Background As a key glycolytic enzyme, Pyruvate kinase M2 (PKM2), which is highly expressed in cancer cells, promotes hepatocellular carcinoma (HCC) proliferation/metastasis. This research investigates the involvement of Ubiquitin protein ligase E3 component N-recognin 7 (UBR7) in HCC progression/glycolysis and its potential mechanisms. Methods UBR7 expressions in HHL-5, Huh-7, and HepG2 cells were investigated using Quantitative Reverse Transcription Polymerase Chain Reaction and Western Blot. Cell counting kit-8, clone formation experiment, scratch-wound assay, and transwell testing were conducted to assess the malignant biological behaviors of HepG2 and Huh-7 cells; the absorption level of glucose and generation levels of lactic acid and ATP were tested by assay kits. Huh-7 cells with stably overexpressed or knocked down UBR7 were inoculated into nude mice. Regular measurements were conducted on the tumor size, the tumors were isolated and weighed on the 35th day, and the glycolytic level in the tumor tissues was determined. Results In HCC cells, UBR7 was significantly downregulated. Notably, UBR7 knockdown promoted HCC progression and glycolysis, increasing the viability of HepG2 and Huh-7 cells by 23%u201324% (p u0026lt; 0.001), whereas UBR7 overexpression exerted the opposite inhibitory effects, resulting in a reduction of about 19%u201331% in cell viability (p u0026lt; 0.001). UBR7 knockdown upregulated PKM2 expression in HCC cells, while UBR7 overexpression led to a marked reduction in PKM2 levels. Importantly, PKM2 overexpression partly abrogated the inhibitory impacts of UBR7 on HCC progression and glycolysis. In vivo experiments further demonstrated that UBR7 overexpression suppressed tumor growth and hindered glycolysis in nude mice. Conclusion UBR7 suppressed PKM2 expression, thus hindering malignant biological progression and glycolysis in HCC, providing a potential therapeutic target for HCC treatment.

Background: As a key glycolytic enzyme, Pyruvate kinase M2 (PKM2), which is highly expressed in cancer cells, promotes hepatocellular carcinoma (HCC) proliferation/metastasis. This research investigates the involvement of Ubiquitin protein ligase E3 component N-recognin 7 (UBR7) in HCC progression/glycolysis and its potential mechanisms. Methods: UBR7 expressions in HHL-5, Huh-7, and HepG2 cells were investigated using Quantitative Reverse Transcription Polymerase Chain Reaction and Western Blot. Cell counting kit-8, clone formation experiment, scratch-wound assay, and transwell testing were conducted to assess the malignant biological behaviors of HepG2 and Huh-7 cells; the absorption level of glucose and generation levels of lactic acid and ATP were tested by assay kits. Huh-7 cells with stably overexpressed or knocked down UBR7 were inoculated into nude mice. Regular measurements were conducted on the tumor size, the tumors were isolated and weighed on the 35th day, and the glycolytic level in the tumor tissues was determined. Results: In HCC cells, UBR7 was significantly downregulated. Notably, UBR7 knockdown promoted HCC progression and glycolysis, increasing the viability of HepG2 and Huh-7 cells by 23%-24% (p < 0.001), whereas UBR7 overexpression exerted the opposite inhibitory effects, resulting in a reduction of about 19%-31% in cell viability (p < 0.001). UBR7 knockdown upregulated PKM2 expression in HCC cells, while UBR7 overexpression led to a marked reduction in PKM2 levels. Importantly, PKM2 overexpression partly abrogated the inhibitory impacts of UBR7 on HCC progression and glycolysis. In vivo experiments further demonstrated that UBR7 overexpression suppressed tumor growth and hindered glycolysis in nude mice. Conclusion: UBR7 suppressed PKM2 expression, thus hindering malignant biological progression and glycolysis in HCC, providing a potential therapeutic target for HCC treatment.

Inflammation significantly affects the progression of gastric cancer (GC). The Systemic Inflammation Response Index (SIRI) is an emerging prognostic marker for various malignant tumors, including GC. However, the mechanisms by which SIRI influences patient outcomes remain unclear. This study employed a multi-omics approach to analyze GC patients with different SIRI levels, aiming to identify factors underlying differences in prognosis. A cohort of 495 GC patients was classified into high SIRI (n = 276) and low SIRI (n = 219) groups using a threshold of 2.6. Kaplan-Meier analysis and Receiver operating characteristic (ROC) curves were used to assess survival outcomes and the predictive accuracy of SIRI compared to other inflammatory markers. Gut microbiota and untargeted metabolomics analyses were conducted on stool samples from 45 GC patients, including 21 with high SIRI and 24 with low SIRI. Elevated SIRI levels were significantly associated with worse OS (P = 0.004, HR = 1.845, 95

Abstract Introduction Conventional library-based antibody display can only explore a small fraction of the sequences generated from animal immunization, not even to exhaust the potential sequence diversity that can be turned into antibody therapies. This is because screening for antibody is limited to sequences that can be displayed, which only constitute a subset of the entire sequences generated by B cells, whereas screening for antibody directly from single B cells can be costly. Here, we introduce a novel Artificial Intelligence-enabling tool to navigate antibody discovery from a broader range of search space with reduced cost. We trained a transformer-based model from sequences of an immunized library to cluster the clones and a generative adversarial network (GAN)-based model to generate novel sequences that can be potentially developed into antibody therapies. Background and significance One limitation in the early discovery of antibody is the number of functional candidates that can be selected. Our work provides an AI-enabling tool to discover and generate a panel of antibodies of differentiated binding strengths to a broad range of epitopes to ensure functional coverage. Methods & Results We extracted 104 sequences from the FACS-enriched yeast pool from a fully immunized alpaca (Lama pacos) using Next Generation Sequencing, from which we assembled 103 unique sdAb sequences. We fine-tuned a transformer-based deep learning model, which was previously trained from our dataset containing 100,000 antibody sequences, on such pre-processed sdAb sequences giving representation that correlates to the sequence homology for the clustering of clonal types. We postulate such representation also encodes long-range amino acid interactions in the 3D structure, making the accuracy exceeds the performance of bioinformatics-based primary sequence homology analysis. This process is fully automated and optimized to require minima computational resources. We selected 15 candidates from AI-clustered clonal groups and experimentally measured their binding activity. Kd of 12 candidates were of 10−9 affinity and 1 candidates were of 10−8 affinity, the rest one candidate was non-binding (hence a hit rate of 87%). The large sequence diversity of the CDR3 show these nanobodies are potentially good binders for a wide range of epitopes. We generated a CDR-diversifying virtual library (103) of each binding candidate by training a GAN-based models using the sequences of the same clonal group of the binder sequences. This method incorporates the probability of amino acid residues on each specific location that provides a more precise mutagenesis route than PCR-based affinity maturation. The generated sequences provided a wider CDR sequence diversity for the selection of antibodies of differentiated affinity and epitopes, which could generate candidates of different functionality. Conclusion Antibody discovery is a central step in early drug development that identification of a wide range of functional candidates could increase the success rate and reduce risks in later developments. We built an AI-enabling tool for the searching and generation of functional antibodies from animal immunization library. We believe this technology would help deliver candidates of fine-tuned affinity and functionality.

Antibody therapies have become an important class of therapeutics in recent years as they have exhibited outstanding efficacy and safety in the treatment of several major diseases including cancers, immune-related diseases, infectious disease and hematological disease. There has been significant progress in the global research and development landscape of antibody therapies in the past decade. In this review, we have collected available data from the Umabs Antibody Therapies Database (Umabs-DB, https://umabs.com) as of 30 June 2022. The Umabs-DB shows that 162 antibody therapies have been approved by at least one regulatory agency in the world, including 122 approvals in the US, followed by 114 in Europe, 82 in Japan and 73 in China, whereas biosimilar, diagnostic and veterinary antibodies are not included in our statistics. Although the US and Europe have been at the leading position for decades, rapid advancement has been witnessed in Japan and China in the past decade. The approved antibody therapies include 115 canonical antibodies, 14 antibody-drug conjugates, 7 bispecific antibodies, 8 antibody fragments, 3 radiolabeled antibodies, 1 antibody-conjugate immunotoxin, 2 immunoconjugates and 12 Fc-Fusion proteins. They have been developed against 91 drug targets, of which PD-1 is the most popular, with 14 approved antibody-based blockades for cancer treatment in the world. This review outlined the global landscape of the approved antibody therapies with respect to the regulation agencies, therapeutic targets and indications, aiming to provide an insight into the trends of the global development of antibody therapies.

Tumor-infiltrating immune cells are a pivotal component of the tumor microenvironment (TME), but their indicative role remains poorly defined. A meta-analysis was performed to reveal the prognostic efficiency of tumor-infiltrating immune cells in gastric cancer (GC). By searching PubMed and Embase, we identified a total of 35 eligible articles that involved 4888 patients. Random or fixed effect models were employed to extract pooled hazard ratios (HRs) with 95% confidence intervals (CIs). Our results indicated that high CD3+ lymphocyte infiltration in all the locations (AG), the tumor nest (TN), and the tumor stroma (TS) predicted better overall survival (OS) (HR=0.71, 95% CI=0.57-0.90; HR=0.58, 95% CI=0.42-0.80; and HR=0.50, 95% CI=0.37-0.68, respectively). CD8+ T cell infiltration in AG and FoxP3+ regulatory T cells (Tregs) in the tumor invasive margin (TM) were also associated with improved OS (HR=0.90, 95% CI=0.83-0.97; HR=0.65, 95% CI=0.48-0.87, respectively). However, contrasting results were found in the macrophage subset, with M2 in AG (HR=1.45, 95% CI=1.13-1.86) and the TN (HR=1.67, 95% CI=1.12-2.48) associated with worse OS. In summary, the combination of the densities and locations of tumor-infiltrating immune cells can be useful for predicting survival for GC patients, but additional research is needed to reinforce the reliability of this study's conclusions.

The capsular polysaccharide (CP) produced by Staphylococcus aureus is a virulence factor that allows the organism to evade uptake and killing by host neutrophils. Polyclonal antibodies to the serotype 5 (CP5) and type 8 (CP8) capsular polysaccharides are opsonic and protect mice against experimental bacteremia provoked by encapsulated staphylococci. Thus, passive immunotherapy using CP antibodies has been considered for the prevention or treatment of invasive antibiotic-resistant S. aureus infections. In this report, we generated monoclonal antibodies (mAbs) against S. aureus CP5 or CP8. Backbone specific mAbs reacted with native and O-deacetylated CPs, whereas O-acetyl specific mAbs reacted only with native CPs. Reference strains of S. aureus and a selection of clinical isolates reacted by colony immunoblot with the CP5 and CP8 mAbs in a serotype-specific manner. The mAbs mediated in vitro CP type-specific opsonophagocytic killing of S. aureus strains, and mice passively immunized with CP5 mAbs were protected against S. aureus bacteremia. Neither CP8-specific mAbs or polyclonal antibodies protected mice against bacteremia provoked by serotype 8 S. aureus clinical isolates, although these same antibodies did protect against a serotype 5 S. aureus strain genetically engineered to produce CP8. We detected soluble CP8 in culture supernatants of serotype 8 clinical isolates and in the plasma of infected animals. Serotype 5 S. aureus released significantly less soluble CP5 in vitro and in vivo. The release of soluble CP8 by S. aureus may contribute to the inability of CP8 vaccines or antibodies to protect against serotype 8 staphylococcal infections.

Objective. This meta-analysis is aimed at assessing the safety and efficiency of colonic self-expanding metallic stents (SEMS) used as a bridge to surgery in the management of left-sided malignant colonic obstruction (LMCO). Methods. A systematic search was conducted in PubMed, Web of Knowledge, OVID, Google Scholar, CNKI, and WANGFANG for relevant randomized trials comparing colonic stenting used as a bridge in semielective surgery versus emergency surgery from January 2001 to September 2013. Result. Five published studies were included in this systematic review, including 273 patients (140 male/133 female). 136 patients received semielective surgery after SEMS installation while 137 patients underwent emergency surgery without SEMS. SEMS intervention resulted in significantly lower overall colostomy rate (41.9% versus 56.2%, P = 0.02), surgical site infection rate (10.2% versus 19.7%, P = 0.03), and overall complication rate (29.2% versus 60.5%, P = 0.05). There was no statistic difference for the rate of primary anastomosis, anastomotic leak and operation-related mortality between two groups. Conclusions. semielective surgery with SEMS as a bridge for proper patients of LMCO can lower the overall rate for colostomy, surgical site infection, and complications.

Trametes versicolor is a mushroom used as a traditional Chinese medicine (Yun-zhi) for a wide array of seemingly disparate conditions. We hypothesized that many of its multiple purported activities could be mediated through stimulation of beneficial mutualist components of the microbiota. Human fecal microbiota was cultured anaerobically to determine its ability to ferment a common extract of T. versicolor, designated polysaccharide peptide (PSP), and the ability of PSP to alter the composition of the microbial community. The presence of PSP and fructooligosaccharides (FOS, a common prebiotic) in the medium, but not cellulose, significantly increased levels of Bifidobacterium spp. PSP also elevated Lactobacillus spp., while reducing Clostridium spp., Staphylococcus spp. and Enterococcus spp. Levels of Streptococcus spp., Bacteroides spp. and Escherichia did not significantly change. Fermentation of PSP increased the concentration of organic acids (lactate and short-chain fatty acids), decreased the pH, and induced β-galactosidase and β-glucosidase activities. The genera of the human microbiota that are promoted by FOS and other prebiotics are also stimulated by the Trametes versicolor extract, PSP. Thus, Trametes versicolor, a common East Asian botanical, contains putative prebiotic agents that alter human gut microbiota and pH. This prebiotic-like activity may help explain some of the plethora of the health benefits attributed to this traditional Chinese medicine.

Breastfed infants have a lower risk of influenza than those weaned early, suggesting that human milk could contain components that inhibit infection by influenza virus. This study was to test whether human milk per se inhibited H1N1 pseudovirus infection in vitro, and to identify the milk components responsible for any inhibition. The inhibition model was Pseudovirus (A/California/04/2009, H1N1) infection of cultured Madin‐Darby Canine Kidney (MDCK) cells. The luciferase plasmid of this pseudovirus allowed infection to be quantified via a luciferase assay. Inhibition was measured for pooled human milk and its fractions, including lactoferrin, mucin 1, mucin 4, human milk oligosaccharides (HMOS), and bile salt stimulated lipase (BSSL). At 80 μg/mL of Lactoferrin, 100 μg/mL of mucin 1, 150 μg/mL of mucin 4 and 50 μg/mL HMOS, these components inhibited H1N1 influenza virus infection in vitro. Thus, multiple milk components at their native concentrations have the ability to protect against H1N1 influenza virus, and perhaps other types of influenza viruses.Grant Funding Source: NIH HD013021, AI075563, Abbott Nutrition

Human milk oligosaccharides are poorly digested and absorbed in the small intestine, allowing their passage into the colon, where they influence colonization by the microbiota. The ability of individual major fucosylated and sialylated human milk oligosaccharides to promote growth and physiologic activity of 25 human intestinal microbes was investigated. 2′‐Fucosyllactose, 3‐ Fucosyllactose, and Lactodifucotetraose stimulated most Bifidobacteria spp. and Bacteroides spp. to grow and produce abundant lactate or short chain fatty acids. 3′‐sialyllactose and 6′‐ sialyllactose promoted moderate growth of B. longum JCM7007, 7009, 7010, 7011, 1272, 11347, ATCC15708, B. thetaiotaomicron ATCC29148, and C. leptum ATCC29065; these bacteria produced lactate or short chain fatty acids or both. In contrast, L. rhamnosus ATCC53103, E. facalis ATCC29200, S. epidermidis ATCC12228 and E. coli K12 did not consume the milk oligosaccharides. In summary, specific Bifidobacteria and Bacteroides differentially digest specific individual human milk oligosaccharides, especially the major fucosylated milk oligosaccharides, and these may prove useful for treating dysbiosis of the microbiota and associated inflammatory disorders. Grant Funding Source : HD013021 , AI075563 , and Abbott Nutrition

Breastfeeding protects the neonate against pathogen infection. Major mechanisms of protection include human milk glycoconjugates functioning as soluble receptor mimetics that inhibit pathogen binding to the mucosal cell surface, prebiotic stimulation of gut colonization by favorable microbiota, immunomodulation, and as a substrate for bacterial fermentation products in the gut. Human milk proteins are predominantly glycosylated, and some biological functions of these human milk glycoproteins (HMGPs) have been reported. HMGPs range in size from 14 kDa to 2,000 kDa and include mucins, secretory immunoglobulin A, bile salt-stimulated lipase, lactoferrin, butyrophilin, lactadherin, leptin, and adiponectin. This review summarizes known biological roles of HMGPs that may contribute to the ability of human milk to protect neonates from disease.

Many human milk glycans inhibit pathogen binding to host receptors and their consumption by infants is associated with reduced risk of disease. Salmonella infection is more frequent among infants than among the general population, but the incidence is lower in breast-fed babies, suggesting that human milk could contain components that inhibit Salmonella. This study aimed to test whether human milk per se inhibits Salmonella invasion of human intestinal epithelial cells in vitro and, if so, to identify the milk components responsible for inhibition. Salmonella enterica serovar Typhimurium SL1344 (SL1344) invasion of FHs 74 Int and Caco-2 cells were the models of human intestinal epithelium infection. Internalization of fluorescein-5-isothiocyanate–labeled SL1344 into intestinal cells was measured by flow cytometry to quantify infection. Human milk and its fractions inhibited infection; the inhibitory activity localized to the high molecular weight glycans. Mucin 1 and mucin 4 were isolated to homogeneity. At 150 μg/L, a typical concentration in milk, human milk mucin 1 and mucin 4 inhibited SL1344 invasion of both target cell types. These mucins inhibited SL1344 invasion of epithelial cells in a dose-dependent manner. Thus, mucins may prove useful as a basis for developing novel oral prophylactic and therapeutic agents that inhibit infant diseases caused by Salmonella and related pathogens.

Breast-fed infant microbiota is typically rich in bifidobacteria. Herein, major human milk oligosaccharides (HMOS) are assessed for their ability to promote the growth of bifidobacteria and to acidify their environment, key features of prebiotics. During in vitro anaerobic fermentation of infant microbiota, supplementation by HMOS significantly decreased the pH even greater than supplementation by fructooligosaccharide (FOS), a prebiotic positive control. HMOS elevated lactate concentrations, increased the proportion of Bifidobacterium spp. in culture, and through their fermentation into organic acids, decreased the proportion of Escherichia and Clostridium perfringens. Three principal components of HMOS, 2'-fucosyllactose, lactodifucotetraose and 3-fucosyllactose, were consumed in these cultures. These three principal oligosaccharides of human milk were then individually tested as supplements for in vitro growth of four individual representative strains of infant gut microbes. Bifidobacterium longum JCM7007 and B. longum ATCC15697 efficiently consumed oligosaccharides and produced abundant lactate and short-chain fatty acids, resulting in significant pH reduction. The specificity of fermentation differed by microbe species and strain and by oligosaccharide structure. Escherichia coli K12 and C. perfringens did not utilize appreciable fucosylated oligosaccharides, and a typical mixture of organic acid fermentation products inhibited their growth. In summary, 2'-fucosyllactose, lactodifucotetraose, and 3-fucosyllactose, when cultured with B. longum JCM7007 and B. longum ATCC15697, exhibit key characteristics of a prebiotic in vitro. If these bifidobacteria are representative of pioneering or keystone species for human microbiota, fucosylated HMOS could strongly promote colonization and maintenance of a mutualist symbiotic microbiome. Thus, these simple glycans could mediate beneficial effects of human milk on infant health.

Salmonella infection is more frequent among infants than the general population, but the incidence is lower in breastfed babies. This study was to test whether human milk per se inhibited salmonella invasion of human intestinal epithelial cells in vitro, and to identify the milk components likely to be responsible for any inhibition. Salmonella typhimurium SL1344 invasion of FHs 74 Int and CaCo‐2 cells were the models of human intestinal epithelium infection. Internalization of FITC‐labeled salmonella into intestinal cells was measured by FACS analysis to quantify infection. Pooled human milk and its fractions were tested for their ability to inhibit infection, including two human milk mucins, mucin 1 and mucin 4. At 150 ng/mL, approximating the amount in milk, human milk mucin 1 and mucin 4 inhibited salmonella invasion of both cell types. They also bound Salmonella typhimurium SL1344 in a dose‐dependent manner. Thus, mucins may prove useful as a basis for the development of novel oral prophylactic and therapeutic agents that inhibit infant diseases caused by salmonella, and perhaps other pathogens. Supported by NIH HD013021 and AI075563.

Despite advances in the development of human papillomavirus (HPV) prophylactic vaccines, little progress has been made in the field of therapeutic vaccines in recent years. In the present study, we found a significant accumulation of immature myeloid cells (ImC) in large TC‐1 tumors and demonstrated that a HPV therapeutic vaccine restored antitumor immune responses with the correction of aberrant myeloid cell differentiation by all‐trans retinoic acid (ATRA). Our study demonstrated that combining ATRA with vaccination not only decreased the number of Gr‐1 + CD11b + ImC, but for the first time also suppressed the function of Gr‐1 + CD11b + ImC with decreased expression of CD80. Furthermore, large numbers of CD11c + CD80 + , CD11c + CD86 + , and CD11c + MHCII + mature dendritic cells were recruited. The combination therapy generated significantly increased numbers of functional E7‐specific T cells with elevated interferon‐γ secretion and enhanced cytotoxic T‐cell activity. These findings suggest potential clinical benefits for the combined use of ATRA and HPV therapeutic vaccines. ( Cancer Sci 2009; 100: 334–340)

Human papillomaviruses (HPV), particularly HPV16, is considered a necessary cause of cervical and oral cancer. Thus, the development of a therapeutic vaccine against HPV is important for the control of cervical cancer. However, therapeutic vaccination has been limited by inadequate antigen-specific immune responses. Heat shock proteins (HSP), including calreticulin (CRT), HSP70 and gp96, have been shown to act as potent immunoadjuvant to enhance antigen-specific tumor immunity. Previous studies have shown that N domain CRT (NCRT) or C-terminal half of HSP70 (hsp) linked with HPV16 E7 are capable of inducing potent antigen-specific CTL activity in experimental animal models. Here we developed a recombinant NCRT/E7/hsp fusion protein to investigate the synergistic effects of NCRT and hsp for enhancing the potency of HPV16 E7 therapeutic vaccine and evaluated the immune responses induced by this fusion protein. Our results demonstrated that NCRT and hsp synergistically exhibited significant increases in E7-specific CD8(+) T cell responses and impressive antitumor effects against E7-expressing tumors. Furthermore, the NCRT/E7/hsp fusion protein also generates potent antiangiogenic effects. These results indicate that NCRT/E7/hsp fusion protein is a promising therapeutic vaccine for treatment of cervical cancer through a combination of antigen-specific immunotherapy and antiangiogenesis, with possible therapeutic potential in clinical settings.

Two dicaffeoylquinic acids, namely 3,5-dicaffeoylquinic acid and 4,5-dicaffeoylquinic acid, have been successfully separated by high-speed counter-current chromatography (HSCCC) from an extract of Ainsliaea fragrans Champ, followed by an initial clean-up step using AB-8 resin. A two-phase solvent system composed of chloroform:methanol:water (8:8:4) was selected for the isolation with the aqueous-rich phase as the stationary phase and the organic-rich phase as the mobile phase. The developed HSCCC method yielded 34 mg of 3,5-dicaffeoylquinic acid and 17 mg of 4,5-dicaffeoylquinic acid from 150 mg of the crude sample in a one-step separation with purities of 98 and 95%, respectively, as determined by HPLC. The structures of the two compounds were identified from ESI/MS, (1)H- and (13)C-NMR spectroscopic data.

Increasing evidence has suggested that infection with high-risk human papillomavirus (HPVs) is closely associated with esophageal squamous cell carcinoma (ESCC) in China. The E6 and E7 oncoproteins expressed in ESCC are considered as attractive tumor-specific antigen targets for immunotherapy. We have reported that the HPV16 mE6delta/mE7/TBhsp70delta fusion protein vaccination induced powerful anti-tumor immunity against TC-1 tumor cells in a C57BL/6 mouse model. In the present study, we further evaluate the protective efficacy of this fusion protein vaccine using an HPV E7-expressing human ESCC cell line (EC9706) and a Hu-PBL-SCID mouse model. We demonstrated that immunization with the fusion protein vaccine caused significant inhibition of tumor growth with the delay time to tumor detection (tests vs. controls, 16 d vs. 9 d, p<0.01) and much smaller tumor size (p<0.01) in vivo. The inhibitory rate was ca. 69.6%, and 25% of the fusion protein vaccinated-mice remained tumor free by the end of the experiment (42 d). Furthermore, the activated lymphocytes (CD8+) were capable of infiltrating into the tumor site, and much more apoptotic cells along with activation of caspase-3 were observed in the tumors from vaccinated-mice. Also, high expression levels of human IFN-gamma, TNF-alpha, granzyme B and perforin were detected in the tumors from vaccinated-mice. Therefore, we concluded that the HPV16 mE6delta/mE7/TBhsp70delta fusion protein vaccine is able to stimulate cellular-mediated immune response against E7-containing ESCC cells through CD8+-dependent CTL-induced apoptosis in Hu-PBL-SCID mice. These findings provide a scientific basis for HPV E7-expressing ESCC active immunotherapy.