Search Results (38)

Oxytetracycline (OTC) has served as an antibiotic to treat various bacterial infections in fish raised in aquaculture. Nonetheless, administering OTC in overly high doses can lead to adverse side effects in fish and also negatively impact on their surroundings. The objective of this work was to evaluate the expression levels of immune markers such as TLR-1, TLR-2, IκB-α, MyD88, NF-κB, IFN-γ, and IL-6 in intestinal cell primary culture (foregut, midgut, and hindgut) using qRT-PCR, and in addition, to assess the in vivo response to different doses of OTC in coho salmon at different times. The expression levels of all genes increased significantly after 1 h on day 1 with high doses of OTC compared with control conditions in all tissues under both approaches (in vivo and in vitro). However, the transcriptional responses decreased to 3, 6, and 12 h in vitro and day 3 in vivo. In conclusion, the transcriptional responses were differentially modulated by OTC in the three intestinal portions under both experimental conditions. These results demonstrate for the first time in primary cell culture fish that the expression of immune biomarkers in all tissues induces a differential response of these genes, depending on the concentration of OTC and the kinetics of time. This study offers valuable insights that can be applied to enhance aquaculture, determine optimal drug doses, and improve fish health. Full article

The liver of fish is an essential metabolic organ that also serves an immune regulatory role. In this study, we constructed a model of largemouth bass (Micropterus salmoides) infected with Nocardia seriolae by injection to explore the immune and antioxidant functions of the liver. The results showed that N. seriolae infection caused severe pathological changes in the liver, including cell necrosis, granuloma formation, and leukocyte infiltration. The level of mRNA expression of immune-related genes in the liver was significantly increased 2 days post-infection. Moreover, the combined analysis of transcriptome and metabolome showed that N. seriolae infection markedly affected liver metabolism, including glutathione metabolism, arginine and proline metabolism, arachidonic acid metabolism, as well as starch and sucrose metabolism. Additionally, multiple key biomarkers were identified as involved in regulating responses to N. seriolae infection, including arginine, glutathione, gpx, GST, PLA2G, GAA, and PYG. To further elucidate the regulatory effects of arginine on the immune and antioxidant processes in the liver, primary hepatocytes were isolated and cultured. The results demonstrated that arginine supplementation significantly reduced the expression of LPS-induced apoptosis-related genes (bax, cas3, cas8, and cas9) by up to 50% while increasing the expression of antioxidant genes (gpx, GST) by up to 700% at 24 h. Through the analysis of metabolic changes and immune responses in the liver following N. seriolae infection, combined with in-vitro experiments, this study elucidated the anti-apoptotic and antioxidant effects of arginine, revealing the immune response mechanisms in fish liver and laying the groundwork for using nutritional strategies to improve fish health. Full article

Abdominal wall endometriosis (AWE) is a clinical disorder with unknown pathogenesis with an incidence between 0.03% and 1% in women affected by cutaneous/scar endometriosis. We investigated the pathological, molecular cytogenetic and cell proliferation features of a primary AWE developed in rectus abdominis muscle in a patient without co-existing pelvic endometriosis. An investigational model of cultured stromal cells was additionally established. Histologically, the lesion revealed areas of endometrial-like glands surrounded by a thick stromal layer in addition to numerous disseminated foci composed exclusively of stromal cells. Beyond the strong expression of Estrogen (ER) and Progesterone receptors (PRs), consistent immunolabeling for several mesenchymal stromal/stem cell antigens and oncoproteins was revealed in both the endometrioma as well as in the cultured stromal cells. The Fluorescence in situ hybridization (FISH) analysis of the endometrioma demonstrated a structural alteration of the c-MYC protooncogene, with a mean of three gene copies in 3% to 5% of both glandular and stromal cells. The FISH assay applied on the cultured cells showed c-MYC gene amplification, with an average number of more than six gene copies in 18% to 25% of the cellular nuclei. Altogether, these results markedly highlight the pathological and molecular features of idiopathic AWE essential for histo-pathogenetic categorization. Full article

Nutritional intake is closely linked to gonadal development, although the mechanisms by which food intake affects gonadal development are not fully understood. Cholecystokinin (CCK) is a satiety neuropeptide derived from the hypothalamus, and the present study observed that hypothalamic CCK expression is significantly influenced by food intake, which is mediated through blood glucose levels. Interestingly, CCK and its receptors were observed to exhibit a high expression in the hypothalamus–pituitary–gonad (HPG) axis of grass carp (Ctenopharyngodon idellus), suggesting that CCK is potentially involved in regulating fish reproduction through the HPG axis. Further investigations revealed that CCK could significantly stimulate the expression of gonadotropin-releasing hormone-3 (GnRH3) in the hypothalamus. In addition, single-cell RNA sequencing showed that cckrb was highly enriched in pituitary follicle-stimulating hormone (FSH) cells. Further study confirmed that CCK can significantly induce FSH synthesis and secretion in primary cultured pituitary cells. Additionally, with primary cultured ovary cells as a model, the in vitro experiment demonstrated that CCK directly induces the expression of lhr, fshr, and cyp19a1a mRNA. This indicates that hypothalamic CCK may act as a nutrient sensor involved in regulating gonadal development in teleosts. Full article

Background/Objectives: Marine collagen peptides (MCPs) and glycosaminoglycans (GAGs) have been described as potential wound-healing (WH) agents. Fish cartilage hydrolysate (FCH) is a natural active food ingredient obtained from enzymatic hydrolysis which combines MCPs and GAGs. Recently, the clinical benefits of FCH supplementation for the skin, as well as its mode of action, have been demonstrated. Some of the highlighted mechanisms are common to the WH process. The aim of the study is therefore to investigate the influence of FCH supplementation on the skin healing processes and the underlying mechanisms. Methods: To this end, an ex vivo clinical approach, which takes into account the clinical digestive course of nutrients, coupled with primary cell culture on human dermal fibroblasts (HDFs) and ultra-deep proteomic analysis, was performed. The effects of human serum enriched in circulating metabolites resulting from FCH ingestion (FCH-enriched serum) were assessed on HDF WH via an in vitro scratch wound assay and on the HDF proteome via diaPASEF (Data Independent Acquisition—Parallel Accumulation Serial Fragmentation) proteomic analysis. Results: Results showed that FCH-enriched human serum accelerated wound closure. In support, proteins with anti-inflammatory and immunomodulatory properties and proteins prone to promote hydration and ECM stability showed increased expression in HDFs after exposure to FCH-enriched serum. Conclusions: Taken together, these data provide valuable new insights into the mechanisms that may contribute to FCH’s beneficial impact on human skin functionality by supporting WH. Further studies are needed to reinforce these preliminary data and investigate the anti-inflammatory and immunomodulatory properties of FCH. Full article

Examinations of ovarian cancer cells require the ability to identify tumor cells. Array-based comparative genome hybridization (aCGH) on 30 ovarian carcinomas (OC) identified three genomic loci (8q24.23; 17p12; 18q22.3) over- or under-represented in OC. A fluorescence in situ hybridization (FISH) probe of these three loci is intended to identify tumor cells by their signal pattern deviating from a diploid pattern. Human DNA from these three loci is isolated from bacterial artificial chromosomes (BAC), amplified and labeled with fluorescent dyes. After a standard FISH procedure, 71 OC suspensions from primary tumors, three OC cell lines, three lymphocyte suspensions, and one mesenchymal cell line LP-3 are analyzed with a fluorescence microscope. On average, 15% of the lymphocytes deviate from the expected diploid signal pattern, giving a cut-off of 36%. If this value is exceeded, tumor cells are detected. The mesenchymal cell line LP-3 shows only 21% as a negative control. The OC cell lines as positive controls exceed this value at 38%, 67%, and 54%. Of the 71 OC primary cultures, four cases fell below this cut-off as false negatives. In the two-sample t-test, the percentages of conspicuous signal patterns differ significantly. Full article

Establishing muscle lineage cell lines from fish will provide a great opportunity to study muscle development, which can eventually contribute to the improvement of the fish quality in the aquaculture industry. However, there has been a lack of the development of proper fish muscle lineage cell lines so far. Here, we report the establishment of a skeletal muscle-derived myogenic cell line from black sea bream (Acanthopagrus schlegelii). For this, we first attempted to find the optimal conditions for the primary explant culture of A. schlegelii muscle tissues and then established muscle-derived cell lines. After that, cell lines were characterized for their muscle-specific gene expression, growth, and myogenic differentiation. We found that the primary explant culture was effective when the tissue fragments were cultured in Dulbecco’s Modified Eagle Medium supplemented with fetal bovine serum and antibiotics on gelatin-coated dishes. Additionally, we confirmed that the addition of basic fibroblast growth factor was necessary to establish the cell lines. One of three cell lines established was capable of long-term culture, expressed three major myogenic regulatory genes including Pax7, MyoD, and Myog, and differentiated to myotubes in the condition using low concentration of horse serum, demonstrating that this cell line was a skeletal muscle-derived myogenic cell line. Full article

As an ancient and endangered species unique to the Yangtze River in China, the wild population of the Dabry’s sturgeon has become scarce. Due to the long time till the first sexual maturity of Dabry’s sturgeon, the population of artificially bred Dabry’s sturgeon recovered slowly. As a member of the tachykinin family, TAC1 has been reported to have a variety of functions in mammals such as pain control, smooth muscle contraction and reproductive cycle regulation, but the function of Tac1 in fish has been rarely reported. In this study, we synthesized two tac1 gene products, Substance P (SP) and neurokinin A (NKA), and further verified the effect of two tac1 gene products on the secretion of related hormones in the pituitary of Dabry’s Sturgeon by intraperitoneal injection and co-culture of primary cells. Expression studies revealed that the newly cloned tac1 were mainly distributed in the hypothalamus and pituitary tissue of the brain. In prepubertal Dabry’s sturgeon, this study showed that the two gonadotropins’ mRNA levels in pituitary tissue can be significantly increased by SP and NKA through intraperitoneal injection, and the LH protein level in serum was also increased. Further study showed that both NKA and SP could promote the two gonadotropins’ mRNA expression in pituitary cells of Dabry’s sturgeon. In addition, we explored the optimal dose and time of SP and NKA on pituitary cells is 24 h and over 10 nM. These results, as a whole, suggested that tac1 gene products play an important role in gonadotropin release and gonadal development in prepubertal Dabry’s sturgeon. Full article

Corticotropin-releasing hormone (CRH) is known for its crucial role in the stress response system, which could induce pituitary adrenocorticotropic hormone (ACTH) secretion to promote glucocorticoid release in the adrenal gland. However, little is known about other pituitary actions of CRH in teleosts. Somatolactin is a fish-specific hormone released from the neurointermediate lobe (NIL) of the posterior pituitary. A previous study has reported that ACTH was also located in the pituitary NIL region. Interestingly, our present study found that CRH could significantly induce two somatolactin isoforms’ (SLα and SLβ) secretion and synthesis in primary cultured grass carp pituitary cells. Pharmacological analysis further demonstrated that CRH-induced pituitary somatolactin expression was mediated by the AC/cAMP/PKA, PLC/IP3/PKC, and Ca²⁺/CaM/CaMK-II pathways. Finally, transcriptomic analysis showed that both SLα and SLβ should play an important role in the regulation of lipid metabolism in primary cultured hepatocytes. These results indicate that CRH is a novel stimulator of somatolactins in teleost pituitary cells, and somatolactins may participate in the stress response by regulating energy metabolism. Full article

Background: SARS-CoV-2 can enter the environment from the feces of COVID-19 patients and virus carriers through untreated sewage. The virus has shown the ability to adapt to a wide range of hosts, so the question of the possible involvement of aquafauna and animals of coastal ecosystems in maintaining its circulation remains open. Methods: the aim of this work was to study the tropism of SARS-CoV-2 for cells of freshwater fish and reptiles, including those associated with aquatic and coastal ecosystems, and the effect of ambient temperature on this process. In a continuous cell culture FHM (fathead minnow) and diploid fibroblasts CGIB (silver carp), SARS-CoV-2 replication was not maintained at either 25 °C or 29 °C. At 29 °C, the continuous cell culture TH-1 (eastern box turtle) showed high susceptibility to SARS-CoV-2, comparable to Vero E6 (development of virus-induced cytopathic effect (CPE) and an infectious titer of 7.5 ± 0.17 log₁₀ TCID₅₀/mL on day 3 after infection), and primary fibroblasts CNI (Nile crocodile embryo) showed moderate susceptibility (no CPE, infectious titer 4.52 ± 0.14 log₁₀ TCID₅₀/mL on day 5 after infection). At 25 °C, SARS-CoV-2 infection did not develop in TH-1 and CNI. Conclusions: our results show the ability of SARS-CoV-2 to effectively replicate without adaptation in the cells of certain reptile species when the ambient temperature rises. Full article

Fish primary cell culture has emerged as a valuable tool for investigating the physiological roles and responses of various cell types found in fish species. This review aims to provide an overview of the advancements and applications of fish primary cell culture techniques, focusing on the profiling of physiological roles exhibited by fish cells in vitro. Fish primary cell culture involves the isolation and cultivation of cells directly derived from fish tissues, maintaining their functional characteristics and enabling researchers to study their behavior and responses under controlled conditions. Over the years, significant progress has been made in optimizing the culture conditions, establishing standardized protocols, and improving the characterization techniques for fish primary cell cultures. The review highlights the diverse cell types that have been successfully cultured from different fish species, including gonad cells, pituitary cells, muscle cells, hepatocytes, kidney and immune cells, adipocyte cells and myeloid cells, brain cells, primary fin cells, gill cells, and other cells. Each cell type exhibits distinct physiological functions, contributing to vital processes such as metabolism, tissue regeneration, immune response, and toxin metabolism. Furthermore, this paper explores the pivotal role of fish primary cell culture in elucidating the mechanisms underlying various physiological processes. Researchers have utilized fish primary cell cultures to study the effects of environmental factors, toxins, pathogens, and pharmaceutical compounds on cellular functions, providing valuable insights into fish health, disease pathogenesis, and drug development. The paper also discusses the application of fish primary cell cultures in aquaculture research, particularly in investigating fish growth, nutrition, reproduction, and stress responses. By mimicking the in vivo conditions in vitro, primary cell culture has proven instrumental in identifying key factors influencing fish health and performance, thereby contributing to the development of sustainable aquaculture practices. Full article

Aquaculture fish are kept for long periods in sea cages or tanks. Consequently, accumulated stress causes the fish to present serious problems with critical economic losses. Fish food has been supplemented to reduce this stress, using many components as amino acids such as tryptophan. This study aims to determine the transcriptional effect of tryptophan and cortisol on primary cell cultures of salmon head and posterior kidney. Our results indicate activation of the kynurenine pathway and serotonin activity when stimulated with tryptophan and cortisol. An amount of 95% of tryptophan is degraded by the kynurenine pathway, indicating the relevance of knowing how this pathway is activated and if stress levels associated with fish culture trigger its activation. Additionally, it is essential to know the consequence of increasing kynurenic acid “KYNA” levels in the short and long term, and even during the fish ontogeny. Full article

Fish cell culture is a common in vitro tool for studies in different fields such as virology, toxicology, pathology and immunology of fish. Fish cell cultures are a promising help to study how to diagnose and control relevant viral and intracellular bacterial infections in aquaculture. They can also be used for developing vaccines and immunostimulants, especially with the ethical demand aiming to reduce and replace the number of fish used in research. This study aimed to isolate head kidney primary cell cultures from three Chilean salmonids: Salmo salar, Oncorhynchus kisutch, and Oncorhynchus mykiss, and characterize the response to bacterial and viral stimuli by evaluating various markers of the innate and adaptive immune response. Specifically, the primary cell cultures of the head kidney from the three salmonids studied were cultured and exposed to two substances that mimic molecular patterns of different pathogens, i.e., Lipopolysaccharide (LPS) (bacterial) and Polyinosinic: polycytidylic acid (POLY I:C). Subsequently, we determined the mRNA expression profiles of the TLR-1, TLR-8, IgM, TLR-5, and MHC II genes. Head kidney primary cell cultures from the three species grown in vitro responded differently to POLY I:C and LPS. This is the first study to demonstrate and characterize the expression of immune genes in head kidney primary cell culture isolated from three salmonid species. It also indicates their potential role in developing immune responses as defense response agents and targets of immunoregulatory factors. Full article

Copper is a common component of industrial heavy metal waste and a major component of some fish medicines, which can cause oxidative stress and damage the health of farmed fish. The Nrf2 signaling pathway is an important pathway related to the oxidative stress on vertebrates. Exploring the effect of copper on the Nrf2 signaling pathway in fish hepatocytes would help improve the understanding of the molecular mechanism of antioxidants in fish hepatocytes and provide theoretical data for relevant toxicological research. Adult tilapia were cultured under properly controlled conditions for two weeks to adapt to laboratory culture conditions. Primary tilapia hepatocytes were obtained by cell culture. MTT (3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide) assay was used to detect the effect of copper ions on the viability of tilapia hepatocytes. The lipid peroxidation level (MDA) and antioxidant ability of tilapia hepatocytes (SOD and CAT activity) were detected. Quantitative PCR (qPCR) was used to detect the differential expression of each gene (Nrf2, Keap1a, Keap1b, CuZnSOD, MnSOD, HO-1, and GSTA) in the Nrf2 signaling pathway. The results suggested that after treatment with 100 μM copper ions for 4 h, 8 h, and 24 h, the viability of hepatocytes significantly decreased (p < 0.05). LDH and MDA after 8 h and 24 h treatment were significantly higher than those in the control group (p < 0.05). CAT activity significantly decreased after 4 h (p < 0.05), and SOD activity significantly decreased after 8 h and 24 h (p < 0.05). The results of qPCR showed that the expression of MnSOD significantly increased after a treatment with copper ions for 4 h, and the expression of Nrf2, Keap1a, CuZnSOD, HO-1 as well as GSTA significantly increased after a treatment with copper ions for 8 h, compared with the control group (p < 0.05). After being treated with copper ions for 24 h, the expression of Nrf2 and CuZnSOD significantly increased compared with the control group (p < 0.05). There was no significant difference in the expression of Keap1b or CAT at each time point. In conclusion, with copper ions exposure, the viability of tilapia hepatocytes was reduced, causing lipid peroxidation, a reduction in the antioxidant capacity of cells, the activation of the Nrf2 signaling pathway, and the increase in the expression of most genes in this pathway, which are defensive responses of hepatocytes to oxidative stress caused by copper ions. This study can provide theoretical data for related toxicological research. Full article

Genetic and drug sensitivity assays on primary cultures are not only of basic but also of translational interest and could eventually aid oncologists in the selection of treatments. However, cancer cells need to be identified and differentiated from the non-tumor cells always present in primary cultures. Also, successive passages can change the proportions of these two subpopulations. In this study, we propose fluorescence in situ hybridization (FISH) analysis on cell smears to determine the presence of tumor cells in primary cultures obtained from patients carrying translocations or copy number gains. FISH proved to be an easy, fast, economic, and reliable method of characterizing cell populations, which could be used repeatedly at different passages to monitor variations and to confirm the maintenance of translocations and copy number gains throughout the culture process. Full article