Physical linkage to drug resistance genes results in conservation of var genes among west pacific Plasmodium falciparum isolates

The multicopy var gene family encoding the variant surface antigen Plasmodium falciparum erythrocyte membrane protein 1 is highly diverse, with little overlap between different P. falciparum isolates. We report 5 var genes (varS1-varS5) that are shared at relatively high frequency among 63 genetically diverse P. falciparum isolates collected from 5 islands in the West Pacific region. The varS1, varS2, and varS3 genes were localized to the internal region on chromosome 4, similar to 200 kb from pfdhfr-ts, whereas varS4 and varS5 were mapped to an internal region of chromosome 7, within 100 kb of pfcrt. The presence of varS2 and varS3 were significantly correlated with the pyrimethamine-resistant pfdhfr genotype, whereas varS4 was strongly correlated with the chloroquine-resistant pfcrt genotype. Thus, the conservation of these var genes is the result of their physical linkage with drug-resistant genes in combination with the antimalarial drug pressure in the region.

The functional effects of ceramide on the monocyte CD36 scavenger receptor and NADPH oxidase

Atherosclerosis is the principal cause of death in the United States, Europe and much of Asia. During the last decade, inflammation has been suggested to play a key role in the development of atherosclerosis. Reactive oxygen species (ROS) released during inflammation additionally oxidize LDL, which is subsequently taken up in an unregulated way through scavenger receptors on macrophages to form foam cells, the hallmark of atherosclerotic lesions. Previous work has shown that the lipid ceramide, which is found in aggregated LDL and in atherosclerotic plaques, decreases intracellular peroxide most likely through reducing NADPH oxidase activity. Ceramide is an important component of membrane microdomains called lipid rafts which are important for membrane protein function. Endogenous ceramide enhances lipid raft f'ormation and alters theirs composition. NADPH oxidase membrane subunits cytochrome b558 (which includes gp91) strongly associates with lipid rafts Therefore present study investigated whether short chain ceramides reduce NADPH oxidase in U937 monocytes by disrurting the membrane component of NADPH oxidase. Results showed that C2 ceramide alters the distribution of raft marker, flottillin and the raft environment. NADPH oxidase membrane component gp9J phox and cytosolic component p47 phox were identified in rafts. C2 ceramide reduces both gp91 and p47 phox in rafts, which leads to the decrease of peroxide production by NADPH oxidase. Ceramide is also an important second messenger involved in many different signaling pathways associated with atherogenesis from the activation of sphingomyelinase (SMase). It has been reported that SMase enhances LDL receptor mediated LDL endocytosis. However, no study has been done to investigate the effect of ceramide on scavenger receptors such as CD36 and oxidized LDL (OxLDL) uptake. CD36 is the major recertor far OxLDL. Reduced CD36 expression results in less foam cell formation and less atherosclerotic lesion without disrupting the clearance of OxLDL from plasma. This thesis shows that ceramides significantly reduce CD36 surface expression on U937 monocytes, macrophages and human primary monocytes. This effect is seen using both synthetic short chain ceramide and SMase catalysed long chain ceramide treatment. To investigate whether the effect of ceramide on CD36 is functional, OxLOL uptake was measured in ceramide treated cells. Ceramide reduces the uptake of OxLOL by both U937 monocytes and PMA-differentiated macrophages. The mechanism of ceramide reduction of CD36 expression was studied by measuring the surface antigen using flow cytometry and fluorescence microscopy, whole cellular CD36 expression and shedding of C036 by Western blotting of cell lysates and cell culture supernatants and mRNA level of CD36 using RT-PCR. Ceramide reduces shedding of CD36, activates mRNA expression of CD36 and induces intracellular CD36 accumulation probably through retaining the receptor inside cells. In summary, ceramides modulate several of the processes involved in LOL oxidation and uptake by CD36 receptors on monocytes/macrophages in a way which may protect against atherosclerosis.

In silico identified CCR4 antagonists target regulatory T cells and exert adjuvant activity in vaccination

Adjuvants are substances that enhance immune responses and thus improve the efficacy of vaccination. Few adjuvants are available for use in humans, and the one that is most commonly used (alum) often induces suboptimal immunity for protection against many pathogens. There is thus an obvious need to develop new and improved adjuvants. We have therefore taken an approach to adjuvant discovery that uses in silico modeling and structure-based drug-design. As proof-of-principle we chose to target the interaction of the chemokines CCL22 and CCL17 with their receptor CCR4. CCR4 was posited as an adjuvant target based on its expression on CD4(+)CD25(+) regulatory T cells (Tregs), which negatively regulate immune responses induced by dendritic cells (DC), whereas CCL17 and CCL22 are chemotactic agents produced by DC, which are crucial in promoting contact between DC and CCR4(+) T cells. Molecules identified by virtual screening and molecular docking as CCR4 antagonists were able to block CCL22- and CCL17-mediated recruitment of human Tregs and Th2 cells. Furthermore, CCR4 antagonists enhanced DC-mediated human CD4(+) T cell proliferation in an in vitro immune response model and amplified cellular and humoral immune responses in vivo in experimental models when injected in combination with either Modified Vaccinia Ankara expressing Ag85A from Mycobacterium tuberculosis (MVA85A) or recombinant hepatitis B virus surface antigen (rHBsAg) vaccines. The significant adjuvant activity observed provides good evidence supporting our hypothesis that CCR4 is a viable target for rational adjuvant design.

Liposome (Lipodine™)-mediated DNA vaccination by the oral route

Plasmid DNA pRc/CMV HBS encoding the S (small) region of hepatitis B surface antigen (HBsAg) was incorporated by the dehydration-rehydration method into Lipodine™ liposomes composed of 16 μmoles phosphatidylcholine (PC) or distearoyl phosphatidylcholine (DSPC), 8 μmoles of (dioleoyl phosphatidylethanolamine (DOPE) or cholesterol and 4 μmoles of the cationic lipid 1,2-dioleoyl-3-(trimethylammonium propane (DOTAP) (molar ratios 1:0.5:0.25). Incorporation efficiency was high (89-93% of the amount of DNA used) in all four formulations tested and incorporated DNA was shown to be resistant to displacement in the presence of the competing anionic sodium dodecyl sulphate molecules. This is consistent with the notion that most of the DNA is incorporated within the multilamellar vesicles structure rather than being vesicle surface-complexed. Stability studies performed in simulated intestinal media also demonstrated that dehydration-rehydration vesicles (DRV) incorporating DNA (DRV(DNA)) were able to retain significantly more of their DNA content compared to DNA complexed with preformed small unilamellar vesicles (SUV-DNA) of the same composition. Moreover, after 4h incubation in the media, DNA loss for DSPC DRV(DNA) was only minimal, suggesting this to be the most stable formulation. Oral (intragastric) liposome-mediated DNA immunisation studies employing a variety of DRV(DNA) formulations as well as naked DNA revealed that secreted IgA responses against the encoded HBsAg were (as early as three weeks after the first dose) substantially higher after dosing with 100 μg liposome-entrapped DNA compared to naked DNA. Throughout the fourteen week investigation, IgA responses in mice were consistently higher with the DSPC DRV(DNA) liposomes compared to naked DNA and correlated well with their improved DNA retention when exposed to model intestinal fluids. To investigate gene expression after oral (intragastric) administration, mice were given 100 μg of naked or DSPC DRV liposome-entrapped plasmid DNA expressing the enhanced green fluorescent protein (pCMV.EGFP). Expression of the gene, in terms of fluorescence intensity in the draining mesenteric lymph nodes, was much greater in mice dosed with liposomal DNA than in animals dosed with the naked DNA. These results suggest that DSPC DRV liposomes containing DNA (Lipodine™) may be a useful system for the oral delivery of DNA vaccines.

Liposome-entrapped plasmid DNA:characterisation studies

Plasmid DNA pRc/CMV HBS (5.6 kb) (100 microg) encoding the S (small) region of hepatitis B surface antigen was incorporated by the dehydration-rehydration method into liposomes composed of 16 micromol egg phosphatidylcholine (PC), 8 micromol dioleoylphosphatidylcholine (DOPE) and 1, 2-diodeoyl-3-(trimethylammonium)propane (DOTAP) (cationic liposomes) or phosphatidylglycerol (anionic liposomes) in a variety of molar ratios. The method, entailing mixing of small unilamellar vesicles (SUV) with the DNA, followed by dehydration and rehydration, yielded incorporation values of 95-97 and 48-54% of the DNA used, respectively. Mixing of preformed cationic liposomes with 100 microg plasmid DNA also led to high complexation values of 73-97%. As expected, the association of DNA with preformed anionic liposomes was low (9%). Further work with cationic PC/DOPE/DOTAP liposomes attempted to establish differences in the nature of DNA association with the vesicles after complexation and the constructs generated by the process of dehydration/rehydration. Several lines of evidence obtained from studies on vesicle size and zeta-potential, fluorescent microscopy and gel electrophoresis in the presence of the anion sodium dodecyl sulphate (SDS) indicate that, under the conditions employed, interaction of DNA with preformed cationic SUV as above, or with cationic SUV made of DOPE and DOTAP (1:1 molar ratio; ESCORT Transfection Reagent), leads to the formation of large complexes with externally bound DNA. For instance, such DNA is accessible to and can be dissociated by competing anionic SDS molecules. However, dehydration of the DNA-SUV complexes and subsequent rehydration, generates submicron size liposomes incorporating most of the DNA in a fashion that prevents DNA displacement through anion competition. It is suggested that, in this case, DNA is entrapped within the aqueous compartments, in between bilayers, presumably bound to the cationic charges.

Fatty acids, monocytes and ageing

Elevated free fatty acids (FFA) are a feature of ageing and a risk factor for metabolic disorders such as cardiovascular disease (CVD) and type-2 diabetes (T2D). Elevated FFA contribute to insulin resistance, production of inflammatory cytokines and expression of adhesion molecules on immune cells and endothelial cells, risk factors for CVD and T2D. Molecular mechanisms of FFA effects on monocyte function and how FFA phenotype is affected by healthy ageing remain poorly understood. This thesis evaluated the effects of the two major FFA in plasma, oleate and palmitate on monocyte viability, cell surface antigen expression, and inflammatory activation in THP-1 monocytes. Palmitate but not oleate increased cell surface expression of CD11b and CD36 after 24h, independent of mitochondrial superoxide, but dependent on de novo synthesis of ceramides. LPS-mediated cytokine production in THP-1 monocytes was enhanced and decreased following incubation with palmitate and oleate respectively. In a model of monocyte-macrophage differentiation, palmitate induced a pro-inflammatory macrophage phenotype which required de novo ceramide synthesis, whilst oleate reduced cytokine secretion, producing a macrophage with enhanced clearance apoptotic cells. Plasma fatty acid analysis in young and mid-life populations revealed age-related increases in both the SFA and MUFA classes, especially the medium and very long chain C14 and C24 fatty acids, which were accompanied by increases in the estimated activities of desaturase enzymes. Changes were independently correlated with increased PBMC CD11b, plasma TNF-a and insulin resistance. In conclusion, the pro-atherogenic phenotype, enhanced LPS responses in monocytes, and pro-inflammatory macrophage in the presence of palmitate but not oleate is reliant upon de novo ceramide synthesis. Age-related increases in inflammation, cell surface integrin expression are related to increases in both the MUFA and SFA fatty acids, which in part may be explained by altered de novo fatty acid synthesis.

HBsAg seroclearance or seroconversion induced by peg-interferon alpha and lamivudine or adefovir combination therapy in chronic hepatitis B treatment: a meta-analysis and systematic review

Background and aims: Seroclearance or seroconversion of hepatitis B surface antigen (HBsAg) is generally considered as a clinical endpoint. The purpose of the present meta-analysis was to evaluate the effect of combined therapy with pegylated interferon alpha (PEG-IFNα) with or without lamivudine (LAM) or adefovir (ADV) on HBsAg seroclearance or seroconversion in subjects with chronic hepatitis B (CHB). Methods: Randomized controlled trials performed through May 30th 2015 in adults with CHB receiving PEG-IFNα and LAM or ADV combination therapy or monotherapy for 48-52 weeks were included. The Review Manager Software 5.2.0 was used for the meta-analysis. Results: No statistical differences in HBsAg seroclearance (9.9% vs. 7.1%, OR = 1.47, 95% CI: 0.75, 2.90; p = 0.26) or HBsAg seroconversion (4.2% vs. 3.7%, OR = 1.17, 95% CI: 0.57, 2.37; p = 0.67) rates were noticed between PEG-IFNα + LAM and PEG-IFN α + placebo during post-treatment follow-up for 24-26-weeks in subjects with hepatitis Be antigen (HBeAg)-positive CHB. No statistical differences in HBsAg clearance (10.5% vs. 6.4%, OR = 1.68, 95% CI: 0.75, 3.76; p = 0.21) were seen, but statistical differences in HBsAg seroconversion (6.3% vs. 0%, OR = 7.22, 95% CI: 1.23, 42.40; p = 0.03) were observed, between PEG-IFNα + ADV and PEG-IFNα for 48-52 weeks of treatment in subjects with HBeAg-positive CHB. A systematic evaluation showed no differences in HBsAg disappearance and seroconversion rates between PEG-IFNα + placebo and PEG-IFNα + LAM for 48-52 weeks in subjects with HBeAg-positive CHB. A systematic assessment found no differences in HBsAg disappearance and seroconversion rates between PEG-IFNα + placebo and PEG-IFNα + LAM during 24 weeks' to 3 years' follow-up after treatment in subjects with HBeAg-negative CHB. Conclusion: Combined therapy with PEG-IFNα and LAM or ADV was not superior to monotherapy with PEG-IFNα in terms of HBsAg seroclearance or seroconversion.

Efficacy of entecavir and tenofovir in chronic hepatitis B under treatment in the public health system in southern Brazil

There are about 350 million hepatitis B virus (HBV) carriers worldwide and chronic HBV is considered a major public health problem. The objective of the present study was to assess the effectiveness of the nucleos(t)ide analogues tenofovir (TDF) and entecavir (ETV) in the treatment of chronic HBV. A cross-sectional study was carried out from March-December 2013, including all patients with chronic HBV, over 18 years of age, undergoing therapy through the public health system in southern Brazil. Only the data relating to the first treatments performed with TDF or ETV were considered. Retreatment, co-infection, transplanted or immunosuppressed patients were excluded. Six hundred and forty patients were evaluated, of which 336 (52.5%) received TDF and 165 (25.8%) ETV. The other 139 (21.7%) used various combinations of nucleos(t)ide analogues and were excluded. The negativation of viral load was observed in 87.3% and 78.8% and the negativation of hepatitis B e antigen was achieved in 79% and 72% of those treated with ETV or TDF, respectively. Negativation of hepatitis B surface antigen was not observed. There was no occurrence of adverse effects. This is a real-life study demonstrating that long-term treatment with ETV and TDF is both safe and effective.

Original Research Article Humoral and cellular immune responses to modified hepatitis B plasmid DNA vaccine in mice

Purpose: To evaluate the immunogenicity and types of immune response of a quality-controlled modified recombinant hepatitis B surface antigen (HBsAg) plasmid encoding HBsAg in mice. Methods: The characterized plasmid DNA was used in the immunization of Balb/c mice. Three groups of mice were intramuscularly injected with three different concentrations (50, 25 and 10 μg/100 μL) of the modified plasmid. Humoral immune response was monitored by enzyme-linked immunosorbent assay (ELISA), while cellular immune response was investigated by analysis of spleen cytokine profile (TNFα, IFN γ and IL2) as well as CD69 expression level in CD4 and CD8 positive cells. Results: In general, the activated CD4 cells showing intracellular cytokines were higher than CD8 positive population of cells (p < 0.05). These findings indicate that the vaccine induced both a humoral and cellular immunity. Cytokine profile also showed high levels of TNFα, IFN γ and IL2 and CD69 expression in the group of animals immunized at a dose of 10 μg when compared to control group (p < 0.05). Conclusion: A 10 μg dose intramuscular injection of the modified DNA-based vaccine encoding HBsAg in mice induces both high humoral and cellular immune responses.

The severity of chorioamnionitis in pregnant sheep is associated with in vivo variation of the surface-exposed multiple-banded antigen/gene of ureaplasma parvum

Ureaplasma species are the bacteria most frequently isolated from human amniotic fluid in asymptomatic pregnancies and placental infections. Ureaplasma parvum serovars 3 and 6 are the most prevalent serovars isolated from men and women. We hypothesized that the effects on the fetus and chorioamnion of chronic ureaplasma infection in amniotic fluid are dependent on the serovar, dose, and variation of the ureaplasma multiple banded antigen (MBA) and mba gene. We injected high- or low dose U. parvum serovar 3, serovar 6, or vehicle intra-amniotically into pregnant ewes at 55 days of gestation (term = 150 days) and examined the chorioamnion, amniotic fluid, and fetal lung tissue of animals delivered by cesarean section at 125 days of gestation. Variation of the multiple banded antigen/mba generated by serovar 3 and serovar 6 ureaplasmas in vivo were compared by PCR assay and Western blot. Ureaplasma inoculums demonstrated only one (serovar 3) or two (serovar 6) MBA variants in vitro, but numerous antigenic variants were generated in vivo: serovar 6 passage 1 amniotic fluid cultures contained more MBA size variants than serovar 3 (P = 0.005),and ureaplasma titers were inversely related to the number of variants (P = 0.025). The severity of chorioamnionitis varied between animals. Low numbers of mba size variants (five or fewer) within amniotic fluid were associated with severe inflammation, whereas the chorioamnion from animals with nine or more mba variants showed little or no inflammation. These differences in chorioamnion inflammation may explain why not all women with in utero Ureaplasma spp. experience adverse pregnancy outcomes.

Baker's yeast expressing the Japanese encephalitis virus envelope protein on its cell surface: induction of an antigen-specific but non-neutralizing antibody response

Live recombinant Saccharomyces cerevisiae yeast expressing the envelope antigen of Japanese encephalitis virus (JEV) on the outer mannoprotein layer of the cell wall were examined for their ability to induce antigen-specific antibody responses in mice. When used as a modelantigen, parenteral immunization of mice with surface-expressing GFP yeast induced a strong anti-GFP antibody response in the absence of adjuvants. This antigen delivery approach was then used for a more stringent system, such as the envelope protein of JEV, which is a neurotropic virus requiring neutralizing antibodies for protection.Although 70% of cells were detected to express the total envelope protein on the surface by antibodies raised to the bacterially expressed protein, polyclonal anti-JEV antibodies failed to react with them. In marked contrast, yeast expressing the envelope fragments 238-398, 373-399 and 373-500 in front of a Gly-Ser linker were detected by anti-JEV antibodies as well as a monoclonal antibody but not by antibodies raised to the bacterially expressed protein. Immunization of mice with these surface-expressing recombinants resulted in a strong antibody response. However, the antibodies failed to neutralize the virus, although the fragments were selected based on neutralizing determinants.

Quantifying Cell Binding Kinetics Mediated By Surface-Bound Blood Type B Antigen To Immobilized Antibodies

Cell adhesion is crucial to many biological processes, such as inflammatory responses, tumor metastasis and thrombosis formation. Recently a commercial surface plasmon resonance (SPR)-based BIAcore biosensor has been extended to determine cell binding mediated by surface-bound biomolecular interactions. How such cell binding is quantitatively governed by kinetic rates and regulating factors, however, has been poorly understood. Here we developed a novel assay to determine the binding kinetics of surface-bound biomolecular interactions using a commercial BIAcore 3000 biosensor. Human red blood cells (RBCs) presenting blood group B antigen and CM5 chip bearing immobilized anti-B monoclonal antibody (mAb) were used to obtain the time courses of response unit, or sensorgrams, when flowing RBCs over the chip surface. A cellular kinetic model was proposed to correlate the sensorgrams with kinetic rates. Impacts of regulating factors, such as cell concentration, flow duration and rate, antibody-presenting level, as well as pH value and osmotic pressure of suspending medium were tested systematically, which imparted the confidence that the approach can be applied to kinetic measurements of cell adhesion mediated by surface-bound biomolecular interactions. These results provided a new insight into quantifying cell binding using a commercial SPR-based BIAcore biosensor.

Interfacial recognition of human prostate-specific antigen by immobilized monoclonal antibody: effects of solution conditions and surface chemistry.

The specific recognition between monoclonal antibody (anti-human prostate-specific antigen, anti-hPSA) and its antigen (human prostate-specific antigen, hPSA) has promising applications in prostate cancer diagnostics and other biosensor applications. However, because of steric constraints associated with interfacial packing and molecular orientations, the binding efficiency is often very low. In this study, spectroscopic ellipsometry and neutron reflection have been used to investigate how solution pH, salt concentration and surface chemistry affect antibody adsorption and subsequent antigen binding. The adsorbed amount of antibody was found to vary with pH and the maximum adsorption occurred between pH 5 and 6, close to the isoelectric point of the antibody. By contrast, the highest antigen binding efficiency occurred close to the neutral pH. Increasing the ionic strength reduced antibody adsorbed amount at the silica-water interface but had little effect on antigen binding. Further studies of antibody adsorption on hydrophobic C8 (octyltrimethoxysilane) surface and chemical attachment of antibody on (3-mercaptopropyl)trimethoxysilane/4-maleimidobutyric acid N-hydroxysuccinimide ester-modified surface have also been undertaken. It was found that on all surfaces studied, the antibody predominantly adopted the 'flat on' orientation, and antigen-binding capabilities were comparable. The results indicate that antibody immobilization via appropriate physical adsorption can replace elaborate interfacial molecular engineering involving complex covalent attachments.

A strategy for sensitivity and specificity enhancements in prostate specific antigen-[alpha] 1-antichymotrypsin detection based on surface plasmon resonance

A biochip based on surface plasmon resonance was fabricated to detect prostate specific antigen-a1-antichymotrypsin (PSA-ACT complex) in both HBS buffer and human serum. To reduce non-specific binding and steric hindrance effect, the chemical surface of the sensor chips was constructed by using various oligo(ethylene glycol) mixtures of different molar ratios of HS(CH2)11(OCH2CH2)6OCH2COOH and HS(CH2)11(OCH2CH2)3OH. The self-assembled monolayers were biotinylated to facilitate the immobilization of streptavidin. Using the chip surfaces, PSA-ACT complex in HBS buffer and human serum was detected at 20.7 and 47.5 ng/ml by primary immunoresponse, respectively. However, the limit of detection could be simply enhanced by a sandwich strategy to improve the sensitivity and specificity of the immunoassay. An intact PSA polyclonal antibody was used as an amplifying agent in the strategy. As a result, PSA-ACT complex concentrations as low as 10.2 and 18.1 ng/ml were found in the HBS buffer and human serum sample, respectively. The result indicates that this approach could satisfy our goal without modifying the secondary interactant.

Sequence diversity and evolutionary dynamics of the dimorphic antigen merozoite surface protein-6 and other Msp genes of Plasmodium falciparum

Immune evasion by Plasmodium falciparum is favored by extensive allelic diversity of surface antigens. Some of them, most notably the vaccine-candidate merozoite surface protein (MSP)-1, exhibit a poorly understood pattern of allelic dimorphism, in which all observed alleles group into two highly diverged allelic families with few or no inter-family recombinants. Here we describe contrasting levels and patterns of sequence diversity in genes encoding three MSP-1-associated surface antigens of P. falciparum, ranging from an ancient allelic dimorphism in the Msp-6 gene to a near lack of allelic divergence in Msp-9 to a more classical multi-allele polymorphism in Msp-7 Other members of the Msp-7 gene family exhibit very little polymorphism in non-repetitive regions. A comparison of P. falciparum Msp-6 sequences to an orthologous sequence from P. reichenowi provided evidence for distinct evolutionary histories of the 5` and 3` segments of the dimorphic region in PfMsp-6, consistent with one dimorphic lineage having arisen from recombination between now-extinct ancestral alleles. In addition. we uncovered two surprising patterns of evolution in repetitive sequence. Firsts in Msp-6, large deletions are associated with (nearly) identical sequence motifs at their borders. Second, a comparison of PfMsp-9 with the P. reichenowi ortholog indicated retention of a significant inter-unit diversity within an 18-base pair repeat within the coding region of P. falciparum, but homogenization in P. reichenowi. (C) 2009 Elsevier B.V. All rights reserved.