A subunit vaccine based on Brucella rBP26 induces Th1 immune responses and M1 macrophage activation.

Brucellosis is a global zoonotic infection caused by Brucella bacteria, which poses a significant burden on society. While transmission prevention is currently the most effective method, the absence of a licenced vaccine for humans necessitates the urgent development of a safe and effective vaccine. Recombinant protein-based subunit vaccines are considered promising options, and in this study, the Brucella BP26 protein is expressed using prokaryotic expression systems. The immune responses are evaluated using the well-established adjuvant CpG-ODN. The results demonstrate that rBP26 supplemented with a CpG adjuvant induces M1 macrophage polarization and stimulates cellular immune responses mediated by Th1 cells and CD8 + T cells. Additionally, it generates high levels of rBP26-specific antibodies in immunized mice. Furthermore, rBP26 immunization activates, proliferates, and produces cytokines in T lymphocytes while also maintaining immune memory for an extended period of time. These findings shed light on the potential biological function of rBP26, which is crucial for understanding brucellosis pathogenesis. Moreover, rBP26 holds promise as an effective subunit vaccine candidate for use in endemic areas.

Omics-based investigation of pathological liver injury induced by Echinococcus multilocularis infection in mice.

BACKGROUND: Alveolar echinococcosis (AE) can cause severe liver injury and be fatal if left untreated. Currently, there are no effective therapeutic options for AE-induced liver injury. Therefore, by exploring the changes of gene proteins in mice with damaged liver, we attempted to identify the key molecules of liver damage, and provide data that will enable the development of drugs targeting hepatic AE. METHODS: BALB/c mice were inoculated with protoscoleces via the hepatic portal vein. Three months later, B-ultrasound examination and Hematoxylin-eosin (H&E) staining were used to confirm liver damage in mice. RNA sequencing and Liquid chromatography-mass spectrometry (LC-MS) were used to screen differentially expressed molecules associated with liver damage through bioinformatics, and Quantitative Real-Time PCR (qRT-PCR) was used to verify their expression. RESULTS: B-ultrasound examination showed liver lesions in the infected group, and H&E staining showed liver inflammation, fibrosis and liver necrosis. RNA sequencing and LC-MS results showed changes in the levels of more than 1000 genes and proteins, with upregulation of immune and inflammation pathways. By contrast, the downregulated genes and proteins were mostly involved in various metabolic reactions. Correlation analysis was conducted between the transcriptome data and proteome data. The results revealed 240 differentially expressed genes, of which 192 were upregulated, and 48 were downregulated. Many of these genes were involved in metabolic reactions, such as Catalase (Cat), fatty acid synthase (Fasn), and IL-16 genes, which may have relevance to liver injury. The results of qRT-PCR were consistent with those of bioinformatics analysis. CONCLUSIONS: The mechanisms of liver injury in mice infected with Echinococcus multilocularis are complex, involving abnormal metabolism, oxidative stress, inflammatory response, and many other factors. This study provides the data for preliminary exploration for the development of targeted therapies against AE.

Changes in intestinal flora of mice induced by rEg.P29 epitope peptide vaccines.

OBJECTIVE: Cystic echinococcosis (CE), a zoonotic parasitic disease caused by Echinococcus granulosus, remains a public health and socioeconomic issue worldwide, making its prevention and treatment of vital importance. The aim of this study was to investigate changes in the intestinal microbiota of mice immunized with three peptide vaccines based on the recombinant antigen of E. granulosus, P29 (rEg.P29), with the hope of providing more valuable information for the development of vaccines against CE. METHODS: Three peptide vaccines, rEg.P29T , rEg.P29B , and rEg.P29T + B , were prepared based on rEg.P29, and a subcutaneous immunization model was established. The intestinal floras of mice in the different immunization groups were analyzed by 16 S rRNA gene sequencing. RESULTS: The intestinal microbiota analysis at both immunization time points revealed that Firmicutes, Bacteroidota, and Verrucomicrobiota were the predominant flora at the phylum level, while at the genus level, Akkermansia, unclassified_Muribaculaceae, Lachnospiraceae_NK4A136_group, and uncultured_rumen bacterium were the dominant genera. Some probiotics in the intestines of mice were significantly increased after immunization with the peptide vaccines, such as Lactobacillus_taiwanensis, Lactobacillus_reuteri, Lachnospiraceae_NK4A136_group, Bacteroides_acidifaciens, and so forth. Meanwhile, some harmful or conditionally pathogenic bacteria were decreased, such as Turicibacter sanguinis, Desulfovibrio_fairfieldensis, Clostridium_sp, and so forth, most of which are associated with inflammatory or infectious diseases. Kyoto Encyclopaedia of Genes and Genomes enrichment analysis revealed that the differential flora were enriched in multiple metabolic pathways, primarily biological systems, human diseases, metabolism, cellular processes, and environmental information processing. CONCLUSION: In this study, we comprehensively analyzed and compared changes in the intestinal microbiota of mice immunized with three peptide vaccines as well as their related metabolic pathways, providing a theoretical background for the development of novel vaccines against E. granulosus.

Immunogenicity of peptide-based vaccine composed of epitopes from Echinococcus granulosus rEg.P29.

Echinococcus granulosus is one of the main causes of economic loss in the livestock industry because of its food-borne transmission. Cutting off the transmission route is a valid prevention method, and vaccines are the most effective means of controlling and eliminating infectious diseases. However, no human-related vaccine has been yet marketed. As a genetic engineering vaccine, recombinant protein P29 of E. granulosus (rEg.P29) could provide protection against deadly challenges. In this study, we generated peptide vaccines (rEg.P29T , rEg.P29B , and rEg.P29T+B ) based on rEg.P29 and an immunized model was established by subcutaneous immunization. Further evaluation showed that peptide vaccine immunization in mice induced T helper type 1 (Th1)-mediated cellular immune responses, leading to high levels of rEg.P29 or rEg.P29B -specific antibodies. In addition, rEg.P29T+B immunization can induce a higher antibody and cytokine production level than single-epitope vaccines, and immune memory is also longer. Collectively, these results suggest that rEg.P29T+B has the potential to be developed as an efficient subunit vaccine for use in areas where E. granulosus is endemic.

Recombinant antigen P29 of Echinococcus granulosus induces Th1, Tc1, and Th17 cell immune responses in sheep.

Echinococcosis is a common human and animal parasitic disease that seriously endangers human health and animal husbandry. Although studies have been conducted on vaccines for echinococcosis, to date, there is no human vaccine available for use. One of the main reasons for this is the lack of in-depth research on basic immunization with vaccines. Our previous results confirmed that recombinant antigen P29 (rEg.P29) induced more than 90% immune protection in both mice and sheep, but data on its induction of sheep-associated cellular immune responses are lacking. In this study, we investigated the changes in CD4+ T cells, CD8+ T cells, and antigen-specific cytokines IFN-γ, IL-4, and IL-17A after rEg.P29 immunization using enzyme-linked immunospot assay (ELISPOT), enzyme-linked immunosorbent assay (ELISA), and flow cytometry to investigate the cellular immune response induced by rEg.P29 in sheep. It was found that rEg.P29 immunization did not affect the percentage of CD4+ and CD8+ T cells in peripheral blood mononuclear cells (PBMCs), and was able to stimulate the proliferation of CD4+ and CD8+ T cells after immunization in vitro. Importantly, the results of both ELISPOT and ELISA showed that rEg.P29 can induce the production of the specific cytokines IFN-γ and IL-17A, and flow cytometry verified that rEg.P29 can induce the expression of IFN-γ in CD4+ and CD8+ T cells and IL-17A in CD4+ T cells; however, no IL-4 expression was observed. These results indicate that rEg.P29 can induce Th1, Th17, and Tc1 cellular immune responses in sheep against echinococcosis infection, providing theoretical support for the translation of rEg.P29 vaccine applications.

Adaptive immune responses and cytokine immune profiles in humans following prime and boost vaccination with the SARS-CoV-2 CoronaVac vaccine.

BACKGROUND: Adaptive immune response has been thought to play a key role in SARS-CoV-2 infection. The role of B cells, CD4+T, and CD8+T cells are different in vaccine-induced immune response, thus it is imperative to explore the functions and kinetics of adaptive immune response. We collected blood samples from unvaccinated and vaccinated individuals. To assess the mechanisms contributing to protective immunity of CoronaVac vaccines, we mapped the kinetics and durability of humoral and cellular immune responses after primary and boost vaccination with CoronaVac vaccine in different timepoints. MATERIALS AND METHODS: We separate PBMC and plasma from blood samples. The differentiation and function of RBD-spcific CD4+T and CD8+T cells were analyzed by flow cytometry and ELISA. Antibodies response was analyzed by ELISA. ELISPOT analysis was perfomed to detected the RBD-spcific memory B cells. CBA analysis was performed to detected the cytokine immune profiles. Graphpad prism 8 and Origin 2021 were used for statistical analysis. RESULTS: Vaccine-induced CD4+T cell responses to RBD were more prominent than CD8+T cell responses, and characterized by a predominant Th1 and weak Th17 helper response. CoronaVac vaccine triggered predominant IgG1 antibody response and effectively recalled specific antibodies to RBD protein after booster vaccination. Robust antigen-specific memory B cells were detected (p < 0.0001) following booster vaccination and maintained at 6 months (p < 0.0001) following primary vaccination. Vaccine-induced CD4+T cells correlated with CD8+T cells (r = 0.7147, 0.3258, p < 0.0001, p = 0.04), memory B cell responses (r = 0.7083, p < 0.0001), and IgG and IgA (r = 0.6168, 0.5519, p = 0.0006, 0.003) after vaccination. In addition, vaccine induced a broader and complex cytokine pattern in plasma at early stage. CONCLUSION: Taken together, these results highlight the potential role of B cell and T cell responses in vaccine-induced long-term immunity.

Alterations in Gut Microbiota Profiles of Mice Infected with Echinococcus granulosus sensu lato Microbiota Profiles of Mice Infected with E. granulosus s.l.

OBJECTIVE: Cystic echinococcosis is a kind of parasitic disease that seriously endangers human and animal health. At present, its prevention and treatment still do not achieve the desired results. The aims of this study were to explore the effect of CE on intestinal microflora in mice. METHODS: In this study, 16S rRNA metagenome sequencing and bioinformatics were used to analyze the intestinal flora of mice infected with E. granulosus s.l. Changes in intestinal microbial community abundance were investigated and the differences in microbial populations of mice infected with E. granulosus s.l. were screened. RESULTS: Our results show that at the phylum level, nine abundant taxa were identified, the relative abundance of Firmicutes and Proteobacteria were enriched in infected mice, whereas Bacteroidetes and Patescibacteria were enriched in control mice (P < 0.01). At the class level, 13 abundant taxa were identified, the relative abundance of Bacilli was enriched in control mice, but decreased in infected mice (P < 0.01). At the order level, 15 abundant taxa were identified, the relative abundance of Lactobacillales was enriched in control mice, but decreased in infected mice (P < 0.01). At the family level, 28 abundant taxa were identified, enriched bacteria in the infected mice was Streptococcaceae, while the enriched bacteria in the control group was Lactobacillaceae (P < 0.01). At the genus level, 79 abundant taxa were identified, enriched bacteria in the infected mice was Streptococcus, while the enriched bacteria in the control group was uncultured_bacterium_f_Eggerthellaceae (P < 0.01). At the species level, 80 abundant taxa were identified, enriched bacteria in the infected mice was uncultured_bacterium_g_Streptococcus, while the enriched bacteria in the control group was uncultured_bacterium_f_Eggerthellaceae (P < 0.01). 39 KEGG pathways were identified that were differentially enriched between the infected and control mice. CONCLUSION: This study comprehensively demonstrates the differential intestinal microbiota of infected mice and analyzes the metabolic pathways related to the specific microbiota. This could provide new targets and research direction for the treatment and prevention of diseases caused by E. granulosus s.l.

Clinical characteristics and antibodies against Echinococcus granulosus recombinant antigen P29 in patients with cystic echinococcosis in China.

OBJECTIVES: Cystic echinococcosis (CE) is a neglected parasitic zoonotic disease caused by the larval stage of the tapeworm Echinococcus granulosus (E. granulosus). This study aimed to understand the clinical characteristics of human CE in Ningxia Hui Autonomous Region (NHAR) located in northwest China and to investigate the antibody profiles against the recombinant E. granulosus antigen P29 (rEg.P29) in plasma of CE patients. METHODS: A total of 37 human CE patients, along with 37 healthy donors enrolled in this study and demographic and clinical data were analyzed, including age, gender, laboratory data, symptoms, and cysts description. Plasma levels of cytokines, total IgG, and total IgE were determined by sandwich ELISA kits. Specific antibodies against rEg.P29 and hydatid cyst fluid (HCF) were assessed by indirect ELISA. RESULTS: The results revealed that females have a higher percentage of CE patients than males. The incidence of CE reached a peak in the 41-50 years-old group. The liver was the most frequent location, accounting for 91.9%. Based on the CT images, cysts of 34 patients who had liver involvement, were classified as 1 (2.9%) CE1, 12 (35.3%) CE2, 5 (14.7%) CE3a, 1 (2.9%) CE3b, and 15 (44.2%) CE5. Twenty-nine (78.4%) patients had a single cyst and 8 (21.6%) had at least two cysts. The most frequently reported symptom was upper abdominal pain. The plasma level of IL-6 and total IgE were significantly increased in CE patients compared with healthy donors. Additionally, IgG response to rEg.P29 in CE patients was significantly higher than in healthy donors, and the dominant IgG subclass was IgG4. Further analysis of different patient groups revealed that rEg.P29-specific IgG and IgG4 were only elevated in CE patients with CE2 type cysts. CONCLUSIONS: This study systematically investigated the clinical characteristics of patients with CE and may provide a reference basis for the diagnosis and treatment of CE in NHAR. Furthermore, tests of specific IgG and IgG4 against rEg.P29 can be used as an assisted method for imaging techniques to identify cystic activity and determine the best therapeutic approach for CE.

Baseline T-lymphocyte and cytokine indices in sheep peripheral blood.

BACKGROUND: Sheep are an important livestock species worldwide and an essential large-animal model for animal husbandry and veterinary research. Understanding fundamental immune indicators, especially T-lymphocyte parameters, is necessary for research on sheep diseases and vaccines, to better understand the immune response to bacteria and viruses for reducing the use of antibiotics and improving the welfare of sheep. We randomly selected 36 sheep of similar ages to analyze cell-related immune indicators in peripheral blood mononuclear cells (PBMCs). The proportions of CD4+ and CD8+ T cells in PBMCs were detected by flow cytometry. We used Concanavalin A (Con A) and Phorbol-12-myristate-13-acetate (PMA)/Ionomycin to stimulate PBMCs, and measured the expression of IFN-γ, IL-4, and IL-17A using enzyme-linked immunosorbent assay (ELISA) and enzyme-linked immunospot assay (ELISpot). Simultaneously, PMA/Ionomycin/brefeldin A (BFA) was added to PBMCs, then the expression of IFN-γ, IL-4, and IL-17A was detected by flow cytometry after 4 h of culturing. In addition, we observed the proliferation of PBMCs stimulated with Con A for 3, 4, and 5 days. RESULTS: The proportions of CD4+ T lymphocytes (18.70 ± 4.21%) and CD8+ T lymphocytes (8.70 ± 3.65%) were generally consistent among individuals, with a CD4/CD8 ratio of 2.40 ± 0.79. PBMCs produced high levels of IFN-γ, IL-4, and IL-17A after stimulation with PMA/Ionomycin and Con A. Furthermore, PMA/Ionomycin stimulation of PBMC yielded significantly higher cytokine levels than Con A stimulation. Flow cytometry showed that the level of IFN-γ (51.49 ± 11.54%) in CD8+ T lymphocytes was significantly (p < 0.001) higher than that in CD4+ T lymphocytes (14.29 ± 3.26%); IL-4 (16.13 ± 6.81%) in CD4+ T lymphocytes was significantly (p < 0.001) higher than that in CD8+ T lymphocytes (1.84 ± 1.33%), There was no difference in IL-17A between CD4+ (2.83 ± 0.98%) and CD8+ T lymphocytes (1.34 ± 0.67%). The proliferation of total lymphocytes, CD4+ T lymphocytes, and CD8+ T lymphocytes continued to increase between days 3 and 5; however, there were no significant differences in proliferation between the cell types during the stimulation period. CONCLUSIONS: Evaluating primary sheep immune indicators, especially T lymphocytes, is significant for studying cellular immunity. This study provided valuable data and theoretical support for assessing the immune response of sheep to pathogens and improving sheep welfare.

Identification of a dominant murine T-cell epitope in recombinant protein P29 from Echinococcus granulosus

Echinococcus granulosus causes echinococcosis, an important zoonotic disease worldwide and a major public health issue. Vaccination is an economical and practical approach for controlling E. granulosus. We have previously revealed that a recombinant protein P29 (rEg.P29) is a good vaccine candidate against E. granulosus. However, T cell immunogenic epitopes have not been identified. In the present study, we use rEg.P29-immunized mice as models to screen immunogenic epitopes for the construction of a novel multi-epitope vaccine. We search for immunodominant epitopes from an overlapping peptide library to screen the peptides of rEg.P29. Our results confirm that rEg.P29 immunization in mice elicits the activation of T cells and induces cellular immune responses. Further analyses show that a T cell epitope within amino acids 86­100 of rEg.P29 elicits significant antigen-specific IFN-γ production in CD4+ and CD8+ T cells and promotes specific T-cell activation and proliferation. Collectively, these results provide a reference for the construction of a novel vaccine against broad E. granulosus genotypes based on epitopes of rEg.P29.

Coding and Noncoding RNA Expression Profiles of Spleen CD4+ T Lymphocytes in Mice with Echinococcosis.

Cystic echinococcosis (CE) is a severe and neglected zoonotic disease that poses health and socioeconomic hazards. So far, the prevention and treatment of CE are far from meeting people's ideal expectations. Therefore, to gain insight into the prevention and diagnosis of CE, we explored the changes in RNA molecules and the biological processes and pathways involved in these RNA molecules as E. granulosus infects the host. Interferon (IFN)-γ, interleukin (IL)-2, IL-4, IL-6, IL-10, IL-17A, and tumor necrosis factor (TNF)-α levels in peripheral blood serum of E. granulosus infected and uninfected female BALB/c mice were measured using the cytometric bead array mouse Th1/Th2/Th17 cytokine kit. mRNA, microRNA (miRNA), long noncoding RNA (lncRNA), and circular RNA (circRNA) profiles of spleen CD4+ T cells from the two groups of mice were analyzed using high-throughput sequencing and bioinformatics. The levels of IFN-γ, IL-2, IL-4, IL-6, IL-10, IL-17A, and TNF-α were significantly higher in the serum of the CE mice than in control mice (P < 0.01). In total, 1,758 known mRNAs, 37 miRNAs, 175 lncRNAs, and 22 circRNAs were differentially expressed between infected and uninfected mice (|fold change| ≥ 0.585, P < 0.05). These differentially expressed molecules are involved in chromosome composition, DNA/RNA metabolism, and gene expression in cell composition, biological function, and cell function. Moreover, closely related to the JAK/STAT signaling pathways, mitogen-activated protein kinase signaling pathways, P53 signaling pathways, PI3K/AKT signaling pathways, cell cycle, and metabolic pathways. E. granulosus infection significantly increased the levels of IFN-γ, IL-2, IL-4, IL-6, IL-10, IL-17A, and TNF-α in mouse peripheral blood of mice and significantly changed expression levels of various coding and noncoding RNAs. Further study of these trends and pathways may help clarify the pathogenesis of CE and provide new insights into the prevention and treatment of this disease.

Identification of B-cell dominant epitopes in the recombinant protein P29 from Echinococcus granulosus.

INTRODUCTION: Echinococcus granulosus (E. granulosus) causes a hazardous zoonotic parasitic disease. This parasite can occupy the liver and several areas of the body, causing incurable damage. Our previous studies have provided evidence that the recombinant protein P29 (rEg.P29) exhibit immune protection in sheep and mice against pathological damage induced by E. granulosus, showing its potential as candidate for vaccine development. However, information on the B-cell epitopes of rEg.P29 has not yet been reported. METHODS: Immunological model was established in mice with rEg.P29. SDS-PAGE and Western blot were used to identify protein. Screening for B-cell dominant epitope peptides of rEg.P29 by enzyme-linked immunosorbent assay (ELISA) and immune serum. Dominant epitopes were validated using ELISA and flow cytometry. Multiple sequence alignment analysis was performed using BLAST and UniProt. RESULTS: Immunization with rEg.P29 induced intense and persistent antibody responses, and the epitope of the dominant antigen of B cells are identified as rEg.P29166-185 (LKNAKTAEQKAKWEAEVRKD). Anti-rEg.P29166-185 -specific antibodies lack epitopes against IgA, IgE, and IgG3, compared to anti-rEg.P29-specific antibodies. However, anti-rEg.P29166-185 IgG showed comparatively higher titers, as determined among those peptides by endpoint titration. In addition, rEg.P29 and rEg.P29166-185 promote B-cell activation and proliferation in vitro. The dominant epitopes are relatively conserved in different subtypes of the rEg.P29 sequence. CONCLUSION: rEg.P29166-185 can act as a dominant B-cell epitope for rEg.P29 and promote cell activation and proliferation in the same way as rEg.P29.

Screening, construction, and serological identification of the diagnostic antigen molecule EG-06283 for the diagnosis of Echinococcus granulosus.

Several strategies exist to prevent and control echinococcosis, a global parasitic disease. However, most treatments are ineffective and adverse effects are common. Therefore, we aimed to screen protoscolex antigen molecules of Echinococcus granulosus to identify a diagnostic biomarker for hydatid disease. Published E. granulosus transcriptome sequencing data were analyzed to screen for antigen molecules that are highly expressed in protoscoleces but not in oncospheres. The membrane protein EG-06283 (annotated as Frizzled-4) was selected from 16 antigens, and its gene fragment was subjected to codon optimization and synthesis. rEG-06283 expression was induced in the pET-24a/EG-06283/BL21 strain; subsequently, the protein was purified and subcutaneously injected into ICR mice at weeks 0, 2, 4, and 6. Blood sampling occurred periodically to quantify serum immunoglobulin G (IgG) levels via enzyme-linked immunosorbent assays (ELISA). Immunogenicity was determined by western blot assays using sera from normal mice and mice with secondary hydatid infections. The antigen's immune reactivity and diagnostic value were validated using sera of patients with hydatid disease. ELISA results confirmed that the antigen molecule induced specific IgG production in mice, resulting in significantly higher levels than those in the adjuvant and control groups (P < 0.05). The western blot results indicated that the protein was recognized by antibodies in the sera of mice with hydatid infection and the antisera of immunized mice. Quantification of protein levels in the sera of patients with hydatid disease significantly differed from levels in healthy participants (P < 0.05). These results indicate that rEG-06283 is a potential diagnostic antigen for E. granulosus infections.

Prognostic value of γ-glutamyl transpeptidase to albumin ratio combined with aspartate aminotransferase to lymphocyte ratio in patients with hepatocellular carcinoma after hepatectomy.

Hepatocellular carcinoma (HCC) is a malignant tumor associated with a high recurrence rate after hepatectomy. Recently, preoperative inflammatory and liver function reserve indices were found to predict increased risk of recurrence and decreased survival in HCC patients. This study aims to evaluate the ability of the γ-glutamyl transpeptidase-to-albumin ratio (GAR) and aspartate aminotransferase-to-lymphocyte ratio (ALRI), individually and in combination, to predict the prognosis of HCC patients after hepatectomy.We retrospectively reviewed 206 HCC patients who underwent radical resection at the General Hospital of Ningxia Medical University from January 2011 to November 2016. Receiver operating characteristic (ROC) curve analysis was performed to determine the optimal cut-off value for GAR and ALRI. The Pearson Chi-Squared test was used to analyze the correlations between GAR, ALRI and clinicopathological characteristics. Univariate and multivariate analyses were used to determine the predictive value of these factors for disease-free survival (DFS) and overall survival (OS). Survival rates were drawn according to the Kaplan-Meier method and differences between subgroups were compared by the log-rank statistics.GAR and ALRI were significantly correlated with gender, history of smoking, prothrombin time, tumor diameter, T stage and early intrahepatic recurrence by the Pearson Chi-Squared test (all P < .05). Univariate analysis indicated that T stage, GAR and ALRI were significantly correlated with DFS and OS in HCC patients after hepatectomy. Multivariate analysis illustrated that GAR and ALRI were independently related to DFS and OS in HCC patients. Preoperative GAR > 0.946 or ALRI > 18.734 predicted poor prognosis in HCC patients after hepatectomy. Additionally, the predictive scope of GAR combined with ALRI was more sensitive than that of either individual measurement alone.Our data indicate that there is a close association between the clinicopathological characteristics in HCC patients and increased GAR or ALRI. Higher levels of GAR and ALRI could sensitively and specifically predict a poor prognosis in HCC patients after hepatectomy. Furthermore, combined usage of GAR and ALRI could improve the accuracy of this prediction.