Changes of the Transcriptional Levels of Molecules Associated with Endogenous Antigen Processing and Presentation in Porcine Skin-derived Dendritic Cells Infected with PCV2 in vivo

[Objective] This study aimed to investigate the changes of the transcriptional levels of molecules associated with endogenous antigen processing and presenta- tion in porcine skin-derived dendritic cells infected with PCV2 in vivo. [Method] Healthy 40-day-old Landrace piglets were infected with porcine circovirus type 2 （PCV2） and euthanized on the 34, 7rd, 14th, 21st and 35th d post inoculation （DPI）. The porcine skin-derived dendritic cells （DCs） were collected to analyze the transcrip- tional levels of molecules （LMP7, UBP, MHC-I, calreticulin） associated with endogenous antigen processing and presentation by using real-time fluorescent quantitative PCR （real-time FQ-PCR）. [Result] The results showed that the level of LMP7 mR- NAs was reduced significantly on the 3DPI （P〈0.05）; the level of UBP mRNAs was consistently up-regulated, which increased significantly on the 21DPI and 35DPI （P〈 0.05）; the level of MHC-I mRNAs was significantly down-regulated on the 7DPI （P〈 0.05）; the level of calreticulin mRNAs was up-regulated slightly without significant dif- ference. [Conclusion] PCV2 can inhibit the endogenous antigen processing and presentation ability of porcine skin-derived DCs at early stages of infection.

Culture of Porcine Peripheral Blood Monocyte-derived Dendritic Cells in vitro

[Objective] This study aimed to establish an in vitro culture model for porcine peripheral blood monocyte-derived dendritic cells （MoDCs）. [Method] Fresh peripheral blood mononuclear cells （PBMCs） were separated from pig, and precursor dendritic cells were obtained by adherence method. The dendritic cells were treated by recombinant porcine granulocyte-monocyte colony stimulating factor （rpGM-CSF） and recombinant porcine interleukin-4 （rplL-4） together, and lipopolysaccharide （LPS） respectively. The cells in different time periods were collected. The morphology of the collected cells was observed by scanning electron microscopy; the expression of surface molecules and phagocytic ability to FITC-dextran were detected by flow cy- tometry; and the stimulating ability for allogeneic T cells was detected by mixed lymphocyte reaction. [Result] The DCs suffering maturation induction in vitro showed typical dendritic morphology; compared with those of DCs untreated by LPS, the cell surface expression of CDla, CD80, CD86, SLAII and CD172a of DCs treated by LPS was significantly increased, the phagocytic ability was reduced slightly, and the stimulating ability for allogeneic T cells was enhanced to some extent. [Conclusion] An in vitro culture method was successfully established for porcine MoDCs in this study, laying a foundation for further study on the role of porcine MoDCs in immunoregulation and anti-virus infection.

Immune therapy including dendritic cell based therapy in chronic hepatitis B virus infection

Hepatitis B virus （HBV） infection is a global public health problem. Of the approximately 2 billion people who have been infected worldwide, more than 400 million are chronic carriers of HBV. Considerable numbers of chronic HBV carriers suffer from progressive liver diseases. In addition, all HBV carriers are permanent source of this virus. There is no curative therapy for chronic HBV carriers. Antiviral drugs are recommended for about 10% patients, however, these drugs are costly, have limited efficacy, and possess considerable side effects. Recent studies have shown that immune responses of the host to the HBV are critically involved at every stage of chronic HBV infection： （1） These influence acquisition of chronic HBV carrier state, （2） They are important in the context of liver damages, （3） Recovery from chronic HBV-related liver diseases is dependent on nature and extent of HBV-specific immune responses. However, induction of adequate levels of HBV-specific immune responses in chronic HBV carriers is difficult. During the last one decade, hepatitis B vaccine has been administered to chronic HBV carriers as a therapeutic approach （vaccine therapy）. The present regimen of vaccine therapy is safe and cheap, but not so effective. A dendritic cell-based therapeutic vaccine has recently been developed for treating chronic HBV infection. In this review, we will discuss about the concept, scientific logics, strategies and techniques of development of HBV- specific immune therapies including vaccine therapy and dendritic cell-based vaccine therapy for treating chronic HBV infection.

Entecavir up-regulates dendritic cell function in patients with chronic hepatitis B

AIM: To investigate the in vitro effect of entecavir (ETV on the function of dendritic cells (DCs) derived from chronic hepatitis B (CHB) patients. METHODS: Mononuclear cells were isolated from peripheral blood of patients with CHB. DCs wer incubated with RPMI-1640 medium supplemented wit fetal bovine serum, IL-4, granulocyte-macrophag colony-stimulating factor (GM-CSF). DCs were treate with or without ETV on the fourth day. Cell surfac molecules, including CD1a, CD80, CD83 and HLA-DR were assessed by flow cytometry. Concentrations of IL- and IL-12 in the supernatant were assayed by enzyme linked immunosorbent assay (ELISA). The ability of th generated DCs to stimulate lymphocyte proliferation wa observed. RESULTS: Compared with CHB control group, th expression levels of CD1a (29.07 ± 3.20 vs 26.85 ± 2.80 CD83 (25.66 ± 3.19 vs 23.21 ± 3.10), CD80 (28.00 ± 2.7 vs 25.75 ± 2.51) and HLA-DR (41.96 ± 3.81 vs 32.20 ± 3.04) in ETV-treated group were higher (P < 0.05). ETV treated group secreted significantly more IL-12 (157.6 ± 26.85 pg/mL vs 132.60 ± 22.00 pg/mL (P < 0.05) an had a lower level of IL-6 in the culture supernatant (83.0 ± 13.88 pg/mL vs 93.60 ± 13.61 pg/mL, P < 0.05) tha CHB control group. The ability of DCs to stimulate th proliferation of allogeneic lymphocytes was increase in ETV-treated group compared with CHB control grou (1.53 ± 0.09 vs 1.42 ± 0.08, P < 0.05).CONCLUSION: Entecavir can enhance the biological activity of DCs derived from CHB patients.

Targeting hepatitis B virus antigens to dendritic cells by heat shock protein to improve DNA vaccine potency

AIM： To investigate a novel DNA vaccination based upon expression of the HBV e antigen fused to a heat shock protein （HSP） as a strategy to enhance DNA vaccine potency.METHODS： A pCMV-HBeAg-HSP DNA vaccine and a control DNA vaccine were generated. Mice were immunized with these different construct. Immune responses were measured 2 wk after a second immunization by a T cell response assay, CTL cytotoxicity assay, and an antibody assay in C57BL/6 and BALB/c mice. CT26-HBeAg tumor cell challenge test in vivo was Performed in BALB/c mice to monitor anti-tumor immune responses.RESULTS： In the mice immunized with pCMV-HBe-HSP DNA, superior CTL activity to target HBV-positive target cells was observed in comparison with mice immunized with pCMV-HBeAg （44% ± 5% vs 30% ± 6% in E： T 〉 50：1, P 〈 0,05）, ELISPOT assays showed a stronger T-cell response from mice immunized with pCMV-HBe- HSP than that from pCMV-HBeAg immunized animals when stimulated either with MHC class I or class Ⅱ epitopes derived from HBeAg （74% ± 9% vs 31% ± 6%, P 〈 0.01）. ELISA assays revealed an enhanced HBeAg antibody response from mice immunized with pCMV- HBe-HSP than from those immunized with pCMV-HBeAg. The lowest tumor incidence and the slowest tumor growth were observed in mice immunized with pCMV- HBe-HSP when challenged with CT26-HBeAg.CONCLUSION： The results of this study demonstrate a broad enhancement of antigen-specific CD4^＋ helper,CD8^＋ cytotoxic T-cell, and B-cell responses by a novel DNA vaccination strategy. They also proved a stronger antigen-specific immune memory, which may be superior to currently described HBV DNA vaccination strategies for the treatment of chronic HBV infection.

Avian hepatitis B viruses： Molecular and cellular biology, phylogenesis, and host tropism

The human hepatitis B virus （HBV） and the duck hepatitis B virus （DHBV） share several fundamental features. Both viruses have a partially double-stranded DNA genome that is replicated via a RNA intermediate and the coding open reading frames （ORFs） overlap extensively. In addition, the genomic and structural organization, as well as replication and biological characteristics, are very similar in both viruses. Host of the key features of hepadnaviral infection were first discovered in the DHBV model system and subsequently confirmed for HBV. There are, however, several differences between human HBV and DHBV. This review will focus on the molecular and cellular biology, evolution, and host adaptation of the avian hepatitis B viruses with particular emphasis on DHBV as a model system.

Uric acid enhances T cell immune responses to hepatitis B surface antigen-pulsed-dendritic cells in mice

AIM： To study the induction of T cellular immune responses in BALB/c mice immunized with uric acid and dendritic cells （DCs） pulsed with hepatitis B virus surface antigen （HBsAg）. METHODS： DCs were generated from bone-marrow cells of BABL/c mice, and then pulsed or unpulsed with HBsAg protein （HBsAg-pulsed-DCs or unpulsed-DCs） in vitro. BABL/c mice were immunized with HBsAg-pulsed- DCs （1 × 10^6） and uric acid, injected through the tail vein of each mouse. The mice in control groups were immunized with HBsAg-pulsed-DCs alone, unpulsed- DCs alone or 200 μg uric acid alone or PBS alone. The immunization was repeated 7 d later. Cytotoxic T lymphocytes （CTLs） in vivo were determined by the CFSE labeled spleen lysis assay. Spleen cells or spleen T cells were isolated, and re-stimulated in vitro with HBsAg for 120 h or 72 h. Production of IFN-γ, and IL-4 secreted by spleen cells were determined by ELISA method; proliferation of spleen T cells were detected by flow cytometry. RESULTS： The cytotoxicities of HBsAg-specific-CTLs, generated after immunization of HBsAg-pulsed-DCs and uric acid, were 68.63% ±11.32% and significantly stronger than that in the control groups （P 〈 0.01）. Compared with control groups, in mice treated with uric acid and HBsAg-pulsed-DCs, the spleen T cell proliferation to HBsAg re-stimulation was stronger （1.34 ± 0.093 vs 1.081±0.028, P 〈 0.01）, the level of IFN-t, secreted by splenocytes was higher （266.575 ± 51.323 vs 135.223 ±32.563, P 〈 0.01） , and IL-4 level wasower （22.385 ± 2.252 vs 40.598 ± 4.218, P 〈 0.01）. CONCLUSION： Uric acid can strongly enhance T cell immune responses induced by HBsAg-pulsed-DCs vaccine. Uric acid may serve as an effective adjuvant of DC vaccine against HBV infection.

Flow cytometry assay of myeloid dendritic cells (mDCs)in peripheral blood during acute hepatitis C:Possible pathogenetic mechanisms

AIM： To asses the expression of myeloid dendritic cells （CD11c＋） subset during acute HCV hepatitis and its possible involvement in natural history of the infection.METHODS： We enrolled 11 patients with acute hepatitis C （AHC） （Group A）, 10 patients with acute hepatitis A （AHA） （as infective control-Group B） and 10 healthy donors （group C） in this study. All patients underwent selective flow cytometry gating strategies to assess the peripheral number of the myeloid dendritic cells （mDCs） to understand the possible role and differences during acute hepatitis.RESULTS： Eight of 11 patients with acute HCV hepatitis did not show any increase of mDCs compared to healthy individuals, while a significant decrease of mDCs was found in absolute cell count （z=-2.37, P〈0.05） and percentage （z=-2.30; P〈 0.05） as compared with AHA. On the contrary, The remaining three patients of the group A had a higher mDCs number and percentage as occur in group B. Interestingly, after six months, those patients did not show any increase of mDCs subset were chronically infected, while the three subjects with an increase of peripheral mDCs, as in HAV acute infection, resolved the illness.CONCLUSION： The lack of increase of mDCs during acute hepatitis C might be an important factor involved in chronicization of the infection.

Therapeutic effect of autologous dendritic cell vaccine on patients with chronic hepatitis B: A clinical study

AIM: To investigate the therapeutic effect of autologous HBsAg-loaded dendritic cells (DCs) on patients with chronic hepatitis B. METHODS: Monocytes were isolated from fresh peripheral blood of 19 chronic HBV-infected patients by Ficoil-Hypaque density gradient centrifugation and cultured by plastic-adherence methods. DCs were induced and proliferated in the culture medium with recombinant human granulocyte-macrophage-colony- stimulating factor (rhGM-CSF) and human interleukin-4 (rhIL-4). DCs pulsed with HBsAg for twelve hours were injected into patients subcutaneously twice at intervals of two weeks. Two patients received 100 mg oral lamivudine daily for 12 mo at the same time. HBV-DNA and viral markers in sera of patients were tested every two months. RESULTS: By the end of 2003, 11 of 19 (57.9%) patients had a clinical response to DC-treatment. HBeAg of 10 (52.6%) patients became negative, and the copies of HBV-DNA decreased 101.77±2.39 averagely (t = 3.13, P<0.01). Two cases co-treated with DCs and lamivudine had a complete clinical response. There were no significant differences in the efficient rate between the cases with ALT level lower than 2xULN and those with ALT level higher than 2xULN before treatment (X2 = 0.0026). CONCLUSION: Autologous DC-vaccine induced in vitro can effectively suppress HBV replication, reduce the virus load in sera, eliminate HBeAg and promote HBeAg/anti-HBe transformation. Not only the patients with high serum ALT levels but also those with normal ALT levels can respond to DC vaccine treatment, and the treatment combining DCs with lamivudine can eliminate viruses more effectively.

Effects of CpG-ODNs on phenotype and function of monocyte-derived dendritic cells in chronic hepatitis B

AIM:To study the effects of synthetic nonmethylated CpG-containing oligodeoxynucleotides(CpG-ODNs) ,either alone or combined with recombinant Hepatitis B surface antigen(HBsAg) polypeptide,on the phenotype,function,and intracellular signaling pathways of monocyte-derived dendritic cells(DCs) in patients with chronic hepatitis B(CHB) .METHODS:Peripheral blood monocytes isolated from CHB patients and healthy volunteers were induced to be dendritic cells by recombinant human granulocyte-monocyte colony stimulating factor and interleukin-4.The DCs were then treated with CpG-ODNs,CpGODNs/HBsAg,or tumor necrosis factor(TNF)-αfor 18 h.The expression of surface molecules including HLA-DR,CD86,and CD1a in DCs were detected by flow cytometry,and the expression of signal transducers and activators of transcription(STAT1,3,4,5,6) and suppressors of cell signaling(SOCS1,3) were determined by Western blotting assay.In addition,the capacity of DCs to stimulate allogeneic T lymphocytes and the amount of IL-12p70 released from DCs were measured.RESULTS:In the DCs derived from patients with CHB,treatment with TNF-α,CpG-ODNs,or CpG-ODNs/HBsAg,as compared to the vector control,significantly increased the expression of HLA-DR,stimulated the release of IL-12p70,and enhanced the capacity of DCs to stimulate allogenic T lymphocytes.The expressions of STAT1/4/6 and SOCS1/3,but not STAT3/5,were upregulated by TNF-α,CpG-ODNs,and CpG-ODNs/HBsAg.In addition,the expression of CD1a was upregulated only in the presence of both CpG-ODNs and HBsAg.CONCLUSION:The treatment with CpG-ODNs,either alone or combined with HBsAg,has a remarkable stimulatory effect on the impaired phenotype and function of DCs in CHB,possibly by regulating the expression of STAT1,4,6 and SOCS1,3.

Influence of epigallocatechin gallate on the immune function of dendritic cells after ultraviolet B irradiation

To investigate the protective effect of epigallocatechin gallate （EGCG） on the immune function of dendritic cells （DCs） after ultraviolet B irradiation （UVB） and its underlying mechanisms, the monocytes were isolated from peripheral blood and cultivated into DCs with cytokines, such as GM-CSF and IL-4. DCs were harvested after cultivation for 7 d and subjected to irradiation with different dosages of UVB. Then, 200 μg/ml of EGCG were added in certain groups immediately after irradiation. DCs simply treated with UVB or treated with both UVB and EGCG were co-cultured with lymphocytes, and MTT assay was used to detect the ability of DCs to stimulate proliferation of lymphocytes. Surface markers CDS0, CD86, HLA-DR and CD40 were detected by flow cytometry, and the levels of IL-10 and IL-12 secreted from DCs 2d h after cultivation were measured by ELISA. It was demonstrated that UVB irradiation could inhibit the ability of DCs to stimulate the proliferation of lymphocytes and surface expressions of CDS0, CD86, HLA-DR and CD40 on DCs in a dose-dependent manner. The inhibition rate of DCs was improved to some extent after treatment with 200μg/ml of EGCG. When the concentra- tion of EGCG exceeded 100 μg/ml, the enhancing effect of EGCG on the expression of the co-stimulating molecules on DCs could be demonstrated in a dose-dependent manner. UVB showed no significant influence on the secretion of IL-10 and IL-12 from DCs, while EGCG could down-regulate the secretion level of IL-12 and up-regulate that of IL-10. It is concluded that EGCG can antagonize the inhibitory effect on DCs induced by UVB irradiation. This function has some relationship with its protecting effect of the expression of the co-stimulating molecule on the surface of DCs and the secretion level of IL-10 and IL-12.

Comparative Leaves Anatomical Studies of Bouea, Mangifera and Spondias （Anacardiaceae） in Malaysia

Leaves anatomy of two species of Bouea, 11 species of Mangifera and two species of Spondias were studied in order to see the differences in stomata type, petiole, midrib and lamina anatomy and leaf venation. This study aims to use anatomical characters for species and genus identification. Common characters observed were the absence of trichomes, closed vascular bundles, uniseriate epidermal layers, resin canal in parenchyma cells, anticline wall pattems and druses crystals in leaf lamina transverse sections. All species displayed closed vascular bundles except Mangiferapajang which showed a combination of medullary vascular bundles. Uniseriate epidermal layer was observed in all species. All the species showed straight-wavy anticlinal walls. Druses crystals were found in the parenchyma cells of all the species. Four types of stomata were observed namely anomocytic, anisocytic, staurocytic and diacytic. Anomocytic, anisocytic and staurocytic stomata were observed in Mangifera, diacytic in Bouea and anomocytic in Spondias.

Protective immunity against Rickettsia heilongjiangensis in a C3H/HeN mouse model mediated by outer membrane protein B-pulsed dendritic cells

Rickettsia heilongjiangensis is an obligate intracellular bacterium that causes Far-Eastern tick-borne spotted fever. Outer membrane protein B(Omp B) is an important surface protein antigen of rickettsiae. In the present study, the omp B gene of R. heilongjiangensis was divided into four fragments, resulting in four recombinant proteins(OmpB-p1, Omp B-p2, Omp B-p3, and Omp B-p4). Each Omp B was used in vitro to stimulate murine bone marrow-derived dendritic cells(BMDCs) of C3H/He N mice, and the Omp B-pulsed BMDCs were transferred to naive C3H/He N mice. On day 14 post-transfer of BMDCs, the mice were challenged with R. heilongjiangensis and the rickettsial loads in the mice were quantitatively determined on day 7 post-challenge. Mice receiving BMDCs pulsed with Omp B-p2, Omp B-p3, or Omp B-p4 exhibited significantly lower bacterial load compared with mice receiving Omp B-p1-pulsed BMDCs. CD4+ and CD8+ T cells isolated from the spleen of C3H/He N mice receiving BMDCs pulsed with each OmpB were co-cultured with BMDCs pulsed with the respective cognate protein. In flow cytometric analysis, the expression level of CD69 on CD4+ or CD8+ T cells from mice receiving BMDCs pulsed with Omp B-p2, OmpB-p3, or Omp B-p4 was higher than that on cells from mice receiving Omp B-p1-pulsed BMDCs, while the expression level of tumor necrosis factor(TNF)-α on CD8+ T cells and interferon(IFN)-γ on the CD4+ and CD8+ T cells from mice receiving Omp B-p2,-p3, or-p4 was significantly higher than on cells from mice receiving Omp B-p1-pulsed BMDCs. Our results suggest that the protective Omp Bs could activate CD4+ and CD8+ T cells and drive their differentiation toward CD4+ Th1 and CD8+ Tcl cells, respectively, which produce greater amounts of TNF-α and, in particular, IFN-γ, to enhance rickettsicidal activity of host cells.

Atomic force microscopy analysis of orientation and bending of oligodeoxynucleotides in polypod-like structured DNA

We previously demonstrated that polypod-like structured DNA, or polypodna, constructed with three or more oligodeoxynucleotides （ODNs）, is efficiently taken up by immune cells such as dendritic cells and macrophages, depending on its structural complexity. The ODNs comprising the polypodna should bend to form the polypod-like structure, and may do so by adopting either a bend- type conformation or a cross-type conformation. Here, we tried to elucidate the orientation and bending of ODNs in polypodnas using atomic force microscopy （AFM）. We designed two types of pentapodnas （i.e., a polypodna with five pods） using 60- to 88-base ODNs, which were then immobilized on DNA origami frames. AFM imaging showed that the ODNs in the pentapodna adopted bend-type conformations. Tetrapodna and hexapodna also adopted bend-type conformations when they were immobilized on frames under unconstrained conditions. These findings provide useful information toward the coherent design of, and the structure-activity relationships for, a variety of DNA nanostructures.

Reduced capacity of dendritic cells from trauma-hemorrhage mice in initiating delayed-type hypersensitivity to fluorescein isothiocyanate

Objective： To study the role of dendritic cells （DCs） in initiating delayed-type hypersensitivity （DTH） to fluores- cein isothiocyanate （FITC） after trauma-hemorrhage in mice. Methods： Inbred BALB/c mice （6-8 weeks old, male） were epicutaneously sensitized with FITC 12 hours, 1 day, 2 days, 4 days and 7 days after closed bilateral femur fractures combined with hemorrhage. And 5 days after sensitization, DTH was evaluated by ear swelling after a challenge of FITC. Draining lymph node cells were examined for the percentages of FITC-positive cells, cluster of differentiation （CD）11c positive cells and major histocom- patibility complex II （MHC II）-positive cells by means of flow cytometry. In vitro proliferative responses of syngeneic lymphocytes and in vivo passive transfer of DTH to naive recipients induced by isolated DCs from the draining lymph nodes were determined. Results： The time of DTH to FITC decreased more significantly in the mice with trauma-hemorrhage （12 hours to 4 days） than in the mice with sham injury. After sensitization, the relative percentages of FITC^＋ cells, FITC^＋/ CD 11c^＋ cells and FITC^＋/CD 11c^＋/MHC II^＋ cells from the draining lymph nodes were all significantly reduced following injury. And the capacity of DCs from the draining lymph nodes in stimulating proliferative responses of lymphocytes and transferring DTH to naive recipients were also inhibited after injury. Conclusions： Trauma-hemorrhage induces repressive DTH in mice, which may be attributed, at least partially, to the reduced trafficking of DCs into the draining lymph nodes and insufficient maturation during DC migration.