Tuning CARs: recent advances in modulating chimeric antigen receptor (CAR) T cell activity for improved safety, efficacy, and flexibility.

Cancer immunotherapies utilizing genetically engineered T cells have emerged as powerful personalized therapeutic agents showing dramatic preclinical and clinical results, particularly in hematological malignancies. Ectopically expressed chimeric antigen receptors (CARs) reprogram immune cells to target and eliminate cancer. However, CAR T cell therapy's success depends on the balance between effective anti-tumor activity and minimizing harmful side effects. To improve CAR T cell therapy outcomes and mitigate associated toxicities, scientists from different fields are cooperating in developing next-generation products using the latest molecular cell biology and synthetic biology tools and technologies. The immunotherapy field is rapidly evolving, with new approaches and strategies being reported at a fast pace. This comprehensive literature review aims to provide an up-to-date overview of the latest developments in controlling CAR T cell activity for improved safety, efficacy, and flexibility.

Differential expression profile between amygdala and blood during chronic lithium treatment in a rat model of depression - a pilot study.

Lithium is a mood stabilizer widely used in the pharmacotherapy of bipolar disorder and treatment­resistant depression. Taking into account dysregulated inflammatory activity in depression and the immunomodulatory role of lithium, we hypothesized that genes associated with inflammatory responses may be potential biomarkers of lithium action. We aimed to compare gene expression changes between the brain and the periphery after chronic lithium administration in an animal model of depression. Depressive behavior was induced by chronic mild stress protocol for 4 weeks. After 2 weeks, rats started to receive lithium (study group) or water (reference group). The control group were rats not exposed to stress. Amygdala, hippocampus, frontal cortex and peripheral blood were analyzed using whole transcriptome expression microarrays. Changes were confirmed with qPCR and ELISA assay. After 2 weeks of lithium administration, we observed significant changes in gene expression between amygdala and peripheral blood. Logistic regression analysis determined Alox15 expression as a predictor of lithium status, as its expression was tissue­specific and increased in amygdala and decreased in blood. Analysis of serum ALOX15 protein revealed its upregulation after two­week lithium administration. Our study suggests that lithium may have therapeutic potential in depressive behaviors. These results indicate immunomodulatory effect of lithium and that Alox15 may be a new potential marker of chronic lithium treatment.

Promising Immunotherapeutic Modalities for B-Cell Lymphoproliferative Disorders.

Over the last few years, treatment principles have been changed towards more targeted therapy for many B-cell lymphoma subtypes and in chronic lymphocytic leukemia (CLL). Immunotherapeutic modalities, namely monoclonal antibodies (mAbs), bispecific antibodies (bsAbs), antibody-drug conjugates (ADCs), and chimeric antigen receptor T (CAR-T) cell therapy, commonly use B-cell-associated antigens (CD19, CD20, CD22, and CD79b) as one of their targets. T-cell engagers (TCEs), a subclass of bsAbs, work on a similar mechanism as CAR-T cell therapy without the need of previous T-cell manipulation. Currently, several anti-CD20xCD3 TCEs have demonstrated promising efficacy across different lymphoma subtypes with slightly better outcomes in the indolent subset. Anti-CD19xCD3 TCEs are being developed as well but only blinatumomab has been evaluated in clinical trials yet. The results are not so impressive as those with anti-CD19 CAR-T cell therapy. Antibody-drug conjugates targeting different B-cell antigens (CD30, CD79b, CD19) seem to be effective in combination with mAbs, standard chemoimmunotherapy, or immune checkpoint inhibitors. Further investigation will show whether immunotherapy alone or in combinatory regimens has potential to replace chemotherapeutic agents from the first line treatment.

Extracellular Nampt (eNampt/visfatin/PBEF) directly and indirectly stimulates ACTH and CCL2 protein secretion from isolated rat corticotropes.

BACKGROUND: Nicotinamide phosphoribosyltransferase (Nampt/visfatin/PBEF) acts both as an enzyme in the nicotinamide adenine dinucleotide (NAD) synthesis pathway as well as an extracellular hormone (eNampt). Among its effects, eNampt exerts potent pro-inflammatory effects. We have recently shown that, in rats, eNampt stimulates corticosterone secretion by acting through the pituitary rather than the hypothalamus. OBJECTIVES: To investigate the mechanism of action of eNampt on the secretion of adrenocorticotropic hormone (ACTH) and chemokine (C-C motif) ligand 2 (CCL2), which are cytokines secreted by pituitary neuroendocrine tumors. MATERIAL AND METHODS: The research was carried out on the AtT-20 murine cell line, primary rat pituitary cell culture, isolated pituitary corticotropes, and in vivo. The effects of the performed experiments were examined using the following methods: gene expression profiling using microarrays, quantitative polymerase chain reaction (qPCR) and enzyme-linked immunosorbent assay (ELISA). RESULTS: The results suggest that eNampt stimulates ACTH secretion from rat corticotropes both directly and indirectly. Indirect action most likely occurs through interleukin (IL)-6 secreted by folliculostellate cells of the pituitary gland. In isolated ACTH cells of the rat pituitary gland, eNampt stimulates the expression of genes involved in the immune response. Among them, the protein encoded by the CCL2 gene seems to also be involved in the regulation of corticotropin-releasing hormone (CRH)-dependent metabolism. Unlike rat corticotropes, murine AtT-20 corticotropic cells do not react to either eNampt or Fk866 (the inhibitor of Nampt enzymatic action). CONCLUSIONS: The eNampt stimulates the secretion of ACTH from rat corticotropes indirectly and directly, likely by stimulating IL-6 secretion from folliculostellate cells of the pituitary gland. This effect was not observed in the AtT-20 corticotropic cell cancer cell line.

[Corrigendum] Novel markers of human ovarian granulosa cell differentiation toward osteoblast lineage: A microarray approach.

Following the publication of this paper, the authors have requested that, on p. 4412 of the above article in the Funding section of the Declarations, the acknowledgement to one of the funding sources should be removed from the paper; essentially, the reference to grant no. 2018/31/B/NZ5/02475, formulated by the Polish National Science Centre (grant providing institution), should be removed from the paper. Therefore, the revised version of the Funding section paragraph should read as follows: Funding: The present study was supported by a grant from Poznan University of Medical Sciences (grant no. 502­14­02227367­10694). The authors confirm that there are no further errors in the study, and all the authors agree to this correction. The authors are grateful to the Editor of Molecular Medicine Reports for granting them this opportunity to publish a Corrigendum, and apologize for any inconvenience caused. [the original article was published in Molecular Medicine Reports 20: 4403-4414, 2019, DOI: 10.3892/mmr.2019.10709].

New Gene Markers Expressed in Porcine Oviductal Epithelial Cells Cultured Primary In Vitro Are Involved in Ontological Groups Representing Physiological Processes of Porcine Oocytes.

Changes that occur within oviducts after fertilization are dependent on post-ovulation events, including oocyte-oviduct interactions. Although general processes are well-defined, the molecular basis are poorly understood. Recently, new marker genes involved in 'cell development', 'cell growth', 'cell differentiation' and 'cell maturation' processes have been identified in porcine oocytes. The aim of the study was to assess the expression profile of genes in primary in vitro cultured oviductal epithelial cells (OECs), clustered in Gene Ontology groups which enveloped markers also identified in porcine oocytes. OECs (from 45 gilts) were surgically removed and cultured in vitro for ≤ 30 days, and then subjected to molecular analyses. The transcriptomic and proteomic profiles of cells cultured during 7, 15 and 30 days were investigated. Additionally, morphological/histochemical analyzes were performed. The results of genes expression profiles were validated after using RT-qPCR. The results showed a significant upregulation of UNC45B, NOX4, VLDLR, ITGB3, FMOD, SGCE, COL1A2, LOX, LIPG, THY1 and downregulation of SERPINB2, CD274, TXNIP, CELA1, DDX60, CRABP2, SLC5A1, IDO1, ANPEP, FST. Detailed knowledge of the molecular pathways occurring in the OECs and the gametes that contact them may contribute both to developments of basic science of physiology, and new possibilities in advanced biotechnology of assisted reproduction.

Transcriptome Changes in Three Brain Regions during Chronic Lithium Administration in the Rat Models of Mania and Depression.

Lithium has been the most important mood stabilizer used for the treatment of bipolar disorder and prophylaxis of manic and depressive episodes. Despite long use in clinical practice, the exact molecular mechanisms of lithium are still not well identified. Previous experimental studies produced inconsistent results due to different duration of lithium treatment and using animals without manic-like or depressive-like symptoms. Therefore, we aimed to analyze the gene expression profile in three brain regions (amygdala, frontal cortex and hippocampus) in the rat model of mania and depression during chronic lithium administration (2 and 4 weeks). Behavioral changes were verified by the forced swim test, open field test and elevated maze test. After the experiment, nucleic acid was extracted from the frontal cortex, hippocampus and amygdala. Gene expression profile was done using SurePrint G3 Rat Gene Expression whole transcriptome microarrays. Data were analyzed using Gene Spring 14.9 software. We found that chronic lithium treatment significantly influenced gene expression profile in both mania and depression models. In manic rats, chronic lithium treatment significantly influenced the expression of the genes enriched in olfactory and taste transduction pathway and long non-coding RNAs in all three brain regions. We report here for the first time that genes regulating olfactory and taste receptor pathways and long non-coding RNAs may be targeted by chronic lithium treatment in the animal model of mania.

Genes involved in glucocorticoid receptor signalling affect susceptibility to mood disorders.

OBJECTIVES: In mood disorders chronic stress contributes to decreased glucocorticoid receptor signalling in the brain and resistance in the periphery. We hypothesised that aberrant glucocorticoid receptor function may result from genetic predisposition and that decreased GR signalling in the brain correlates with the expression of genes regulating GR complex formation. METHODS: We performed the association analysis of 698 patients: 490 patients with bipolar disorder and 208 patients with major depressive disorder and 564 control subjects. We genotyped 11 variants using TaqMan assays. Gene expression in the brain tissue was done in male Wistar rats after chronic mild stress protocol. The SRSF5 serum concentration was performed using ELISA. Data were analysed in Statistica and GraphPad. RESULTS: We found an association of STIP1 and SRSF5 variants with major depressive disorder and BAG1 variant with bipolar disorder. Gene expression analysis in a rat model of depression confirmed significant changes in the expression of SRSF5, BAG1, and FKBP4 in the brain. For SRSF5, we observed significantly increased expression in the serum of depressed females and male rats exposed to chronic stress. CONCLUSIONS: Our results indicate the involvement of genes associated with GR function, SRSF5, BAG1, and FKBP4 with susceptibility to mood disorders.

Cellular Processes in Human Ovarian Follicles Are Regulated by Expression Profile of New Gene Markers-Clinical Approach.

In the growing ovarian follicle, the maturing oocyte is accompanied by cumulus (CCs) and granulosa (GCs) cells. Currently, there remain many unanswered questions about the epithelial origin of these cells. Global and targeted gene transcript levels were assessed on 1, 7, 15, 30 days of culture for CCs and GCs. Detailed analysis of the genes belonging to epithelial cell-associated ontological groups allowed us to assess a total of 168 genes expressed in CCs (97 genes) and GCs (71 genes) during long-term in vitro culture. Expression changes of the analyzed genes allowed the identification of the group of genes: TGFBR3, PTGS2, PRKX, AHI1, and IL11, whose expression decreased the most and the group of ANXA3, DKK1, CCND1, STC1, CAV1, and SFRP4 genes, whose expression significantly increased. These genes' expression indicates CCs and GCs epithelialization processes and their epithelial origin. Expression change analysis of genes involved in epithelization processes in GCs and CCs during their in vitro culture made it possible to describe the most significantly altered of the 11 genes. Detailed analysis of gene expression in these two cell populations at different time intervals confirms their ovarian surface epithelial origin. Furthermore, some gene expression profiles appear to have tumorigenic properties, suggesting that granulosa cells may play a role in cancerogenesis.

Transcriptomic Profile of Genes Encoding Proteins Involved in Pathogenesis of Sjögren's Syndrome Related Xerostomia-Molecular and Clinical Trial.

Sjögren's syndrome (SS) is characterized by xerostomia. We aimed to investigate and compare gene expressions in the labial salivary glands of SS patients with xerostomia SS (sicca) and without xerostomia SS (non-sicca) and of healthy subjects (HS) by means of microarray analysis, and to find genes involved in xerostomia. The study group comprised 11 SS patients (3 SS (sicca) and 8 SS (non-sicca)) and 9 HS. The relative gene expression changes were validated with RT-qPCR in the larger study group. Among the differently expressed genes belonging to the "secretion" ontology group with a fold change >2 and with a p value < 0.05, the Transmembrane P24 Trafficking Protein 10 (TMED10), Protein Disulfide Isomerase Family A Member 4 (PDIA4), Calnexin (CANX), Amyloid Beta Precursor Protein (APP), and Transmembrane BAX Inhibitor Motif Containing 6 (TMBIM6) gene expressions in both SS (sicca) and SS (non-sicca) groups were lower than in HS. Significant correlations were observed between TMED10, PDIA4, and CANX gene expression in SS (sicca) patients compared to the controls. There were no differences between the SS (sicca) and SS (non-sicca) study groups in the expression of the aforementioned genes. Results indicate their role in the endoplasmic reticulum system, their overlapping function and the loss of the APP neuroprotective function in xerostomia. It has a multifactorial origin and can be triggered by disturbances to the various signaling pathways in saliva secretion.

Cortical Granule Distribution and Expression Pattern of Genes Regulating Cellular Component Size, Morphogenesis, and Potential to Differentiation are Related to Oocyte Developmental Competence and Maturational Capacity In Vivo and In Vitro.

Polyspermia is an adverse phenomenon during mammalian fertilization when more than one sperm fuses with a single oocyte. The egg cell is prepared to prevent polyspermia by, among other ways, producing cortical granules (CGs), which are specialized intracellular structures containing enzymes that aim to harden the zona pellucida and block the fusion of subsequent sperm. This work focused on exploring the expression profile of genes that may be associated with cortical reactions, and evaluated the distribution of CGs in immature oocytes and the peripheral density of CGs in mature oocytes. Oocytes were isolated and then processed for in vitro maturation (IVM). Transcriptomic analysis of genes belonging to five ontological groups has been conducted. Six genes showed increased expression after IVM (ARHGEF2, MAP1B, CXCL12, FN1, DAB2, and SOX9), while the majority of genes decreased expression after IVM. Using CG distribution analysis in immature oocytes, movement towards the cortical zone of the oocyte during meiotic competence acquisition was observed. CGs peripheral density decreased with the rise in meiotic competence during the IVM process. The current results reveal important new insights into the in vitro maturation of oocytes. Our results may serve as a basis for further studies to investigate the cortical reaction of oocytes.

Expression of genes involved in neurogenesis, and neuronal precursor cell proliferation and development: Novel pathways of human ovarian granulosa cell differentiation and transdifferentiation capability in vitro.

The process of neural tissue formation is associated primarily with the course of neurogenesis during embryonic life. The source of neural­like cells is stem cells, which, under the influence of appropriate differentiating factors, may differentiate/transdifferentiate towards a neural­like lineage. The present study suggested that, under long­term in vitro culture conditions, human ovarian granulosa cells (GCs), obtained from granulosa­rich follicular fluid, acquired new properties and expressed genes characteristic of the ontological groups 'neurogenesis' (GO:0022008), 'neuronal precursor cell proliferation' (GO:0061351) and 'nervous system development' (GO:0007399), which are closely related to the formation of neurons. The present study collected GCs from 20 women referred for the procedure of in vitro fertilization. Cells were maintained in long­term in vitro culture for 30 days, and RNA was isolated after 1, 7, 15 and 30 days of culture. The expression profile of individual genes was determined using the Affymetrix microarray method. The 131 genes with the highest expression change in relation to day 1 of culture were then selected; the 10 most affected genes found to be primarily involved in nerve cell formation processes were chosen for consideration in this study: CLDN11, OXTR, DFNA5, ATP8B1, ITGA3, CD9, FRY, NANOS1, CRIM1 and NTN4. The results of the present study revealed that these genes may be considered potential markers of the uninduced differentiation potential of GCs. In addition, it was suggested that GCs may be used to develop a cell line showing neuronal characteristics after 30 days of cultivation. In addition, due to their potential, these cells could possibly be used in the treatment of neurodegenerative diseases, not only in the form of 'cultured neurons' but also as producers of factors involved in the regeneration of the nervous system.

Genes regulating hormone stimulus and response to protein signaling revealed differential expression pattern during porcine oocyte in vitro maturation, confirmed by lipid concentration.

Genes influencing oocyte maturation may be valuable for predicting their developmental potential, as well as discerning the mechanistic pathways regulating oocyte development. In the presented research microarray gene expression analysis of immature and in vitro matured porcine oocytes was performed. Two groups of oocytes were compared in the study: before (3 × n = 50) and after in vitro maturation (3 × n = 50). The selection of viable oocytes was performed using the brilliant cresyl blue (BCB) test. Furthermore, microarrays and RT-qPCR was used to analyze the transcriptome of the oocytes before and after IVM. The study focused on the genes undergoing differential expression in two gene-ontology groups: "Cellular response to hormone stimulus" and "Cellular response to unfolded protein", which contain genes that may directly or indirectly be involved in signal transduction during oocyte maturation. Examination of all the genes of interest showed a lower level of their expression after IVM. From the total number of genes in these gene ontologies ten of the highest change in expression were identified: FOS, ID2, BTG2, CYR61, ESR1, AR, TACR3, CCND2, EGR2 and TGFBR3. The successful maturation of the oocytes was additionally confirmed with the use of lipid droplet assay. The genes were briefly described and related to the literature sources, to investigate their potential roles in the process of oocyte maturation. The results of the study may serve as a basic molecular reference for further research aimed at improving the methods of oocyte in vitro maturation, which plays an important role in the procedures of assisted reproduction.

Transcriptomic analysis of expression of genes regulating cell cycle progression in porcine ovarian granulosa cells during short-term in vitro primary culture.

The primary function of ovarian granulosa cells (GCs) is the support of oocytes during maturation and development. Molecular analyses of granulosa cell-associated processes, leading to improvement of understanding of the cell cycle events during the formation of ovarian follicles (folliculogenesis), may be key to improve the in vitro fertilization procedures. Primary in vitro culture of porcine GCs was employed to examine the changes in the transcriptomic profile of genes belonging to "cell cycle", "cell division", "cell cycle process", "cell cycle phase transition", "cell cycle G1/S phase transition", "cell cycle G2/M phase transition" and "cell cycle checkpoint" ontology groups. During the analysis, microarrays were employed to study the transcriptome of GCs, analyzing the total RNA of cells from specific periods of in vitro cultures. This research was based on material obtained from 40 landrace gilts of similar weight, age and the same living conditions. RNA was isolated at specific timeframes: before the culture was established (0 h) and after 48 h, 96 h and 144 h in vitro. Out of 133 differentially expressed genes, we chose the 10 most up-regulated (SFRP2, PDPN, PDE3A, FGFR2, PLK2, THBS1, ETS1, LIF, ANXA1, TGFB1) and the 10 most downregulated (IGF1, NCAPD2, CABLES1, H1FOO, NEK2, PPAT, TXNIP, NUP210, RGS2 and CCNE2). Some of these genes known to play key roles in the regulation of correct cell cycle passage (up-regulated SFRP2, PDE3A, PLK2, LIF and down-regulated CCNE2, TXNIP, NEK2). The data obtained provide a potential reference for studies on the process of mammalian folliculogenesis, as well as suggests possible new genetic markers for cell cycle progress in in vitro cultured porcine granulosa cells.

New markers for regulation of transcription and macromolecule metabolic process in porcine oocytes during in vitro maturation.

Oocyte maturation is essential for proper fertilization, embryo implantation and early development. While the physiological conditions of these processes are relatively well­known, its exact molecular mechanisms remain widely undiscovered. Oocyte growth, differentiation and maturation are therefore the subject of scientific debate. Precious literature has indicated that the oocyte itself serves a regulatory role in the mechanisms underlying these processes. Hence, the present study performed expression microarrays to analyze the complete transcriptome of porcine oocytes during their in vitro maturation (IVM). Pig material was used for experimentation, as it possesses similarities to the reproductive processes and general genetic proximities of Sus scrofa to human. Oocytes, isolated from the ovaries of slaughtered animals were assessed via the Brilliant Cresyl Blue test and directed to IVM. A number of oocytes were left to be analyzed as the 'before IVM' group. Oocyte mRNA was isolated and used for microarray analysis, which was subsequently validated via RT­qPCR. The current study particularly focused on genes belonging to 'positive regulation of transcription, DNA­dependent', 'positive regulation of gene expression', 'positive regulation of macromolecule metabolic process' and 'positive regulation of transcription from RNA polymerase II promoter' ontologies. FOS, VEGFA, ESR1, AR, CCND2, EGR2, ENDRA, GJA1, INHBA, IHH, INSR, APP, WWTR1, SMARCA1, NFAT5, SMAD4, MAP3K1, EGR1, RORA, ECE1, NR5A1, KIT, IKZF2, MEF2C, SH3D19, MITF and PSMB4 were all determined to be significantly altered (fold change, >|2|; P<0.05) among these groups, with their downregulation being observed after IVM. Genes with the most altered expressions were analyzed and considered to be potential markers of maturation associated with transcription regulation and macromolecule metabolism process.

Novel markers of human ovarian granulosa cell differentiation toward osteoblast lineage: A microarray approach.

Under physiological conditions, human ovarian granulosa cells (GCs), are responsible for a number of processes associated with folliculogenesis and oogenesis. The primary functions of GCs in the individual phases of follicle growth are: Hormone production in response to follicle stimulating hormone (FSH), induction of ovarian follicle atresia through specific molecular markers and production of nexus cellular connections for communication with the oocyte. In recent years, interest in obtaining stem cells from particular tissues, including the ovary, has increased. Special attention has been paid to the novel properties of GCs during long­term in vitro culture. It has been demonstrated that the usually recycled material in the form of follicular fluid can be a source of cells with stem­like properties. The study group consisted of patients enrolled in the in vitro fertilization procedure. Total RNA was isolated from GCs at 4 time points (after 1, 7, 15 and 30 days of culture) and was used for microarray expression analysis (Affymetrix® Human HgU 219 Array). The expression of 22,480 transcripts was examined. The selection of significantly altered genes was based on a P­value <0.05 and expression higher than two­fold. The leucine rich repeat containing 17, collagen type I α1 chain, bone morphogenetic protein 4, twist family bHLH transcription factor 1, insulin like growth factor binding protein 5, GLI family zinc finger 2 and collagen triple helix repeat containing genes exhibited the highest changes in expression. Reverse­transcription­quantitative PCR was performed to validate the results obtained in the analysis of expression microarrays. The direction of expression changes was validated in the majority of cases. The presented results indicated that GCs have the potential of cells that can differentiate towards osteoblasts in long­term in vitro culture conditions. Increased expression of genes associated with the osteogenesis process suggests a potential for uninduced change of GC properties towards the osteoblast phenotype. The present study, therefore, suggests that GCs may become an excellent starting material in obtaining stable osteoblast cultures. GCs differentiated towards osteoblasts may be used in regenerative and reconstructive medicine in the future.

Human Ovarian Granulosa Cells Isolated during an IVF Procedure Exhibit Differential Expression of Genes Regulating Cell Division and Mitotic Spindle Formation.

Granulosa cells (GCs) are a population of somatic cells whose role after ovulation is progesterone production. GCs were collected from patients undergoing controlled ovarian stimulation during an in vitro fertilization procedure, and they were maintained for 1, 7, 15, and 30 days of in vitro primary culture before collection for further gene expression analysis. A study of genes involved in the biological processes of interest was carried out using expression microarrays. To validate the obtained results, Reverse Transcription quantitative Polymerase Chain Reaction (RT-qPCR) was performed. The direction of changes in the expression of the selected genes was confirmed in most of the examples. Six ontological groups ("cell cycle arrest", "cell cycle process", "mitotic spindle organization", "mitotic spindle assembly checkpoint", "mitotic spindle assembly", and "mitotic spindle checkpoint") were analyzed in this study. The results of the microarrays obtained by us allowed us to identify two groups of genes whose expressions were the most upregulated (FAM64A, ANLN, TOP2A, CTGF, CEP55, BIRC5, PRC1, DLGAP5, GAS6, and NDRG1) and the most downregulated (EREG, PID1, INHA, RHOU, CXCL8, SEPT6, EPGN, RDX, WNT5A, and EZH2) during the culture. The cellular ultrastructure showed the presence of structures characteristic of mitotic cell division: a centrosome surrounded by a pericentric matrix, a microtubule system, and a mitotic spindle connected to chromosomes. The main goal of the study was to identify the genes involved in mitotic division and to identify the cellular ultrastructure of GCs in a long-term in vitro culture. All of the genes in these groups were subjected to downstream analysis, and their function and relation to the ovarian environment are discussed. The obtained results suggest that long-term in vitro cultivation of GCs may lead to their differentiation toward another cell type, including cells with cancer-like characteristics.

Analysis of Transcriptome, Selected Intracellular Signaling Pathways, Proliferation and Apoptosis of LNCaP Cells Exposed to High Leptin Concentrations.

Leptin, the first discovered adipokine, has been connected to various physiological and pathophysiological processes, including cancerogenesis. Increasing evidence confirms its influence on prostate cancer cells. However, studies on the effects of leptin on the proliferation and apoptosis of the androgen-sensitive LNCaP line of prostate cancer cells brought conflicting results. Therefore, we performed studies on the effects of high LEP concentration (1 × 10-6 M) on gene expression profile, change of selected signaling pathways, proliferation and apoptosis of LNCaP cells. RTCA (real-time cell analyzer) revealed inhibitory effect of LEP on cell proliferation, but lower LEP concentrations (10-8 and 10-10 M) did not affect cell division. Moreover, flow cytometry with a specific antibody for Cleaved PARP-1, an apoptosis marker, confirmed the activation of apoptosis in leptin-exposed LNCaP line of prostate cancer cells. Within 24 h LEP (10-6 M) increases expression of 297 genes and decreases expression of 119 genes. Differentially expressed genes (DEGs) were subjected to functional annotation and clusterization using the DAVID bioinformatics tools. Most ontological groups are associated with proliferation and apoptosis (seven groups), immune response (six) and extracellular matrix (two). These results were confirmed by the Gene Set Enrichment Analysis (GSEA). The leptin's effect on apoptosis stimulation was also confirmed using Pathview library. These results were also confirmed by qPCR method. The results of Western Blot analysis (exposure to LEP 10 min, 1, 2, 4 and 24 h) suggest (after 24 h) decrease of p38 MAPK, p44-42 mitogen-activated protein kinase and Bcl-2 phosphorylated at threonine 56. Moreover, exposure of LNCaP cells to LEP significantly stimulates the secretion of matrix metallopeptidase 7 (MMP7). Obtained results suggest activation of apoptotic processes in LNCaP cells cultured at high LEP concentration. At the same time, this activation is accompanied by inhibition of proliferation of the tested cells.

New Molecular Markers Involved in Regulation of Ovarian Granulosa Cell Morphogenesis, Development and Differentiation during Short-Term Primary In Vitro Culture-Transcriptomic and Histochemical Study Based on Ovaries and Individual Separated Follicles.

Nowadays, science has a lot of knowledge about the physiology of ovarian processes, especially folliculogenesis, hormone production and ovulation. However, the molecular basis for these processes remains largely undiscovered. The cell layer surrounding the growing oocyte-granulosa cells-are characterized by high physiological capabilities (e.g., proliferation, differentiation) and potential for growth in primary cultures, which predisposes them for analysis in the context of possible application of their cultures in advanced methods of assisted reproduction. In this study, we have used standard molecular approaches to analyze markers of these processes in primarily in vitro cultured porcine granulosa, subjected to conditions usually applied to cultures of similar cells. The material for our research came from commercially slaughtered pigs. The cells were obtained by enzymatic digestion of tissues and in vitro culture in appropriate conditions. The obtained genetic material (RNA) was collected at specific time intervals (0 h-before culture; reference, 48, 98, 144 h) and then analyzed using expression microarrays. Genes that showed a fold change greater than |2| and an adjusted p value lower than 0.05 were described as differentially expressed. Three groups of genes: "Cell morphogenesis", "cell differentiation" and "cell development" were analyzed. From 265 differently expressed genes that belong to chosen ontology groups we have selected DAPL1, CXCL10, NEBL, IHH, TGFBR3, SCUBE1, DAB1, ITM2A, MCOLN3, IGF1 which are most downregulated and PDPN, CAV1, TMOD1, TAGLN, IGFBP5, ITGB3, LAMB1, FN1, ITGA2, POSTN genes whose expression is upregulated through the time of culture, on which we focused in downstream analysis. The results were also validated using RT-qPCR. The aim of our work was to conduct primary in vitro culture of granulosa cells, as well as to analyze the expression of gene groups in relation to the proliferation of follicular granulosa cells in the model of primary culture in real time. This knowledge should provide us with a molecular insight into the processes occurring during the in vitro cultures of porcine granulosa cells, serving as a basic molecular entry on the extent of the loss of their physiological properties, as well as gain of new, culture-specific traits.

"Biological Adhesion" is a Significantly Regulated Molecular Process during Long-Term Primary In Vitro Culture of Oviductal Epithelial Cells (Oecs): A Transcriptomic and Proteomic Study.

Oviductal epithelial cells (OECs) actively produce stimulating and protecting factors, favoring survival and viability of gametes and early embryos. The oviduct participates in the initial reproductive events, which strongly depends on adhesion. The analysis of differential gene expression in OECs, during long-term in vitro culture, enables recognition of new molecular markers regulating several processes, including "biological adhesion". Porcine oviducts were stained with hematoxylin and eosin, as well as with antibodies against epithelial markers. Then, OECs were long-term in vitro cultured and after 24 h, 7, 15, and 30 days of culture were subjected to transcriptomic and proteomic assays. Microarrays were employed to evaluate gene expression, with Matrix-assisted laser desorption/ionization-time of light (MALDI-TOF) mass spectrometry applied to determine the proteome. The results revealed proper morphology of the oviducts and typical epithelial structure of OECs during the culture. From the set of differentially expressed genes (DEGs), we have selected the 130 that encoded proteins detected by MALDI-TOF MS analysis. From this gene pool, 18 significantly enriched gene ontology biological processes (GO BP) terms were extracted. Among them we focused on genes belonging to "biological adhesion" GO BP. It is suggested that increased expression of studied genes can be attributed to the process of intensive secretion of substances that exhibit favorable influence on oviductal environment, which prime gametes adhesion and viability, fertilization, and early embryo journey.