Functional Expression of TRPV1 Ion Channel in the Canine Peripheral Blood Mononuclear Cells.

TRPV1, known as a capsaicin receptor, is the best-described transient receptor potential (TRP) ion channel. Recently, it was shown to be expressed by non-excitable cells such as lymphocytes. However, the data regarding the functional expression of the TRPV1 channel in the immune cells are often contradictory. In the present study, we performed a phylogenetical analysis of the canine TRP ion channels, we assessed the expression of TRPV1 in the canine peripheral blood mononuclear cells (PBMC) by qPCR and Western blot, and we determined the functionality of TRPV1 by whole-cell patch-clamp recordings and calcium assay. We found high expression of TRPV2, -M2, and -M7 in the canine PBMCs, while expression of TRPV1, -V4 and, -M5 was relatively low. We confirmed that TRPV1 is expressed on the protein level in the PBMC and it localizes in the plasma membrane. The whole-cell patch-clamp recording revealed that capsaicin application caused a significant increase in the current density. Similarly, the results from the calcium assay show a dose-dependent increase in intracellular calcium level in the presence of capsaicin that was partially abolished by capsazepine. Our study confirms the expression of TRPV1 ion channel on both mRNA and protein levels in the canine PBMC and indicates that the ion channel is functional.

The Expression of Selected Factors Related to T Lymphocyte Activity in Canine Mammary Tumors.

Crosstalk between neoplastic and immune cells in the tumor microenvironment (TME) influences the progression of disease in human and canine cancer patients. Given that canine mammary tumors are a useful model to study breast cancer biology, we aimed to evaluate the expression of genes associated with T lymphocyte activity in benign, malignant, and metastatic canine mammary tumors. Interestingly, metastatic tumors exhibit increased expression of CXCR3, CCR2, IL-4, IL-12p40, and IL-17. In particular, we focused on IL-17, a key interleukin associated with the Th17 lymphocyte phenotype. Th17 cells have been shown to play a contradictory role in tumor immunity. Although IL-17 showed a high expression in the metastatic tumors, the expression of RORγt, a crucial transcription factor for Th17 differentiation was barely detected. We further investigated IL-17 expression using immunohistochemistry, through which we confirmed the increased expression of this interleukin in malignant and metastatic mammary tumors. Finally, we compared the plasma levels of IL-17 in healthy and malignant mammary tumor-bearing dogs using ELISA but found no differences between the groups. Our data indicate that the IL-17 in metastatic tumors may be produced by other cell types, but not by Th17 lymphocytes. Overall, our results broaden the available knowledge on the interactions in canine mammary tumors and provide insight into the development of new therapeutic strategies, with potential benefits for human immune oncology.

Synthesis and biological evaluation of 3-functionalized 2-phenyl- and 2-alkylbenzo[b]furans as antiproliferative agents against human melanoma cell line.

The key function of microtubules and mitotic spindle in cell division make them attractive targets in anticancer therapy. In the present study, functionalized in 3 position 2-phenyl- and 2-alkylbenzo[b]furans were synthesized and evaluated as antitumor agents. Among the synthesized derivatives 13a, 13b and 14 exhibited the most potent antiproliferative activity against human melanoma A375 cell line with IC50 values of 2.85 µM, 0.86 µM, 0.09 µM, respectively. The most promising compound defined was 14 with three methoxy groups in the 3-aroyl substituent and 7-methoxy group in 2-phenylbenzo[b]furan skeleton. Tubulin polymerization assay, confocal microscopy imaging and flow cytometry analysis revealed that 2-phenyl-3-aroylbenzo[b]furans (13a, 13b and 14) inhibited tubulin polymerization leading to disruption of mitotic spindle formation, cell cycle arrest in G2/M phase and apoptosis.

Effect of FTO and IGF2BP2 gene polymorphisms on duration of pregnancy and Apgar scores in women with gestational diabetes.

Gestational diabetes mellitus (GDM) is a metabolic disorder occurring in pregnant women. The main risk factors include advanced age and obesity. FTO and IGF2BP2 are the genetic loci associated with an increased risk of diabetes type 2 as well as being involved in lipid and carbohydrate metabolism. The aim of this study was to examine the association of FTO rs8050136, IGF2BP2 rs4402960 and rs11705701 gene polymorphisms with GDM risk as well as with clinical parameters of women with GDM and their newborns. This study included 204 pregnant women with GDM and 207 pregnant women with normal glucose tolerance. The diagnosis of GDM was based on a 75 g oral glucose tolerance test administered at 24-28 weeks of gestation. There were no statistically significant differences in the distribution of the FTO rs8050136 and IGF2BP2 rs4402960 and rs11705701 genotypes between women with GDM and normoglycemic women. In the women with the IGF2BP2 rs4402960 TT and rs11705701 AA genotypes, we observed a longer gestation and higher Apgar scores than in the women with other genotypes. The results of this study suggest that FTO rs8050136 and IGF2BP2 rs4402960 and rs11705701 gene polymorphisms are not associated with the risk of GDM in our population, whereas IGF2BP2 rs4402960 and rs11705701 genotype status may affect the length of gestation and the Apgar scores of newborns. IMPACT STATEMENT What is already known on this subject? Gestational diabetes mellitus (GDM) is the glucose intolerance detected during pregnancy. The main risk factors include an advanced age and obesity. FTO and IGF2BP2 are the genetic loci associated with an increased risk of diabetes type 2, as well as being involved in lipid and carbohydrate metabolism. What do the results of this study add? In this study, we examined the association between FTO and IGF2BP2 gene polymorphisms and GDM. There were no statistically significant differences in the distribution of these genotypes between healthy women and women with GDM. However, we observed a longer duration of pregnancy and higher Apgar scores in women with IGF2BP2 rs4402960 TT and rs11705701 AA genotypes. What are the implications of these findings for clinical practice and/or further research? Although the FTO rs8050136 and IGF2BP2 rs4402960 and rs11705701 gene polymorphisms are not associated with the risk of GDM in our population, further studies of these genes may allow for better neonatal care and prediction of wellbeing of newborns.

Picropodophyllin (PPP) is a potent rhabdomyosarcoma growth inhibitor both in vitro and in vivo.

BACKGROUND: Insulin-like growth factors and insulin are important factors promoting cancer growth and metastasis. The molecules act through IGF1 (IGF1R) and insulin (InsR) receptors. Rhambodmyosarcomas (RMS) overproduce IGF2 - a potent ligand for IGF1R and, at the same time, highly express IGF1 receptor. The purpose of the study was to evaluate possible application of picropodophyllin (PPP) - a potent IGF1R inhibitor. METHODS: In our study we used a number of in vitro assays showing influence of IGF1R blockage on RMS cell lines (both ARMS and ERMS) proliferation, migration, adhesion, cell cycling and signal transduction pathways. Additionally, we tested possible concomitant application of PPP with commonly used chemotherapeutics (vincristine, actinomycin-D and cisplatin). Moreover, we performed an in vivo study where PPP was injected intraperitoneally into RMS tumor bearing SCID mice. RESULTS: We observed that PPP strongly inhibits RMS proliferation, chemotaxis and adhesion. What is more, application of the IGF1R inhibitor attenuates MAPK phosphorylation and cause cell cycle arrest in G2/M phase. PPP increases sensitivity of RMS cell lines to chemotherapy, specifically to vincristine and cisplatin. In our in vivo studies we noted that mice treated with PPP grew smaller tumors and displayed significantly decreased seeding into bone marrow. CONCLUSIONS: The cyclolignan PPP effectively inhibits RMS tumor proliferation and metastasis in vitro and in an animal model.

Transepithelial resistance and claudin expression in trout RTgill-W1 cell line: effects of osmoregulatory hormones.

In the present study, we examined the trout gill cell line RTgill-W1 as a possible tool for in vitro investigation of epithelial gill function in fish. After seeding in transwells, transepithelial resistance (TER) increased until reaching a plateau after 1-2 days (20-80Ω⋅cm(2)), which was then maintained for more than 6 days. Tetrabromocinnamic acid, a known stimulator of TER via casein kinase II inhibition, elevated TER in the cell line to 125% of control values after 2 and 6h. Treatment with ethylenediaminetetraacetic acid induced a decrease in TER to <15% of pre-treatment level. Cortisol elevated TER after 12-72 h in a concentration-dependent manner, and this increase was antagonized by growth hormone (Gh). The effects of three osmoregulatory hormones, Gh, prolactin, and cortisol, on the mRNA expression of three tight junction proteins were examined: claudin-10e (Cldn-10e), Cldn-30, and zonula occludens-1 (Zo-1). The expression of cldn-10e was stimulated by all three hormones but with the strongest effect of Gh (50-fold). cldn-30 expression was stimulated especially by cortisol (20-fold) and also by Gh (4-fold). Finally, zo-1 was unresponsive to hormone treatment. Western blot analysis detected Cldn-10e and Cldn-30 immunoreactive proteins of expected molecular weight in samples from rainbow trout gills but not from RTgill-W1 cultures, possibly due to low expression levels. Collectively, these results show that the RTgill-W1 cell layers have tight junctions between cells, are sensitive to hormone treatments, and may provide a useful model for in vitro study of some in vivo gill phenomena.

Aquaporin expression in the Japanese medaka (Oryzias latipes) in freshwater and seawater: challenging the paradigm of intestinal water transport?

We investigated the salinity-dependent expression dynamics of seven aquaporin paralogs (aqp1a, aqp3a, aqp7, aqp8ab, aqp10a, aqp10b and aqp11a) in several tissues of euryhaline Japanese medaka (Oryzias latipes). All paralogs except aqp7 and aqp10a had a broad tissue distribution, and several were affected by salinity in both osmoregulatory and non-osmoregulatory tissues. In the intestine, aqp1a, aqp7, aqp8ab and aqp10a decreased upon seawater (SW) acclimation in both long-term acclimated fish and during 1-3 days of the transition period. In the gill, aqp3a was lower and aqp10a higher in SW than in freshwater (FW). In the kidney no aqps were affected by salinity. In the skin, aqp1a and aqp3a were lower in SW than in FW. In the liver, aqp8ab and aqp10a were lower in SW than in FW. Furthermore, six Na(+),K(+)-ATPase α-subunit isoform transcripts were analysed in the intestine but none showed a consistent response to salinity, suggesting that water transport is not regulated at this level. In contrast, mRNA of the Na(+),K(+),2Cl(-)-cotransporter type-2 strongly increased in the intestine in SW compared with FW fish. Using custom-made antibodies, Aqp1a, Aqp8ab and Aqp10a were localized in the apical region of enterocytes of FW fish. Apical staining intensity strongly decreased, vanished or moved to subapical regions, when fish were acclimated to SW, supporting the lower mRNA expression in SW. Western blots confirmed the decrease in Aqp1a and Aqp10a in SW. The strong decrease in aquaporin expression in the intestine of SW fish is surprising, and challenges the paradigm for transepithelial intestinal water absorption in SW fishes.

Creating an Innovative Artificial Intelligence-Based Technology (TCRact) for Designing and Optimizing T Cell Receptors for Use in Cancer Immunotherapies: Protocol for an Observational Trial.

BACKGROUND: Cancer continues to be the leading cause of mortality in high-income countries, necessitating the development of more precise and effective treatment modalities. Immunotherapy, specifically adoptive cell transfer of T cell receptor (TCR)-engineered T cells (TCR-T therapy), has shown promise in engaging the immune system for cancer treatment. One of the biggest challenges in the development of TCR-T therapies is the proper prediction of the pairing between TCRs and peptide-human leukocyte antigen (pHLAs). Modern computational immunology, using artificial intelligence (AI)-based platforms, provides the means to optimize the speed and accuracy of TCR screening and discovery. OBJECTIVE: This study proposes an observational clinical trial protocol to collect patient samples and generate a database of pHLA:TCR sequences to aid the development of an AI-based platform for efficient selection of specific TCRs. METHODS: The multicenter observational study, involving 8 participating hospitals, aims to enroll patients diagnosed with stage II, III, or IV colorectal cancer adenocarcinoma. RESULTS: Patient recruitment has recently been completed, with 100 participants enrolled. Primary tumor tissue and peripheral blood samples have been obtained, and peripheral blood mononuclear cells have been isolated and cryopreserved. Nucleic acid extraction (DNA and RNA) has been performed in 86 cases. Additionally, 57 samples underwent whole exome sequencing to determine the presence of somatic mutations and RNA sequencing for gene expression profiling. CONCLUSIONS: The results of this study may have a significant impact on the treatment of patients with colorectal cancer. The comprehensive database of pHLA:TCR sequences generated through this observational clinical trial will facilitate the development of the AI-based platform for TCR selection. The results obtained thus far demonstrate successful patient recruitment and sample collection, laying the foundation for further analysis and the development of an innovative tool to expedite and enhance TCR selection for precision cancer treatments. TRIAL REGISTRATION: ClinicalTrials.gov NCT04994093; https://clinicaltrials.gov/ct2/show/NCT04994093. INTERNATIONAL REGISTERED REPORT IDENTIFIER (IRRID): DERR1-10.2196/45872.

Effective Activation and Expansion of Canine Lymphocytes Using a Novel Nano-Sized Magnetic Beads Approach.

Expansion protocols for human T lymphocytes using magnetic beads, which serve as artificial antigen presenting cells (aAPCs), is well-studied. Yet, the efficacy of magnetic beads for propagation and functionality of peripheral blood lymphocytes (PBLs) isolated from companion dogs still remains limited. Domestic dog models are important in immuno-oncology field. Thus, we built the platform for induction of canine PBLs function, proliferation and biological activity using nano-sized magnetic beads (termed as MicroBeads) coated with anti-canine CD3 and CD28 antibodies. Herein we reveal that activation of canine PBLs via MicroBeads induces a range of genes involved in immediate-early response to T cell activation in dogs. Furthermore, canine T lymphocytes are effectively activated by MicroBeads, as measured by cluster formation and induction of activation marker CD25 on canine T cells as quickly as 24 h post stimulation. Similar to human T cells, canine PBLs require lower activation signal strength for efficient proliferation and expansion, as revealed by titration studies using a range of MicroBeads in the culture. Additionally, the impact of temperature was assessed in multiple stimulation settings, showing that both 37°C and 38.5°C are optimal for the expansion of canine T cells. In contrast to stimulation using plant mitogen Concanavalin A (ConA), MicroBead-based activation did not increase activation-induced cell death. In turn, MicroBeads supported the propagation of T cells with an effector memory phenotype that secreted substantial IL-2 and IFN-γ. Thus, MicroBeads represent an accessible and affordable tool for conducting immunological studies on domestic dog models. Similarities in inducing intracellular signaling pathways further underscore the importance of this model in comparative medicine. Presented herein MicroBead-based expansion platforms for canine PBLs may benefit adoptive immunotherapy in dogs and facilitate the design of next-generation clinical trials in humans.

Inflammation, Cancer and Immunity-Implication of TRPV1 Channel.

Process of inflammation and complex interactions between immune and cancer cells within tumor microenvironment are known to drive and shape the outcome of the neoplastic disease. Recent studies increasingly show that ion channels can be used as potential targets to modulate immune response and to treat inflammatory disorders and cancer. The action of both innate and adaptive immune cells is tightly regulated by ionic signals provided by a network of distinct ion channels. TRPV1 channel, known as a capsaicin receptor, was recently documented to be expressed on the cells of the immune system but also aberrantly expressed in the several tumor types. It is activated by heat, protons, proinflammatory cytokines, and associated with pain and inflammation. TRPV1 channel is not only involved in calcium signaling fundamental for many cellular processes but also takes part in cell-environment crosstalk influencing cell behavior. Furthermore, in several studies, activation of TRPV1 by capsaicin was associated with anti-cancer effects. Therefore, TRPV1 provides a potential link between the process of inflammation, cancer and immunity, and offers new treatment possibilities. Nevertheless, in many cases, results regarding TRPV1 are contradictory and need further refinement. In this review we present the summary of the data related to the role of TRPV1 channel in the process of inflammation, cancer and immunity, limitations of the studies, and directions for future research.

Adoptive cell transfer: new perspective treatment in veterinary oncology.

Cancer immunotherapy is recently considered the most promising treatment for human patients with advanced tumors and could be effectively combined with conventional therapies such as chemotherapy or radiotherapy. Patients with hematological malignancies and melanoma have benefited greatly from immunotherapies such as, adoptive cell transfer therapy, experiencing durable remissions and prolonged survival. In the face of increasing enthusiasm for immunotherapy, particularly for the administration of tumor-specific T lymphocytes, the question arises whether this method could be employed to improve treatment outcomes for canine patients. It is warranted to determine whether veterinary clinical trials could support comparative oncology research and thus facilitate the development of new cell-based therapies for humans. Herein, we discuss adoptive transfer of T lymphocytes and lymphokine-activated cells for application in veterinary oncology, in the context of human medicine achievements. Furthermore, we discuss potential benefits of using domestic dog as a model for immunotherapy and its advantages for translational medicine. We also focus on an emerging genome-editing technology as a useful tool to improve a T cells' phenotype.