Expression of IL-17RA in Innate Cells of Patients with Common Variable Immunodeficiency (CVID) and its Clinical Implications.

Summary: Background. Common Variable Immunodeficiency (CVID) is a primary immunodeficiency disorder characterized by B-cell dysfunction and immunoglobulin production deficiency. Dysregulation of interleukin-17 (IL-17) and its receptor IL-17RA have been reported in various immune disorders. This study aimed to investigate the expression of IL-17RA in innate immune cells of CVID patients and its correlation with clinical manifestations. Methods. A cross-sectional study included 22 CVID patients and 14 age- and sex-matched healthy controls. IL-17RA expression was assessed in various immune cell subsets using flow cytometry. Demographic and clinical data were collected, and statistical analysis was performed. Results. CVID patients had elevated IL-17RA expression in neutrophils, non-classical monocytes, and dendritic cells compared to healthy controls. Patients with a history of intestinal microbial colonization, particularly with Campylobacter jejuni and Giardia intestinalis, showed significantly higher IL-17RA expression in innate cells. Elevated IL-17RA expression in monocytes and dendritic cells also correlated with higher fecal calprotectin levels in CVID patients, regardless of microbial colonization. Conclusions. The study suggests that despite previous reports of reduced circulating Th17 cells and IL-17 levels in CVID patients, IL-17RA expression in innate cells may be elevated, potentially indicating altered IL-17 signaling. This heightened IL-17RA expression could contribute to a persistent pro-inflammatory state, possibly due to microbial translocation or other inflammatory factors. The association of IL-17RA expression with gastrointestinal microbial colonization and its correlation with fecal calprotectin underscores the complexity of IL-17RA's role in CVID pathophysiology. Further research in larger cohorts could elucidate the implications of IL-17RA expression in both infectious and non-infectious inflammatory aspects of CVID.

Insights into the Release Mechanisms of ITZ:HPMCAS Amorphous Solid Dispersions: The Role of Drug-Rich Colloids.

Understanding the dissolution mechanisms of amorphous solid dispersions (ASDs) and being able to link enhanced drug exposure with process parameters are key when formulating poorly soluble compounds. Thus, in this study, ASDs composed by itraconazole (ITZ) and hydroxypropylmethylcellulose acetate succinate (HPMCAS) were formulated with different polymer grades and drug loads (DLs) and processed by spray drying with different atomization ratios and outlet temperatures. Their in vitro performance and the ability to form drug-rich colloids were then evaluated by a physiologically relevant dissolution method. In gastric media, drug release followed a diffusion-controlled mechanism and drug-rich colloids were not formed since the solubility of the amorphous API at pH 1.6 was not exceeded. After changing to intestinal media, the API followed a polymer dissolution-controlled release, where the polymer rapidly dissolved, promoting the immediate release of API and thus leading to liquid-liquid phase separation (LLPS) and consequent formation of drug-rich colloids. However, the release of API and polymer was not congruent, so API surface enrichment occurred, which limited the further dissolution of the polymer, leading to a drug-controlled release. ASDs formulated with M-grade showed the highest ability to maintain supersaturation and the lowest tendency for AAPS due to its good balance between acetyl and succinoyl groups, and thus strong interactions with both the hydrophobic drug and the aqueous dissolution medium. The ability to form colloids increased for low DL (15%) and high specific surface area due to the high amount of polymer released until the occurrence of API surface enrichment. Even though congruent release was not observed, all ASDs formed drug-rich colloids that were stable in the solution until the end of the dissolution study (4 h), maintaining the same size distribution (ca. 300 nm). Drug-rich colloids can, in vivo, act as a drug reservoir replenishing the drug while it permeates. Designing ASDs that are prone to form colloids can overcome the solubility constraints of Biopharmaceutics Classification System (BCS) II and IV drugs, posing as a reliable formulation strategy.

The rs5743836 polymorphism in TLR9 confers a population-based increased risk of non-Hodgkin lymphoma.

Non-Hodgkin lymphoma (NHL) has been associated with immunological defects, chronic inflammatory and autoimmune conditions. Given the link between immune dysfunction and NHL, genetic variants in toll-like receptors (TLRs) have been regarded as potential predictive factors of susceptibility to NHL. Adequate anti-tumoral responses are known to depend on TLR9 function, such that the use of its synthetic ligand is being targeted as a therapeutic strategy. We investigated the association between the functional rs5743836 polymorphism in the TLR9 promoter and risk for B-cell NHL and its major subtypes in three independent case-control association studies from Portugal (1160 controls, 797 patients), Italy (468 controls, 494 patients) and the US (972 controls, 868 patients). We found that the rs5743836 polymorphism was significantly overtransmitted in both Portuguese (odds ratio (OR), 1.85; P=7.3E-9) and Italian (OR, 1.84; P=6.0E-5) and not in the US cohort of NHL patients. Moreover, the increased transcriptional activity of TLR9 in mononuclear cells from patients harboring rs5743836 further supports a functional effect of this polymorphism on NHL susceptibility in a population-dependent manner.

Apoptosis deregulation influences chemoresistance to azaguanine in human leukemic cell lines.

The involvement of apoptosis in the cytotoxicity mediated by nucleoside analogues, namely azaguanine, and its implication in resistance are not well understood. Using human T-cell acute lymphoblastic leukemia cell lines, sensitive (CEM cells) and resistant to azaguanine (CM3 cells), we observe a decrease in the expression of proapoptotic proteins in CM3 cells, which may be related to the resistance to cell death induced by azaguanine. On the other hand, CM3 cells lack cross resistance with other anticarcinogenic drugs, suggesting that azaguanine may be used alternatively in the presence of chemoresistance. A better knowledge of the apoptotic pathways involved in leukemic cell death resistance may contribute to the development of therapeutic strategies, aimed to prevent chemotherapy resistance.

Protective effects of the dipeptidyl peptidase IV inhibitor sitagliptin in the blood-retinal barrier in a type 2 diabetes animal model.

AIM: The aim of this study was to evaluate the efficacy of sitagliptin, a dipeptidyl peptidase IV inhibitor (DPP-IV), in preventing the deleterious effects of diabetes on the blood-retinal barrier in male Zucker Diabetic Fatty (ZDF) rats. METHODS: ZDF rats at 20 weeks of age were treated with sitagliptin (10 mg/kg/day) during 6 weeks. The effect of the drug on glycaemia was assessed by evaluating glycated haemoglobin (HbA1c). The content and/or distribution of tight junction (TJ) proteins occludin and claudin-5, as well as nitrotyrosine residues, interleukin (IL)-1ß, BAX and Bcl-2 was evaluated in the retinas by western blotting and/or immunohistochemistry. Retinal cell apoptosis was assessed by the terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling (TUNEL) assay. The number of CD34+ cells present in peripheral circulation was assessed by flow cytometry, and endothelial progenitor cells (EPC) adhesion ability to the retinal vessels was evaluated by immunohistochemistry. RESULTS: Sitagliptin improved glycaemic control as reflected by a significant decrease in HbA1c levels by about 1.2%. Treatment with sitagliptin prevented the changes in the endothelial subcellular distribution of the TJ proteins induced by diabetes. Sitagliptin also decreased the nitrosative stress, the inflammatory state and cell death by apoptosis in diabetic retinas. Diabetic animals presented decreased levels of CD34+ cells in the peripheral circulation and decreased adhesion ability of EPC to the retinal vessels. Sitagliptin allowed a recovery of the number of CD34+ cells present in the bloodstream to levels similar to their number in controls and increased the adhesion ability of EPC to the retinal vessels. CONCLUSIONS: Sitagliptin prevented nitrosative stress, inflammation and apoptosis in retinal cells and exerted beneficial effects on the blood-retinal barrier integrity in ZDF rat retinas.

T cell receptor excision circles (TREC) and recent thymic migrant cells in specific immunotherapy and respiratory allergy to Dermatophagoides pteronyssinus.

INTRODUCTION: T cell receptor excision circles (TREC) on CD31+ T cells are related to recent thymic emigrant cells (RTEs). The involvement of the functional thymic tissue occurs early in the IgE-mediated allergic reaction, and in response to specific immunotherapy (SIT). AIM: Evaluation of specific immunotherapy effects on TREC number in peripheral T cells in patients allergic to Dermatophagoides pteronyssinus (Dpt). METHOD: 85 respiratory allergic patients (both genders), 41 of them (Group II) under maintenance treatment to Dpt SIT (21 sublingual-SLIT, and 20 subcutaneous-SCIT), were selected. The allergic patients (Group I) without specific treatment were submitted to an allergen challenge test (22 nasal and 22 conjunctival). Peripheral cell analysis was performed immediately before treatment and 60 or 240 minutes after allergenic extract administration. TREC quantification was performed in CD4+CD31+ and CD8+CD31+. The results were expressed per 100.000 cells related to RTEs. Samples from 10 healthy individuals (Control - Group III) were obtained with the same method. RESULTS: The value of TRECs on RTEs was constant in control groups. For Group I patients (nasal or conjunctival test), TREC quantification in CD31+ T cells showed relevant individual changes, even in the patients tested earlier (60 minutes), and statistical significant at 240 minutes. Both SCIT and SLIT had also demonstrated enormous individual changes, particularly on TRECs/CD4+CD31+ cells assay. Basal values in Group III were significantly higher than those observed in active patients groups. CONCLUSION: Thymic functional activity is earlier involved in the allergic reaction and SIT IgE-mediated allergy is able to induce RTEs in the periphery, particularly TRECs/CD4+CD31+ cells. Both SLIT and SCIT showed reduced RETs in the periphery, probably due to maturation of regulatory T cells. Our results suggest a crucial role of the functional thymic tissue on the central mechanism of this therapy.

Socioeconomic aspects and profile of fishing according to fishers of commercial edible fish in the municipality of Barcelos, middle Negro River, Amazonas, Brazil.

Fishing plays a prominent role in the socioeconomic scope of the Negro River basin and is considered one of the main extractive activities in the region. The objective of this study was to describe the socioeconomic aspects of commercial fishers of edible fish who work in the municipality of Barcelos, in the middle Negro River region, as well as to highlight the scenario of the fishing activity from the point of view of the fishers. The information was collected between January and December 2016 in the municipality of Barcelos, state of Amazonas, via semi-structured questionnaires. Most interviewees were male (71.14%), aged between 18 and 82 years and a mean age of 48 years. With a low level of education, 45.0% had only incomplete elementary education and 15% were illiterate. The fishing tackle most used by fishermen was the gillnet, especially by urban fishermen (70%). Among the fishing sites, the main one was the Demeni River (50%). Commercial fishing for edible fish in Barcelos focused on Characiformes, Perciformes and Siluriformes fish. The main difficulties faced by the activity are related to the seasonality of the level of rivers and local tributaries, as well as the lack of buyers and low selling price of fish, in addition to conflicts over the use of fishing resources. Commercial fishing for edible fish is an activity of great socioeconomic importance for riverside families in the Middle River Negro basin, which needs greater attention from the public authorities. Therefore, it is expected that the information contained in this study can help in the decision-making process for the management of local fisheries resources and contribute to the resumption of growth and sustainability of commercial edible fish fisheries.

Are Pesticides Used to Control Thrips Harmonious with Soil-Dwelling Predatory Mite Cosmolaelaps sabelis (Mesostigmata: Laelapidae)?

Edaphic predatory mites could be introduced in pest management programs of pests that live, or spend part of their life cycle, in the soil. Some mesostigmatic mites have been widely used for the management of different species of thrips (Thysanoptera), especially in protected cultivation. The edaphic predator Cosmolaelaps sabelis (Mesostigmata: Laelapidae) was a model species in this study, being exposed to the most applied insecticides for the control of thrips in Brazil. After lethal, sublethal and transgenerational effects were evaluated. The pesticides acephate, acetamiprid + etofenprox, azadirachtin, spinetoram, formetanate hydrochloride, and imidacloprid were classified according to the IOBC/WPRS (International Organization for Biological Control-West Paleartic Regional Section) recommendation, considering the acute toxicity and the effects on adult females' reproduction, in the maternal and first generation. The pesticides acetamiprid + etofenprox and azadirachtin were classified as slightly harmful (Class 2), while spinetoram was classified as moderately harmful (Class 3). Acephate and formetanate hydrochloride were classified as harmful (Class 4). Only imidacloprid didn't cause negative effects on the females. Regarding effects on the first generation, acetamiprid + etofenprox, azadirachtin, and spinetoram caused reduction in the oviposition rates. Therefore, we suggest that complimentary bioassays should be done under semi-field and field conditions using the pesticides that were considered harmful in this study, to assess their effects on this predator in other environments prior to recommending not to use them in integrated programs to manage soil-based pests using chemical and biological tools.

Cauda equina syndrome following combined spinal-epidural anesthesia with levobupivacaine for cesarean section.

Cauda equina syndrome (CES) is a rare but possible complication of neuroaxial anesthesia. Damage to the nerve roots may occur due to compression, inflammation, stretching, direct trauma, spinal ischemia or neurotoxicity, usually with lidocaine or bupivacaine. We describe a case of a 33-year-old patient that underwent an uneventful cesarean section with a combined spinal-epidural technique anesthesia, with levobupivacaine. 48 h after the procedure, she presented diminished muscular strength and abolished osteotendinous reflexes in the left lower limb, limited flexion of the right hallux, urinary retention and saddle anesthesia. Imaging exams excluded hematoma, thickening or compression of the cauda equina nerve roots. CES was suspected and treatment was initiated. 9-month follow up revealed diminished osteotendinous reflexes on the left lower limb and perianal hypoesthesia. Despite being unusual, neurological complications require prompt recognition and management to avoid permanent damage.

Expression patterns of HLA-DR+ or HLA-DR- on CD4+/CD25++/CD127low regulatory T cells in patients with allergy.

BACKGROUND: Allergic rhinoconjunctivitis induced by pollen is a highly prevalent chronic inflammatory disease in Europe. Parietariajudaica is a frequent trigger in the Mediterranean area. The function of regulatory T cells (Treg cells) in allergy has recently been investigated, but further data are necessary to better understand their role and to find new strategies to treat allergic diseases such as allergic rhinoconjunctivitis. OBJECTIVE: To characterize gene expression of HLA-DR+ or HLA-DR- on peripheral CD4+/CD25++/CD127low Treg cells in patients with allergy. METHODS: Peripheral Treg cells (CD4+/CD25++/CD127low) were quantified using flow cytometry and sorted according to HLA-DR expression during the pollen season in patients with allergic rhinoconjunctivitis caused by P. judaica. The results were compared with those of nonatopic controls. Expression of associated cytokines and their receptors was measured using quantitative reverse transcription-polymerase chain reaction after extraction of mRNA in sorted populations. RESULTS: During the pollen season, no significant differences were observed between allergic patients with rhinoconjunctivitis and healthy controls in terms of the absolute number or the percentage of Treg cells in peripheral blood. All patients had a higher number/percentage of HLA-DR- Treg cells than HLA-DR+ Treg cells. In both groups we found high levels of FOXP3 mRNA expression. Despite being lower in number, HLA-DR+ Treg cells presented higher expression of CD28, PRF1, GZMB, and FASL than HLA-DR-Treg cells. CONCLUSIONS: The most relevant results obtained suggest that HLA-DR+ Treg cells tend to present higher gene expression of molecules associated with contact-dependent cell activation and cytotoxicity.

Subcutaneous emphysema as a complication of the use of a laryngeal mask in a child. / Enfisema subcutáneo como complicación del uso de una mascarilla laríngea en un niño.

The use of supraglottic airway devices has been increasing in popularity, mostly due to their high success rate and low complications. However, there is very little information available about the potential and group specific concerns regarding their use in children. We present the first description of a child that developed subcutaneous emphysema after the use of a laryngeal mask. We believe that more awareness to the risk of perioperative adverse events with laryngeal mask insertion in the paediatric population is needed.

Electrodialytic removal of tungsten and arsenic from secondary mine resources - Deep eutectic solvents enhancement.

Tungsten is a critical raw material for European and U.S. economies. Tungsten mine residues, usually considered an environmental burden due to e.g. arsenic content, are also secondary tungsten resources. The electrodialytic (ED) process and deep eutectic solvents (DES) have been successfully and independently applied for the extraction of metals from different complex environmental matrices. In this study a proof of concept demonstrates that coupling DES in a two-compartment ED set-up enhances the removal and separation of arsenic and tungsten from Panasqueira mine secondary resources. Choline chloride with malonic acid (1:2), and choline chloride with oxalic acid (1:1) were the DES that in batch extracted the average maximum contents of arsenic (16%) and tungsten (9%) from the residues. However, when ED was operated at a current intensity of 100 mA for 4 days, the extraction yields increased 22% for arsenic and 11% for tungsten, comparing to the tests with no current. From the total arsenic and tungsten extracted, 82% and 77% respectively were successfully removed from the matrix compartment, as they electromigrated to the anolyte compartment, from where these elements can be further separated. This achievement potentiates circular economy, as the final treated residue could be incorporated in construction materials production, mitigating current environmental problems in both mining and construction sectors.

Elevated soluble TNFα levels and upregulated TNFα mRNA expression in purified peripheral blood monocyte subsets associated with high-grade hepatocellular carcinoma.

BACKGROUND: Chronic inflammation is involved in the initiation and progression of various cancers, including liver cancer. The current study focuses on the characterization of the peripheral immune response in hepatocellular carcinoma (HCC) and cholangiocarcinoma (CCA) patients, before and after surgical procedure, in order to assess the effect of tumor resection in the immune system homeostasis and to determine possible prognostic factors associated with high-grade tumors. We developed a whole-blood assay to monitor immune alterations and functional competence of peripheral monocytes in a group of 10 healthy individuals (HG), in 20 HCC patients and 8 CCA patients, by multi-color flow cytometry, qRT-PCR, and ELISA techniques. RESULTS: The qRT-PCR analysis showed an upregulation of TNFα expression by classical and intermediate monocytes purified from HCC patients presenting tumors in grade G3-G4 as compared to G1-G2 HCC patients. Moreover, ELISA assay confirmed elevated serum levels of TNFα in G3-G4 compared to G1-G2 HCC patients. A significant decrease of circulating non-classical monocytes was detected in both CCA and HCC patients before and after surgical procedure. In addition, a functional defect in circulating classical and intermediate monocytes was observed in both groups of cancer patients when compared to the HG, with partial recovery after the surgical intervention. CONCLUSIONS: This integrated analysis permitted the identification of altered functional competence of monocyte subsets in CCA and HCC patients. In addition, our results point to a potential role of TNFα as a prognostic peripheral biomarker in HCC patients, indicating the presence of high-grade tumors that should be further validated.

Ornamental fishing in the region of Barcelos, Amazonas: socioeconomic description and scenario of activity in the view of "piabeiros".

The Negro river basin is considered the largest area of extractive of ornamental fish in Brazil. This area has fundamental importance for the populations from the Amazon. The present study aimed to describe socioeconomic profile of ornamental fishermen known as "piabeiros" in the Municipality of Barcelos, as well as the ornamental fishery, fisheries area, target species, environments, fishing techniques, equipment, capture techniques and main difficulties faced by the current activity. This study was carried out in municipality of Barcelos, through semi - structured interviews, with artisanal ornamental fishermen (N= 89). The main families of ornamental fish caught and traded were: Characidae, Lebiasinidae, Gasteropelecidae, Cichilidae, Anostomidae, Loricaridae, Potamotrygonidae and Gymnotidae. The main catchment areas were igarapés, lakes, flooded fields, beaches, river banks and igapó forest. Rapiché was the most used equipment in the fisheries both by the fishermen of the urban areas (43.81%) and rural (41.89%). Most of the fishermen are associated with the colony of fishermen of Barcelos (Z33). The data showed that the dynamics of ornamental fishing have changed in a short time and directly affected fishermen, in addition to the low age renewal with the participation of younger fishermen, threatening the transmission of ecological knowledge to future generations. As a result, the increase of the problems related to the productive chain and absence of public power to the activity, since ornamental fishing has already been treated as one of the main economic activities more important for the local communities and for the State of Amazonas.

The effect of carbon and nitrogen sources on bovicin HC5 production by Streptococcus bovis HC5.

AIMS: To investigate the effect of media composition and agroindustrial residues on bovicin HC5 production by Streptococcus bovis HC5. METHODS AND RESULTS: Batch cultures of S. bovis HC5 were grown in basal medium containing different carbon and nitrogen sources. The activity of cell-free and cell-associated bovicin HC5 was determined in culture supernatants and acidic extracts obtained from cell pellets, respectively. Streptococcus bovis HC5 produced bovicin using a variety of carbon and nitrogen sources. The highest specific activity was obtained in media containing 16 g l(-1) of glucose, after 16 h of incubation. The peak in cell-free and cell-associated bovicin HC5 activity was detected when S. bovis HC5 cultures reached stationary phase. The bovicin HC5 specific activity and bacterial cell mass increased approximately 3-fold when yeast extract and trypticase (0.5 and 1.0 g l(-1), respectively) were added together to the basal medium. Streptococcus bovis HC5 cultures produced bovicin HC5 in cheese whey and sugar cane juice and maximal volumetric productivity was obtained after 12 h of incubation. CONCLUSIONS: Streptococcus bovis HC5 is a versatile lactic acid bacterium that can utilize several carbon and nitrogen sources for bovicin HC5 production. This bacterium could be a useful model to study bacteriocin production in the rumen ecosystem. SIGNIFICANCE AND IMPACT OF THE STUDY: The use of agroindustrial residues as carbon sources could have an economical impact on bovicin HC5 production. To our knowledge, this is the first report to show the use of sugar cane juice for bacteriocin production by lactic acid bacteria.

Quantification and immunophenotypic characterization of bone marrow and umbilical cord blood mesenchymal stem cells by multicolor flow cytometry.

In recent years, mesenchymal stem cells (MSC) have been attracting the greatest interest in the regeneration of injured tissues, autoimmune diseases, and transplantation of hematopoietic progenitor cells. Bone marrow (BM) represents the major source of MSC; however, umbilical cord blood (UCB) MSC has some advantages over BM, such as the higher differentiation capability and noninvasive collection methods. We sought to establish a 7-color, single-tube flow cytometric assay to quantify MSC in fresh tissues, namely BM and UCB, based on phenotypic markers of these cells. Moreover, we evaluated the differential expression of these markers in BM and UCB MSC. We used 5 UCB samples and 5 BM samples obtained from individuals without hematologic disease. To characterize MSC we used the following combination of monoclonal antibodies: CD71-FITC; CD105-PE; CD184-PE-Cy5; CD34-PE-Cy7; CD133-APC; CD45-APC-H7; CD44-Pacific blue, acquiring at least 1 million nucleated cells. We observed a greater number of BM MSC when compared with UCB MSC as well as some differences in the expression of some MSC antigens, particularly CD105 and CD44. Based on our preliminary results, phenotypic identification of MSC by flow cytometry is possible using a 7-color, single-tube assay. However, culture assays after sorting of cells characterized in this study are required to prove that they correspond to MSC.

Lipoxidation and cancer immunity.

Lipoxidation is a well-known reaction between electrophilic carbonyl species, formed during oxidation of lipids, and specific proteins that, in most cases, causes an alteration in proteins function. This can occur under physiological conditions but, in many cases, it has been associated to pathological process, including cancer. Lipoxidation may have an effect in cancer development through their effects in tumour cells, as well as through the alteration of immune components and the consequent modulation of the immune response. The formation of protein adducts affects different proteins in cancer, triggering different mechanism, such as proliferation, cell differentiation and apoptosis, among others, altering cancer progression. The divergent results obtained documented that the formation of lipoxidation adducts can have either anti-carcinogenic or pro-carcinogenic effects, depending on the cell type affected and the specific adduct formed. Moreover, lipoxidation adducts may alter the immune response, consequently causing either positive or negative alterations in cancer progression. Therefore, in this review, we summarize the effects of lipoxidation adducts in cancer cells and immune components and their consequences in the evolution of different types of cancer.

Functional and Phenotypic Characterization of Tumor-Infiltrating Leukocyte Subsets and Their Contribution to the Pathogenesis of Hepatocellular Carcinoma and Cholangiocarcinoma.

Hepatocellular carcinoma (HCC) and cholangiocarcinoma (CCA) represent the most common primary liver malignancies whose outcome is influenced by the immune response. In the present study, we evaluated the tumor-infiltrating leukocyte (TIL) populations in 21 HCC patients and 8 CCA patients by flow cytometry immediately after the surgical procedure. Moreover, CD4+ T cells, CD8+ T cells, monocytes, and macrophages were purified by cell sorting for further analysis of gene expression by quantitative reverse-transcription polymerase chain reaction. Regarding tumor-infiltrating macrophages, we observed a significantly higher expression of markers associated with M2 phenotype and a higher expression of PD-L1 in patients with HCC in comparison to CCA. In addition, for HCC patients, we found a significant increase in the expression of CD200R in macrophages from tumors that were in grade G3-G4 as compared to tumors in grade G1-G2. Besides, a significantly higher frequency of tumor-infiltrating lymphocytes, CD8+CD56+ T cells, and natural killer cells was detected in HCC biopsies in comparison to CCA. In summary, this study has revealed functional and phenotypic differences in TIL cell subpopulations between CCA and HCC, as well as among different histopathological grades and tumor aggressiveness degrees, and it has provided evidence to better understand the tumor immune microenvironment of CCA and HCC.