Evaluation of the Nano-TiO2 as a Novel Deswelling Material.

Nano-TiO2 is widely applied in the automobile exhaust hose reels as a catalyst to reduce oxynitride emissions, including nitric oxide (NO). In the biomedicine field, NO plays an important role in vasodilation and edema formation in human bodies. However, the deswelling activity of nano-TiO2 has not been reported. Here, we demonstrated that nano-TiO2 can significantly degrade the production of NO in LPS-induced RAW264.7 mouse macrophages. Further study indicated that nano-TiO2 exhibited an effect on vascular permeability inhibition, and prevented carrageenan-induced footpad edema. Therefore, we prepared a nano-TiO2 ointment and observed similar deswelling effects. In conclusion, nano-TiO2 might act as a novel deswelling agent related with its degradation of NO, which will aid in our ability to design effective interventions for edema involved diseases.

Role of berberine in anti-bacterial as a high-affinity LPS antagonist binding to TLR4/MD-2 receptor.

BACKGROUND: Berberine is an isoquinoline alkaloid mainly extracted from Rhizoma Coptidis and has been shown to possess a potent inhibitory activity against bacterial. However, the role of berberine in anti-bacterial action has not been extensively studied. METHODS: The animal model was established to investigate the effects of berberine on bacterial and LPS infection. Docking analysis, Molecular dynamics simulations and Real-time RT-PCR analysis was adopted to investigate the molecular mechanism. RESULTS: Treatment with 40 mg/kg berberine significantly increased the survival rate of mice challenged with Salmonella typhimurium (LT2), but berberine show no effects in bacteriostasis. Further study indicated that treatment with 0.20 g/kg berberine markedly increased the survival rate of mice challenged with 2 EU/ml bacterial endotoxin (LPS) and postpone the death time of the dead mice. Moreover, pretreatment with 0.05 g/kg berberine significantly lower the increasing temperature of rabbits challenged with LPS. The studies of molecular mechanism demonstrated that Berberine was able to bind to the TLR4/MD-2 receptor, and presented higher affinity in comparison with LPS. Furthermore, berberine could significantly suppressed the increasing expression of NF-κB, IL-6, TNFα, and IFNß in the RAW264.7 challenged with LPS. CONCLUSION: Berberine can act as a LPS antagonist and block the LPS/TLR4 signaling from the sourse, resulting in the anti-bacterial action.

T cell receptor ß repertoires in patients with COVID-19 reveal disease severity signatures.

Background: The immune responses to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are crucial in maintaining a delicate balance between protective effects and harmful pathological reactions that drive the progression of coronavirus disease 2019 (COVID-19). T cells play a significant role in adaptive antiviral immune responses, making it valuable to investigate the heterogeneity and diversity of SARS-CoV-2-specific T cell responses in COVID-19 patients with varying disease severity. Methods: In this study, we employed high-throughput T cell receptor (TCR) ß repertoire sequencing to analyze TCR profiles in the peripheral blood of 192 patients with COVID-19, including those with moderate, severe, or critical symptoms, and compared them with 81 healthy controls. We specifically focused on SARS-CoV-2-associated TCR clonotypes. Results: We observed a decrease in the diversity of TCR clonotypes in COVID-19 patients compared to healthy controls. However, the overall abundance of dominant clones increased with disease severity. Additionally, we identified significant differences in the genomic rearrangement of variable (V), joining (J), and VJ pairings between the patient groups. Furthermore, the SARS-CoV-2-associated TCRs we identified enabled accurate differentiation between COVID-19 patients and healthy controls (AUC > 0.98) and distinguished those with moderate symptoms from those with more severe forms of the disease (AUC > 0.8). These findings suggest that TCR repertoires can serve as informative biomarkers for monitoring COVID-19 progression. Conclusions: Our study provides valuable insights into TCR repertoire signatures that can be utilized to assess host immunity to COVID-19. These findings have important implications for the use of TCR ß repertoires in monitoring disease development and indicating disease severity.

Corrigendum to "Role of Baicalin in Anti-Influenza Virus A as a Potent Inducer of IFN-Gamma".

[This corrects the article DOI: 10.1155/2015/263630.].

[Therapeutic Effect of Spleen Low Molecular Weight Extracts on Leukopenia Caused by Epirubicin in Mice and Its Mechanism].

OBJECTIVE: To investigate the therapeutic effect of spleen low molecular weight extracts on epileptics hydrochloride-induced leukopenia in mice and explore its mechanism. METHODS: The model of leukopenia in mice was established by the injection of epirubicin hydrochloride (10 mg/kg). After the injection of chemotherapeutic drugs, leukocytopenia mice were treated with different doses of spleen low molecular weight extract, Ganoderma oral solution and recombinant granulocyte colony stimulating factor (rhG-CSF). The general survival status indicators such as body weight, coat color and athletic ability of mice in each group were recorded; the tail vein blood of mice in each group was collected and the white blood cell count in them was calculated; bone marrow of mice was taken and bone marrow smears were observed. RESULTS: In the model group, the weight of the mice gradually decreased in the later period, their coat became dark and rough, and the ability to exercise decreased, while the mice in the treatment groups showed different degrees of improvement in their survival status except for the mice treated by rhG-CSF. There was no significant fluctuation in the white blood cell count of the blank control mice. After injection of epirubicin, the white blood cell count of peripheral blood in the model mice and treated mice were decreased. The white blood cell count was lower in the mice treated with high-dose low molecular weight extract and rhG-CSF than that in other experimental groups. Bone marrow smear showed that the proportion of bone marrow nucleated cells in the mice treated with the low molecular weight extract of the spleen was significantly higher than that of model mice (P<0.05). CONCLUSION: The low molecular weight spleen extracts can significantly improve the hematopoietic state of mouse bone marrow, promote the proliferation of inhibited bone marrow cells, and thus has the effect of treating leukopenia in mice.

Salmonella expressing a T-cell epitope from Sendai virus are able to induce anti-infection immunity.

Bacterial fimbriae can accept foreign peptides and display them on the cell surface. A highly efficient gene replacement method was used to generate peptide vaccines based on Salmonella enterica subsp. enterica serovar Typhimurium LT2. DNA encoding an epitope from Sendai virus, SV9 (Sendai virus nucleoprotein peptide 324-332, FAPGNYPAL), which is known to induce cytotoxic T lymphocytes, was incorporated into the gene encoding AgfA (the major subunit protein of thin aggregative fimbriae of Salmonella) by replacing an equal length DNA segment. To improve cytotoxic T lymphocyte recognition, both termini of the peptide were flanked by double alanine (AA) or arginine (RR) residues. Western blotting and immunofluorescence microscopy using AgfA-specific antiserum verified the expression of chimeric AgfA; expression was also proved by a Congo red binding assay. Oral immunizations of C57BL/6 mice with the four strains induced an epitope-specific T-cell response (detected by enzyme-linked immunosorbent spot assay). When the mice were challenged with the Sendai virus, the magnitude of the infection was significantly reduced in the immunized groups compared with the controls. The Salmonella fimbrial display system efficiently induces a cellular immune response and anti-infection immunity in vivo, providing a new strategy for the development of efficient peptide vaccination.

[Preparation and preliminary application of rabbit polyclonal antibodies against intracellular peptides of human platelet glycoprotein GPIbalpha].

OBJECTIVE: To prepare rabbit polyclonal antibodies against intracellular peptides of human platelet glycoprotein GPIbalpha. METHODS: Two peptides corresponding to human platelet GPIbalpha C-terminus were synthesized and purified by high-performance liquid chromatography (HPLC). The peptides were cross-linked with keyhole limpet hemocyanin (KLH). Two New Zealand white rabbits were immunized with conjugated peptides for 3 times. The polyclonal antibodies were purified by Ammonium Sulfate Precipitation and identified by dot blotting and ELISA. GPIbalpha intracellular peptides phosphorylation was tested with these polyclonal antibodies by ELISA. RESULTS: The titers of the two polyclonal antibodies against the GPIbalpha C-terminus peptides were 1:32 000 and 1:64 000 respectively and both of these antibodies reacted with purified GPIbalpha. CONCLUSIONS: Two rabbit polyclonal antibodies against C-terminal peptides of human platelet GPIbalpha have been prepared successfully, providing a way for the preparation of these kinds of antibody. Both phosphorylation and dephosphorylation states exist in the intracellular peptide of human platelets.

[Effect of exposure to allergen through different duration on a rat asthmatic model].

OBJECTIVE: To investigate the effect of exposure to allergen (ovalbumin, OVA) at different times in infancy on the asthmatic airway inflammation of adult rats, and its possible mechanisms. METHODS: Newborn rats were classified as asthma model group, day 3, day 7, day 14 after birth allergen exposure groups and control group, and were injected with OVA 1 mg (containing aluminum hydroxide gel 5 mg) subcutaneously on days 3, 7 and 14 after birth respectively. When all rats were kept till six weeks old, all groups but the control group were immunized and provoked via OVA to make asthma model. The pathologic changes of lung tissue and the hyperplasia of mucous cells per bronchiole were observed. The percentage of CD4(+)CD25(+)T cells and forkhead box P3 (Foxp3) transcription factor mRNA in the splenocytes and thymocytes were also measured. RESULTS: The airway inflammation alleviated significantly and number of mucous cells per bronchiole decreased significantly in day 3 group [(2.29 +/- 0.49) vs(3.50 +/- 0.76),P<0.01] and day 7 group[(1.25+/-0.46) vs (3.50 +/-0.76), P<0.01] compared with asthma group. However, the hyperplasia of mucous cells did not alleviate significantly in day 14 group [(3.00+/-1.16) vs( 3.50+/-0.76), P>0.05]. The CD4(+)CD25(+)T cells and Foxp3 mRNA in the splenocytes increased in day 3 group[(13.68+/-3.54)% vs (7.33+/-3.39)%, P<0.01; (18.46+/-5.01) vs (5.49+/-1.99), P<0.01] and day 7 group [(16.65+/-4.91)% vs (7.33+/-3.39)%,P<0.01; (26.37+/-4.68 )vs( 5.49plusmn;1.99), P<0.01]compared with control group, so did Foxp3 mRNA in thymus in day 7 group [(18.73+/-3.66) vs( 11.13 +/-1.75), P<0.01].But neither the CD4(+)CD25(+)T cells[(11.04+/-2.88)% vs (7.33+/-3.39)%, P>0.05] nor Foxp3 mRNA expression[(8.71 +/- 2.19) vs( 5.49+/-1.99), P>0.05] had a statistically significant increase in day 14 group. CONCLUSION: There exists a "time-window" for inhibiting airway inflammation by early exposure to allergen in the rat asthma model. The possible mechanism could be associated with induced generation of regulatory T cells.

TFDP3 regulates the apoptosis and autophagy in breast cancer cell line MDA-MB-231.

Cancer/testis antigen TFDP3 belongs to the transcription factor DP(TFDP) family. It can bind to E2F family molecules to form a heterodimeric transcription factor E2F/TFDP complex. The complex is an important regulatory activator of cell cycle, involved in the regulation of cell proliferation, differentiation, apoptosis and other important physiological activities. In addition, TFDP3 has also been found to be a tumor-associated antigen that only expresses in malignant tumor tissue and normal testicular tissue; Thus, it is closely related to tumor occurrence and development. In this study, our group investigated the expression of TFDP3 in mononuclear cell samples from a variety of tissue-derived malignant tumors, breast cancer and benign breast lesions. The results show that TFDP3 is expressed in the malignant form of various tissues. Moreover, our recent research had focused on the ability of TFDP3 to influence the drug resistance and apoptosis of tumor cells. To further clarify the mechanisms involved in tumor resistance, this study also examined the expression of TFDP3 and tumor cell autophagy regulation; Autophagy helps cells cope with metabolic stress (such as in cases of malnutrition, growth factor depletion, hypoxia or hypoxia) removes erroneously folded proteins or defective organelles to prevent the accumulation of abnormal proteins; and removes intracellular pathogens. Our results showed that TFDP3 expression can induce autophagy by up-regulating the expression of autophagic key protein LC3(MAP1LC3) and increasing the number of autophagosomes during chemotherapy of malignant tumors. Then, DNA and organelles damage caused by the chemotherapy medicine are repaired. Thus, TFDP3 contributes toward tumor cell resistance. When siRNA inhibits TFDP3 expression, it can reduce cell autophagy, improving the sensitivity of tumor cells to chemotherapy drugs.

Role of Berberine in the Treatment of Methicillin-Resistant Staphylococcus aureus Infections.

Berberine is an isoquinoline alkaloid widely used in the treatment of microbial infections. Recent studies have shown that berberine can enhance the inhibitory efficacy of antibiotics against clinical multi-drug resistant isolates of methicillin-resistant Staphylococcus aureus (MRSA). However, the underlying mechanisms are poorly understood. Here, we demonstrated that sub-minimum inhibitory concentrations (MICs) of berberine exhibited no bactericidal activity against MRSA, but affected MRSA biofilm development in a dose dependent manner within the concentration ranging from 1 to 64 µg/mL. Further study indicated that berberine inhibited MRSA amyloid fibrils formation, which consist of phenol-soluble modulins (PSMs). Molecular dynamics simulation revealed that berberine could bind with the phenyl ring of Phe19 in PSMα2 through hydrophobic interaction. Collectively, berberine can inhibit MRSA biofilm formation via affecting PSMs' aggregation into amyloid fibrils, and thereby enhance bactericidal activity of antibiotics. These findings will provide new insights into the multiple pharmacological properties of berberine in the treatment of microbial-generated amyloid involved diseases.

Cooperation between heparin-binding EGF-like growth factor and interleukin-6 in promoting the growth of human myeloma cells.

Interleukin-6 (IL-6) is a major survival and proliferation factor of human malignant plasma cells and IL-6 dependent myeloma cell lines can be obtained from patients with terminal disease. We show here that mutated diphtheria toxin, a specific inhibitor of heparin-binding epidermal growth factor-like growth factor (HB-EGF), blocked the IL-6-induced growth of two myeloma cell lines (XG-1 and XG-14) and did not significantly affect that of two other cell lines (XG-6 and XG-13). The IL-6 mediated growth of myeloma cells was also inhibited by antibodies to ErbB1, a receptor for HB-EGF. The XG-1 and XG-14 cell lines that are sensitive to HB-EGF inhibitors overexpressed HB-EGF and EGF receptor (ErbB1) genes. They also overexpressed CD9, a tetraspanin that binds to the heparin-binding domain of HB-EGF and is critical for promoting ErbB1 activation by HB-EGF. The XG-6 and XG-13 myeloma cells that were not significantly sensitive to HB-EGF antagonists, poorly expressed HB-EGF, ErbB1 and CD9 genes or proteins. We demonstrated that recombinant HB-EGF supported the long-term growth of myeloma cells, as did IL-6. The myeloma cell growth factor activity of HB-EGF was completely inhited by antibodies to ErbB1, but also by antibodies to gp130 IL-6 transducer or to IL-6. These data indicate that in the XG-1 and XG-14 IL-6-dependent myeloma cell lines, the CD9/HB-EGF/erbB1 and the IL-6/IL-6R/gp130 pathways cooperate synergistically to trigger myeloma cell growth. They suggest that inhibitors of the EGF receptor or HB-EGF may be useful for inducing myeloma cell apoptosis in patients with multiple myeloma.

Role of Baicalin in Anti-Influenza Virus A as a Potent Inducer of IFN-Gamma.

Baicalin (BA) is a flavonoid compound purified from Scutellaria baicalensis Georgi and has been shown to possess a potent inhibitory activity against viruses. However, the role of BA in anti-influenza virus has not been extensively studied, and the immunological mechanism of BA in antiviral activity remains unknown. Here, we observed that BA could protect mice from infection by influenza virus A/PR/8/34 (H1N1), associated with increasing IFN-γ production, but presented no effects in IFN-γ or IFN-γ receptor deficient mice. Further study indicated that BA could inhibit A/PR/8/34 replication through IFN-γ in human PBMC. Moreover, BA can directly induce IFN-γ production in human CD4(+) and CD8(+) T cells and NK cells, and activate JAK/STAT-1 signaling pathway. Collectively, BA exhibited anti-influenza virus A (H1N1) activity in vitro and in vivo as a potent inducer of IFN-γ in major IFN-γ producing cells.

Expression of Human Soluble CD40 Ligand in Pichia pastoris and Its Effects on Dendritic Cells and Malignant B Cells.

CD40 ligand (CD40L) is a member of the tumor necrosis factor (TNF) superfamily and is expressed primarily on the activated CD4( )T lymphocytes. The CD40 molecule, the cognate receptor of CD40L presents on many immunocytes such as B lymphocytes, dendritic cells (DCs) as well as on some neoplastic cells. Triggering of CD40 through CD40L plays a central role in the initiation and regulation of the human immune response. In order to further investigate the possible biological roles of CD40 signaling triggered by CD40L, we subcloned the DNA fragment encoding the extracellular region of human CD40L into the pSK plasmid. After being sequenced, the target fragment was introduced into the pPICZalphaA plasmid to construct the pPICZalphaA-sCD40L expressing vector which was then transduced into Pichia pastoris GS115 cells by electroporation. The tansformant expressed sCD40L in culture supernatants with a maximum yield of about 35 mg/L. Furthermore, we found that the recombinant human soluble CD40 ligand (rhsCD40L) could effectively induced human peripheral blood monocytes(PBMCs) in vitro in the absence of TNFalpha into dendritic cells (DCs) with the typical morphology and special surface markers of dendritic cells including CD1a, CD80, CD83, and HLA-DR etc. To our surprise, the rhsCD40L also could inhibit directly in vitro proliferation of the CD40-positive multiple myeloma cell line XG-2 and the B lymphoma cell line Daudi significantly at an optimal concentration from 2.5 to 15.0 mg/L, while CD40 negative ovarian carcinoma cell lines, SKB and SKR, were not effected by either high or low concentration of rhsCD40L. Moreover, rhsCD40L had the same effects as CD40L-transfected cell in inducing XG2 cell apoptosis. Our results demonstrated that functional human soluble CD40L could be successfully expressed in the Pichia pastoris system and that the recombinant human soluble CD40L might be a potential immune adjuvant and a new powerful molecule for tumor bio-therapy.

[Expression and purification of recombinant N-terminal protein of SARS S1 subunit expressed in E. Coli].

OBJECTIVE: To express and purify the recombinant N-terminal protein of SARS virus S1 subunit and to study its role in SARS immune response. METHODS: The gene encoding N-terminal 334 amino acid residuals of SARS virus S1 subunit was cloned and expressed in E. Coli. After purification, the recombinant protein was identified by anti-SARS positive sera from recovered SARS patients. The sera from health donors, which were collected before the out-break of SARS, were used as negative control in the study. RESULTS: Sequencing analysis confirmed that the desired DNA sequence in recombinant plasmid was correct and had the same sequence of natural N-terminal of SARS virus S1 subunit. The molecular weight of recombinant fusion protein is about 64 000. The recombinant S1 protein could react with three antibody positive samples from recovered SARS patients, which showed specific bands at 64 000, but not with the control samples according to results of western blot. CONCLUSION: The recombinant N-terminal protein of SARS virus S1 subunit displays specific reaction with SARS antibody and may provide a good tool for further research of immune response to SARS virus.

[Role of PD153035 in the induction of apoptosis of XG-1 myeloma cell line].

OBJECTIVE: To study the role of epidermal growth factor receptor (EGFR) in the proliferation and survival of human myeloma cells. METHODS: The inhibitor of EGFR, PD153035, was used to block the signal transduction of EGFR. The proliferation and apoptosis of myeloma cell line, XG-1, were detected by 3H-TCR incorporation assay and Annexin V staining analysis, respectively. The phosphatation of STAT3, the key activate signal to the myeloma cell proliferation, was detected with Western blot. RESULTS: PD153035 decreased the proliferation of XG-1 and induced an obvious apoptosis in XG-1. The phosphatation of STAT3 induced by HB-EGF but not by IL-6 was blocked by PD153035. CONCLUSION: The proliferation and survival of myeloma cells may be suppressed by PD153035 due to the blockage of phosphatation of STAT3 induced by the activation of EGFR.

The "time-window" effect of early allergen exposure on a rat asthma model.

BACKGROUND: The hygiene hypothesis has been proposed to explain the pathogenesis of asthma. Allergen exposure was shown to inhibit asthma in an animal model. But the optimal timing of allergen exposure remains unclear. This study aims to explore the time effcct of allergen exposure and the possible mechanisms. METHODS: Neonate Wistar rats were randomly divided into asthma group, control group and day 1, day 3, day 7, and day 14 groups. The day 1, day 3, day 7 and day 14 groups were injected with ovalbumin (OVA) subcutaneously on days 1, 3, 7 and 14 after birth, respectively. Six weeks later, all groups, except the control group, were sensitized and stimulated with OVA to make the asthma model. We observed the pulmonary pathologic changes, detected the regulatory T cells, and CD28 expression level in thymus and spleen by flow cytometry. RESULTS: The asthmatic inflammation in the day 1, day 3 and day 7 groups, but not the day 14 group, was alleviated. The asthma group and day 14 group had lower proportions of regulatory T cells in the thymus compared with the control group, day 1, day 3, and day 7 groups. There was no significant difference in the CD28 expression levels on the regulatory and conventional T cells among groups. But the control group and the day 1, day 3, and day 7 groups had relatively higher proportions of CD28 positive regulatory T cells in the thymus than the day 14 group and the asthma group. CONCLUSIONS: There is a "time-window" for early allergen exposure. The impairment of regulatory T cells may promote the development of asthma. Allergen exposure in the "time-window" can make the thymus produce normal quantity of regulatory cells. The CD28 signal on regulatory T cells may participate in the production of regulatory T cells.

Effects of epithelial cell injury of the lower respiratory tract in the pathogenesis of allergic responses in a rat model.

BACKGROUND: Asthma is a complex disease involving genetic and environment interactions. Atopy is a strong risk factor for asthma. The airway epithelium not only forms a physical barrier but also provides immune defense against harmful materials. To explore the effects of airway epithelium on asthma, we hypothesized that environmental injuries could act on bronchial epithelial cells and damage the physical barrier, which might facilitate allergens to stimulate immunoreactions and play an important role in the pathogenesis of asthma. METHODS: Thirty eight-week-old male Wistar rats were randomly divided into five groups with six rats in each group: control group, asthma group, ovalbumin (OVA) + OVA group, lipopolysaccharide (LPS) group and LPS + OVA group. In the control group, 0.9% saline was injected intraperitoneally on day 1. Fourteen days later, the rats were exposed to aerosolized 0.9% saline. In the asthma group, the rats were sensitized with an injection of 10 mg of OVA, followed by an aerosolized 2% OVA challenge 14 days later. The OVA + OVA group was sensitized by an inhalation 2% OVA, 20 minutes a day, from day 1 to day 7, and then OVA challenged in the same way as the asthma group. In the LPS group, LPS (200 µl, 1 µg/µl) was given by airway on day 1 and day 3, with a simultaneous aerosol inhalation of 2% OVA for 20 minutes a day from day 1 to day 7. Fourteen days later, the rats were challenged with saline as in the control group. While in the LPS + OVA group, LPS (200 µl, 1 µg/µl) was given by airway on day 1 and day 3, with a simultaneous aerosol inhalation of 2% OVA for 20 minutes a day from day 1 to day 7. Fourteen days later, the rats were challenged with OVA as in the asthma group. The expression of interleukin (IL)-4, interferon-gamma (IFN-γ) and thymic stromal lymphopoietin (TSLP) in the lungs was detected by reverse transcription polymerase chain reaction (RT-PCR) and the pulmonary pathological changes were also observed. The level of IL-4, IFN-γ and IgE in plasma was detected by enzyme-linked immunosorbent assay (ELISA). Bronchoalveolar lavage fluid (BALF) was collected to conduct differential cell counts. Flow cytometry analysis was also used to count Th1 and Th2 cells. RESULTS: The pathological changes in the LPS + OVA group were similar to the asthma group, while in other groups, the pathological changes were not obvious. The ratio of lymphocytes in BALF, IL-4/IFN-γ in plasma and the expression of the TSLP and IL-4 in the asthma and LPS + OVA groups were higher than in the control group and the OVA + OVA group (P < 0.05). The level of IgE was higher in the asthma, LPS and LPS + OVA groups than in the control group and the OVA + OVA group (P < 0.05). By flow cytometry analysis, the Th1/Th2 ratio was lower in the LPS + OVA and asthma groups than in other groups (P < 0.05). CONCLUSIONS: The experiment results show that the injury to the bronchial epithelial layer may be the initial event of allergic responses. This finding implies that a rational approach to therapeutics would be to increase the resistance of the airways to environmental injuries rather than concentrating on suppressing inflammation.

Oral vaccination with attenuated Salmonella enterica strains encoding T-cell epitopes from tumor antigen NY-ESO-1 induces specific cytotoxic T-lymphocyte responses.

Bacterial fimbriae can accept foreign peptides and display them on the cell surface. A highly efficient gene replacement method was used to generate peptide vaccines based on Salmonella enterica serovar Typhimurium SL3261. The T-cell epitopes (NY-ESO-1 p157-165 and p157-167) from NY-ESO-1, which is a promising target antigen in patients for the specific immune recognition of cancer, were incorporated into the gene encoding AgfA (the major subunit protein of thin aggregative fimbriae of Salmonella) by replacing an equal length of the DNA segment. To improve cytotoxic T-lymphocyte recognition, both termini of the peptide were flanked by double alanine (AA) residues. Immunofluorescence microscopy with AgfA-specific antiserum verified the expression of chimeric AgfA, which was also proved by a Congo red binding assay. Oral immunizations of HLA-A*0201 transgenic mice with recombinant SL3261 strains encoding NY-ESO-1 p157-165 or p157-167 induced NY-ESO-1 p157-165-specific CD8(+) T cells, detected by an HLA-A*0201 pentamer, and induced a T-cell response detected by an enzyme-linked immunospot assay. The Salmonella fimbrial display system was efficient at the induction of an antitumor cellular immune response in vivo, providing a new strategy for the development of efficient cancer vaccinations.

CHP2 activates the calcineurin/nuclear factor of activated T cells signaling pathway and enhances the oncogenic potential of HEK293 cells.

CHP2 (calcineurin B homologous protein 2) was initially identified as a tumor-associated antigen highly expressed in hepatocellular carcinoma. Its biological function remains largely unknown except for a potential role in transmembrane Na(+)/H(+) exchange. In the present study, we observed that ectopic expression of CHP2 promoted the proliferation of HEK293 cells, whereas knockdown of endogenous CHP2 expression in HepG2 inhibited cell proliferation. When inoculated into nude mice, CHP2 transfected HEK293 cells displayed markedly increased oncogenic potential. In analysis of the underlying molecular mechanisms, we found that like calcineurin B, CHP2 was able to bind to and stimulate the phosphatase activity of calcineurin A. In accord with this, CHP2-transfected cells showed increased nuclear presence of NFATc3 (nuclear factor of activated T cells) and enhanced NFAT activity. Finally, both accelerated cell proliferation and NFAT activation following CHP2 transfection could be suppressed by the calcineurin inhibitor cyclosporine A, suggesting an intrinsic connection between these events. Taken together, our results highlighted a potential role of CHP2 in tumorigenesis and revealed a novel function of CHP2 as an activator of the calcineurin/NFAT signaling pathway.