Recent insights into the mucosal reactions associated with Giardia lamblia infections.

Giardia lamblia is an intestinal protozoan parasite infecting humans and various other mammalian hosts. The most important clinical signs of giardiasis are diarrhoea and malabsorption. Giardia lamblia is able to undergo continuous antigenic variation of its major surface antigen, named VSP (variant surface protein). While intestinal antibodies, and more specifically anti-VSP IgA antibodies, were proven to be involved in modulating antigenic variation of the parasite the participation of the local antibody response in control of the parasite infection is still controversial. Conversely, previous studies based on experimental infections in mice showed that cellular immune mechanisms are essential for elimination of the parasite from its intestinal habitat. Furthermore, recent data indicated that inflammatory mast cells have a potential to directly, or indirectly, interfere in duodenal growth of G. lamblia trophozoites. However, this finding was challenged by other reports, which did not find a correlation between intestinal inflammation and resistance to infection. Since intestinal infiltration of inflammatory cells and/or CD8+T-cells were demonstrated to coincide with villus-shortening and crypt hyperplasia immunological reactions were considered to be a potential factor of pathogenesis in giardiasis. The contribution of physiological factors to pathogenesis was essentially assessed in vitro by co-cultivation of G. lamblia trophozoites with epithelial cell lines. By using this in vitro model, molecular (through surface lectins) and mechanical (through ventral disk) adhesion of trophozoites to the epithelium was shown to be crucial for increased epithelial permeability. This phenomenon as well as other Giardia-induced intestinal abnormalities such as loss of intestinal brush border surface area, villus flattening, inhibition of disaccharidase activities, and eventually also overgrowth of the enteric bacterial flora seem to be involved in the pathophysiology of giardiasis. However, it remains to be elucidated whether at least part of these pathological effects are causatively linked to the clinical manifestation of the disease.

Quantitative assessment of sense and antisense transcripts from genes involved in antigenic variation (vsp genes) and encystation (cwp 1 gene) of Giardia lamblia clone GS/M-83-H7.

Antigenic variation of the intestinal protozoan parasite Giardia lamblia is caused by an exchange of the parasite's variant surface protein (VSP) coat. Many investigations on antigenic variation were performed with G. lamblia clone GS/M-83-H7 which produces surface antigen VSP H7. To generate novel information on giardial vsp gene transcription, vsp RNA levels were assessed by quantitative reverse transcription-(RT)-PCR in both axenic VSP H7-type trophozoites and subvariants obtained after negative selection of GS/M-83-H7 trophozoites by treatment with a cytotoxic, VSP H7-specific monoclonal antibody. Our investigation was not restricted to the assessment of the sense vsp transcript levels but also included an approach aimed at the detection of complementary antisense vsp transcripts within the two trophozoite populations. We found that sense vsp H7 RNA predominated in VSP H7-type trophozoites while sense RNA from only one (vsp IVg) of 8 subvariant vsp genes totally analysed predominated in subvariant-type trophozoites. Interestingly, the two trophozoite populations exhibited a similar relative distribution regarding the vsp H7 and vsp IVg antisense RNA molecules. An analogous sense versus antisense RNA pattern was also observed when the transcripts of gene cwp 1 (encoding cyst wall protein 1) were investigated. Here, both types of RNA molecules only appeared after cwp 1 had been induced through in vitro encystation of the parasite. These findings for the first time demonstrated that giardial antisense RNA production did not occur in a constitutive manner but was directly linked to complementary sense RNA production after activation of the respective gene systems.

Experimental infections of neonatal mice with cysts of Giardia lamblia clone GS/M-83-H7 are associated with an antigenic reset of the parasite.

Transmission of the protozoan parasite Giardia lamblia from one to another host individuum occurs through peroral ingestion of cysts which, following excystation in the small intestine, release two trophozoites each. Many studies have focused on the major surface antigen, VSP (for variant surface protein), which is responsible for the antigenic variability of the parasite. By using trophozoites of G. lamblia clone GS/M-83-H7 (expressing VSP H7) and the neonatal mouse model for experimental infections, we quantitatively assessed the process of antigenic variation of the parasite on the transcriptional level. In the present study, variant-specific regions identified on different GS/M-83-H7 vsp sequences served as targets for quantitative reverse transcription-PCR to monitor alterations in vsp mRNA levels during infection. Respective results demonstrated that antigenic switching of both the duodenal trophozoite and the cecal cyst populations was associated with a massive reduction in vsp H7 mRNA levels but not with a simultaneous increase in transcripts of any of the subvariant vsp genes analyzed. Most importantly, we also explored giardial variant-type formation and vsp mRNA levels after infection of mice with cysts. This infection mode led to an antigenic reset of the parasite in that a VSP H7-negative inoculum "converted" into a population of intestinal trophozoites that essentially consisted of the original VSP H7 type. This antigenic reset appears to be associated with excystation rather than with a selective process which favors expansion of a residual population of VSP H7 types within the antigenically diversified cyst inoculum. Based on these findings, the VSP H7 type has to be regarded as a predominant variant of G. lamblia clone GS/M-83-H7 which (re-)emerges during early-stage infection and may contribute to an optimal establishment of the parasite within the intestine of the experimental murine host.

HBV core sequence: definition of genotype-specific variability and correlation with geographical origin

There are eight genotypes and nine subtypes of HBV. Small differences in geographical origin are associated with sequence changes in the surface gene. Here, we compared core gene sequences from different genotypes and geographical regions. Specific combinations of 24 amino acid substitutions at nine residues allowed allocation of a sequence to a subtype. Six of these nine residues were located in different T cell epitopes depending on HBV geographical area and/or genotype. Thirty-seven nucleotide changes were associated uniquely with specific genotypes and subtypes. Unique amino acid and nucleotide variants were found in a majority of sequences from specific countries as well as within subtype ayw2 and adr. Specific nucleotide motifs were defined for Korean, Indian, Chinese, Italian and Pacific region isolates. Finally, we observed amino acid motifs that were common to either South-east Asian or Western populations, irrespective of subtype. We believe that HBV strains spread within constrained ethnic groups, result in selection pressures that define sequence variability within each subtype. It suggests that particular T cell epitopes are specific for geographical regions, and thus ethnic groups; this may affect the design of immunomodulatory therapies.

Prevalence of HBsAg mutants and impact of hepatitis B infant immunisation in four Pacific Island countries

The prevalence rate of hepatitis B virus (HBV) infection in Pacific Island countries is amongst the highest in the world. Hepatitis B immunisation has been incorporated into national programmes at various times, often with erratic supply and coverage, until a regionally co-ordinated programme, which commenced in 1995 ensured adequate supply. The effectiveness of these programmes was recently evaluated in four countries, Vanuatu and Fiji in Melanesia, Tonga in Polynesia and Kiribati in Micronesia. That evaluation established that the programmes had a substantial beneficial impact in preventing chronic hepatitis B infection [Vaccine 18 (2000) 3059]. Several studies of hepatitis B vaccination programmes in endemic countries have identified the potential significance of surface gene mutants as a cause for failure of immunisation. In the study outlined in this paper, we screened infected children and their mothers for the emergence and prevalence of these variants in specimens collected from the four country evaluation. Although the opportunity for the emergence of HBV vaccine escape mutants in these populations was high due to the presence of a considerable amount of the virus in the population and the selection pressure from vaccine use, there were no a determinant vaccine escape mutants found. This suggests that vaccine escape variants are not an important cause for failure to prevent HBV transmission in this setting. Other HBsAg variants were detected, but their functional significance remains to be determined. The failure to provide satisfactory protection during such immunisation programmes reflects the need for achieving and sustaining high vaccine coverage, improving the timeliness of doses as well as improving 'cold-chain' support, rather than the selection of vaccine-escape mutants of HBV. (C) 2004 Elsevier Ltd. All rights reserved.

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.