CpG Groups Research Articles

Metal-Organic Coordination Polymer for Delivery of a Subunit Broadly Acting Influenza Vaccine.

A zinc-carnosine (ZnCar) metal-organic coordination polymer was fabricated in biologically relevant N-(2-hydroxyethyl)piperazine-N'-ethanesulfonic acid (HEPES) buffer for use as a vaccine platform. In vitro, ZnCar exhibited significantly less cytotoxicity than a well-established zeolitic imidazolate framework (ZIF-8). Adsorption of CpG on the ZnCar surface resulted in enhanced innate immune activation compared to soluble CpG. The model antigen ovalbumin (OVA) was encapsulated in ZnCar and exhibited acid-sensitive release in vitro. When injected intramuscularly on days 0 and 21 in C57BL/6 mice, OVA-specific serum total IgG and IgG1 were significantly greater in all groups with ZnCar and antigen compared to soluble controls. Th1-skewed IgG2c antibodies were significantly greater in OVA and CpG groups delivered with ZnCar for all time points, regardless of whether the antigen and adjuvant were co-formulated in one material or co-delivered in separate materials. When broadly acting Computationally Optimized Broadly Reactive Antigen (COBRA) P1 influenza hemagglutinin (HA) was ligated to ZnCar via its His-tag, significantly greater antibody levels were observed at all time points compared to soluble antigen and CpG. ZnCar-formulated antigen elicited increased peptide presentation to B3Z T cells in vitro and production of IL-2 after ex vivo antigen recall of splenocytes isolated from vaccinated mice. Overall, this work displays the formation of a zinc-carnosine metal-organic coordination polymer that can be applied as a platform for recombinant protein-based vaccines.

The Association Between RAPSN Methylation in Peripheral Blood and Early Stage Lung Cancer Detected in Case-Control Cohort.

BackgroundEarly detection is essential to improve the survival and life quality of lung cancer (LC) patients. Changes of peripheral blood DNA methylation could be associated with malignancy but were mostly studied in Caucasians.MethodsHere, in a Chinese population, we performed mass spectrometry assays to investigate the association between very early stage LC and methylation levels of RAPSN in the peripheral blood by a case–control cohort using of 221 LC patients (93.2% LC at stage I) and 285 unrelated cancer free control individuals.ResultsThe odds ratios (ORs) of all CpG sites were evaluated for their risk to LC using inter-quartile analyses by logistic regression. In general, we observed an association between very early LC and decreased methylation of RAPSN_CpG_1.15 and RAPSN_CpG_3.4 (referring to Q4, OR range from 1.64 to 1.81, p<0.05). Stratified by gender, while hypomethylation of RAPSN_CpG_1.15, RAPSN_CpG_3.4 and RAPSN_CpG_7.14 were associated with LC in males (referring to Q4, ORs range from 1.94 to 2.31, p<0.05), RAPSN_CpG_2 and RAPSN_CpG_5 showed significantly lower methylation in female LC patients comparing to controls (referring to Q4, ORs range from 2.49 to 3.60, p<0.05). The risk of RAPSN hypomethylation to LC was enhanced by aging, and typically for people older than 55 years (referring to Q4, ORs range from 2.17 to 3.61 in six out of all 10 analyzed CpG groups, p<0.05).ConclusionOur study reveals an association between RAPSN hypomethylation in peripheral blood and LC and suggests the occurrence of altered blood-based methylation at the early stage of cancer.

Clearance of Brucella with formulation rCagA, TN-OMPs & LPS in mice

The development of vaccines against Brucella is necessary. The formulations rCagA/OMP/LPS were evaluated against the clearance of Brucella. All of the mouse groups were immunized three times orally and followed two times by IM. Two weeks after the last immunization mice were challenged with B. abortus strain 544. Blood and spleen samples were collected from mice.Antigen-specific interleukine responses were measured in the spleen of immunized mice before and post-challenge by ELISA. Specific IgG1,2 isotopes were measured from sera before and after challenge. Sandwich ELISA assays were used to determine IFN-γ, IL-4 and IL-10 levels in the supernatants of the splenocyte cells. Statistical analysis of IFN-γ titers indicated a significant difference between all groups with Mock (p = 0.00). However, there was no significant difference between other groups. Furthermore, the analysis of IL-10 titers had similar results. The statistical analysis for IL-4 showed a significant difference between rCagA with LPS-rCagA conjugate and LPS-rCagA+ CpG groups (p = 0.001). Statistical analysis between IFN-γ and IL-4 titers showed a significant difference among all groups (p = 0.00). Statistical analysis between IFN and IL-10 titers showed a significant difference among all groups (p = 0.00). Statistical analysis between IL-10 and IL-4 titers showed no significant difference in any group. Clearance finally was 7 log with OMP/rCagA against B. abortus strain 544. Immunization with the current combination was safe in animal models and was able to induce appropriate responses and also decrease Brucella colonization.

In silico studies reveal RSc1154 and RhlE as temperature-related pathogenic proteins of Ralstonia solanacearum.

This study aimed to explore the temperature-related pathogenic mechanism of Ralstonia solanacearum infection in tomato (Lycopersicon esculentum Mill.). Based on bioinformatics analysis of microarray dataset (GSE33657), the co-differentially expressed genes (co-DEGs) ribonucleic acids were identified in R. solanacearum GMI1000-infected L. esculentum Mill., which was cultured at 20°C and 28°C, in rich medium containing casamino acids, peptone, and glucose (CPG) and planta. In total, 63 upregulated co-DEGs and 57 downregulated co-DEGs were identified between 20°C and 28°C in the CPG and planta groups. Protein-protein interaction network revealed 70 protein interaction pairs and 59 nodes. Notably, iolG, iolE, ioll and RSc1248 played critical roles in the network. The subcellular localization and functional annotation showed that the increased expressed proteins were mainly localized in the inner cell membrane, while those with decreased expression were localized in the cytoplasm. Furthermore, these proteins were mainly enriched in regulation of DNA-templated transcription. RSc1154 and RhlE were predicted to be temperature-related pathogenic genes for R. solanacearum in tomato. Furthermore, phosphorelay signal transduction system function might play an important role in R. solanacearum infection. The candidate genes were verified by quantitative real-time PCR, and the results were consistent with gene expression profile.

Low-Dose Prasugrel vs Clopidogrel-Based Tailored Premedication for Endovascular Treatment of Cerebral Aneurysms.

Antiplatelet premedication is widely accepted for interventional treatment of cerebral aneurysms to prevent thromboembolism. However, antiplatelet resistance sometimes limits the effectiveness of premedication. To compare 2 groups administered low-dose prasugrel (PSG group) or clopidogrel-based tailored antiplatelet (CPG group) in terms of platelet function and procedure-related complications. A total of 411 patients with 505 unruptured aneurysms who underwent endovascular treatment within the past 17 mo were retrospectively enrolled in this study. The PSG (225 patients with 277 aneurysms) and CPG groups (186 patients with 228 aneurysms) were administered the respective medication prior to endovascular treatment. We measure the response to the antiplatelet medication with a laboratory test. Episodes of periprocedural bleeding and thromboembolism were compared between the 2 groups. There were significant differences between the 2 groups in terms of the mean P2Y12 reaction unit values (125.7 in the PSG group vs 251.0 in the CPG group; P<.001) and percentage inhibition (57.8% vs 18.7%, respectively; P<.001). Drug resistance was 29.6% per patient in the CPG group and 2.7% per patient in the PSG group. The PSG group reported 1 thromboembolism and bleeding each; meanwhile, the CPG group reported 7 thromboembolism and 3 bleeding. Compared to clopidogrel administration, prasugrel administration significantly decreased the risk of thromboembolism (weighted hazard ratio, 0.17; 95% confidence interval, 0.03-0.99). However, the risk of bleeding was not significant. Prasugrel was found to be more effective in reducing periprocedural thromboembolism compared to clopidogrel.

Immunomodulatory asthma therapy in the equine animal model: A dose-response study and evaluation of a long-term effect.

IntroductionEquine asthma represents a naturally occurring animal model for human allergic neutrophilic asthma. Inhalative nanoparticle‐bound cytosine‐phosphate‐guanosine (CpG‐GNP) immunotherapy, independent of specific allergens, has already shown promising clinical and immunological results in previous studies and offers the possibility to treat the underlying cause of the disease. This study analyses the relationship between dose and response, and evaluates a possible long‐term effect.MethodsIn the prospective, randomised, double‐blind clinical field study, 29 horses suffering from equine asthma received 10 inhalation treatments with either 187.5 µg CpG‐GNP (CpG single dose [CpGsd]; n = 11), 375 µg CpG‐GNP double dose (CpG double dose [CpGdd]; n = 9) (q48h for 20 days) or 1600 µg beclomethasone (n = 9) (q24h for 10 days). Each horse was examined three times: before the treatment (I), immediately after the 10 inhalations (II), and 8 weeks after the final inhalation (III). The three groups were compared according to clinical and laboratory parameters. The study examined the sustainability of the long‐term effect of the treatment after 8 weeks, as well as the tolerability of the formula as a double dose.ResultsThe CpGsd resulted in a significant improvement in 82% of the parameters, the CpGdd in 72%. In the long‐term evaluation, the CpGsd showed a significant improvement in 100% of the parameters in comparison to the initial values, the CpGdd in 67%. On the immunological level, the bronchoalveolar lavage revealed a significant reduction of IL‐4, IL‐8, and interferon‐γ.ConclusionBoth CpG groups displayed significant improvements in clinical and laboratory parameters, especially regarding the long‐term effect of CpGsd. Doubling the CpG dose did not result in any improvement in comparison to the original single dose. On the immunological level, an anti‐inflammatory, as well as an immunomodulatory effect, apart from a Th2‐dominated immune response, could be observed. This immunomodulatory inhalation treatment could indicate a new possibility for human allergic asthma therapy.

The Cambridge Prognostic Groups for improved prediction of disease mortality at diagnosis in primary non-metastatic prostate cancer: a validation study

BackgroundThe purpose of this study is to validate a new five-tiered prognostic classification system to better discriminate cancer-specific mortality in men diagnosed with primary non-metastatic prostate cancer.MethodsWe applied a recently described five-strata model, the Cambridge Prognostic Groups (CPGs 1-5), in two international cohorts and tested prognostic performance against the current standard three-strata classification of low-, intermediate- or high-risk disease. Diagnostic clinico-pathological data for men obtained from the Prostate Cancer data Base Sweden (PCBaSe) and the Singapore Health Study were used. The main outcome measure was prostate cancer mortality (PCM) stratified by age group and treatment modality.ResultsThe PCBaSe cohort included 72,337 men, of whom 7162 died of prostate cancer. The CPG model successfully classified men with different risks of PCM with competing risk regression confirming significant intergroup distinction (p < 0.0001). The CPGs were significantly better at stratified prediction of PCM compared to the current three-tiered system (concordance index (C-index) 0.81 vs. 0.77, p < 0.0001). This superiority was maintained for every age group division (p < 0.0001). Also in the ethnically different Singapore cohort of 2550 men with 142 prostate cancer deaths, the CPG model outperformed the three strata categories (C-index 0.79 vs. 0.76, p < 0.0001). The model also retained superior prognostic discrimination in the treatment sub-groups: radical prostatectomy (n = 20,586), C-index 0.77 vs. 074; radiotherapy (n = 11,872), C-index 0.73 vs. 0.69; and conservative management (n = 14,950), C-index 0.74 vs. 0.73. The CPG groups that sub-divided the old intermediate-risk (CPG2 vs. CPG3) and high-risk categories (CPG4 vs. CPG5) significantly discriminated PCM outcomes after radical therapy or conservative management (p < 0.0001).ConclusionsThis validation study of nearly 75,000 men confirms that the CPG five-tiered prognostic model has superior discrimination compared to the three-tiered model in predicting prostate cancer death across different age and treatment groups. Crucially, it identifies distinct sub-groups of men within the old intermediate-risk and high-risk criteria who have very different prognostic outcomes. We therefore propose adoption of the CPG model as a simple-to-use but more accurate prognostic stratification tool to help guide management for men with newly diagnosed prostate cancer.

Effects of a subunit vaccine (FlaA) and immunostimulant (CpG-ODN 1668) against Vibrio anguillarum in tilapia (Oreochromis niloticus)

Vibriosis, caused by Vibrio anguillarum, is a common bacterial disease in cultured fish. Treatment of vibriosis can lead to the development of antibiotic resistance or tolerance. Therefore, alternative methods for combatting vibriosis are needed. In this study, we investigated the effects of a subunit vaccine (recombinant flagellin-A [rFlaA]) and adjuvant (CpG-ODN 1668 [CpG]) candidate against V. anguillarum in tilapia (Oreochromis niloticus). In an in vivo experiment, fish in the rFlaA+CpG and rFlaA-only groups were first treated with CpG or PBS, followed by vaccination with rFlaA the next day. In the control group, both vaccine and adjuvant were replaced by PBS. The highest survival rate (60%) was observed in fish treated with rFlaA+CpG, compared with 0% in PBS-treated fish or 35% in fish immunized with rFlaA-only. Moreover, sera purified from tilapia treated with rFlaA+CpG exhibited significantly stronger agglutination activity (approximately 3-fold higher) than sera obtained from control fish, whereas sera from fish treated with rFlaA-only showed values 2.5-fold higher than those in control fish. The bacterial survival rates were also lower in sera from tilapia treated with rFlaA+CpG (41%) than in sera from fish treated with rFlaA alone (61%) or control fish (100%). Thus, fish treated with both rFlaA and CpG exhibited the highest immune activation in terms of agglutination and bactericidal activity. In summary, FlaA could be a promising subunit vaccine, and CpG-ODN 1668 could be applied as an adjuvant for immunization against V. anguillarum.

The effect of ataxia-telangiectasia mutated kinase-dependent hyperphosphorylation of checkpoint kinase-2 on oligodeoxynucleotide 7909 containing CpG motifs-enhanced sensitivity to X-rays in human lung adenocarcinoma A549 cells.

ObjectiveThe aim of the study reported here was to further investigate the potential effect of ataxia-telangiectasia mutated (ATM) kinase-dependent hyperphosphorylation of checkpoint kinase-2 (Chk2) on radiosensitivity enhanced by oligodeoxynucleotide 7909 containing CpG motifs (CpG ODN7909) in human lung adenocarcinoma A549 cells.MethodsIn vitro A549 cells were randomly separated into control, CpG, X-ray, CpG+ X-ray, ATM kinase-small interfering RNA (siRNA)+CpG+X-ray (ATM-siRNA), and Chk2-siRNA+CpG+X-ray (Chk2-siRNA) groups. siRNAs were adopted to silence the ATM and Chk2 genes. Expression and phosphorylation of ATM kinase and Chk2 were detected by Western blot assay. Cell colonies were observed under inverted phase-contrast microscopy. Cellular survival curves were fitted using a multi-target single-hitting model. Cell cycle and apoptosis were analyzed by flow cytometry.ResultsExpression of ATM kinase and Chk2 was similar among the control, CpG, X-ray, and CpG+X-ray groups. Phosphorylated ATM kinase and Chk2 were significantly increased in the CpG+X-ray group compared with in the X-ray group (t=6.00, P<0.01 and t=3.13, P<0.05, respectively), though these were hardly detected in the control and CpG groups. However, expression of ATM kinase and Chk2 was clearly downregulated in the ATM-siRNA and Chk2-siRNA groups, respectively. Similarly, their phosphorylation levels were also significantly decreased in the ATM-siRNA group (t=14.35, P<0.01 and t=8.46, P<0.01, respectively) and a significant decrease in phosphorylated Chk2 was observed in the Chk2-siRNA group (t=7.28, P<0.01) when compared with the CpG+X-ray group. Further, the number of A549 cells at Gap 2/mitotic phase and the apoptosis rate were clearly increased in the CpG+X-ray group compared with in the other groups (all P<0.05). The multi-target single-hitting model showed that the sensitization enhancement ratio calculated by mean death dose was 1.39 in CpG+X-ray group (vs 1.04 and 1.03 in the ATM-siRNA and Chk2-siRNA groups, respectively).ConclusionThis study provides the first evidence, as far as we are aware, that CpG ODN7909 can potentiate A549 cell radiosensitivity via increasing ATM kinase-dependent phosphorylation of Chk2, suggesting activation of the ATM kinase/Chk2 signal pathway. However, the mechanism of ATM kinase activation is worth further exploration.

The Effectiveness of recombinant OL fusion protein (ovalbumin-LHRH-7) in suppressing reproductive functions when injected in single-dose vaccination protocols with different adjuvants

The objective of this study was to evaluate the effectiveness of recombinant LHRH fusion protein, Ovalbumin-LHRH-7 (OL), using a single-dose vaccination protocol in combination with different adjuvants in suppressing reproductive functions in buck kids. For this purpose, either a mixture of free OL antigen and encapsulated OL antigen, or encapsulated OL antigen was used. Thirty-nine native buck kids at 12 weeks of age were divided into control (n=7) and treatment groups (n=8 bucks/group). The four treatment groups were formed according to the different vaccine formulations: Group CpG received 0.5mg free OL protein together with 1.0mg of encapsulated protein with CpG adjuvant. Group mFCA received 0.5mg free OL protein together with 1.0mg of encapsulated protein with modified Freund's complete adjuvant. Group IS received 1.5mg encapsulated OL protein with a mix of inulin and saponin adjuvants. Group ISmFCA received 1.5mg encapsulated OL protein with a mix of inulin, saponin and modified Freund's complete adjuvants. Scrotal circumference in CpG and mFCA groups were significantly smaller than that of Control, IS and ISmFCA groups (P<0.05). Numbers and percentage of bucks having spermatozoa in their ejaculate were significantly lower in CpG and mFCA groups (P<0.05). OL immunization completely suppressed sperm production, except one buck, in CpG and mFCA groups (P<0.05). These results imply that it is possible to use OL protein in a single injection protocol for the purpose of immunocastration. Further investigation with a larger number of animals should be carried out to determine the longevity of response to a single injection.

The development and preliminary evaluation of a newMycobacterium tuberculosisvaccine comprising Ag85b, HspX and CFP-10:ESAT-6 fusion protein with CpG DNA and aluminum hydroxide adjuvants

Ag85b and HspX of Mycobacterium tuberculosis (Mtb) (H37Rv) were expressed and purified in this study. These two proteins were combined with another fusion protein CFP-10:ESAT-6 (C/E) (Ag), then mixed with the adjuvants CpG DNA and aluminum hydroxide and used to vaccinate mice and guinea pigs challenged with Mtb (H37Rv). The number of spleen lymphocytes secreting Ag85b, HspX and C/E-specific interferon-gamma were significantly higher in the Ag+Al+CpG group than in the Ag and CpG groups. The combination of Ag, Al and CpG induced the highest concentrations of anti-Ag85b, anti-HspX and anti-C/E immunoglobulin G in mouse serum. Mouse peritoneal macrophages from the Ag+Al+CpG group secreted significantly higher levels of interleukin-12 compared with macrophages from the other groups. The total mean liver, lung and spleen lesion scores and bacterial loads in the spleen in guinea pigs vaccinated with Ag+Al+CpG were lower than those of the other groups, but no significant difference was found. These results show that the mixture of Ag85b, HspX and C/E with a combination of CpG and aluminum adjuvants can induce both humoral and cellular immune responses in mice, whereas it plays only a small role in the control of disease progression in guinea pigs challenged with Mtb.

Phase 1 Trial of the Plasmodium falciparum Blood Stage Vaccine MSP142-C1/Alhydrogel with and without CPG 7909 in Malaria Naïve Adults

BackgroundMerozoite surface protein 142 (MSP142) is a leading blood stage malaria vaccine candidate. In order to induce immune responses that cover the major antigenic polymorphisms, FVO and 3D7 recombinant proteins of MSP142 were mixed (MSP142-C1). To improve the level of antibody response, MSP142-C1 was formulated with Alhydrogel plus the novel adjuvant CPG 7909.MethodsA Phase 1 clinical trial was conducted in healthy malaria-naïve adults at the Center for Immunization Research in Washington, D.C., to evaluate the safety and immunogenicity of MSP142-C1/Alhydrogel +/− CPG 7909. Sixty volunteers were enrolled in dose escalating cohorts and randomized to receive three vaccinations of either 40 or 160 µg protein adsorbed to Alhydrogel +/− 560 µg CPG 7909 at 0, 1 and 2 months.ResultsVaccinations were well tolerated, with only one related adverse event graded as severe (Grade 3 injection site erythema) and all other vaccine related adverse events graded as either mild or moderate. Local adverse events were more frequent and severe in the groups receiving CPG. The addition of CPG enhanced anti-MSP142 antibody responses following vaccination by up to 49-fold two weeks after second immunization and 8-fold two weeks after the third immunization when compared to MSP142-C1/Alhydrogel alone (p<0.0001). After the third immunization, functionality of the antibody was tested by an in vitro growth inhibition assay. Inhibition was a function of antibody titer, with an average of 3% (range −2 to 10%) in the non CPG groups versus 14% (3 to 32%) in the CPG groups.Conclusion/SignificanceThe favorable safety profile and high antibody responses induced with MSP142-C1/Alhydrogel + CPG 7909 are encouraging. MSP142-C1/Alhydrogel is being combined with other blood stage antigens and will be taken forward in a formulation adjuvanted with CPG 7909.Trial RegistrationClinicalTrials.gov Identifier: NCT00320658

Preparation of human papillomavirus 16 E7 peptide vaccine and its effectiveness in vitro and in vivo.

To prepare the human papillomavirus (HPV) 16 peptide vaccine and explore the effect in vitro and in vivo. (1) Prediction of the major histocompatibility complex (MHC) class I restricted T cell epitopes by bioinformatics target at transporter associated with antigen processing (TAP) and named by E7Pa, E7Pb, E7Pc separately. (2) In vivo, the C57BL/6 mice were divided into five groups with same amounts randomly after loading with TC-1 cells (HPV 16 positive tumor cells from C57BL/6 mouse), named as E7Pa + CpG, E7Pb + CpG, E7Pc + CpG (as experiment groups, and added 50 microg/ml E7Pa, E7Pb, E7Pc, respectively), CpG (as positive control group and added Con A with 12 mg/L final concentration) and blank control group (without any treatment). The T cell proliferation was detected by methyl thiazolyl tetrazolium (MTT) assay at different time points; the lactate dehydrogenase (LDH) delivery method was used to test the cytolytic T lymphocyte (CTL) activity of mouse splenic lymphocyte in different ratio of effector cells and target cells (E:T); the related cytokines in tumor tissue and mouse peripheral blood were evaluated by real-time PCR and enzyme-linked immunosorbent assay (ELISA), respectively. The tumor volumes were measured to contrast the therapeutic effect in different groups. (1) Three peptide named E7Pa, E7Pb, E7Pc were successfully preparated which had high affinity and specificity. (2) After vaccination of 24, 48, 72, 96 hours, MTT results shown that the proliferation rate in E7Pa + CpG group were (131 +/- 32)%, (302 +/- 15)%, (552 +/- 28)%, (731 +/- 24)% individually, which were much higher than those in blank control [(72 +/- 15)%, (120 +/- 57)%, (176 +/- 41)%, (288 +/- 29)%; P < 0.01], and the other groups i.e. E7Pb + CpG, E7Pc + CpG and CpG groups all proliferated much higher than those in blank control group with statistic signification (P < 0.05), but there was no significant difference between groups (P > 0.05); the LDH delivery assay showed that when the ratio of E:T was 100:1, the activity of CTL in the E7Pa + CpG group was most powerful than the other groups with statistic signification (P < 0.01).Meanwhile, the ratio of E:T was concentration-dependent. Compared E7Pb + CpG, E7Pc + CpG or CpG groups with blank control group, there were significantly difference (P < 0.05), while there was no significant difference between groups (P > 0.05). The mRNA levels of interferon gamma (IFN-gamma), interleukin-2 (IL-2) in tumor tissue and peripheral blood in E7Pa + CpG group were significantly higher than those in blank control group (P < 0.01), which was the similar results when compared E7Pb + CpG, E7Pc + CpG or CpG groups with control group (P < 0.05), and without significant difference between groups (P > 0.05). The tumor volumes were suppressed obviously in all the experiment groups, especially at the 60th days, the volumes in E7Pa + CpG group were much smaller than that in blank control group with statistic signification (P < 0.01), which was the similar results that E7Pb + CpG, E7Pc + CpG or CpG groups had difference than blank control group with statistic signification (P < 0.05), and without significant difference between groups (P > 0.05). The HPV16 E7 peptide target at TAP combination with CpG as a vaccine could treat effectively the HPV16 E7 positive tumor in experiment.

An empirical typology of lifetime and current gambling behaviors: Association with health status of older adults

Purpose: Despite the low prevalence of gambling problems, older adults experience poorer health status given certain vulnerabilities associated with aging. Thus, we aimed to classify lifetime (LPG) and current (CPG) problem gambling patterns, identify determinants of gambling patterns, and examine their association with current health status Methods: Using older adult gamblers (n = 489) in the Gambling Impact and Behavior Study, Latent Class Analysis classified LPG and CPG subgroups based on 10 DSM-IV criteria: preoccupation, tolerance, withdrawal, loss of control, escape, chasing losses, lying, illegal acts, relationship impairment and financial bailout. Results: A two-class solution was the best fitting for LPG and CPG groups. Except for illegal acts, the remaining criteria endorsed the distinguishing patterns. We observed 10.8% LPGs, 8.4% CPGs and 2.2% with both. Participation in religious services was protective of both groups. Gambling for excitement and to win money were related to CPG. Further, CPG was significantly related to worse self-rated health. Implications: Although problem gambling is strongly characterized by number and type of diagnostic criteria, findings support a focus to include targeted assessment of additional clinically meaningful gambling correlates. Research on the moderator of participation in faith-based communities on problem gambling is also warranted.

CpG-ODN combined with Neospora caninum lysate, but not with excreted-secreted antigen, enhances protection against infection in mice

CpG oligodeoxynucleotides (ODN) have shown to be potent immunoadjuvants for several pathogens, but there is limited information concerning their use in immunization protocols against neosporosis. This study aimed to evaluate the potential of CpG-ODN combined with Neospora lysate antigen (NLA) or excreted-secreted antigen (NcESA) to induce protective immune response against Neospora caninum infection in mice. C57BL/6 mice were vaccinated subcutaneously three times at 2-week intervals with NLA, NLA + CpG, NcESA, NcESA + CpG, CpG (adjuvant control) or PBS (infection control). Serological assays showed an increased specific IgG2a response in animals immunized with either antigen plus adjuvant and elevated levels of the IgG1 isotype in those vaccinated with antigens alone. Splenocyte proliferative responses upon antigen stimulation were higher in groups immunized with NLA or NcESA combined with CpG, showing increased IL-12 levels. Also, mice vaccinated with NcESA or NcESA + CpG demonstrated higher IFN-γ levels and IFN-γ/IL-10 ratio. After lethal challenge, mice immunized with NLA + CpG or NLA had lower morbidity score and body weight changes in comparison to other groups, and animals did not succumb during acute infection. In contrast, NcESA + CpG or NcESA groups exhibited the highest morbidity scores, body weight impairment and mortality rates, associated with greatest brain parasite burden and inflammation. In conclusion, CpG-ODN was able to induce a Th1-type humoral immune response with predominant IgG2a levels for either NLA or NcESA, but resulting in an effective Th1-driven cellular immune response and total protection only when combined with NLA. Vaccination with NcESA alone or combined with CpG resulted in a strong cellular immune response associated with high levels of IFN-γ and inflammation, rendering mice more susceptible to parasite challenge.

26 DNA METHYLATION PATTERNS ARE APPROPRIATELY ESTABLISHED IN THE SPERM OF BULLS GENERATED BY SOMATIC CELL NUCLEAR TRANSFER AFTER PASSAGE THROUGH THE GERMLINE

Cloning of domestic animals by somatic cell nuclear transfer (SCNT) has permitted the rescue of valuable genetics and has the potential to allow rapid dissemination of desirable traits in production animals through the use of cloned sires. Whilst cloned animals may show developmental deviations and aberrant DNA methylation suggestive of incomplete nuclear reprogramming, it is widely accepted that their offspring are normal, as any aberrant epigenetic marks are believed to be corrected on passage of the genome through the germline. We assessed the extent of reprogramming by comparing DNA methylation patterns in sperm of SCNT bulls (n = 4) with sperm from bulls generated by AI (n = 5) and with the nuclear donor somatic cells (adult skin fibroblasts). The genomic regions examined were 3 repetitive sequences (satellites 1, 2, and alpha) and CpG islands in 5 genes [HAND1, LIT1, MASH2, IGF2, Dickkopf-1(DKK-1)]. Semen was collected from 16-month-old bulls and assessed for volume, sperm number, morphology, and motility. DNA was extracted from washed sperm and somatic donor cells, bisulfite-treated and processed for quantification of CpG methylation using the Sequenom MassArray system. Methylation levels at individual CpG sites/groups of CpGs were compared between sample groups using the t-test with pooled variances. No apparent difference was detected in semen characteristics between SCNT and AI bulls. Sperm DNA methylation levels were very low in single copy genes with the exception of the CpG island in IGF2, which has previously been shown to be completely methylated in sperm. At all genomic regions examined, each CpG site or CpG groups were methylated to different levels, and each region had a distinctive profile, which was almost invariant between individual sperm samples from either the SCNT or AI bulls. In all sites examined, there were no significant differences in methylation profiles between sperm from SCNT and AI bulls. In contrast, DNA methylation profiles were significantly different between SCNT bull sperm and the donor cells. The exception was the CpG island in MASH2, which was essentially unmethylated in both. For the 3 satellite sequences along with LIT1, HAND1, and to a lesser extent, the DKK-1 region, DNA was significantly less methylated in sperm than in the donor cells. Only IGF2 was significantly more methylated in SCNT and AI sperm than in the donor cells at 10/25 CpG sites (P &lt; 0.02). The results indicate that gametes from SCNT bulls had different epigenotypes from the donor somatic cells. This is the first molecular evidence that donor cell genomes have been reprogrammed in these SCNT bulls and that after going through the germline had acquired DNA methylation profiles that were similar to AI-derived bulls. It also suggests that any epigenetic aberrations that SCNT bulls may harbor are unlikely to be passed on to their offspring through their gametes. Supported by FRST contract C10X0311.

HCV core protein immunization with Montanide/CpG elicits strong Th1/Th2 and long-lived CTL responses

An efficient vaccine against Hepatitis C virus (HCV) infection requires induction of strong humoral and cellular responses against viral proteins. We evaluated the immunogenicity of HCV core protein (HCVcp), a prime vaccine candidate, formulated in various human compatible adjuvants. An Escherichia coli-expressed HCVcp, purified in native conditions was used for murine immunization in separate groups of: free HCVcp (Ag), Ag+C/IFA (Freunds), Ag+CpG, Ag+M720 (Montanide ISA 720), Ag+F127 (Pluronic acid) and cocktails of Ag+F127+CpG and Ag+M720+CpG. Mice immunized with M720(+CpG) developed the highest HCVcp-specific titers of total IgG, IgG1, 2a, 2b, and that of IFN-γ and IL-4 cytokines compared to all other groups. HCVcp-specific-CTLs against relevant MHC class I peptides were detected only for Ag+M720+CpG, Ag+M720, and Ag+CpG groups and could be blocked by antimouse-CD8 antibodies. While CTLs were stable, only F127 formulated groups demonstrated detectable IgG antibodies one year post-immunization. Hence, HCVcp formulated in M720 (with a synergistic effect by inclusion of CpG) could induce balanced and strong Th1/Th2 responses with long-lived CD4 −CD8 + CTLs.

The human papillomavirus-18 genome is efficiently targeted by cellular DNA methylation

Human papillomaviruses (HPVs) infect epithelia, including the simple and the squamous epithelia of the cervix, where they can cause cancer and precursor lesions. The molecular events leading from asymptomatic HPV infections to neoplasia are poorly understood. There is evidence that progression is modulated by transcriptional mechanisms that control HPV gene expression. Here, we report the frequent methylation of HPV-18 genomes in cell culture and in situ. DNA methylation is generally known to lead to transcriptional repression due to chromatin changes. We investigated two cell lines derived from cervical cancers, namely, C4-1, which contains one HPV-18 genome, and different clones of HeLa, with 50 HPV-18 genomes. By restriction cleavage, we detected strong methylation of the L1 gene and absence of methylation of parts of the long control region (LCR). A 3-kb segment of the HPV-18 genomes downstream of the oncogenes was deleted in both cell lines. Bisulfite sequencing showed that in C4-1 cells and two HeLa clones, 18 of the 19 CpG residues in the 1.2-kb terminal part of the L1 gene were methylated, whereas a third HeLa clone had only eight methylated CpG groups, indicating changes of the methylation pattern after the establishment of the HeLa cell line. In the same four clones, none of the 12 CpG residues that overlapped with the enhancer and promoter was methylated. In six HPV-18 containing cancers and five smears from asymptomatic patients, most of the CpG residues in the L1 gene were methylated. There was complete or partial methylation, respectively, of the HPV enhancer in three of the cancers, and lack of methylation in the remaining eight samples. The promoter sequences were methylated in three of the six cancers and four of the six smears, and unmethylated elsewhere. Our data show that epithelial cells efficiently target HPV-18 genomes for DNA methylation, which may affect late and early gene transcription.

Humoral immunoreaction induced by TCR DNA vaccine for β chain of T cell lymphoma

We exploited the humoral immunoreaction of mice induced by TCR DNA vaccine of β chain of T cell lymphoma. The plasmids of pcDNA3.1/TCR V β 8 was constructed. The BALB/c mice were randomly divided into four groups which were pcDNA3.1, pcDNA3.1/TCR V β 8, pcDNA3.1/TCR V β 8 + CpG + liposome and phosphorothioate CpG groups with six mice in each group. Vaccines were injected in bilateral musculus quadriceps femoris of mice in the 0, second, and fourth week, respectively. The antibody formation was tested by indirect immuofluorescence in the 0, second, fourth, sixth and eighth weeks, respectively, before and after immunization. Production of antibody against TCR V β 8 antigen was observed in the groups of pcDNA3.1/TCR V β 8 and pcDNA3.1/TCR V β 8 + CpG + liposome. The antibody titer began to rise in the fourth week and attain the maximal value in the sixth week. The antibody titer in the group of pcDNA3.1/TCR V β 8 + CpG + liposome was higher than that in the group of pcDNA3.1/TCR V β 8 in the fourth and eighth weeks (both P<0.01); the antibody titer in the group of pcDNA3.1/TCR V β 8 + CpG + liposome was markedly higher than that in the group of pcDNA3.1/TCR V β 8 in the sixth week ( P<0.001). The result indicate that TCR V β 8 antigen can induce formation of special antibody in mice. CpG and liposome can improve the humoral immunoreaction induced by TCR V β 8 gene vaccine.