Critical Care Resources in Guangdong Province of China: Three Surveys from 2005 to 2015.

OBJECTIVES: Data about the critical care resources in China remain scarce. The purpose of this study was to investigate the variation and distribution of critical care resources in Guangdong province from 2005 to 2015. DESIGN: Data in regard to critical care resources were collected through questionnaires and visits every 5 years from 2005. SETTING: All hospitals in Guangdong province were screened and hospitals that provide critical care services were enrolled. INTERVENTION: None. MEASUREMENTS AND MAIN RESULTS: One hundred eleven, 158, and 284 hospitals that provide critical care services were enrolled in the three consecutive surveys respectively. The number of ICUs, ICU beds, intensivists, and nurses increased to 324, 3,956, 2,470, and 7,695, respectively, by 2015. Adjusted by population, the number of ICU beds per 100,000 (100,000) population increased by 147.7% from 2005 to 2015, and the number of intensivists and nurses per 100,000 population increased by 35.3% and 55.1% from 2011 to 2015. However, the numbers in the Pearl River Delta, a richer area, were higher than those in the non-Pearl River Delta area (ICU beds: 4.64 vs 2.58; intensivists: 2.90 vs 1.61; nurses: 9.30 vs 4.71 in 2015). In terms of staff training, only 17.85% of intensivists and 14.29% of nurses have completed a formal accredited critical care training program by 2015. CONCLUSIONS: Our study was the first one to investigate the trend and distribution of critical care resources in China. The quantity of ICU beds and staff has been increasing rapidly, but professional training for staff was inadequate. The distribution of critical care resources was unbalanced. Our study can be beneficial for healthcare policymaking and the allocation of critical care resources in Guangdong province and other provinces in China.

ADAMTS-7 exhibits elevated expression in cartilage of osteonecrosis of femoral head and has a positive correlation with TNF- α and NF- κ B P65.

ADAMTS-7 has been reported to exaggerate cartilage degeneration and to be associated with TNF-α and NF-κB signaling pathway. In this study we compared the expression of ADAMTS-7, TNF-α, and Phospho-NF-κB in patients with femoral neck fracture (FNF) and osteonecrosis of femoral head (ONFH) at different stages. We found that expression of ADAMTS-7, TNF-α, and Phospho-NF-κB was significantly upregulated in ONFH patients' articular cartilage and related to the pathogenesis of ONFH. Thus we conclude that ADAMTS-7 level appears to be positively associated with expression of TNF-α and Phospho-NF-κB P65 in cartilage, which may imply its association with cartilage destruction of ONFH.

H19 inhibits RNA polymerase II-mediated transcription by disrupting the hnRNP U-actin complex.

BACKGROUND: H19 was one of the earliest identified, and is the most studied, long noncoding RNAs. It is presumed that H19 is essential for regulating development and disease conditions, and it is associated with carcinogenesis for many types. However the biological function and regulatory mechanism of this conserved RNA, particularly with respect to its effect on transcription, remain largely unknown. METHODS: We performed RNA pulldown, RNA immunoprecipitation and deletion mapping to identify the proteins that are associated with H19. In addition, we employed EU (5-ethynyl uridine) incorporation, immunoprecipitation and Western blotting to investigate the functional aspects of H19. RESULTS: Our research further verifies that H19 is bound to hnRNP U, and this interaction is located within the 5' 882 nt region of H19. Moreover, H19 disrupts the interaction between hnRNP U and actin, which inhibits phosphorylation at Ser5 of the RNA polymerase II (Pol II) C-terminal domain (CTD), consequently preventing RNA Pol II-mediated transcription. We also showed that hnRNP U is essential for H19-mediated transcription repression. CONCLUSIONS: In this study, we demonstrate that H19 inhibits RNA Pol II-mediated transcription by disrupting the hnRNP U-actin complex. GENERAL SIGNIFICANCE: These data suggest that H19 regulates general transcription and exerts wide-ranging effects in organisms.

Hepatitis B virus X protein (HBx)-related long noncoding RNA (lncRNA) down-regulated expression by HBx (Dreh) inhibits hepatocellular carcinoma metastasis by targeting the intermediate filament protein vimentin.

UNLABELLED: The hepatitis B virus X protein (HBx) has been implicated as an oncogene in both epigenetic modifications and genetic regulation during hepatocarcinogenesis, but the underlying mechanisms are not entirely clear. Long noncoding RNAs (lncRNAs), which regulate gene expression with little or no protein-coding capacity, are involved in diverse biological processes and in carcinogenesis. We asked whether HBx could promote hepatocellular carcinoma (HCC) by regulating the expression of lncRNAs. In this study we investigated the alteration in expression of lncRNAs induced by HBx using microarrays and real-time quantitative polymerase chain reaction (PCR). Our results indicate that HBx transgenic mice have a specific profile of liver lncRNAs compared with wildtype mice. We identified an lncRNA, down-regulated expression by HBx (termed lncRNA-Dreh), which can inhibit HCC growth and metastasis in vitro and in vivo, act as a tumor suppressor in the development of hepatitis B virus (HBV)-HCC. LncRNA-Dreh could combine with the intermediate filament protein vimentin and repress its expression, and thus further change the normal cytoskeleton structure to inhibit tumor metastasis. We also identified a human ortholog RNA of Dreh (hDREH) and found that its expression level was frequently down-regulated in HBV-related HCC tissues in comparison with the adjacent noncancerous hepatic tissues, and its decrement significantly correlated with poor survival of HCC patients. CONCLUSION: These findings support a role of lncRNA-Dreh in tumor suppression and survival prediction in HCC patients. This discovery contributes to a better understanding of the importance of the deregulated lncRNAs by HBx in HCC and provides a rationale for the potential development of lncRNA-based targeted approaches for the treatment of HBV-related HCC.

Effects of self-blood on the molding process of polymethyl methacrylate bone cement.

OBJECTIVE: To evaluate whether the self-blood has influence on the molding process of polymethyl methacrylate (PMMA) bone cement, and to make sure whether it is valuable for the clinical practice. METHODS: An in vitro study was performed to evaluate the prolonging-effect of self-blood on PMMA bone cement. The effect of prolonging was evaluated by the dough time (TD) and operable time (TO). Moreover, hardness test, squeezing value test and peak temperature test were also conducted to complete the evaluation of this program. RESULTS: The self-blood, especially the plasma, could greatly prolong the handling time of PMMA bone cement without affecting its basic characteristics including hardness, leakage level and peak temperature. On the other hand, we found that in some abnormal conditions, for example with hyperlipemia, self-blood though can also prolong the handling time, would cause some side-effects. CONCLUSION: We report a new effective way to prolong the handling time of PMMA bone cement by adding moderate amount of self-blood. But "individualized medicine" should be noticed because some abnormal conditions like hyperlipemia would cause undesired side-effects.

Lentiviral-mediated transfer of CDNF promotes nerve regeneration and functional recovery after sciatic nerve injury in adult rats.

Peripheral nerve injury is often followed by incomplete and unsatisfactory functional recovery and may be associated with sensory and motor impairment of the affected limb. Therefore, a novel method is needed to improve the speed of recovery and the final functional outcome after peripheral nerve injuries. This report investigates the effect of lentiviral-mediated transfer of conserved dopamine neurotrophic factor (CDNF) on regeneration of the rat peripheral nerve in a transection model in vivo. We observed notable overexpression of CDNF protein in the distal sciatic nerve after recombinant CDNF lentiviral vector application. We evaluated sciatic nerve regeneration after surgery using light and electron microscopy and the functional recovery using the sciatic functional index and target muscle weight. HE staining revealed better ordered structured in the CDNF-treated group at 8 weeks post-surgery. Quantitative analysis of immunohistochemistry of NF200 and S-100 in the CDNF group revealed significant improvement of axonal and Schwann cell regeneration compared with the control groups at 4 weeks and 8 weeks after injury. The thickness of the myelination around the axons in the CDNF group was significantly higher than in the control groups at 8 weeks post-surgery. The CDNF group displayed higher muscle weights and significantly increased sciatic nerve index values. Our findings suggest that CDNF gene therapy could provide durable and stable CDNF protein concentration and has the potential to enhance peripheral nerve regeneration, morphological and functional recovery following nerve injury, which suggests a promising strategy for peripheral nerve repair.

Hepatitis C virus single-stranded RNA induces innate immunity via Toll-like receptor 7.

BACKGROUND/AIMS: Innate immune responses to HCV infection are triggered through host recognition of pathogen-associated molecular patterns. Interferons are critical for the protection against HCV infection. However, the pathways linking virus recognition to IFN induction remain poorly understood. METHODS: Immune cells and Huh-7 cells were infected with HCV cell culture (HCVcc) or transfected with HCV-derived immunostimulatory RNA oligonucleotides (ORNs), and immune activation was assessed. RESULTS: We found that HCVcc suppressed immune responses because the HCVcc protein impaired the PBMC and pDC responses. However, HCVcc genomic RNA had an immunostimulatory effect. HCV encodes G/U-rich ssRNA TLR7 ligands that significantly activate innate immunity, and induced IFN-alpha production. Moreover, HCV-derived ORNs also activated IRF7 and NF-kappaB in Huh-7 cells. In particular, the HCV 3'-UTR strongly induced cytokine production. Different lengths of polyuridine tract in the 3'-UTR of different HCV strains induced IFN-alpha production. These data demonstrate that the HCV-specific G/U fragment is a motif sequence, and is recognized by TLR7 as a PAMP. The requirement for TLR7 to recognize HCV RNA was confirmed using specific inhibitors, RNAi and by TLR7overexpression. CONCLUSION: These results provide an insight into the development of immune adjuvant for vaccines and for the production of new antiviral drugs.

EVI1 promotes cell proliferation in HBx-induced hepatocarcinogenesis as a critical transcription factor regulating lncRNAs.

UNLABELLED: The involvement of the hepatitis B virus X (HBx) protein in epigenetic modifications during hepatocarcinogenesis has been previously characterized. Long noncoding RNAs (lncRNAs), a kind of epigenetic regulator molecules, have also been shown to play crucial roles in HBx-related hepatocellular carcinoma (HCC). In this study, we analyzed the key transcription factors of aberrantly expressed lncRNAs in the livers of HBx transgenic mice by bioinformatics prediction, and found that ecotropic viral integration site 1 (Evi1) was a potential main transcription regulator. Further investigation showed that EVI1 was positively correlated to HBx expression and was frequently up-regulated in HBV-related HCC tissues. The forced expression of HBx in liver cell lines resulted in a significant increase of the expression of EVI1. Furthermore, suppression of EVI1 expression decreased the proliferation of HCC cells overexpressing HBx in vitro and in vivo. CONCLUSION: Our findings suggest that EVI1 is frequently up-regulated and regulates a cluster of lncRNAs in HBV-related hepatocellular carcinoma (HCC). These findings highlight a novel mechanism for HBx-induced hepatocarcinogenesis through transcription factor EVI1 and its target lncRNAs, and provide a potential new approach to predict the functions of lncRNAs.

Th17 cell frequency and IL-17 concentration correlate with pre- and postoperative pain sensation in patients with intervertebral disk degeneration.

Numerous studies have revealed the presence of T helper 17 (Th17) cells in pathologic intervertebral disk (IVD) tissues and the contribution of Th17-associated cytokines to the development of this disease. However, the pre- and postoperative changes in the proportion of Th17 cells and the concentration of IL-17 in the peripheral blood of patients with IVD degeneration are not clear. The levels of Th17 frequency and the interleukin-17 (IL-17) concentration in peripheral blood from patients and volunteers were examined by flow cytometry and by enzyme-linked immunosorbent assay (ELISA), respectively. The clinical results were evaluated using the visual analogue scale (VAS). These results were subjected to a correlation analysis. Compared with the normal controls, the proportion of Th17 cells and the concentration of IL-17 were significantly increased preoperatively in patients with IVD degeneration. Postoperatively, the levels of Th17 cells and the expression of IL-17 were dramatically decreased. The correlation analysis of the VAS pain scores, Th17 cell frequency, and IL-17 concentration, including the pre- and postoperative levels and the changes induced by the surgery, revealed a positive correlation. The authors' results explain the contribution of Th17 cells and IL-17 to the pain sensation experienced by patients with IVD degeneration. These 2 factors may be good indicators for the evaluation of the surgical outcome of patients with lumbar disk herniation.

A long noncoding RNA activated by TGF-ß promotes the invasion-metastasis cascade in hepatocellular carcinoma.

The role of TGF-ß-induced epithelial-mesenchymal transition (EMT) in cancer cell dissemination is well established, but the involvement of lncRNAs in TGF-ß signaling is still unknown. In this study, we observed that the lncRNA-activated by TGF-ß (lncRNA-ATB) was upregulated in hepatocellular carcinoma (HCC) metastases and associated with poor prognosis. lncRNA-ATB upregulated ZEB1 and ZEB2 by competitively binding the miR-200 family and then induced EMT and invasion. In addition, lncRNA-ATB promoted organ colonization of disseminated tumor cells by binding IL-11 mRNA, autocrine induction of IL-11, and triggering STAT3 signaling. Globally, lncRNA-ATB promotes the invasion-metastasis cascade. Thus, these findings suggest that lncRNA-ATB, a mediator of TGF-ß signaling, could predispose HCC patients to metastases and may serve as a potential target for antimetastatic therapies.

Mesencephalic astrocyte-derived neurotrophic factor inhibits oxygen-glucose deprivation-induced cell damage and inflammation by suppressing endoplasmic reticulum stress in rat primary astrocytes.

Astrocyte inflammation plays important roles both in physiological and pathological processes in the central nervous system (CNS). Ischemic injury in the CNS causes damage to astrocytes and the release of proinflammatory cytokines, such as tumor necrosis factor-α, interleukin-1ß, and interleukin-6. This current study investigates whether mesencephalic astrocyte-derived neurotrophic factor (MANF) inhibits oxygen-glucose deprivation (OGD)-induced cell damage and inflammatory cytokine secretion by suppressing endoplasmic reticulum stress in rat primary astrocytes. We found that MANF alleviated OGD-induced astrocyte damage and rescued the cell viability, and the upregulation of GRP78 (endoplasmic reticulum (ER) stress marker) and NF-κB p65 (one of the central mediators of proinflammatory pathways) induced by OGD were significantly reduced by preincubation of MANF. In addition, the increases of secretion and mRNA expression levels of the proinflammatory cytokines IL-1ß, IL-6, and TNF-α in astrocytes induced by OGD were significantly suppressed by MANF. These findings demonstrate that MANF shows the potential to alleviate cell damage and inflammation in rat primary astrocytes by suppressing ER stress, indicating that MANF plays an important role in astrocyte inflammation and functioning and may suggest a promising strategy for neuroprotection in the CNS.

Identification of an HLA-A*0201-restricted CD8(+) T-cell epitope encoded within Leptospiral immunoglobulin-like protein A.

Leptospirosis is an important zoonosis in humans. Immunity against leptospiral infection was thought to be primarily humoral, and limited studies have addressed the role of CD8(+)T cells. Leptospiral immunoglobulin-like protein A (LigA) is an important protective antigen of Leptospira and a potential target for Leptospira-specific cell-mediated immunity. In this study, twenty LigA-derived peptides were tested their binding affinity and stability for the HLA-A*0201 molecule. Peptides with high binding affinity and stability for HLA-A*0201 were then assessed their capacity to elicit specific cytotoxic T-lymphocyte (CTL) responses using cytotoxicity, ELISPOT assays for IFN-gamma and HLA-A*0201-peptide tetramer assays. We identified a HLA-A*0201-restricted epitope, LigA(305-313) KLIVTPAAL in Leptospira LigA. CTLs specific for LigA(305-313) were elicited both in HLA-A2.1/K(b) transgenic mice and in patients with a clinical and/or laboratory diagnosis of leptospirosis. Staining of the HLA-A*0201-LigA(305-313) tetramer revealed the presence of LigA(305-313)-specific CTLs in peripheral blood mononuclear cells (PBMCs) sourced from five patients infected with three different serovars of Leptospira. In conclusion, we report the existence of specific cytotoxic CD8(+)T cells in patients with leptospirosis and we suggest that the newly identified epitope, LigA(305-313), will be helpful in enhancing the understanding of the mechanism of immunity to leptospirosis.

A preliminary study on the activation and antigen presentation of hepatitis B virus core protein virus-like particle-pulsed bone marrow-derived dendritic cells.

Hepatitis B virus core protein virus-like particles (HBc-VLPs) act as a strong immunogen and are suitable for uptake by dendritic cells (DCs), in which they directly promote DC maturation and migration. To illustrate the utility of global proteomic analysis techniques in elucidating the molecular events that are altered in HBc-VLP-pulsed bone marrow-derived DCs (BMDCs) and to gain a better understanding of the molecular mechanisms of capture and processing of HBc-VLP-pulsed BMDCs, an antigen (Ag) delivery system based on HBc-VLP-pulsed BMDCs was developed. Two-dimensional electrophoresis (2-DE) and tandem mass spectrometry (MS/MS) analyses were utilized to analyze the differential protein expression patterns between HBc-VLP-pulsed and untreated BMDCs. Protein spots with significantly altered expression levels were detected, identified and validated. The results showed that exogenous HBc-VLPs were phagocytosed efficiently by BMDCs and enhanced the efficacy of BMDC maturation and Ag presentation, VLPs also induced high levels of Ag-specific CD8(+) T cells that displayed high cytotoxic T lymphocyte (CTL) activity in vivo. Several differentially expressed proteins, including growth factor receptor bound protein 2 (Grb2) and annexin A2 (AnxA2), were detected by proteomic analysis, identified by mass spectrometry and validated by western blot.

Optimization strategy for plasmid DNAs containing multiple-epitopes of foot-and-mouth disease virus by cis-expression with IL-2.

To optimize previous candidate DNA vaccine, a cis-expression plasmid DNA encoding two genes, human IL-2 and multiple-epitopes genes of foot-and-mouth disease virus (FMDV) was constructed with internal ribosome entry site (IRES) from encephalomyocarditis virus (EMCV) and intramuscularly inoculated into mice at 1-week interval. Specific antibodies in serum and cytokines (IL-2, IL-4 and IFN-gamma) from splenocytes were detected by indirect ELISA. Splenocytes proliferation rate was determined by a standard 3-(4,5-dimethylthiazol-2-yl) 2,5-diphenyltetrazolium bromide (MTT) method. The results showed that higher specific antibody, proliferate rate and cytokines were induced by plasmid DNA cis-expression with IL-2 compared to non-cis-expression plasmid DNA. Another series of mice were inoculated with plasmid DNA and boosted with antigenic protein. Specific antibody, proliferation rate and cytokines were induced significantly higher than those of mice immunized with protein or plasmid DNA only. However, only the cis-expression plasmid DNA elicited higher neutralization antibody in mice and provided one third protection against homologous virus in guinea pigs. In conclusion, cis-expression strategy with IL-2 up-regulated specific immunological response and provide protection against homologous virus.

Combined DNA vaccination against three animal viruses elicits decreased immunogenicity of a single plasmid in mice.

In order to investigate whether combined DNA vaccines are an ideal way to combine antigens in a single vaccine formulation, we immunized mice with three plasmids (pVSG, pVgD and pVE2), respectively, encoding the antigen of foot-and-mouth disease virus (FMDV), pseudorabies virus (PRV) and classic swine fever virus (CSFV), either alone or in a combined vaccine regimen. We also investigated the immune responses induced by a series of mixtures in which three plasmids were mixed in pairs. Then we further immunized mice with three different plasmids in separate sites and preformed an adoptive transfer experiment. While being given alone, each of the vaccine plasmids induced significant virus-specific antibody responses and splenocytes proliferative activity. But reduced immunogenicity of the pVSG plasmid was found in combined DNA vaccination, no matter whether it was injected in a single or a separate site. Removal single plasmid (pVgD or pVE2) from combined DNA vaccine led to significant increase in the immunogenicity of the pVSG plasmid (P<0.05). And the induction of immune suppression was not mediated by suppressor T cells, as demonstrated by an adoptive transfer experiment. Furthermore, by boosting with whole virus protein of FMDV, mice primed with either pVSG alone or combined DNA vaccine produced statistically significant increase in the FMDV-specific antibody titers (P<0.05). But after boosting, FMDV-specific splenocytes proliferative activity of mice primed with combined DNA vaccine was even lower than that of mice primed with pVSG alone (P<0.05). Taken together, this study reflected the immunogenicity of a single plasmid may be decreased in combined DNA immunization strategy, which still needs to be carefully evaluated before practical application.

Annexin B1 from Taenia solium metacestodes is a newly characterized member of the annexin family.

We previously reported cloning of the Taenia solium annexin B1 gene from a metacestode cDNA expression library and demonstrated that it acts as a protective antigen for effective vaccine development against cysticercosis. In the present study we produced recombinant annexin B1 and antiserum against the protein to investigate its structural and functional properties. Western blotting of metacestode fractions indicated that T. solium annexin B1, similar to vertebrate annexins, associates with acid phospholipids in the presence of Ca(2+). This property was confirmed by the recognition of apoptotic cells by labeled annexin B1. CD spectroscopy results demonstrated that alpha-helices are the main secondary structures of the protein. Ca(2+) binding increases the alpha-helix content and causes significant thermal stabilization with a melting temperature increase of approximately 10 degrees C. Functional Ca(2+)-dependent phospholipid binding sites of annexin B1 were investigated using mutant proteins. By changing a conserved acidic amino acid residue that putatively combines Ca(2+) in each domain of annexin B1 singly or in combination, we found that Ca(2+) binding in the first domain is more important than that at the other Ca(2+) binding sites. Annexin B1 is a metacestode stage-specific antigen, with the protein being mainly localized in the teguments and surrounding cyst wall of T. solium metacestodes, suggesting a role in the parasite-host interaction.

Comparative proteomics analysis to annexin B1 DNA and protein vaccination in mice.

DNA vaccines have been widely reported to elicit both effective humoral and cellular immune responses, but the mechanisms of antigen processing and presentation in DNA immunization is still ambiguous. Aiming to molecular mechanisms involved in DNA immunization, comparative serum proteomics was introduced to discover differentially expressed proteins after different immunizations. Using two-dimensional electrophoresis and matrix-assisted laser desorption ionisation-time-of-flight mass spectrometry, 23 three-fold or greater up-regulated proteins were separated and identified, including 14 from ANXB1 DNA immunized mice and 9 from annexin B1 protein immunized mice. The histocompatibility class I molecule H2-Q10 (HA10_MOUSE) and proteasome activator PA28 alpha-subunit (PSME1_MOUSE) were found up-regulated in ANXB1 DNA immunized mice, which may contribute to the augmented activation of T lymphocytes. These proteins may serve as potential surrogate markers of successful vaccination and provide research targets for molecular mechanisms of vaccinology.

16S rRNA gene probe quantitates residual host cell DNA in pharmaceutical-grade plasmid DNA.

The development and widespread use of DNA-based vaccination against infectious pathogens have been a great triumph of medical science. Quality control of DNA vaccines as biopharmaceutical productions is a problem to solve. Residual genomic DNA of engineering bacteria has been identified as a potential risk factor, so whose level must be controlled under the regulatory standards. We report a dot-blot hybridization method to detect residual host cell DNA in purified DNA vaccines. The assay utilizes PCR amplified and digoxigenin-labeled Escherichia coli 16S rRNA gene as probe. The sensitivity of the dot-blot hybridization assay with E. coli 16S rRNA gene probe was evaluated in comparison with single copy UidR gene probe. The optimized dot-blot hybridization assay had both low background and a suitable sensitivity, detecting 10 pg of residual E. coli DNA. The method is suitable in the routine use of measuring the levels of residual E. coli DNA in the pharmaceutical-grade DNA vaccine.

Effects of epitopes combination and adjuvants on immune responses to anti-Alzheimer disease DNA vaccines in mice.

Alzheimer disease (AD) is a neurodegenerative disorder characterized by neuropathological hallmarks including deposits of the beta-amyloid peptide (AssP). Studies have shown that immunization with Abeta42 peptide reduces both the spatial memory impairments and Alzheimer disease-like neuropathologic changes in Alzheimer disease transgenic mice, but can cause side effect of a cell-mediated autoimmune meningoencephalitis. Recently, some studies showed that DNA vaccination could be used to generate an antibody response to Abeta without the adverse cell-mediated immune effect. In the current study, we generate four DNA vaccine plasmids (pV-GE1, pV-GE2, pV-GE3, and pV-GE4) against Alzheimer disease by separately fusing Abeta epitope sequences (coding for EFGH, DAEFGH, EFGH+EFGH, and EFGH+DAEFGH) with IgG heavy chain coding region of mouse. Meanwhile, the full-length gene Abeta encoding plasmid (pV-Abeta), empty vector (pVAX) and synthetic AssP were also included as control. The sera of BALB/c mice immunized via intramuscular with plasmids and peptide were tested by indirect ELISA for auto-AssP immunoreactivity. The results showed that all the DNA vaccine plasmids induced AssP-specific antibodies; moreover pV-GE2 and pV-Abeta constructs elicited higher antibody titers than other constructs (P < 0.05). To further enhance the immune response, GM-CSF encoding plasmid (pGM-CSF) and purified BCG-DNA were used as molecular adjuvants. BCG-DNA could enhance humoral and cellular immune responses simultaneously and did not alter the phenotype of the immune responses, whereas pGM-CSF showed no obvious effect on immune response. These results suggest that this immunization strategy of using Abeta epitope encoding plasmid plus BCG-DNA adjuvant may serve as the basis for developing anti-Alzheimer disease vaccines.

Induction of mucosal and systemic immune response by single-dose oral immunization with biodegradable microparticles containing DNA encoding HBsAg.

The purpose of this work was to assess the ability of plasmid DNA encoding hepatitis B virus (HBV) HBsAg encapsulated in poly(DL-lactide-co-glycolic acid) (PLGA) microparticles to induce local and systemic HBsAg-specific immunity following a single dose of oral immunization. RT-PCR analysis demonstrated prolonged transcription of plasmid DNA, consistent with the sustained expression and presentation of target antigen observed by confocal laser scanning microscopy, in gut-associated lymphocyte tissue (GALT) from mice immunized orally with plasmid DNA encapsulated into PLGA microparticles. Oral administration of PLGA-DNA microparticles induced a long-lasting and stable antigen-specific antibody response, both serum total antibody and intestinal IgA, in BALB/c mice. Mice immunized orally exhibited antigen-specific gamma interferon production and cytotoxic T lymphocyte responses in spleen and GALT after restimulation in vitro with HBsAg or tumour cells stably expressing HBsAg. In contrast, naked DNA vaccines given by intramuscular injection induced only systemic cellular and humoral responses to HBsAg, which were much lower than the responses elicited by oral DNA encapsulated in PLGA microparticles at equivalent doses. The results are encouraging with regard to obtaining good compliance and vaccination coverage with candidate plasmid DNA vaccines, especially in developing countries.