Indoleamine 2,3-dioxygenase 1 (IDO1) inhibitors have been developed with the aim of reinvigorating antitumor T-cell responses in the tumor microenvironment by blocking the conversion of the essential amino acid tryptophan into immunoregulatory kynurenines. The lack of efficacy demonstrated in the clinical trials prompts us to revise the “on-target” mechanism of these molecules. By studying the turnover of IDO1 protein in human tumor cells exposed to various IDO1 catalytic inhibitors, such as epacadostat, linrodostat, and navoximod, we show here that these molecules stabilize a non-enzymatic protein conformation of IDO1, independently of their mechanism of inhibition. In the thyroid carcinoma cell line FTC-133, the stabilized and non-enzymatic IDO1 protein promotes the proliferation and migration of the tumor, resulting in an adverse pro-tumorigenic effect. These results uncover an unexpected adverse effect of IDO1 inhibitors in the tumor microenvironment that overcomes the enzymatic inhibition of IDO1, and suggest protein degradation, rather than enzymatic inhibition, as a more effective approach to target IDO1 in the tumor microenvironment.

Nerve growth factor (NGF) plays a critical role in reproduction through paracrine and endocrine mechanisms. However, its autocrine effects on uterine receptivity and inflammatory pathways remain unknown. This study is the first to demonstrate NGF’s direct autocrine action on sheep endometrial luminal epithelial cells (SELECs), primary cultures treated with NGF for 12, 24, and 48 h, with or without the NTRK1 antagonist. NGF significantly increased PGE2 (p < 0.0001) and PGF2α (p < 0.0001) levels only at 12 h, with no significant changes at 24 and 48 h. NGF also upregulated the expression of NGF, COX2, and NTRK1 (p < 0.0001), and p75NTR and STAR (p < 0.001), at 12 h, with the effects reversed by NTRK1 inhibition, while no significant changes were observed for TLR4 (p > 0.05). Western blot (WB) analysis was performed exclusively to confirm the presence of NGF protein, revealing no significant differences (p > 0.05) across experimental conditions. These findings highlight NGF’s role in directly regulating SELEC activity through autocrine mechanisms, emphasizing its importance in uterine receptivity and reproductive readiness. This study provides novel insights into NGF’s role in sheep reproduction and its potential applications in fertility treatments.

The grey squirrel, an invasive species, threatens the Eurasian red squirrel’s conservation, particularly in Umbria, Italy. Understanding its reproductive biology is essential to limiting its reproductive success. This study investigates the NGF system and its receptors (NTRK1 and p75NTR) in the testes of male grey squirrels, following prior research on female reproductive biology. NGF plays a role in testicular morphogenesis and spermiogenesis in animals and humans. As part of the LIFE Project U-SAVEREDS, eighteen squirrels were captured and classified into three morphotypes (immature, pubertal, and active spermatogenesis). NGF and its receptors were analyzed using real-time PCR, western blotting, immunohistochemistry, and plasma levels measured via ELISA. NGF qPCR expression levels were significantly higher during puberty compared to the immature and spermatogenesis stages (p < 0.01). Immunohistochemistry revealed NGF in Leydig cells, with stronger staining in pubertal and mature squirrels, while NTRK1 was found in Leydig cells in immature squirrels and germ cells in pubertal and mature ones. NGF receptors were observed in Sertoli cells in pubertal and mature squirrels. Plasma NGF levels showed a significant upregulation in pubertal squirrels (135.80 ± 12 pg/mL) compared to those in the immature (25.60 ± 9.32 pg/mL) and spermatogenesis stages (34.20 ± 6.06 pg/mL), with a p value < 0.01. The co-localization of NGF and its receptors suggests that NGF, produced by Leydig cells, regulates testis development and reproductive success through autocrine or paracrine mechanisms, potentially involving an unidentified pathway.

The tryptophan-degrading enzyme indoleamine 2,3-dioxygenase 1 (IDO1) is a plastic immune checkpoint molecule that potently orchestrates immune responses within the tumor microenvironment (TME). As a heme-containing protein, IDO1 catalyzes the conversion of the essential amino acid tryptophan into immunoactive metabolites, called kynurenines. By depleting tryptophan and enriching the TME with kynurenines, IDO1 catalytic activity shapes an immunosuppressive TME. Accordingly, the inducible or constitutive IDO1 expression in cancer correlates with a negative prognosis for patients, representing one of the critical tumor-escape mechanisms. However, clinically trialed IDO1 catalytic inhibitors disappointed the expected anti-tumor efficacy. Interestingly, the non-enzymatic apo-form of IDO1 is still active as a transducing protein, capable of promoting an immunoregulatory phenotype in dendritic cells (DCs) as well as a pro-tumorigenic behavior in murine melanoma. Moreover, the IDO1 catalytic inhibitor epacadostat can induce a tolerogenic phenotype in plasmacytoid DCs, overcoming the catalytic inhibition of IDO1. Based on this recent evidence, IDO1 plasticity was investigated in the human ovarian cancer cell line, SKOV-3, that constitutively expresses IDO1 in a dynamic balance between the holo- and apo-protein, and thus potentially endowed with a dual function (i.e., enzymatic and non-enzymatic). Besides inhibiting the catalytic activity, epacadostat persistently stabilizes the apo-form of IDO1 protein, favoring its tyrosine-phosphorylation and promoting its association with the phosphatase SHP-2. In SKOV-3 cells, both these early molecular events activate a signaling pathway transduced by IDO1 apo-protein, which is independent of its catalytic activity and contributes to the tumorigenic phenotype of SKOV-3 cells. Overall, our findings unveiled a new mechanism of action of epacadostat on IDO1 target, repositioning the catalytic inhibitor as a stabilizer of the apo-form of IDO1, still capable of transducing a pro-tumorigenic pathway in SKOV-3 tumor. This mechanism could contribute to clarify the lack of effectiveness of epacadostat in clinical trials and shed light on innovative immunotherapeutic strategies to tackle IDO1 target.

Indoleamine 2,3-dioxygenase 1 (IDO1) and IDO2 originated from gene duplication before vertebrate divergence. While IDO1 has a well-defined role in immune regulation, the biological role of IDO2 remains unclear. Discovered in 2007, IDO2 is located near the IDO1 gene. Because of their high sequence similarity, IDO2 was initially thought to be a tryptophan (Trp)-degrading enzyme like IDO1. Differently from what expected, IDO2 displays extremely low catalytic activity toward Trp. Nevertheless, many studies, often contradictory, have tried to demonstrate that IDO2 modulates immune responses by catabolizing Trp into kynurenine, an unconvincing hypothesis linked to an incomplete understanding of IDO2’s activity. In this study, we review IDO2’s functional role beyond Trp metabolism. IDO2’s evolutionary persistence across species, despite being almost inactive as an enzyme, suggests it has some relevant biological importance. IDO2 expression in human normal cells is poor, but significant in various cancers, with two prevalent SNPs. Overall, the comparison of IDO2 to IDO1 as a Trp-degrading enzyme may have led to misunderstandings about IDO2’s true physiological and pathological roles. New insights suggest that IDO2 might function more as a signaling molecule, particularly in cancer contexts, and further studies could reveal its potential as a target for cancer therapy.

Small synthetic oligodeoxynucleotides (ODNs) can mimic microbial nucleic acids by interacting with receptor systems and promoting immunostimulatory activities. Nevertheless, some ODNs can act differently on the plasmacytoid dendritic cell (pDC) subset, shaping their immunoregulatory properties and rendering them suitable immunotherapeutic tools in several clinical settings for treating overwhelming immune responses. We designed HIV–1–derived, DNA- and RNA-based oligonucleotides (gag, pol, and U5 regions) and assessed their activity in conferring a tolerogenic phenotype to pDCs in skin test experiments. RNA-but not DNA-oligonucleotides are capable of inducing tolerogenic features in pDCs. Interestingly, sensing the HIV–1–derived single-stranded RNA-gag oligonucleotide (RNA-gag) requires both TLR3 and TLR7 and the engagement of the TRIF adaptor molecule. Moreover, the induction of a suppressive phenotype in pDCs by RNA-gag is contingent upon the induction and activation of the immunosuppressive enzyme Arginase 1. Thus, our data suggest that sensing of the synthetic RNA-gag oligonucleotide in pDCs can induce a suppressive phenotype in pDCs, a property rendering RNA-gag a potential tool for therapeutic strategies in allergies and autoimmune diseases.

Indoleamine 2,3-dioxygenase 1 (IDO1) and IDO2, originated from gene duplication before vertebrate diver-gence. While IDO1 has a well-defined role in immune regulation, the biological role of IDO2 remains unclear. Discovered in 2007, IDO2 is located near the IDO1 gene. Because of their high sequence similarity, IDO2 was initially thought to be a tryptophan (Trp)-degrading enzyme like IDO1. Differently from what expected, IDO2 displays extremely low catalytic activity toward Trp. Nevertheless, many studies, often contradictory, tried to demonstrate that IDO2 modulates immune responses by catabolizing Trp into kynurenine, a hypothesis un-convincing and, let’s say, linked to the first life of IDO2. Here, we review its “new life”, the one in which IDO2 has a functional role beyond Trp metabolism. The IDO2’s evolutionary persistence across species, despite be-ing almost inactive as an enzyme, suggests it has some relevant biological importance. IDO2 expression in human normal cells is poor, but significant in various cancers, with two prevalent SNPs. Overall, the com-parison of IDO2 to IDO1 as a Trp-degrading enzyme may have led to misunderstandings about IDO2’s true physiological and pathological roles. New insights suggest that IDO2 might function more as a signaling molecule, particularly in cancer contexts, and further studies could reveal its potential as a target for cancer therapy.

Indoleamine 2,3-dioxygenase 1 (IDO1) is a tryptophan metabolizing enzyme chronically activated in many cancer patients and its expression and activity correlate with a poor prognosis. In fact, it acts as an immune regulator and contributes to tumor-induced immunosuppression by determining tryptophan deprivation and producing immunosuppressive metabolites named kynurenines. These findings made IDO1 an attractive target for cancer immunotherapy and small-molecule inhibitors, such as epacadostat, have been developed to block its enzymatic activity. Although epacadostat was effective in preclinical models and in early phase trials, it gave negative results in a metastatic melanoma randomized phase III study to test the benefit of adding epacadostat to the reference pembrolizumab therapy. However, the reason for the epacadostat failure in this clinical trial has never been understood. Our data suggest that a possible explanation of epacadostat ineffectiveness may rely on the ability of this drug to enhance the other IDO1 immunoregulatory mechanism, involving intracellular signaling function. These findings open up a new perspective for IDO1 inhibitors developed as new anticancer drugs, which should be carefully evaluated for their ability to block not only the catalytic but also the signaling activity of IDO1.

Multiple sclerosis (MS) is a neurodegenerative and autoimmune disease affecting the central nervous system (CNS). The precise etiology of MS is still undeciphered, and signs and symptoms of the disease are varied and complex, ranging from axonal degeneration, synaptic, and neuronal loss to demyelination. Inflammation plays a critical role in determining the onset and the progression of MS, but there is still a lot of information missing before scientists come to understand what are the factors that contribute to the establishment of the neuroinflammation. Thus, various murine models, each representative of a specific hallmark of MS, are used to study the processes underlying the pathogenetic mechanisms of the disease in an attempt to find effective drugs for its treatment. Among the many causes of MS, viral infections appear to be one of the most prominent ones. In this scenario, the comprehension of the role of receptors activated upon the recognition of viral, and in general microbial, components in determining onset and progression of the neuroinflammation is of paramount importance. Toll-like receptors (TLRs) are evolutionarily conserved receptors that recognizeMolecular recognition several pathogen-associated molecular patterns (PAMPs), common structures of the pathogens, or the damage caused by the pathogens within the host. TLRs are thus directly involved in the regulation of inflammatory reactions and in the activation of the innate and, subsequently, the adaptive immune responses crucial for the elimination of infectious pathogens. The role of TLR activation in the development of MS is widely studied in various murine models of MS, as well as in MS patients. In this chapter, we will summarize the current knowledge about the contribution of TLRs to the development or progression of MS, and we will illustrate different methods commonly used for the investigation of the role of different TLRs in various murine models of the disease.

Apelin, a member of the adipokine family, is a novel endogenous peptide which regulates the male reproductive system of mammals by interacting with a specific receptor. Recent studies have highlighted that apelin may play a role in the regulation of reproduction by reducing testosterone production and inhibiting LH secretion. To the best of our knowledge, there is no available data on the presence of the apelin and its receptor in canine testes. Therefore, the aim of this study was to reveal the presence of apelin and evaluate its distribution in the canine testes using immunohistochemical and RT-PCR techniques. For this purpose, five fertile and healthy male dogs were subjected to elective orchiectomy. The immunohistochemical reaction revealed the presence of apelin and its receptor in the canine testes. Apelin was localized in spermatids and spermatozoa with a positive signal in the "acrosomal bodies". As regards the apelin receptor, a positive immunoreaction was detected in the cytoplasm of the cells localized near to the basal membrane of the seminiferous tubules and in the cytoplasm of Leydig cells. The RT-PCR analysis showed the presence of transcripts for apelin and apelin receptor in all of the samples under study. A 35kDa band confirmed apelin receptor protein expression in all of the samples analysed. In conclusion, the paracrine and endocrine role of apelin and its cognate receptor on male reproduction reported in humans and laboratory animals could also be hypothesized in dogs.

Indoleamine 2,3-dioxygenase 2 (IDO2) is a paralog of Indoleamine 2,3-dioxygenase 1 (IDO1), a tryptophan-degrading enzyme producing immunomodulatory molecules. However, the two proteins are unlikely to carry out the same functions. IDO2 shows little or no tryptophan catabolic activity and exerts contrasting immunomodulatory roles in a context-dependent manner in cancer and autoimmune diseases. The recently described potential non-enzymatic activity of IDO2 has suggested its possible involvement in alternative pathways, resulting in either pro- or anti-inflammatory effects in different models. In a previous study on non-small cell lung cancer (NSCLC) tissues, we found that IDO2 expression revealed at the plasma membrane level of tumor cells was significantly associated with poor prognosis. In this study, the A549 human cell line, basally expressing IDO2, was used as an in vitro model of human lung adenocarcinoma to gain more insights into a possible alternative function of IDO2 different from the catalytic one. In these cells, immunocytochemistry and isopycnic sucrose gradient analyses confirmed the IDO2 protein localization in the cell membrane compartment, and the immunoprecipitation of tyrosine-phosphorylated proteins revealed that kinase activities can target IDO2. The different localization from the cytosolic one and the phosphorylation state are the first indications for the signaling function of IDO2, suggesting that the IDO2 non-enzymatic role in cancer cells is worthy of deeper understanding.

The recovery of polyphenols from grape pomace (GP) supports the promotion of sustainable bioeconomy. Accordingly, the development of pre-treatment and extraction techniques using low-solvent amounts and energy-efficient processes is highly desirable. In this work, a comprehensive strategy to maximize phenol extraction from two Umbrian red wine cultivars, Cabernet Sauvignon and Merlot, was proposed. Focus was paid to sample pre- (fresh, air-, oven- and freeze-dried pomace) and post-extraction (chemical- and enzymatic-hydrolysis) treatments. An experimental design was applied to optimize phenol recovery by ultrasound-assisted extraction: the variables water content in ethanol (20–80% v/v) and time (10–30 min) highlighted a critical influence on the total phenol content (TPC) selected as the response. β-glucuronidase hydrolysis provided the highest TPC and in vitro total antioxidant capacity also improving phenol identification and quantification via HPLC–DAD (particularly evident for freeze-dried Merlot and Cabernet GP, which gave a total phenolic content measured by HPLC equal to 1904.08 mg/g and 2064.64 mg/g, respectively). LC-HRMS/MS analysis allowed identity confirmation of the major phenols, and cytotoxicity assay highlighted the safety of the selected β-glucuronidase extracts towards Caco2 cell line. Ultimately, in the present work, we have addressed the importance of the recovery of bioactive compounds from grape pomace by proposing a comprehensive analytical strategy in which different methodological alternatives have been evaluated to preserve these compounds, in a frame of sustainability on a larger industrial scale.

PurposeCurrently, bladder cancer (BC) surveillance consists of periodic white light cystoscopy and urinary cytology (UC). However, both diagnostic tools have limitations. Therefore, to improve the management of recurrent BC, novel, innovative diagnostic tests are needed. The primary aim of this study was to determine the diagnostic performance of Bladder EpiCheck (BE) and photodynamic diagnosis (PDD) guided cystoscopy in the surveillance of high-risk BC. A secondary aim was to compare Bladder EpiCheck (BE) and PDD-guided cystoscopy findings with whose of UC to design a diagnostic algorithm that facilitates clinical decision making.Patients and MethodsThis was a prospective, blinded, single-arm, single-visit cohort study. All patients were under surveillance for high-risk non-muscle-invasive bladder cancer, and underwent cystoscopy with PDD and a BE test. Those who received a histological diagnosis were used as a reference population. Receiver operating characteristic curve analysis was performed to evaluate the diagnostic performance of BE, PDD-guided cystoscopy, and UC for identifying biopsy-confirmed BC lesions. The diagnostic power of the test was assessed by determining the area under the curve (AUC).ResultsForty patients were enrolled. For BE, the AUC was 0.95, and BC recurrence was detected at a sensitivity of 100% and specificity of 90.9%. For PDD, the AUC was 0.51, with a sensitivity and specificity of 61% and 41%, respectively. BE was combined with UC to create a decision-making algorithm capable of reducing the number of follow-up cystoscopies needed.ConclusionBE is a very accurate diagnostic tool that has the potential to be useful in the surveillance of high-risk BC patients. Especially when combined with UC, it may be used to reduce the number of cystoscopies needed throughout follow-up. Conversely, the use of PDD as a diagnostic tool in such patients should be reconsidered. However, due to the small sample size of this study, a larger prospective clinical trial should be performed to confirm findings.

Over the last two decades, indoleamine 2,3-dioxygenase 1 (IDO1) has attracted wide interest as a key player in immune regulation, fostering the design and development of small molecule inhibitors to restore immune response in tumor immunity. In this framework, biochemical, structural, and pharmacological studies have unveiled peculiar structural plasticity of IDO1, with different conformations and functional states that are coupled to fine regulation of its catalytic activity and non-enzymic functions. The large plasticity of IDO1 may affect its ligand recognition process, generating bias in structure-based drug design campaigns. In this work, we report a screening campaign of a fragment library of compounds, grounding on the use of three distinct conformations of IDO1 that recapitulate its structural plasticity to some extent. Results are instrumental to discuss tips and pitfalls that, due to the large plasticity of the enzyme, may influence the identification of novel and differentiated chemical scaffolds of IDO1 ligands in structure-based screening campaigns.

Indoleamine 2,3-dioxygenase 1 (IDO1) catalyzes the initial rate-limiting step in the degradation of the essential amino acid tryptophan along the kynurenine pathway. When discovered more than 50 years ago, IDO1 was thought to be an effector molecule capable of mediating a survival strategy based on the deprivation of bacteria and tumor cells of the essential amino acid tryptophan. Since 1998, when tryptophan catabolism was discovered to be crucially involved in the maintenance of maternal T-cell tolerance, IDO1 has become the focus of several laboratories around the world. Indeed, IDO1 is now considered as an authentic immune regulator not only in pregnancy, but also in autoimmune diseases, chronic inflammation, and tumor immunity. However, in the last years, a bulk of new information-including structural, biological, and functional evidence-on IDO1 has come to light. For instance, we now know that IDO1 has a peculiar conformational plasticity and, in addition to a complex and highly regulated catalytic activity, is capable of performing a nonenzymic function that reprograms the expression profile of immune cells toward a highly immunoregulatory phenotype. With this state-of-the-art review, we aimed at gathering the most recent information obtained for this eclectic protein as well as at highlighting the major unresolved questions.

The aim of this study was to evaluate the effect of dietary polyphenols on the expression of the effectors involved in inflammation and apoptosis in rabbit ovary. New Zealand White female rabbits were fed a basal control diet (CTR), or the same diet supplemented with a polyphenolic concentrate (POL, 282.4 mg/kg) obtained from olive mill waste waters. The follicle counts and the relative mRNA (RT-qPCR) and protein (immunohistochemistry) expression of the effectors involved in inflammation (cyclooxygenase-2; interleukin-1beta; tumor necrosis factor-alpha, TNFA) and apoptosis (BCL2-associated X protein, BAX), detected in the ovaries of both groups, were examined. The POL diet increased the primary and total follicles number. Cyclooxygenase-2 gene expression was higher (p < 0.05) in the POL group than in the CTR group, whereas BAX was lower (p < 0.05) in POL than CTR. Immunohistochemistry revealed the presence of all the proteins examined, with weaker (p < 0.05) COX2 and BAX signals in POL. No differences between the CTR and POL groups were observed for IL1B and TNFA gene and protein expression. These preliminary findings show that dietary polyphenols modulate inflammatory and apoptotic activities in rabbit ovary, regulating cyclooxygenase-2 and BAX expression, thus suggesting a functional involvement of these dietary compounds in mammalian reproduction.

Immune checkpoint inhibitors have revolutionized the clinical approach of untreatable tumors and brought a breath of fresh air in cancer immunotherapy. However, the therapeutic effects of these drugs only cover a minority of patients and alternative immunotherapeutic targets are required. Metabolism of l-tryptophan (Trp) via the kynurenine pathway represents an important immune checkpoint mechanism that controls adaptive immunity and dampens exaggerated inflammation. Indoleamine 2,3-dioxygenase 1 (IDO1), the enzyme catalyzing the first, rate-limiting step of the pathway, is expressed in several human tumors and IDO1 catalytic inhibitors have reached phase III clinical trials, unfortunately with disappointing results. Although much less studied, the IDO1 paralog IDO2 may represent a valid alternative as drug target in cancer immunotherapy. Accumulating evidence indicates that IDO2 is much less effective than IDO1 in metabolizing Trp and its functions are rather the consequence of interaction with other, still undefined proteins that may vary in distinct inflammatory and neoplastic contexts. As a matter of fact, the expression of IDO2 gene variants is protective in PDAC but increases the risk of developing tumor in NSCLC patients. Therefore, the definition of the IDO2 interactome and function in distinct neoplasia may open innovative avenues of therapeutic interventions.

The lack of symptoms at the early stages of clear cell renal cell carcinoma (ccRCC) allows the tumour to metastasize, leading to a dramatic reduction in patient survival. Therefore, we studied and set up a method based on urinary microRNAs (miRNAs) for the diagnosis of ccRCC. First, miRNA expression in ccRCC specimens and kidney tissues from healthy subjects (HSs) was investigated through analysis of data banks and validated by comparing expression of miRNAs in ccRCC and adjacent non-cancerous kidney tissue specimens by RT-qPCR. Subsequently, we developed an algorithm to establish which miRNAs are more likely to be found in the urine of ccRCC patients that indicated miR-122, miR-1271, and miR-15b as potential interesting markers. The evaluation of their levels and three internal controls in the urine of 13 patients and 14 HSs resulted in the development of a score (7p-urinary score) to evaluate the presence of ccRCC in patients. The resulting area under the Receiver Operating Characteristic (ROC) curve, sensitivity, and specificity were equal to 0.96, 100% (95% CI 75–100%), and 86% (95% CI 57–98%), respectively. In conclusion, our study provides a proof of concept that combining the expression values of some urinary miRNAs might be useful in the diagnosis of ccRCC.

Many different genetic alterations, as well as complex epigenetic interactions, are the basis of the genesis and progression of prostate cancer (CaP). This is the reason why until now the molecular pathways related to development of this cancer were only partly known, and even less those that determine aggressive or indolent tumour behaviour. MicroRNAs (miRNAs) represent a class of about 22 nucleotides long, small non-coding RNAs, which are involved in gene expression regulation at the post-transcriptional level. MiRNAs play a crucial role in regulating several biological functions and preserving homeostasis, as they carry out a wide modulatory activity on various molecular signalling pathways. MiRNA genes are placed in cancer-related genomic regions or in fragile sites, and they have been proven to be involved in the main steps of carcinogenesis as oncogenes or oncosuppressors in many types of cancer, including CaP. We performed a narrative review to describe the relationship between miRNAs and the crucial steps of development and progression of CaP. The aims of this study were to improve the knowledge regarding the mechanisms underlying miRNA expression and their target genes, and to contribute to understanding the relationship between miRNA expression profiles and CaP.