secretion, whereas only depletion of SMPD3 or ESCRT protein TSG101, but not the tetraspanin protein CD63, led to ISR activation in HSPCs (p<0.05). As a functional corollary, clonogenicity and long-term transplantation outcomes were enhanced by KO of SMPD3 (1.5-fold; p<0.01)and TSG101 (2-fold; p<0.001), but reduced after CD63-KO (0.6-fold; p<0.05). One function of EVs is the export of protein and nucleic acid cargo from the cell. We reasoned the observed differences in stress response and HSPC fitness may reflect uniquely altered secretomes. Quantitative proteomic analysis of the vesicle secretome across KO cells revealed impaired secretion of mitochondrial (n=160, FDR=8.3E-22) and oxidative stress-related (n=82, FDR=2.8E-12) proteins from SMPD3- and TSG101- KO, but not CD63-KO HSPCs, highlighting the heterogeneity and specificity of EV cargo trafficking. We validated this observation by pharmacologic blockade of SMPD3 with GW4869, which similarly led to transient decreases in mitochondrial DNA (0.36-fold, p<0.01) and protein (0.6-fold, p<0.05) export. Finally, we confirmed that impaired mitochondrial cargo secretion into EVs (mtEV) after SMPD3 or TSG101, but not CD63, depletion led to transient accumulation of mitochondrial-sourced reactive oxygen species (p<0.01) that specifically triggered the observed ISR activation and downstream mTOR inhibition. Our study reveals a novel mechanism by which secretory cargo trafficking into EVs guards HSPC homeostasis and fitness. We further demonstrate that transient modulation of this pathway, wherein EV cargo retention activates a molecularly-defined stress response pathway, can durably improve long-term transplantation outcomes. Sphingolipid biology inextricably links cellular metabolism and EV biogenesis, making it likely that prior descriptions of Cer-dependent adaptive stress response involve modulation of EV secretion. Finally, our work underscores the high degree of specificity that governs EV secretion in regulating intrinsic cell function. In aggregate, we show that differential EV biogenesis pathways regulate HSPC fate and fitness, with important translational implications in safeguarding stemness and regenerative capacity during ex vivo expansion and therapeutic transplantation.

Platinum Talen-Mediated Knock-in of Single-Stranded DNA Templates Facilitates Manufacturing of Clinical-Scale Non-Virally Gene-Edited T Cells from Peripheral Blood

Background: During the past decade, technologies that manufacture gene-modified immune cells, such as chimeric antigen receptor T cells, have been rapidly evolved and introduced in human clinics. T cells reprogrammed to express antigen-specific T-cell receptors (TCR-T) are an up-and-coming tool to treat intractable infection or malignant neoplasm in a personalized manner. However, several caveats still exist in the production of TCR-T for clinical use, because competitive surface expression of endogenous and transduced TCRs might hamper desired expression of transduced TCR. Moreover, mispairing of TCR subunits could mediate unexpected harmful immune complications. Therefore, in this study, we aimed to develop a method to introduce any desired TCRs in human primary T cells independent of interference with endogenous TCR by way of targeted knockout of TRA and TRB gene loci using highly-efficient transcription activator-like effector nuclease (TALEN), named Platinum TALEN (Sakuma T, Sci Rep 2013). Methods: We designed and synthesized two pairs of 5'-Cap-1 Platinum TALEN mRNA targeting the gene sequences that encode constant regions of human TRA ( TRAC) and TRB ( TRBC). TCR knockout efficiencies by these TALEN pairs were assessed by flow cytometric analysis using anti-CD3/TCR antibodies. Next, we constructed single-stranded DNA (ssDNA) homology-directed repair templates for the knock-in of desired TCR transgene into the TALEN target sequence of the TRAC locus. As a model TCR, we selected a 1G4 clone that reacts with NY-ESO-1-derived peptide in an HLA-A*02-restricted manner. We delivered these TALEN mRNA pairs and 1G4-encoding ssDNA by electroporation into primary human T cells collected from healthy volunteer adult donors after 72 hours of anti-CD3/CD28 beads activation. Genome-edited T cells were subsequently cultured in Therapeak TM X-VIVO15 (Lonza) supplemented with 10% human AB serum in the presence of 2-mercaptoethanol and interleukin-2 followed by second anti-CD3/CD28 beads activation. Density of T cells were maintained approximately at 1.0-3.0 x10 6/mL and transferred to larger flasks with additional fresh media as appropriate according to cell counts throughout the culture.1G4-transduced T cells were detected and counted by HLA-A*02:01 NY-ESO-1 tetramers at 17-22 days after electroporation. Additionally, we performed optical genome mapping analysis (Bionano Genomics) of Platinum TALEN-treated cells to screen genome-editing-associated large chromosomal structural variations (SVs) including unintended translocation. Results: Platinum TALEN mRNA pairs targeting TRAC and TRBC yielded a high proportion (>70%) of CD3/TCR negative T cells without the cost of compromised cell viability. By co-electroporation of 1G4 ssDNA and these Platinum TALEN mRNA pairs, we successfully obtained an average of 10.5 (range 2.2-19.0) million (n=5)NY-ESO-1-tetramer-positive cells with high viability from 3 millionprimary T cells until 3 weeks after electroporation. By flow cytometric analysis, a proportion of 1G4-tetramer positive cells that express Vβ chains other than Vβ13 (Vβ of 1G4) was approximately 10%. The PD-1-positive fraction in the expanded 1G4-expressing T cells was less than 10%, while levels of TIM-3 expression were 40-60%. These tetramer-positive cells showed specific cytotoxicities against HLA-A*02-positive cells pulsed with cognate NY-ESO-1 peptides. In an optical genome mapping analysis of genomic DNA obtained from TALEN-treated T cells, we found unique SVs with relatively small sizes and low coverage but no large unique SVs with high coverage when compared with control genomic DNA. Additionally, no interchromosomal translocation was observed. Conclusions: We have successfully designed Platinum TALEN mRNA pairs targeting TRAC and TRBC which facilitate the production of genome-edited human primary T cells. We also developed an electroporation and expansion culture protocol that enables us to produce viable genome-edited 1G4-transduced T cells at a large cell dose equivalent to a clinical scale. Collectively, these results suggest that targeted orthotopic knock-in of antigen-specific TCR-encoding ssDNA into the TCR locus by the use of Platinum TALEN is a promising strategy that can be applied for the clinical manufacturing of therapeutic TCR-T cells.

Enhancement of Mitochondrial Membrane Potential and Pro-Survival Pathways with Azole Compound C7 Resulting in Expansion of Hematopoietic Stem and Progenitor Cells

Ex-vivo expansion of hematopoietic stem & progenitor cells (HSPC) could improve hematopoietic recovery and reduce post-transplant complications, especially for umbilical cord blood (UCB) grafts where limited total cell numbers result in delayed engraftment. Freshly-thawed UCB mononucleated cells (MNCs) or purified CD34 + cells were cultured in animal component free medium supplemented with C7 and a basal cytokine cocktail. The effects of the expansion protocol were measured with phenotypic, in-vitro, and in-vivo functional assays. A range of molecular techniques were also performed to elucidate the mechanisms of action of C7. In previous studies, expansion cultures initiated with UCB-MNCs and supplemented with C7 resulted in significant expansions of UCB HSPC over 11 days and these grafts demonstrated elevated and sustained multi-lineage engraftment of human cells in primary NSG mice (Bari et al., 2018). In UCB-CD34+ enriched cultures, the addition of C7 to a basal cytokine cocktail also boosted absolute CD45+CD34(BR)+CD38-CD45RA- progenitor expansion by 283±29.4 fold over 11 days which was 1.86±0.12 fold higher than control cultures (p<0.001; n=6). Furthermore, C7 induced significant expansion of primitive HSPC in UCB-MNC defined by phenotype CD45+CD34(BR)+CD38-CD45RA-CD90+CD49f+ by more than 633±8.5 fold, which is 7.5±0.16 fold higher compared to control cultures (p<0.01; n=3). The addition of C7 to CD34+ cells from mobilized peripheral blood (PB) and bone marrow (BM) also resulted in a significant expansion of HSPCs (p<0.05; n=3) (Fig 1A). When expanded UCB-CD34+ were engrafted in NSG mice, NSG PB analysis at week 3 and week 28 post-transplantation showed significantly higher (p<0.01) human CD45+ cell engraftment in C7 expanded graft (16.4%±2.6) than cytokine expanded graft (7.8%±0.9). NSG BM analysis at week 31 also exhibited similar trends for human CD45+ cell engraftment in C7 expanded grafts with multilineage reconstitution (p<0.01; n=6). Significantly enhanced CD45+ cell viability (p<0.01; n=3) was observed in C7 cultures compared to control cultures. Mitochondrial health was also improved as confirmed by lower mitochondrial superoxides and higher mitochondrial membrane potential in C7-cultured UCB-MNCs (p<0.05; n=3) that is reflective of reduced reactive oxygen species (ROS) (Fig 1B). As C7 is a chemical analogue of a p38 inhibitor (SB203580), p-p38 (T180/Y182) was significantly reduced in C7-expanded CD34+ culture compared to controls. Significant increases in HIF-1α in C7 expanded CD34+ cells were also observed, potentially indicative of a hypoxic state induced by C7 to benefit HSPC together with reduced levels of ROS. Kinase inhibition profiling studies on C7 identified CK1δ, a kinase involved in Wnt and p53 signalling pathways, as one of the activation targets directly activated by C7. CK1δ was boosted in C7-expanded cells and this was further validated with western blots on C7 expanded pooled CD34+. CK1δ plays a putative role in p53 activation which is stipulated to promote HSC quiescence favouring long-term maintenance of HSPCs. Western blot analysis also revealed that p53 and BCL-2 were increased with C7 expanded CD34+. GSK3β was also boosted in C7 expanded pooled CD34+ cultures possibly as a result of CK1δ signalling leading to increased proliferation via the Wnt signalling pathway. Multiple safety studies were conducted on C7 to ensure its safety in clinical applications. This included the BIOMAP phenotypic safety and toxicology tests on human primary cells, AMES test and fluctuation assays for bacteria and SAFETYscan47 TM In Vitro Pharmacological Profiling for biochemical safety, all of which were negative for any cytotoxicity or off-targets associated with acute toxicity. No changes were detected in cytogenetics and fluorescence in situ hybridization with UCB-MNC for major leukemia-related genetics as well when they were cultured with C7. As a small molecule capable of significant HSPC expansion from both UCB and PB sources, C7 is promising for the future of HSPC transplantation. The mechanism behind C7 is likely to be a multi-pronged one with p38 inhibition, increase in HIF-1α, CK1δ activation, improved mitochondrial health, and induction of pro-survival pathways for expanding HSPC populations.

Pengaruh Motivasi Hedonisme dan Korean Wave Terhadap Impulsive Buying dengan Personal Branding Syariah Sebagai Variabel Intervening

In the last two decades, Korean culture has experienced rapid development and has spread globally to various countries in the world, including Indonesia. The Korean Wave or in Korean usually called hallyu according to (Suh, Cho, & Kwon, 2006) is defined as "The rapid expansion of the Korean contemporary culture and cultural products throughout East Asia since the mid-1990's" or the expansion of Korean culture and products carried out in a large and rapid manner to East Asia in the mid-1990s. The massive development of information technology due to globalization is the main factor in the growth of public enthusiasm for the Korean Wave in Indonesia itself, especially among the younger generation (millennial generation) in the 2000s and the current young generation (generation Z). The aim of this research is to determine the influence of Hedonistic Motivation and the Korean Wave on Impulsive Buying with Personal Branding as an intervening variable. This research uses quantitative methods to test and search for evidence for the hypotheses that have been established. As many as 133 respondents were generated from the questionnaire distributed by researchers. The purposive sampling method was chosen in this research because the researcher determined the criteria and attributes of the individuals who were used as research respondents. Judging from the objectives of the research conducted, it is known that hedonistic motivation is related to impulsive buying, but the Korean wave and personal branding are not related or have an influence on impulsive buying. However, hedonic motivation and Korean wave each have a significant influence on personal branding. The research model construct focuses on impulsive buying among consumers who are fans of K-pop groups in Indonesia. The research was carried out with a sample size that was stated to be sufficient using PLS SEM as a data processing tool. Next, findings related to the research hypotheses presented will be revealed.

The Influence of Western Fantasy Fiction on Contemporary Bengali OTT and TV Series

The publication of the Lord of the Rings trilogy marks a significant watershed moment in the literary history of the twentieth century. After the publication of this seminal text, it was “translated into numerous languages”(Manlove 156) and it also began to weave its influence into the various literary and cultural traditions of the world. Though the state of West Bengal possessed a powerful and rich literary and cultural background, in terms of supernatural or fantastic literature, the Bengali cultural field also started to take the colour of Tolkien’s magnum opus. Subsequently, the Harry Potter phenomenon in the 1990s, and the widely watched Peter Jackson film adaptations of the Lord of the Rings, added to the popularity of Western fantasy in West Bengal. This can be traced in a number of articles in Bengali magazines, journals, and blogs, such as “Angti Niye Abar Lorai,” “Dorja Jure Bisal Sap Er Murti! Khoj Millo,” which reveal the investment of Bengali audiences in the genre. Gradually, as this paper will argue in some detail, some of the newly emergent Bengali OTT and TV series, coming out in the post 2010s - were also coloured by the cultural expansion of Western fantasy. This paper will particularly demonstrate the influence of Western fantasy on Bengali OTT and TV Series with a special emphasis on the features of Western fantasy like the evocation of wonder through specific devices, mythopoeic tendencies, the dynamics of wish fulfilment, and the like. Side by side, the resemblance of scenes, actions, and ideas of the Lord of the Rings trilogy and the Harry Potter series in Bengali OTT and TV Series will also be depicted in this paper. The primary texts that will be studied, include one web series and two serials: Rudrabinar Obhishaap, Kiranmala, and Saat Bhai Champa.

The Path of Meishan Cultural Renewal and Expansion Based on Data Association Rules

Abstract Metadata and data association rules are combined in this paper to create an aggregation framework for Meishan cultural digital resources. Meishan cultural digital resources are standardized through the use of metadata standards. The accuracy of metadata is evaluated in conjunction with trustworthiness. Metadata association rules are employed to evaluate the degree of association in Meishan cultural data. Enhance the efficiency of data association analysis by enhancing the Apriori algorithm. The algorithm is applied to the association analysis of Meishan culture data, and the degree of association between different digital resources and Meishan culture is determined by combining the data. The results show that the highest correlation with Meishan culture is the digital resources of documentaries and skills, with a correlation degree of 0.9, and the lowest correlation is the book resources, with a correlation degree of 0.65. The number of nodes in the period of 1-6 and the time consumed by each dataset is in the interval of (7000,1100).

Utilizing YouTube for The Local Film Culture Expansion

This article explore the role of YouTube in developing local film culture. The Covid-19 pandemic has disrupted various local short film festivals and screenings. Local filmmakers are trying to find alternative channels to show their short films via YouTube. Using the media industries studies perspective, this research offers two cases, Tilik and Loz Jogjakartoz, to study the role of YouTube for local film the promotion and distribution. Based on in-depth interviews, Ravacana Films and Kebon Ijo Studio implement specific strategies such as film upload timing, social media promotion and audience attention management so that their films trend on YouTube. This research reveals that YouTube can play an important role in connecting local filmmakers with audiences and the national film industry.

THE EFFECT OF CHRONOLOGICAL AGE AND GENDER AND DIFFERENT SOURCE OF TISSUE ON THE IN VITRO PROPERTIES OF MESENCHYMAL STROMAL CELLS

AbstractObjectivesOsteoarthritis is a common articular cartilage disorder and causes a significant global disease burden. Articular cartilage has a limited capacity of repair and there is increasing interest in the use of cell-based therapies to facilitate repair including the use of Mesenchymal Stromal Cells (MSCs). There is some evidence in the literature that suggests that advancing age and gender is associated with declining MSC function, including reduced proliferation and differentiation potential, and greater cellular apoptosis. In our study, we first performed a systematic review of the literature to determine the effects of chronological age and gender on the in vitro properties of MSCs, and then performed a laboratory study to investigate these properties.Methods and ResultsWe initially conducted a PRISMA systematic review of the literature to review the evidence base for the effects of chronological age and gender on the in vitro properties of MSCs including cell numbers, expansion, cell surface characterization and differentiation potential. This was followed by laboratory-based experiments to assess these properties. Compare the extent of the effect of age on MSC cell marker expression, proliferation and pathways. Tissue from patients undergoing total knee replacement surgery was used to isolate MSCs from the synovium, fat pad and bone fragments using a method developed in our laboratory. The growth kinetics was determined by calculating the population doublings per day. Following expansion in culture, MSCs at P2 were characterised for a panel of cell surface markers using flow cytometry. The cells were positive for CD73, CD90 and CD105, and negative for antibody cocktail (eg included CD34, CD45). The differentiation potential of the MSCs was assessed through tri-lineage differentiation assays. At P2 after extracting RNA, we investigate the gene analysis using Bulk seq. Clear differences between the younger and older patients and gender were indicated.ConclusionsChronological age and gender-related changes in MSC function have important implications on the use of these cells in clinical applications for an ageing population. The results from this study will be used to plan further work looking at the effects of chronological age and gender on cellular senescence and identify pathways that could be targeted to potentially reverse any age and gender-related changes.Declaration of Interest(b) declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported:I declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research project.

Foreign Policy Issues of the Republic of Turkey and the United States in the Period after the End of the Cold War until the 2020

At the beginning of the 21st century, in 2002, a new political era began with the arrival of Recep Tayyip Erdogan and the Islam-oriented Justice and Development Party (AKP) at the head of the Republic of Turkey, which was gradually followed by changes in both foreign and domestic policy of the country.
 The main ideologist of modern Turkey’s foreign policy is one of the founders of the Justice and Development Party and Erdogan’s ally, former Foreign Minister and Prime Minister Professor Ahmet Davutoglu, who outlined the strategic priorities of Turkey’s foreign policy in his pioneering work Strategic Depth. In his doctrine, Ahmet Davutoglu argues that Turkey has “strategic depth” that allows it to pursue an independent foreign policy and claims to be the leading state in the region.
 As we know, the Republic of Turkey is located at the crossroads of Europe and Asia. According to the doctrine, precisely because of its geographical position and historical ties, it has a desire to influence all these regions (the Middle East, the Balkans, the Caucasus, Central Asia, the Mediterranean, the Persian Gulf, and the Black Sea), what is considered the core of neo-Ottoman ideology.
 Neo-Ottomanism implies a shift of the Turkish political vector from West to East. Since the beginning of the 21st century, neo-Ottomanism as an ideology has become a force responsible for the shape of political life of Turkey. According to the ideology of neo-Ottomanism, the Republic of Turkey must get rid of the subordination of the United States, which will allow it to put itself on a par with such states as Britain, France, Russia, and China.
 Neo-Ottomanism is the main ideological direction of the new foreign policy of the Republic of Turkey. One of the arguments used by Erdogan and his party in moving from a parliamentary to a presidential model is the fact that the country needs a government that is almost as strong and centralized as the Ottoman Empire. At the same time, the president himself and his entourage constantly emphasize that the Turks are the “heirs of the Ottomans” and that the country must return to its former glory and strengthen its political, economic and cultural influence in the former territories of the Ottoman Empire.
 Thus, neo-Ottomanism is part of the official ideology of the Justice and Development Party and the basis of the country’s new identity, shaped by the political elite over the years. Although there is no direct indication of Turkish regional hegemony in the “strategic depth”, most analysts, especially in the West, believe that the expansion of spheres of influence mentioned in the doctrine is, in fact, nothing more than the restoration of Turkey’s monopoly hegemony in the region, but this times by means adapted to the modern era, in particular through the use of political, economic and cultural expansion and other “soft power” tools.

The Historical Evolution of Russophilia in Europe: a Study of the Development and Transformation of Positive Attitudes Towards Russia

The purpose of this paper is to investigate the historical trajectory, spread, and evolution of russophilia, a sociological phenomenon that exists in contemporary Europe. The article conducts a comprehensive analysis of the developmental process of russophilia in Europe, tracing its origins from the early stages of primitive sympathy towards the Russian state during the Middle Ages to its subsequent conscious ideological-cultural and political-economic manifestations in the 20th century. Furthermore, the article examines the contemporary projection of russophilia, which reached its pinnacle in the aforementioned region during the 20th century, while also exploring the factors contributing to the current decline of russophilia.
 The article introduces a significant scientific contribution by being the pioneering study to comprehensively explore the historical evolution of russophilia in Europe. Previous research on russophilia has been limited in scope, with only a few articles addressing the topic in a fragmented manner. Prior to this article, there has been a lack of comprehensive studies examining the overall development of russophilia in Europe. Therefore, this article stands as the first comprehensive endeavor in this field of research, filling a notable gap in the existing literature.
 Conclusions. The results of the study shed light on the origins and development of russophilia in Europe, examining both Western and Eastern regions. Key factors contributing to russophilia include the influence of communism, particularly the USSR, as well as cultural expansion. Effective propaganda campaigns and the establishment of economic and social connections play crucial roles in fostering admiration for another culture. Ethnic kinship alone is insufficient to foster deep affinity without widespread and systematic propaganda integrated into state policy. Nostalgia for communism, fuelled by shared experiences and the positive aspects of former communist systems, also contributes to russophilia. Countries such as East Germany, Poland, and Serbia etc., which underwent communist rule, exhibit heightened russophilia. The common fate shared by these nations, including the same political ideology, communist culture, and perceived defeat by the capitalist USA, further strengthens russophilia sentiments among communist nostalgics.

Human left ventricular cardiac fibroblasts undergo a dynamic shift in secretome and gene expression toward a cardiac myofibroblast phenotype during early passage in typical culture expansion conditions

Cardiac fibroblasts (CFs) are critical components of the cardiac niche and primarily responsible for assembly and maintenance of the cardiac extracellular matrix (ECM). CFs are increasingly of interest for tissue engineering and drug development applications, as they provide synergistic support to cardiomyocytes through direct cell-to-cell signaling and cell-to-ECM interactions via soluble factors, including cytokines, growth factors and extracellular vesicles. CFs can be activated to a cardiac myofibroblast (CMF) phenotype upon injury or stimulation with transforming growth factor beta 1. Once activated, CMFs assemble collagen-rich ECM, which is vitally important to stabilize scar formation following myocardial infarction, for example. Although there is greater experience with culture expansion of CFs among non-human strains, very little is known about human CF-to-CMF transitions and expression patterns during culture expansion. In this study, we evaluated for shifts in inflammatory and angiogenic expression profiles of human CFs in typical culture expansion conditions. Understanding shifts in cellular expression patterns during CF culture expansion is critically important to establish quality benchmarks and optimize large-scale manufacturing for future clinical applications.

European Capital of Culture and creative industries: Real impact or unproven belief? The case of Wrocław

This study investigates the impact of the European Capital of Culture (ECoC) Wrocław 2016 on the growth of creative industries in the city. The research involved surveying managers of local enterprises in Wrocław's creative sectors, focusing on their opinions regarding the expected benefits of the ECoC and its potential influence on the companies' economic development in the future.The findings indicate that most respondents believed the ECoC positively impacted local development by fostering new artistic endeavours and promoting local entertainment companies. They also perceived significant economic effects, including increased demand for cultural goods and services and increased spending on culture and entertainment. Instead, opinions on company development were primarily influenced by the long-term investment climate that involves multiple factors. The ECoC indirectly contributed to this climate by redirecting local policies towards further cultural expansion, creating a more welcoming environment for cultural industries, and triggering spillover effects in this sector.Thus, the study argues that the ECoC title is not a direct catalyst for developing cultural industries in Wrocław. Rather, it represents the culmination of a long-term strategy to establish the city as a cultural hub, effectively enhancing this sector of the local economy.

Impact of Human Recombinant Irisin on Tissue-Engineered Skeletal Muscle Structure and Function.

Tissue engineering of exogenous skeletal muscle units (SMUs) through isolation of muscle satellite cells from muscle biopsies is a potential treatment method for acute volumetric muscle loss (VML). A current issue with this treatment process is the limited capacity for muscle stem cell (satellite cell) expansion in cell culture, resulting in a decreased ability to obtain enough cells to fabricate SMUs of appropriate size and structural quality and that produce native levels of contractile force. This study determined the impact of human recombinant irisin on the growth and development of three-dimensional (3D) engineered skeletal muscle. Muscle satellite cells were cultured without irisin (control) or with 50, 100, or 250 ng/mL of irisin supplementation. Light microscopy was used to analyze myotube formation with particular focus placed on the diameter and density of the monotubes during growth of the 3D SMU. Following the formation of 3D constructs, SMUs underwent measurement of maximum tetanic force to analyze contractile function, as well as immunohistochemical staining, to characterize muscle structure. The results indicate that irisin supplementation with 250 ng/mL significantly increased the average diameter of myotubes and increased the proliferation and differentiation of myoblasts in culture but did not have a consistent significant impact on force production. In conclusion, supplementation with 250 ng/mL of human recombinant irisin promotes the proliferation and differentiation of myotubes and has the potential for impacting contractile force production in scaffold-free tissue-engineered skeletal muscle.

Cyclosporine and Voclosporin Resistant Immune Effector Cells to Improve Outcomes after Stem Cell Transplantation

Introduction: Relapse of a patient's underlying malignancy, and complications related to viral reactivation are prominent causes of morbidity and mortality after allogeneic hematopoietic cell transplantation (HCT). Immune effector cells (IEC) are increasingly deployed to prevent and treat these complications. These include anti-CD19 CAR-T-cells used for post-HCT B-ALL relapse, as well as viral specific T cells (VSTs) targeting EBV, CMV, and others. However, the efficacy of these IECs early after HCT may be compromised by the inhibitory effects of immunosuppressive agents used to prevent graft-versus host-disease (GVHD). A commonly employed immunosuppressant used for GVHD prophylaxis is the calcineurin inhibitor (CNI) cyclosporine (CsA). A structural analog, voclosporin (VCS), is now FDA approved for lupus nephritis and in trials to prevent rejection after solid organ transplantation. Given its favorable pharmacodynamic and toxicity profile versus CsA and tacrolimus, VCS is poised to become an important option for HCT patients. Both CsA and VCS interact with calcineurin only in the presence of their immunophilin binding partner - cyclophilin A ( PPIA). Tacrolimus and sirolimus interact with a distinct immunophilin, FKBP12. This creates the opportunity to delete a single immunophilin (eg. PPIA) to create IECs resistant to one class of CNI (CsA/VCS) but sensitive to other immunosuppressive agents, an important safety feature. Here we developed CsA/VCS-resistant IECs by CRISPR/Cas9 knockout (KO) of PPIA. These cells exhibited superior proliferation and preserved effector function in the presence of CsA and VCS. Methods: We developed IECs that are simultaneously resistant to both CsA and VCS via CRISPR-mediated KO of PPIA. Proliferation, cytotoxicity, and cytokine production of PPIA KO conventional T cells (Tcon), CMV (pp65)-specific VSTs, and CD19 CAR-T cells were tested against mock-edited (MOCK) cells in functional in vitro assays with escalating doses of CsA and VCS. Results: PPIA KO Tcon demonstrated high editing efficiency (88-92%), high viability, and robust expansion in culture. Indeed, PPIA KO efficiency was maintained over several weeks in culture, suggestive of no survival disadvantage of cells bearing this deletion. Importantly, when PPIA KO cells were used as Responders in a mixed lymphocyte reaction in the presence of CsA, the edited cells demonstrated significant resistance to drug-mediated suppression of CD4+ and CD8+ T cell proliferation across the dose-response curve (p < 0.01; Fig. 1). Similar results were observed with VCS, with PPIA KO cells significantly resistant to VCS-mediated suppression across otherwise highly suppressive doses (p<0.0001; Fig. 1). Having demonstrated that edited Tcon were resistant to CsA and VCS, we next sought to make PPIA KO IECs. CRISPR-edited VSTs and CD19 CAR-T cells showed similar editing efficiencies to Tcon. Consistent with prior results, CMV-specific PPIA KO responder VSTs were significantly resistant to suppression of proliferation by CsA vs MOCK cells at all doses tested (p=0.0004, not shown). Proliferation in the presence of drug was then assessed in KO CAR-T cells using the CD19+ NALM6 line as stimulators. Division of PPIA KO responder CD19 CAR-T cells was almost completely unaffected by CsA and VCS across escalating doses ( Fig. 2). For example, at an effector-to-target (E:T) ratio of 1:0.1 and a CsA dose of 50 ng/mL, there was 40.6 ± 2.3 % suppression of division in MOCK CD8+ CAR-T cells vs. -0.2 ± 0.4 % in PPIA KO CD8+ CAR-T cells (p<0.0001). Similar findings were observed with CD4+ responder CAR-T cells (p<0.0001, not shown). PPIA KO CAR-T cells also demonstrated intact cytotoxic function with no significant differences in % killing between the MOCK and PPIA KO groups at 3 different E:T ratios. For example, at a 1:1 E:T ratio, there was 59 ± 15% killing vs. 55 ± 12% killing in MOCK vs PPIA KO groups, respectively (not shown). Conclusion: KO of PPIA is an effective strategy for engineering resistance to CsA and VCS incellular therapies. Our results demonstrate that PPIA KO conventional T cells, VSTs, and CAR-T cells were markedly resistant to the otherwise suppressive effects on T cell proliferation that are hallmarks of both CsA and VCS. Engineering CNI-resistance into IECs will enhance the ability to deploy these cells early after transplant to prevent or treat both malignant relapse and viral reactivation.

Single Cell Analyses Indicate SRSF2 and TET2 Co-Mutation Results in Skewed Haematopoiesis in Myelodysplastic Syndromes

We used single-cell ATAC-seq (scATAC-seq) to study how synchronous SRSF2 and TET2 co-mutations result in skewed myelopoiesis compared with SRSF2 mutation only in subjects with myelodysplastic neoplasms (MDNs). We identified 621 differentially expressed genes in comparing of transcriptome sequencing data of bone marrow mononuclear cells ( p < 0.01; |log2FC| > 2; Figure 1 A). Up-regulated genes in the co-mutation cohort involved those expressed in pluripotent haematopoietic stem cells (HSCs), cytokine receptor interactions and cancer-related signaling pathways ( Figure 1 B). We extended our observation by interrogating data from the GSE58831 public dataset where we identified 18 similar pathways in subjects with SRSF2 and TET2 co-mutations ( Figure 1 C ). We then used scATAC-seq to verify regulatory networks with genes and transcription factors. 49,532 cells had high-quality scATAC-seq profiles and passed stringent quality filtering with a median of 12,858 fragments per cell 39% of which mapped to peaks. 16 cell types were annotated according to cell marker genes and aligned with the public single cell RNA-seq gene expression matrix of haematopoietic cell types ( Figure 1 D-E). Next, we compared transcription factor (TF) and cis-elements active during differentiation of HSC to myelopoiesis betweenthe SRSF2 and TET2 co-mutation and SRSF2 mutation-only cohorts. In the SRSF2 and TET2 co-mutation cohort we found GATA4, RUNX1, SNAI1, REL and JDP2 favour myeloid differentiation and in NF-KB related inflammatory responses. Related gene activity correlated with high activity of inflammatory cytokines such as BCL2A1, EGR1, IL-1β, and NLRP3( Figure 1 F). SRSF2 and TET2 co-mutations result in early clonal dominance at the haematopoietic stem cell stage. 19,836 peaks were accessible in SRSF2 and TET2 co-mutated samples and annotated for putative functions by GREAT ontology enrichment analysis ( Figure 1 G-H). More accessibility peaks in SRSF2 and TET2 co-mutation were restricted to cancer-related process (reactive oxygen species biosynthetic process, regulation of phagocytosis, intrinsic apoptotic signaling pathway, mRNA catabolism) and cytokine related pathways (IL-1 mediated signaling pathway) compared with SRSF2 mutation-only samples. Next, we focused on the IL-1-mediated signaling pathway, a regulator of HSC function. We isolated umbilical cord blood HSCs (CD34+) monitored their expansion in liquid culture with or without (IL-1A or IL-1B (25 ng/ml). HSCs cultured with IL-1A or IL-1B differentiated and expanded significantly faster than untreated HSCs over 7-day period ( Figure 1 I). To directly address the effects of IL-1 on HSC differentiation we used colony formation assays in methylcellulose ± IL-1. Strikingly, IL-1-treated HSCs produced almost exclusively myeloid-committed granulocyte/macrophage (GM)-type colonies containing abundant macrophages (CFU-GM), a higher proportion of immature multi-lineage granulocyte/ erythrocyte /macrophage/megakaryocyte (CFU-GEMM)-type colonies containing mostly immature myeloblasts and a lower proportion of burst-forming unit-erythroid (BFU-E). IL-1-treated HSCs also had decreased re-placing capacity ( Figure 1 J-K). Next, we constructed a SRSF2 P95H single mutation and SRSF2 P95H mutation with TET2 knockout cell lines. We found that co-mutant cells had significantly increased NLRP3 and IL-1β expression in LPS (1 ug/ml) and ATP (5 mM) treated cultures whereas no change was detected IL-1α expression ( Figure 1 L-M). In conclusion, our data preliminary indicate SRSF2 and TET2 co-mutated causes increased NLRP3-mediated IL-1β secretion resulting in skewed differentiation of HSCs in persons with MDNs.

Olympic Ceremonial Costume in the Context of Cultural Expansion/Occupation: Defining the Concept

The purpose of the article is to form the conceptual and terminological component of the study of the Olympic ceremonial costume in the context of cultural expansion. The research methodology involves the use of general logical methods of objective analysis of the design of the Olympic ceremonial costume and analysis of scientific sources, which became the basis for the formation of the conceptual and terminological apparatus of the study. The scientific novelty consists in the fact that, for the first time in the scientific field, an attempt has been made to form a conceptual and terminological apparatus for the study of the Olympic ceremonial costume in the context of cultural expansion. Conclusions. The Olympic ceremonial costume is a transmitter of iconic and symbolic messages that reflects the essence of a country’s culture and serves as a means of its presence at international competitions. Unlike professional sports equipment (uniforms), the main function of this costume is to demonstrate the country’s culture. Given the specifics of the Olympics, designing a costume for the Parade of Nations is a creative endeavour that reflects the historical state of the era – its political, economic, cultural, and social context. The Olympic ceremonial costume is not only a fashion product but also an object of art history, with the help of artistic and informational identification, it reproduces the state of society in its certain historical context. The conceptual and terminological apparatus is a fundamental component of any research. The absence of definitions of concepts leads to a “blurring” of the meanings of the terms used, allows for situational speculation, and makes it impossible to implement mechanisms to bring to justice propaganda adherents who use visual language as a means of instilling the necessary narratives of the political order of a country pursuing an expansionary policy. Cultural expansion, within the framework of which the Olympic ceremonial costume is studied, is part of the subversive activities of the stage of demoralising the enemy. This phenomenon is characterised by the promotion of false information messages in a cultural product, the implementation process of which does not have a specific time frame. Cultural expansion is realised through diffusionism, appropriation, and extrapolation of the cultural heritage of one state by another and has an official or unofficial character, which is a threat to national security in the field of culture and information.

Co-infections of Aeromonas dhakensis and Chryseobacterium indologenes in largemouth bass (Micropterus salmoides)

As one of the most important economic fishes, largemouth bass (Micropterus salmoides) is being widely farmed around the world. With the improper expansion and high-density culture, largemouth bass is susceptible to an increasing number of pathogens. Particularly, co-infection of multiple pathogens is ubiquitous, which has become a main obstacle to the development of largemouth bass industry. In the study, a co-infected case caused by Aeromonas dhakensis (A. dhakensis) and Chryseobacterium indologenes (C. indologenes) in largemouth bass was firstly reported. Diseased fish with clinical signs such as exophthalmia, hemorrhage on gill cover, rot or ulceration on gill, skin and fin were collected for further diagnostics. Tissue lesions of liver, spleen and kidney were observed, such as necrosis, hemorrhage, vacuolization and disintegration. Two bacteria strains were isolated from liver, one appeared as a white or yellowish colony, and the other was characterized by golden yellow color. Based on the conjoint analysis of 16S rRNA and gyrB gene sequences, the isolated strains were identified as A. dhakensis and C. indologenes, respectively. Antibiotic sensitivity testing indicated that both A. dhakensis and C. indologenes were sensitive to flumequine, enrofloxacin, ceftazidime and minocycline, and resistant to penicillin, sulfamethoxine sodium, ampicillin and cefamezin. Largemouth bass showed similar clinical signs and tissue lesions to naturally diseased fish following co-infection with the isolated strains, indicating that the isolated strains fulfill Koch's postulates. Besides, a slightly increased pathogenicity was observed following co-infection compared with single infection. The data firstly reveal a co-infected case caused by A. dhakensis and C. indologenes in largemouth bass, not only enriching the relevant information of largemouth bass diseases, but also promoting the diagnosis, prevention and control of A. dhakensis and C. indologenes co-infection.