[Determination of Th1/Th2 cytokines and chemokines in patients with allergic diseases and its significance].

OBJECTIVE: To determine Th1/Th2 cytokines and chemokines in patients with allergic diseases and its clinic significance. METHODS: Serum levels of IFN-gamma, IL-4, IL-5, IL-13, Eotaxin, RANTES and LTB4 were determined from peripheral blood of 64 allergic patients and 21 healthy controls with ELISA. RESULTS: IL-4, IL-5, IL-13 and Eotaxin, LTB4 were increased significantly in serum of allergic patients compared with those of controls (P<0.05). There were no significant differences in serum levels of IFN-gamma and RANTES between patients and controls (P>0.05). Serum levels of IL-4, IL-5, IL-13 and LTB4 were correlated with each other (P<0.01). Eotaxin, RANTES and IFN-gamma levels were also significantly correlated with each other (P<0.05). LTB4 was correlated with Eotaxin as well (P<0.01). CONCLUSION: A wide range of cytokines and chemokines is involved in allergic diseases,which may play their roles in a complex interactive manner.

Mapping of cytotoxic T lymphocytes epitopes in E7 antigen of human papillomavirus type 11.

One of the critical steps in the progression to condyloma acuminatum (CA) is the establishment of a persistent human papillomavirus (HPV) infection, majority of HPV type 6 and 11. Cytotoxic T lymphocytes (CTL), which can be induced by the epitope-based peptides in vitro, are thought to be able to recognize and destroy virus-infected cells. In order to screen and identify HLA-A*0201 restricted HPV-11E7 CTL epitopes, five epitope peptides and tetramers were selected including HPV-11E7 7-15 (TLKDIVLDL), 15-23 (LQPPDPVGL), 47-55 (PLTQHYQIL), 81-89 (DLLLGTLNI) and 82-90 (LLLGTLNIV). Human monocyte-derived dendritic cells (DCs) from HLA-A*0201 healthy individuals were pulsed with these peptides to assess the expression of CD83, CD86, HLA-DR and the secretion of IL-12. The ability of peptide-loaded mature DCs to activate autologous T cells was evaluated by analyzing the frequency of specific tetramer(+) CD8(+) T cells using flow cytometry, and the level of IFN-gamma secretion by ELISA. The ability of the epitope-specific CTLs to kill the target cells was also analysed. It was found that the immature DCs could be fully activated by all the five HPV-11E7 peptides and peptide-loaded mature DCs were able to stimulate the epitope-specific T cells in vitro. There was an increased frequency of CD8(+) T cells specific for the E7 7-15 epitope when compared to other four predicted epitopes of HPV-11E7 (P < 0.05). The epitope-specific CTLs for E7 7-15 induced the strongest cytotoxicity to HPV-11E7 expressing cell line at an E:T ratio of 50:1 (P < 0.05). Taken together, these findings demonstrate that E7 7-15 (TLKDIVLDL) is an HLA-A*0201-restricted CTL epitope of HPV type 11. We propose that this epitope could be more helpful in the characterization of HPV control mechanism and be useful for the development of immunotherapeutic approaches for low-risk HPV infectious diseases such as CA.

[Modulatory effect of fumaric acid esters on superoxide-anion generation in human phagocytes].

Fumaric acid esters (FAE), mainly dimethylfumarate (DMF), have been shown to be highly efficacious in the treatment of psoriasis. Among the potential side effects of FAE therapy, lymphocytopenia is sometimes observed. In order to address the question whether FAE may interfere with systems of the innate defense, the modulatory role of FAE on the generation of superoxide-anion by human monocytes and neutrophils was studied by measuring the reduction of cytochrome c. Various concentrations of DMF and its metabolite methylhydrogenfumarate (MHF) were used to observe their modulatory effect on superoxide-anion generation by monocytes and neutrophils in response to bacteria (S. aureus and E. coli) and candida (C. albicans). Dexamethasone (DXM, 1 x 10(-7) mol x L(-1)) was also studied at the same time. We found that DXM significantly inhibited superoxide-anion generation from monocytes in response to bacteria and C. albicans, whereas DMF and MHF (10-20 microg x mL(-1)) significantly increased the production of superoxide-anion in monocytes in response to the above mentioned bacteria. DXM, DMF and MHF did not affect superoxide-anion generation of neutrophils. Our data indicate that DMF and MHF enhance superoxide-anion generation in human monocytes as one of the important mechanisms of innate defense against microorganisms.

[Effects of drugs known to trigger psoriasis on HaCaT keratinocytes].

To investigate whether lithium carbonate, propranolol or chloroquine aggravate psoriasis through influencing cytokines of the psoriatic cytokine network, HaCaT keratinocytes were stimulated with TNF-a after treatment with these drugs. Protein secretion of a set of multiple different cytokines and growth factors in culture supernatants were measured by using a cytokine antibody array technology. Expression of IL-8 and IL-6 mRNA was determined by real-time PCR. In culture supernatants of TNF-alpha-stimulated HaCaT cells, production of IL-6 and TNF-alpha could be enhanced by lithium carbonate; production of IL-6 and a panel of cytokines and growth factors could be enhanced by propranolol hydrochloride; and IL-6 was up-regulated by chloroquine diphosphate as well. Real-time PCR analysis showed a significantly dose-dependent increase of IL-8 and IL-6 mRNA expression in HaCaT cells stimulated with TNF-a as compared to cells without TNF-alpha-stimulation, the mRNA expression of IL-8 was higher than that of IL-6 with the same concentration of TNF-alpha (P < 0.01). Compared with HaCaT cells cultured with medium alone, propranolol hydrochloride at the concentration of 1 x 10(-6) mol x L(-1) could stimulate HaCaT cells to express higher level of IL-6 mRNA (P < 0.05). The drugs investigated show a modulatory effect on certain cytokines and growth factors which are able to modulate inflammatory type of immune reaction present in psoriatic lesions.

[The construction and immune response of fusion DNA vaccine CRT180/HPV6E7].

OBJECTIVE: To investigate the immune response, in particular, the anti-virus cellular immune response induced by the constructed fusion DNA vaccine CRT (Calreticulin) 180/HPV6E7 in vivo and vitro. METHODS: The HPV6E7 open reading frame was amplified by polymerase chain reaction from pUC/HPV6 plasmid. The CRT180 gene was cloned by reverse transcription from human muscle tissues. The PCR products of CRT180 and HPV6E7 were subcloned into pcDNA3. 1-GFP eukaryotic vector. The recombinant plasmids CRT180/ HPV6E7 was authenticated by restrict enzyme digestion and confirmed by DNA sequencing. The DNA plasmid HPV6E7 with report gene GFP was transfected to murine B16 cells by lipofectamine kit to establish the target cells. The HPV6E7-expressing cell line was selected by G418 and identified by RT-PCR. The C57BL/6 mice were vaccinated via intramuscular injection in the right legs with 100 microg plasmid encoding CRT180-HPV6E7, CRT180 or HPV6E7, empty plasmid without insert, and PBS group respectively. The changes of the T lymphocyte subsets in the peripheral blood of the mice were evaluated by flow cytometric analysis. The lymphocytes from the spleen and lymph nodes were harvested and co-cultured with HPV6E7-expressing cell lines. The CTLs kill activity and the cytokines IL-2, IFN-gamma secretion levels of the lymphocytes were assessed by LDH and ELISA respectively. RESULTS: The constructed recombinant plasmids pcDNA3. 1-CRT180/HPV6E7, pcDNA3. 1-CRT180 and pcDNA3. 1-HPV6E7 were authenticated by restrict enzyme digestion and confirmed by DNA sequencing. Green fluorescence located in the cellular nucleus and plasma could be detected by fluorescent microscope after transfection with plasmids. The results of RT-PCR from the selected positive cell line showed the expected fragments of HPV6E7 mRNA. After immunization, the percentage of CD8+ or TCRgamma delta tau cells in the peripheral blood, the CTLs kill activity of the spleen cells and lymphocytes against HPV6E7-expressing cells, and the secretion levels of IL-2, IFN-gamma in CRT180/HPV6E7 vaccinated group increased significantly compared with the control groups (P < 0.05). CONCLUSION: Vaccination with fusion DNA vaccine CRT180/HPV6E7 could elicit a more efficient HPV6E7-specific immune response than with HPV6E7 plasmid, indicating the potential of CRT180/HPV6E7 vaccine to enhance the antigen presentation. The recombinant CRT180/HPV6E7 might help the elimination of virus in animal models and accordingly be used as a vaccine candidate in the therapy of CA.

Recombined DNA vaccines encoding calreticulin linked to HPV6bE7 enhance immune response and inhibit angiogenic activity in B16 melanoma mouse model expressing HPV 6bE7 antigen.

Calreticulin (CRT) has been reported to have an effect of upregulating MHC class I presentation as well as inhibiting angiogenesis in vitro and in vivo. Combination of dual mechanisms of enhanced immunogenicity of human papillomavirus (HPV) 6bE7 antigen and antiangiogenesis may be introduced in the strategy of vaccines against condyloma acuminatum (CA) resulting from HPV infection. Therefore, we constructed DNA vaccines by employing different lengths of CRT chimerically linked to a model antigen HPV6bE7 and investigated the immunological and antiangiogenic effects of these vaccines in a B16 melanoma model that express HPV6bE7 antigen. Our results showed that vaccination with CRT180/HPV6bE7 or CRT120/HPV6bE7 enhanced the presence of CD8(+) T cells and TCRgammadelta T cells in vivo, increased the specific lysis activity against E7-expressing cells and secretion levels of IL-2 and IFN-gamma by activating T cells in vitro significantly. Moreover, recombined CRT180 or CRT120 with HPV6bE7 vaccines could elicit a more efficient E7-specific immune response than HPV6bE7 alone. The similarity of immunological enhancement of CRT180/HPV6bE7 and CRT120/HPV6bE7 implies that the immunologically active region mainly exist in fragment 1-120 aa. Furthermore, CRT180/HPV6bE7 and CRT180 displayed remarkable superiority over CRT120/HPV6bE7 in vivo angiogenesis assay, suggesting that the antiangiogenic activity of CRT resides in a domain between aa 120 and 180. Vaccination with CRT180/HPV6bE7 generated the best protective effect of delaying tumor formation and reduction of tumor size in tumor growth inhibition experiment among all DNA constructs. Therefore, CRT180/HPV6bE7 vaccine may enhance the immunological response to HPV6bE7 and inhibit angiogenesis. This construct may be useful in preventing HPV-associated dermatosis and may be developed as a promising strategy to control CA.

Increased endocytic activity in monocyte-derived dendritic cells in patients with psoriasis vulgaris.

BACKGROUND & OBJECTIVE: The understanding of the pathogenesis of psoriasis vulgaris has focused on T cell mediated immune disorder for many years. Recent studies provide evidence that dendritic cells may be of major importance as regulatory cells driving the psoriasis tissue reaction, and they are one of the therapeutic targets. In order to further characterize the role of dendritic cells in psoriasis, this study was designed to assess the differentiation of dendritic cells from monocytes (MoDC), the expression of phagocytosis related receptors by MoDC, their endocytic activity for fluorescent beads and lucifer yellow as well as their superoxide generation in patients with psoriasis. METHODS: Twenty eight patients with psoriasis vulgaris and 12 healthy controls were included in the study. MoDC were obtained by culturing monocytes with granulocyte macrophage-colony stimulating factor (GM-CSF) and interleukin-4 (IL-4) for 5 days. Cell surface expression of CD1a, CD14, CD40, CD80, CD83, CD86, HLA-DR, mannose receptor (MR) and Fcg receptors by MoDC and their endocytosis of dextran and lucifer yellow were analyzed by flow cytometry. Zymosan ingestion was measured to access the phagocytosis of MoDC. RESULTS: Differentiation of monocytes to dendritic cells was upregulated in patients manifested as significantly increased expression of CD40, CD80, CD86 and HLA-DR compared with that in healthy controls (P<0.01). Expression of MR and Fcg receptor II (CD32) by MoDC was significantly increased in patients with psoriasis as well (P<0.01). Endocytosis of dextran but not lucifer yellow in patients was significantly higher than controls (P<0.01), and significantly enhanced phagocytosis by increasing zymosan ingestion was also observed (P<0.01) in patients. Taken together, endocytic and phagocytic activity of MoDC in psoriasis was increased than normal persons. INTERPRETATION & CONCLUSION: Enhanced activity of dendritic cells binding and capturing foreign antigens for subsequent antigen presentation and the initiation of immune responses in psoriasis may contribute to the pathogenesis of the disease. The upregulated expression of MR and the enhanced endocytic activity of DC might be an explanation for the absence of skin infection observed in psoriasis.

Triptolide affects the differentiation, maturation and function of human dendritic cells.

Triptolide is a purified component from a traditional Chinese herb Tripterygium wilfordii Hook F. It has been shown to have anti-inflammatory and immunosuppressive activities by its inhibitory effect on T cells. But the effect of triptolide on dendritic cells (DC) is unknown. Dexamethasone (Dex) is a classic immunosuppressive agent known to suppress the immune response at different levels and has recently found to modulate the development of DC, thereby influencing the initiation of the immune response. In this study, we investigated the affect of triptolide on the differentiation, maturation and function of DC differentiated from human monocytes (MoDC) in vitro in the presence of GM-CSF and IL-4. Dex was included in the study as a reference. Our data show that both triptolide and Dex prevented the differentiation in immature MoDC by inhibiting CD1a, CD40, CD80, CD86 and HLA-DR expression but upregulating CD14 expression, as well as by reducing the capacity of MoDC to stimulate lymphocyte proliferation in the allogeneic mixed lymphocyte reaction. They blocked the maturation of MoDC as totally blocked induction of CD83 expression and absent upregulation of CD40, CD80, CD86 and HLA-DR. In addition, higher concentration of triptolide (20 ng/ml) and 10(-6) M Dex induced apoptosis in MoDC as measured by expression of APO2*7 and DNA fragmentation (TUNEL assay). However, the phagocytic capacity of MoDC was enhanced by triptolide but not Dex. Therefore, the suppression of DC differentiation, the function in immature DCs as well as the inhibition of DC maturation by triptolide may explain some of its immunosuppressive properties. It is suggested that DCs are a primary target of the immunosuppressive activity of triptolide.

[1 alpha, 25-dihydroxyvitamin D3 and its analogues modulate the phagocytosis of human monocyte-derived dendritic cells].

AIM: To investigate the role of 1 alpha, 25-dihydroxyvitamin D3 (calcitriol) and its analogues tacalcitol and 24, 25(OH)2D3 on the phagocytosis of human monocyte-derived dendritic cells (MoDC). METHODS: MoDC were generated in vitro by differentiating monocytes in the presence of GM-CSF and IL-4 for 5 days. Expression of mannose receptor (MR) and Fc gamma receptors (Fc gamma Rs) by MoDC was analysed by flow cytometry. Zymosan ingestion was measured to assess the phagocytosis of MoDC. RESULTS: MoDC expressed high level of MR and Fc gamma Rs and showed the capacity of zymosan ingestion. Calcitriol and tacalcitol but no 24, 25(OH)2D3 not only upregulated the expression of MR and Fc gamma Rs on MoDC but also correspondingly enhanced their phagocytosis by increasing zymoasan ingestion. Furthermore, the upregulatory role occurred in the early stage of MoDC differentiation and was irreversible. The upregulatory role of calcitriol was dose dependent. CONCLUSION: Calcitriol and its analogue tacalcitol may play an important role in dendritic cell binding and capturing foreign antigens at the initiation of immune response.

[Effects of fumaric acid esters on differentiation of dendritic cells in vitro]

OBJECTIVE: To investigate the effect of anti-psoriatic drug fumaric acid esters (FAE) on the differentiation of dendritic cells (DC). METHODS: Dendritic cells were obtained by differentiating human monocytes in vitro. Flow cytometry was used to analyse the effect of FAE on cell surface expression of CD1a, CD14, CD40, CD80, CD86 and HLA-DR by monocyte-derived dendritic cells (MoDC). Mixed lymphocyte reaction (MLR) was made to demonstrate the influence of FAE on T cell stimulatory activity of MoDC. RESULTS: Dimethylfumarate and methylhydrogenfumarate-calcium-salt (0.01 approximate, equals 100 mg/L) inhibited MoDC differentiation as well as reducing the capacity of MoDC to stimulate lymphocytic proliferation in MLR. CONCLUSION: The mode of action of FAE in pso riasis may be mediated by inhibition of DC differentiation.

Hemophagocytic syndrome secondary to adult-onset Still's disease but very similar to lymphoma.

Hemophagocytic syndrome (HPS) is a clinicopathologic entity characterized by increased proliferation and activation of benign macrophages with hemophagocytosis throughout the reticuloendothelial system. HPS may be primary, or secondary to malignancy, infections, auto-immune diseases and pharmacotherapy. In patients with adult-onset Still's disease (AOSD), HPS is a rare but life-threatening complication. Herein, we described a female patient with HPS secondary to AOSD. During the therapy, giant gastric ulcer similar to lymphoma developed after treatment with corticosteroid and nonsteroidal anti-inflammatory drugs.

Toll like receptor agonists augment HPV 11 E7-specific T cell responses by modulating monocyte-derived dendritic cells.

Impaired local cellular immunity is one of the mechanisms responsible for condyloma acuminatum (CA) recurrence. The activation of dendritic cells (DCs) is important in vaccine development. We investigated the effect of different toll like receptor (TLR) agonists including LPS (TLR4 agonist), polyinosinic acid-polycytidylic acid (PIC, TLR3 agonist), CpG oligonucleotide (TLR9 agonist), and imiquimod (TLR7 agonist) on human monocyte-derived dendritic cells (mdDCs) loading of human papillomavirus (HPV) type 11 E7 epitope. As a result, we found that mdDCs loading HLA-A*0201-restricted HPV 11 E7 CTL epitope peptide could respond to the TLR agonists, especially LPS and PIC. This was characterized by an enhanced expression of CD40, CD80, CD86, CD83 and HLA-DR, and a high level of IL-12 production. TLR agonists, especially PIC, enhanced the ability of E7-loaded mdDCs to induce IFN-gamma-secretion CD4(+) naïve T cells. Moreover, E7-loaded mdDCs exposed to TLR agonists augmented autologous T cell responses including effector cytokines production and specific cytotoxic T lymphocyte (CTL) responses. In addition, the inhibitory effect of IL-10 on mdDCs maturation could be partially restored by LPS, PIC or imiquimod. Taken together, these results demonstrate that TLR agonists promoted the maturation of E7-loaded mdDCs and their ability to induce T help type 1 polarization and augment E7-specific T cell responses. These data also indicated that TLR3/4 agonists might be effective adjuvants of mdDC-based vaccines against CA.

[Detection of peripheral blood Th1/Th2 and Tc1/Tc2 subsets in patients with condyloma acuminatum and its significance].

OBJECTIVE: To investigate the role of T-helper (Th) and cytotoxic T (Tc) lymphocyte polarization in the pathogenesis of condyloma acuminatum (CA) and its correlation with recurrence. METHODS: Three-colour immunofluorescent flow cytometry was used to detect the proportion of CD3+ CD8- /IFN-gamma+ (Th1), CD3+ CD8- /IL-4+ (Th2), CD3+ CD8+/IFN-gamma+ (Tc1) and CD3+ CD8+ /IL-4+ (Tc2) cells in the peripheral blood of CA patients and health controls. RESULTS: Compared to health controls, CA patients showed a decreased number of Th1 (P < 0.01) and Tc1 cells (P < 0.05), as well as a decreased Th1/Th2 and Tc1/Tc2 ratio (P < 0.05). Furthermore, in 15 recurrent CA patients the ratio of Th1/Th2 was remarkably decreased (P < 0.01), while the ratio of Tc1/Tc2 had no significant change in comparison with health controls. CONCLUSION: The decrease of Th1 and Tc1 subsets results in relative Th2 and Tc2 predominance, and this tendency is more significant in recurrent CA patients. The Th1 to Th2 and Tc1 to Tc2 shifts in CA patients could be responsible for the fact that human papilloma virus (HPV) is hard to be eliminated.

Imiquimod inhibits the differentiation but enhances the maturation of human monocyte-derived dendritic cells.

Imiquimod is a topically used immune response modifier effective in the treatment of genital warts caused by HPV. Its therapeutic effects are believed to be the release of proinflammatory cytokines from the monocyte-macrophage lineage resulting in a cascade of events abating the HPV replication. Dendritic cell maturation and activation have also been found to be induced by imiquimod. We hypothesized that imiquimod may promote the development of DC at all levels of their life cycle. In this study, in vitro cultured monocyte-derived dendritic cells (MoDC) were used to evaluate the effect of imiquimod regarding the modulation of DC differentiation, terminal maturation and their function by phenotypic cell surface molecules expression, cytokine secretion and T cell stimulation in allogeneic system. We demonstrate that imiquimod exerts differential effects on DC biology at different stages of DC development. It inhibits the differentiation of DC, which may indicate a more potent antigen uptake activity. DC maturation is induced by imiquimod with an enhanced antigen presenting activity and IL-12 production. These evidence might be relevant with the clinically proven effectiveness of imiquimod in the treatment of genital warts.

[HPV DNA vaccines expressing recombinant CRT/HPV6bE7 fusion protein inhibit tumor growth and angiogenic activity].

This paper was to study the angiogenic inhibitory effect and the potential antitumor effect of the constructed recombinant DNA vaccine CRT/HPV6bE7 in vivo. The C57BL/6 mice were vaccinated respectively with recombinant CRT/HPV6bE7 DNA plamids. The inhibitory effects on angiogenesis of generated vaccines in vivo were evaluated by a bFGF-induced angiogenesis assay using the Matrigel kit. To investigate the potential antitumor effect, the mean tumor weights, sizes and tumor appearing times were measured in C57BL/6 mice treated with HPV6bE7-expressing B16 cells. The results indicated that the recombinants CRT180/HPV6bE7 and CRT180 showed strong anti-angiogenic effects in bFGF-induced angiogenesis in vivo. Moreover, CRT180/HPV6bE7 and CRT180 DNA vaccines could significantly inhibit the tumor growth in tumor challenge experiment, and CRT180/HPV6bE7 was superior to other vaccines in delaying tumor formation time, limiting tumor size and weight in tumor protection experiment. In conclusion, recombinant CRT180/HPV6bE7 DNA could elicit a most efficient anti-angiogenic effect and inhibit tumor growth in mice inoculated with DNA vaccines. The antiangiogenic activity of CRT were suggested residing in a domain between CRT 120-180 aa.