Prediction of Prognostic Features Based on Neutrophil-Related Genes for Lung Squamous Cell Carcinoma Reveals Immune Landscape and Drug Candidates.

UNASSIGNED: Background: Since to the prognosis of lung squamous cell carcinoma is generally poor, there is an urgent need to innovate new prognostic biomarkers and therapeutic targets to improve patient outcomes. Objectives: Our goal was to develop a novel multi-gene prognostic model linked to neutrophils for predicting lung squamous cell carcinoma prognosis. Methods: We utilized messenger RNA expression profiles and relevant clinical data of lung squamous cell carcinoma patients from the Cancer Genome Atlas database. Through K-means clustering, least absolute shrinkage and selection operator regression, and univariate/multivariate Cox regression analyses, we identified 12 neutrophil-related genes strongly related to patient survival and constructed a prognostic model. We verified the stability of the model in the Cancer Genome Atlas database and gene expression omnibus validation set, demonstrating the robust predictive performance of the model. Results: Immunoinfiltration analysis revealed remarkably elevated levels of infiltration for natural killer cells resting and monocytes in the high-risk group compared to the low-risk group, while macrophages had considerably lower infiltration in the high risk group. Most immune checkpoint genes, including programmed cell death protein 1 and cytotoxic T-lymphocyte-associated antigen 4, exhibited high expression levels in the high risk group. Tumor immune dysfunction and exclusion scores and immunophenoscore results suggested a potential inclination toward immunotherapy in the "RIC" version V2 revised high risk group. Moreover, prediction results from the CellMiner database revealed great correlations between drug sensitivity (e.g., Vinorelbine and PKI-587) and prognostic genes. Conclusion: Overall, our study established a reliable prognostic risk model that possessed significant value in predicting the overall survival of lung squamous cell carcinoma patients and may guide personalized treatment strategies. (Rev Invest Clin. 2024;76(2):116-31).

Ag-carbon dots with peroxidase-like activity for colorimetric and SERS dual mode detection of glucose and glutathione.

Currently, nanozymes have made important research progress in the fields of catalysis, biosensing and tumor therapy, but most of nanozymes sensing systems are single-mode detection, which are easily affected by environment and operation, so it is crucial to construct nanozymes sensing system with dual-signal detection to obtain a more stable and reliable performance. In this paper, Ag-carbon dots (Ag-CDs) bifunctional nanomaterials were synthesized using carbon dots as reducing agent and protective agent by a facile and green one-step method. A simple and sensitive colorimetric-SERS dual-mode sensing platform was constructed for the detection of glucose and glutathione(GSH) in body fluids by taking advantage of good peroxidase-like and SERS activities of Ag-CDs. Ag-CDs catalyzes H2O2 to hydroxyl radicals(•OH), which oxidized TMB to form ox-TMB blue solution with characteristic absorption peak at 652 nm and Raman characteristic peak at 1607 cm-1. Ag-CDs sensing method exhibited high performance for glucose and GSH with detection limits for colorimetric and SERS as low as 11.30 µM and 3.54 µM, 0.38 µM and 0.24 µM respectively (S/N = 3). In addition, Ag-CDs have good stability and uniformity, ensuring long-term applicability of catalytic system. This colorimetric-SERS dual-mode sensing platform can be used for the determination of glucose and GSH in saliva and urine, and has the advantages of simple, low cost, rapid, and high accuracy, which has a potential application prospect in biosensor and medical research.

Genome-wide identification of Oryza sativa: A new insight for advanced analysis of ABC transporter genes associated with the degradation of four pesticides.

ATP-binding cassette (ABC) transporter is a large genes superfamily. It involves transportation of diverse substrates (e.g., heavy metal, amino acids, pesticides, metabolites). The ABC transporters can be strongly induced by environmental stress and responsible for the phase III metabolic process of toxic compounds in plants. To investigate the potential molecular and biochemical function of ABC transporters in response to pesticides, we used bioinformatics and high-throughput sequencing to identify 107 loci from rice (Oryza sativa) exposed to different pesticides (ametryn, AME; bentazone, BNTZ; fomesafen, FSA; mesotrione, MTR) and annotated as ABC transporter genes. ABC transporter genes were categorized to eight subfamilies including ABCA-G and ABCI. ABCG subfamily was the largest group in rice genome followed by ABCC subfamily and ABCB subfamily. The distribution of each ABC transporter on twelve chromosomes was identified. The result showed that a large number of genes were scattered around chromosome. Differentially expressed genes (DEGs) were selected for cis-acting analysis under pesticide stress. Multiple cis-elements for biological functions such as hormone-sensitive elements and defense-related elements were found to involve the initiation and regulation of transcription. Comprehensive phylogenetic analysis and domain prediction of all ABC DEGs from rice and Arabidopsis were carried out. The docking analysis of ABC transporters and pesticides provided insights into the key amino acid residues involved in the binding of the pesticides. Consequently, the results provided applicable information and reference for a more functional analysis of ABC transporter genes on regulation of pesticide metabolism and transport in plants.

Different effects of bronchoscopic interventions on children and adults with tracheobronchial mucoepidermoid carcinoma.

AIMS: To investigate the efficacy and safety of minimally invasive bronchoscopic interventions for patients with tracheobronchial mucoepidermoid carcinoma (MEC). METHODS: Patients with tracheobronchial MEC were included in this retrospective study, and the clinical features, histologic grading, treatments, and cumulative survival rates were calculated. Patients were categorized into child (n = 16) and adult (n = 19) group according to their ages. Histologic grading, treatments, and survival status were compared between the two groups. RESULTS: In pathology, high-grade MEC counts for 6.77% and 42.10% in the child and adult group, respectively. As tumor growth pattern was concerned, 93.33% and 21.05% tumors in the child and adult group present intratracheal type. Multiple bronchoscopic interventions were conducted, including rigid bronchoscopy, argon plasma coagulation (APC), dioxide carbon cryotherapy, and electric loop. Tumors could be removed by multiple bronchoscopic interventions. Bronchoscopy-associated complications were rare, including an oral mucosa injury and a glottis edema. In the child group, one patient underwent left upper lung lobectomy. In the adult group, lobectomy and/or chemotherapy and/or radiotherapy were conducted in seven patients. The 5-year survival rate was 100% and 68.90% in the child and the adult group, respectively. CONCLUSIONS: Almost all children have low-grade and intratracheal MEC; 2/5 adults have invasive high-grade MEC. Multiple bronchoscopic interventions are effective in erasing low-grade intratracheal MEC without severe complications. For high-grade invasive MEC, aggressive and comprehensive therapy should be considered.

Identification of differentially expressed genes associated with aluminum resistance in the soybean plant.

Aluminum (Al) toxicity is a major limitation to crop production in countries where acidic soil is abundant. In China, soybean production is constrained by Al stress-induced toxicity. As such, there is growing interest to develop Al-resistant varieties. In the present study, we sought to determine potential genes, functions and pathways for screening and breeding of Al-resistant varieties of soybean. First, we mined the E-GEOD-18517 dataset and identified 729 differentially expressed genes (DEGs) between untreated and Al-treated groups. Next, we performed Gene Ontology and Kyoto Encyclopedia of Genes and Genome pathways enrichment analysis and observed that most of the screened genes were mainly enriched in defense response, plasma membrane and molecular transducer activity. They were also enriched in three important pathways, the phenylpropanoid biosynthesis, plant-pathogen interaction, and cutin, suberine and wax biosynthesis. Utilizing weighted gene co-expression network analysis of 815 DEGs screened by Venn diagram, we identified DEGs that were the most disparate between treated and untreated groups. LOC100793667 (probable protein phosphatase 2C 60, GLYMA_17G223800), LOC100780576 (ethylene-responsive transcription factor 1B, GLYMA_02G006200), and LOC100785578 (protein ESKIMO 1, GLYMA_02G258000) were the most differentially expressed, which were consistent with the qRT-PCR results. As these genes are known to participate in essential functions, such as cell junction and phenylpropanoid biosynthesis, these genes may be important for breeding Al-resistant varieties. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s12298-021-01018-x.

The Predictive Efficacy of Tumor Mutation Burden (TMB) on Nonsmall Cell Lung Cancer Treated by Immune Checkpoint Inhibitors: A Systematic Review and Meta-Analysis.

BACKGROUND: Nonsmall cell lung cancer (NSCLC) is the most common type of lung cancer, and the majority of NSCLC patients are diagnosed at the advanced stage. Chemotherapy is still the main treatment at present, and the overall prognosis is poor. In recent years, immunotherapy has developed rapidly. Immune checkpoint inhibitors (ICIs) as the representative have been extensively applied for treating various types of cancers. Tumor mutation burden (TMB) as a potential biomarker is used to screen appropriate patients for treatment of ICIs. To verify the predictive efficacy of TMB, a systematic review and meta-analysis were conducted to explore the association between TMB and ICIs. METHOD: PubMed, EMBASE, Cochrane Library, and son on were systematically searched from inception to April 2020. Objective response rate (ORR), progression-free survival (PFS), and overall survival (OS) were estimated. RESULTS: A total of 11 studies consisting of 1525 nonsmall cell lung cancer (NSCLC) patients were included. Comparison of high and low TMB: pooled HRs for OS, 0.57 (95% CI 0.32 to 0.99; P = 0.046); PFS, 0.48 (95% CI 0.33 to 0.69; P < 0.001); ORR, 3.15 (95% CI 2.29 to 4.33; P < 0.001). Subgroup analysis values: pooled HRs for OS, 0.75 (95% CI 0.29 to 1.92, P = 0.548) for blood TMB (bTMB), 0.44 (95% CI 0.26 to 0.75, P = 0.003) for tissue TMB (tTMB); for PFS, 0.54 (95% CI 0.29 to 0.98, P = 0.044) and 0.43 (95% CI 0.26 to 0.71, P = 0.001), respectively. CONCLUSIONS: These findings imply that NSCLC patients with high TMB possess significant clinical benefits from ICIs compared to those with low TMB. As opposed to bTMB, tTMB was thought more appropriate for stratifying NSCLC patients for ICI treatment.

Safety profiles of terahertz scanning in ophthalmology.

Terahertz (THz) technology has emerged recently as a potential novel imaging modality in biomedical fields, including ophthalmology. However, the ocular biological responses after THz electromagnetic exposure have not been investigated. We conducted a rabbit study to evaluate the safety profiles of THz scanning on eyes, at a tissue, cellular, structural and functional level. Eight animals (16 eyes) were analysed after excessive THz exposure (control, 1 h, 4 h, and 1 week after continuous 4-h exposure; THz frequency = 0.3 THz with continuous pulse generated at 40 µW). We found that at all the time points, the corneas and lens remained clear with no corneal haze or lens opacity formation clinically and histopathologically. No thermal effect, assessed by thermographer, was observed. The rod and cone cell-mediated electroretinography responses were not significantly altered, and the corneal keratocytes activity as well as endothelial viability, assessed by in-vivo confocal microscopy, was not affected. Post-exposed corneas, lens and retinas exhibited no significant changes in the mRNA expression of heat shock protein (HSP)90AB1), DNA damage inducible transcript 3 (DDIT3), and early growth response (EGR)1. These tissues were also negative for the inflammatory (CD11b), fibrotic (fibronectin and α-smooth muscle actin), stress (HSP-47) and apoptotic (TUNEL assay) responses on the immunohistochemical analyses. The optical transmittance of corneas did not change significantly, and the inter-fibrillar distances of the corneal stroma evaluated with transmission electron microscopy were not significantly altered after THz exposure. These results provide the basis for future research work on the development of THz imaging system for its application in ophthalmology.

Anti inflammatory effect of asiaticoside on human umbilical vein endothelial cells induced by ox-LDL.

Early diagnosis and changes associated with atherosclerosis are crucial in clinical medicine. However, atherosclerosis is a multifactorial disease. Asiaticoside (AA), a triterpenoid derived from Centella asiatica, has anti-inflammatory activity. Endothelium-derived nitric oxide is important in modulating vascular tone in a distinct vessel size-dependent manner; it plays a dominant role in conduit arteries and endothelium-dependent hyperpolarisation in resistance vessels. This study evaluated the effects of AA administration on human umbilical endothelial cells with oxidised low-density lipoprotein-induced inflammation. We measured the levels of intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1). Our results indicated that 10-30 µM AA modulated endothelial hyper permeability, adenosine triphosphate levels, ICAM-1 expression, VCAM-1 expression, E-selectin levels, and PECAM-1 expression to 90% (p < 0.005), 80% (p < 0.05), 105% (p < 0.01), 65% (p < 0.005), 70% (p < 0.05), and 105% (p < 0.01), respectively. Taken together, our data suggest that AA inhibits the augmentation of endothelial permeability, thus preventing the early events of atherosclerosis.

MicroRNA-24 Modulates Staphylococcus aureus-Induced Macrophage Polarization by Suppressing CHI3L1.

Macrophages play a crucial role in host innate anti-Staphylococcus aureus defense, which is tightly regulated by multiple factors, including microRNAs. A recent study showed that miR-24 plays an important role in macrophage polarization. Here, we investigated the biological function of miR-24 in S. aureus-stimulated macrophages. The results revealed that miR-24 expression was significantly decreased in both human and mouse macrophage cell lines with S. aureus stimulation in a time-dependent manner. Moreover, miR-24 overexpression significantly decreased the production of M1 phenotype markers, such as IL-6, iNOS, TNF-α, CD86, and CD80, whereas it increased the production of M2 markers, such as Arg1, CCL17, CCL22, CD163, and CD206, in S. aureus-stimulated macrophages. Conversely, knockdown of miR-24 promoted M1 macrophage polarization but diminished M2 macrophage polarization in S. aureus-stimulated macrophages. Furthermore, CHI3L1 was predicted as a target gene of miR-24 using bioinformatics software and identified by luciferase reporter assay. Additionally, miR-24 overexpression inhibited CHI3L1 expression and downregulated the downstream MAPK pathway in S. aureus-stimulated macrophages. Finally, CHI3L1 overexpression rescued macrophage polarization and MAPK pathway inhibition induced by miR-24 mimic transfection in S. aureus-stimulated macrophages. In conclusion, the data suggest that miR-24 serves as a molecular regulator in S. aureus-induced macrophage polarization through targeting of CHI3L1 and regulation of the MAPK pathway, which may provide a promising therapeutic target for S. aureus-related infections and inflammatory diseases.

Investigation on the role of VEGF gene polymorphisms in the risk of osteosarcoma.

OBJECTIVE: The VEGF in low oxygen conditions are reported to prolong the survival of malignant cell, and thus this gene has a critical role in tumor growth and invasion as well as development of malignant tumor. We aimed to assess the association between the six common SNPs and the risk of osteosarcoma, and their association with environmental factors. METHODS: 176 subjects with osteosarcoma and 176 gender- and sex-matched healthy control individuals were enrolled into our study. The VEGF -2578C/A, -1156G/A, +1612G/A, +936C/T, -634G/C and -460T/C gene polymorphisms were determined using a polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assay according to manufacturer's instructions. RESULTS: By conditional logistic regression analysis, AA and CA+AA genotypes of VEGF -2578C/A were associated with significant increased risk of osteosarcoma compared with CC genotype, and the ORs(95%CI) were 2.32(1.18-4.60) and 1.68(1.07-2.64), respectively. Moreover, individuals with CC and TC+CC genotypes of VEGF-460T/C had significant increased risk of osteosarcoma compared with those carrying with the TT genotype, and ORs(95%CI) were 2.15(1.10-4.21) and 1.60(1.0-2.58), respectively. By stratified analysis, we did not find statistically significant associated between VEGF -2578C/A and -460T/C gene polymorphisms and cancer risk by stratification analysis. CONCLUSION: Our results suggested that VEGF -2578C/A and -460T/C gene polymorphisms may be association with an increased risk of osteosarcoma.

Dendritic cells transduced with CPEB4 induced antitumor immune response.

Much evidence leads to the exploration of immunologic approaches for eliminating tumor cells. Cytoplasmic polyadenylation element binding protein 4 (CPEB4) is considered to be a novel therapeutical target for glioblastoma. In this study, we transduced DCs with CPEB4 to explore the immune response in vivo. We found that DCs transduced with recombinant adenovirus encoding CPEB4 could induce specific cytotoxic T lymphocytes (CTLs) to lyse glioma cells and augment the number of IFN-γ secreting T-cells in mice. In addition, the modified DCs could effectively protect mice from lethal challenges against glioma cells, reduce tumor growth and increase the mice life span. These results suggest that the DC transduced with CPEB4 may induce anti-tumor immunity against glioma cells and might be used as an efficient tumor vaccine in clinical applications.

Immunopathology features of chronic rhinosinusitis in high-altitude dwelling Tibetans.

Chronic rhinosinusitis (CRS) presents distinct inflammatory and remodeling patterns in different populations and environments. Tibetan ethnic groups live at high altitudes and in cold weather conditions. We sought to examine whether Tibetans exhibit distinct CRS pathology or characteristics. Sinonasal polyps and mucosal tissue were obtained from 14 Tibetan patients with CRS and nasal polyps (CRSwNPs), 13 patients with CRS without nasal polyps (CRSsNPs), and 12 Tibetan controls. Tissue homogenates and serum samples were assayed for several T-helper (TH) cell cytokines and mediators using enzyme linked immunosorbent assay profiles were measured using quantity polymerase chain reaction. Several key inflammatory cells were examined for immunohistochemical markers. CRSwNPs were characterized by increased mediator promoting eosinophilic inflammation (interleukin [IL]-5, eosinophil cationic protein, and total immunoglobulin E) and slight synergism with expression of IL-8, IL-2sRa, IL-1beta, IL-6, and myeloperoxidase, and a predominance of eosinophils, mast cells, and neutrophils. GATA-3 transcription factor was significantly increased and Foxp3 showed a tendency to be impaired in CRSwNPs compared with controls. CRSsNPs were characterized by significantly high levels of transforming growth factor beta1, increased interferon γ, and a significant enhancement of Foxp3 and T-beta compared with CRSwNPs. There were reduced numbers of inflammatory cells but increased levels of macrophages in CRSsNPs. Compared with CRSsNPs, CRSwNPs present a severe inflammatory reaction and show a TH2 milieu with apparently impaired regulatory T cells (Treg) function and increased inflammatory cells infiltration predominated by eosinophilic and mast cells. In contrast, TH1 polarization with enhanced Treg function and increased levels of macrophages appear in CRSsNPs.

Economic impact of combination therapy with infliximab plus azathioprine for drug-refractory Crohn's disease: a cost-effectiveness analysis.

BACKGROUND: Combination therapy with infliximab (IFX) and azathioprine (AZA) is significantly more effective for treatment of active Crohn's disease (CD) than IFX monotherapy. However, AZA is associated with an increased risk of lymphoma in patients with inflammatory bowel disease. AIM: To evaluate the cost-effectiveness of combination therapy with IFX plus AZA for drug-refractory CD. METHODS: A decision analysis model is constructed to compare, over a time horizon of 1year, the cost-effectiveness of combination therapy with IFX plus AZA and that of IFX monotherapy for CD patients refractory to conventional non-anti-TNF-α therapy. The treatment efficacy, adverse effects, quality-of-life scores, and treatment costs are derived from published data. One-way and probabilistic sensitivity analyses are performed to estimate the uncertainty in the results. RESULTS: The incremental cost-effectiveness ratio (ICER) of combination therapy with IFX plus AZA is 24,917 GBP/QALY when compared with IFX monotherapy. The sensitivity analyses reveal that the utility score of nonresponding active disease has the strongest influence on the cost-effectiveness, with ICERs ranging from 17,147 to 45,564 GBP/QALY. Assuming that policy makers are willing to pay 30,000 GBP/QALY, the probability that combination therapy with IFX plus AZA is cost-effective is 0.750. CONCLUSIONS: Combination therapy with IFX plus AZA appears to be a cost-effective treatment for drug-refractory CD when compared with IFX monotherapy. Furthermore, the additional lymphoma risk of combination therapy has little significance on its cost-effectiveness.

Staphylococcus aureus superantigens and their role in eosinophilic nasal polyp disease.

Nasal polyposis is a chronic disease of the upper airways which adversely affects the quality of life of patients. Its pathophysiology is still unclear. Recently, several studies have shown different inflammatory pathways which relate to both innate and adaptive immune responses. Moreover, different phenotypes may exist in different ethnic groups of patients. This article will review recent data regarding the type of inflammation, cytokine profiles, involvement of macrophages and dendritic cells, and the impact of various organisms (especially Staphylococcus aureus and its superantigens) and their association with lower airway disease (especially asthma).

Identification and characterization of myeloma-associated antigens in Trichinella spiralis.

To investigate the presence of myeloma-associated antigens in Trichinella spiralis and their anti-tumor effect, cross-immune responses between antigens of the myeloma cell SP2/0 versus positive sera to T. spiralis, and antigens of T. spiralis versus positive sera to myeloma cell SP2/0 were determined using T. spiralis and myeloma specific enzyme-linked immunosorbent assays (ELISA). The myeloma-associated antigens in T. spiralis were separated by ultrafiltration and 2-D electrophoresis, and the amino acid sequences and molecular weights were determined by spectrometry. An obvious reaction was found between a 33 kDa antigen and positive sera, and the major component of the antigen was tropomyosin (TM), which is an surface acidic protein with 284 amino acids. Mice were immunized with TM to determine the anti-tumor effect in vivo. The results showed that CD4(+), CD8(+) T lymphocyte, and CD19(+) B lymphocyte were significantly increased (P<0.05). The anti-tumor effects were significantly different between mice immunized with the antigens or adjuvant alone (P<0.05), while the difference between mice immunized with antigens and whole T. spiralis was not significant (P>0.05). The results indicated that TM identified in this study may play a role in eliciting cross-protective immunity.

Posterior rotating rod reduction strategy for irreducible atlantoaxial subluxations with congenital odontoid aplasia.

STUDY DESIGN: Applying rotating rod techniques to reduce irreducible atlantoaxial dislocation. OBJECTIVE: To spare the occipital-C1 motion by the strategy in reduction of before surgery irreducible atlantoaxial dislocation with obvious neurologic symptoms and congenital odontoid aplasia. SUMMARY OF BACKGROUND DATA: The treatment of atlantoaxial dislocation (AAD) is a challenging problem for most surgeons. Posterior surgical stabilization of C1 and C2 include C1-C2 transarticular screws, or C1 lateral with C2 pars screws. These constructs, however, are based on preoperative reductions. When preoperative skull reduction fails and myelopathic symptoms coexist, long-segment cervico-occipital fusion and decompression are usually the only practical choice. METHODS: The authors explored a different surgical technique to spare the axial occipital joints by rotating rods in polyaxial C1, C3 lateral mass, and C2 pars screws, functioning as a lever analogue. Three before surgery irreducible AAD cases with obvious neurologic symptoms and congenital odontoid aplasia were successfully reduced and fused with this procedure. The authors used intraoperative somatosensory-evoked potential monitoring and intraoperative fluoroscopy. Preoperative skull traction was employed to distract and help extend the atlantoaxial complexes. RESULTS: Three C1-C2 dislocations were reduced completely without any deterioration of neurologic signs. Cervical myelopathic symptoms recovered soon after the operation. No atlantoaxial subluxation recurred. They returned to their normal work and/or activities. CONCLUSION: The rotating rod strategy is a viable option to reduce and fuse C1-C3 for AAD with odontoid aplasia. It spares the occipital-C1 motion.

Evaluation of the anti-inflammatory activity of luteolin in experimental animal models.

Luteolin, a flavonoid abundant in plants worldwide, demonstrates a spectrum of biological activities. This study is aimed at evaluating its inhibiting effects on inflammatory responses in vivo. We investigated the anti-inflammatory activity of luteolin in acute and chronic models in mice. We found that oral administration of luteolin (10 and 50 mg/kg) efficiently suppressed paw edema when induced by injecting carrageenan, and a similar tendency was also observed in the cotton pellet granuloma test. In the air pouch test, luteolin markedly reduced the number of infiltrated leukocytes and the elevated level of 6-keto-prostaglandin F1alpha (6-keto-PGF1alpha) in the exudate. The results derived from the whole blood assay for cyclooxygenase (COX) and from the reverse transcription-polymerase chain reaction (RT-PCR) assay indicate that luteolin may be a potent selective inhibitor of cyclooxygenase-2 (COX-2) and that the inhibition is attributable to its down-regulation of the mRNA expression of COX-2 in inflammatory responses.

Augmented humoral and cellular immune responses of a hepatitis B DNA vaccine encoding HBsAg by protein boosting.

Several reports have indicated that combinatorial regimens with DNA and protein vaccines can elicit both strong immune responses, to circumvent the limits of each vaccine. Surprisingly little was known on HBV vaccine. Here, we investigated the immunization effects of several regimens in BALB/c mice. The level of total antibody and isotypes of IgG were determined by ELISA. Cellular immune responses were assayed by measuring the production of cytokines and CTL activity after re-stimulation for 7 days in vitro with tumor cells CT26/S stably expressing HBsAg. The efficacy of immunoprotection against the challenge of transplanted CT26/S was also examined. The regimen involving twice priming pVAX(S) encoding HBsAg and once recombinant HBsAg protein (rHBsAg) boosting, induced strong and homogenous antibody responses. This regimen induced significant stronger responses of interleukin-12 and gamma interferon (IFN-gamma) in splenocytes, and elicited stronger CD8+ CTL responses and greater immunopretectional efficacy than those elicited by immunization with rHBsAg or pVAX(S) alone. Our regimen may thus provide a strategy for developing an improved immunization against HBV and many other pathogens.