Mamao Pomace Extract Alleviates Hypertension and Oxidative Stress in Nitric Oxide Deficient Rats

Reactive oxygen species (ROS)-induced oxidative stress plays a major role in pathogenesis of hypertension. Antidesma thwaitesianum (local name: Mamao) is a tropical plant distributed in the tropical/subtropical areas of the world, including Thailand. Mamao pomace (MP), a by-product generated from Mamao fruits, contains large amounts of antioxidant polyphenolic compounds. The aim of this study was to investigate the antihypertensive and antioxidative effects of MP using hypertensive rats. For this purpose, male Sprague-Dawley rats were given N^ω-nitro-l-arginine methyl ester (l-NAME), an inhibitor of endothelial nitric oxide synthase (eNOS), in drinking water (50 mg/kg) for three weeks. MP extract was orally administered daily at doses of 100 and 300 mg/kg. l-NAME administration induced marked increase in blood pressure, peripheral vascular resistance, and oxidative stress. MP treatment significantly prevented the increase in blood pressure, hindlimb blood flow and hindlimb vascular resistance of l-NAME treated hypertensive rats (p < 0.05). The antihypertensive effect of MP treatment was associated with suppression of superoxide production from carotid strips and also with an increase in eNOS protein expression and nitric oxide bioavailability. The present results provide evidence for the antihypertensive effect of MP and suggest that MP might be useful as a dietary supplement against hypertension.     

Pre-Existing Cross-Reactive Antibodies to Avian Influenza H5N1 and 2009 Pandemic H1N1 in US Military Personnel

We studied cross-reactive antibodies against avian influenza H5N1 and 2009 pandemic (p) H1N1 in 200 serum samples from US military personnel collected before the H1N1 pandemic. Assays used to measure antibodies against viral proteins involved in protection included a hemagglutination inhibition (HI) assay and a neuraminidase inhibition (NI) assay. Viral neutralization by antibodies against avian influenza H5N1 and 2009 pH1N1 was assessed by influenza (H5) pseudotyped lentiviral particle-based and H1N1 microneutralization assays. Some US military personnel had cross-neutralizing antibodies against H5N1 (14%) and 2009 pH1N1 (16.5%). The odds of having cross-neutralizing antibodies against 2009 pH1N1 were 4.4 times higher in subjects receiving more than five inactivated whole influenza virus vaccinations than those subjects with no record of vaccination. Although unclear if the result of prior vaccination or disease exposure, these pre-existing antibodies may prevent or reduce disease severity.Outbreaks of 1997 avian influenza H5N1 and 2009 pandemic (p) H1N1 in humans have provided an opportunity to gain insight into cross-reactive immunity. The US military periodically collects and stores serum samples from service members linked to medical records.¹ We measured cross-reactive antibodies in stored serum to avian influenza H5N1 and 2009 pH1N1 from US military personnel and identified factors associated with presence of neutralizing antibodies.Two hundred archived serum samples were obtained from the US Department of Defense Serum Repository. They were representative of a wide cross-section of active military personnel at the times of collection, whereas specific geographic information was not available on the individual selected; the cohort represents the general US military population, which is deployed throughout the United States and globally. Fifty samples each were selected from four birth cohorts: (1) < 1949, (2) 1960–1965, (3) 1966–1971, and (4) 1972–1977. Within each cohort, 25 samples were collected in the year 2000 (before the introduction of intranasal live attenuated influenza vaccine [LAIV]), and 25 samples were collected in 2008 (where 51% of donors had received LAIV). It has been suggested that LAIV elicits cross-reactive immunity.^2,3 The samples were all collected before the outbreak of 2009 pH1N1, and there have not been any reported outbreaks of H5N1 in US military personnel.Assays used to measure antibodies included a hemagglutination inhibition (HI) assay and a neuraminidase inhibition (NI) assay.⁴ Viral neutralization by antibodies against H5N1 and 2009 pH1N1 was assessed by influenza (H5) pseudotyped lentiviral particle-based (H5pp)⁵ and microneutralization assays, respectively. Electronic medical and vaccination records from the Defense Medical Surveillance System (DMSS), which captured records before the serum sample date, were linked to samples and compared with the in vitro results.¹The odds ratios (ORs) and 95% confidence intervals (95% CIs) of univariate and multivariate binary logistic regression analyses were used to determine the association between donor characteristics and positive antibody responses. A multiple logistic regression model was constructed, and it included independent variables with a P value of < 0.05 in univariate logistic regression. A P value of < 0.05 was considered to indicate statistical significance. SPSS 12.0 for Windows (SPSS Inc., Chicago, IL) was used to perform all statistical analysis.Cross-reactivity is summarized in 5 and 22.5% for the NI assay. H5pp and NI antibody titers to H5N1 were evenly distributed among birth cohorts and did not differ substantially based on history of vaccination or prior respiratory infections. Of those individuals with neutralizing antibodies to H5N1 (N = 28), 32.1% also had neutralizing antibodies to pH1N1, whereas 19.3% of those individuals with any H5N1-specific antibody response also had neutralizing antibodies to pH1N1 (

Discovery of GAMA, a Plasmodium falciparum merozoite micronemal protein, as a novel blood-stage vaccine candidate antigen

One of the solutions for reducing the global mortality and morbidity due to malaria is multivalent vaccines comprising antigens of several life cycle stages of the malarial parasite. Hence, there is a need for supplementing the current set of malaria vaccine candidate antigens. Here, we aimed to characterize glycosylphosphatidylinositol (GPI)-anchored micronemal antigen (GAMA) encoded by the PF08_0008 gene in Plasmodium falciparum. Antibodies were raised against recombinant GAMA synthesized by using a wheat germ cell-free system. Immunoelectron microscopy demonstrated for the first time that GAMA is a microneme protein of the merozoite. Erythrocyte binding assays revealed that GAMA possesses an erythrocyte binding epitope in the C-terminal region and it binds a nonsialylated protein receptor on human erythrocytes. Growth inhibition assays revealed that anti-GAMA antibodies can inhibit P. falciparum invasion in a dose-dependent manner and GAMA plays a role in the sialic acid (SA)-independent invasion pathway. Anti-GAMA antibodies in combination with anti-erythrocyte binding antigen 175 exhibited a significantly higher level of invasion inhibition, supporting the rationale that targeting of both SA-dependent and SA-independent ligands/pathways is better than targeting either of them alone. Human sera collected from areas of malaria endemicity in Mali and Thailand recognized GAMA. Since GAMA in P. falciparum is refractory to gene knockout attempts, it is essential to parasite invasion. Overall, our study indicates that GAMA is a novel blood-stage vaccine candidate antigen.     

Evaluation of the safety and immunogenicity in rhesus monkeys of a recombinant malaria vaccine for Plasmodium vivax with a synthetic Toll-like receptor 4 agonist formulated in an emulsion

Plasmodium vivax is the major cause of malaria outside sub-Saharan Africa and inflicts debilitating morbidity and consequent economic impacts in developing countries. In order to produce a P. vivax vaccine for global use, we have previously reported the development of a novel chimeric recombinant protein, VMP001, based on the circumsporozoite protein (CSP) of P. vivax. Very few adjuvant formulations are currently available for human use. Our interest is to evaluate second-generation vaccine formulations to identify novel combinations of adjuvants capable of inducing strong, long-lasting immune responses. In this study rhesus monkeys were immunized intramuscularly three times with VMP001 in combination with a stable emulsion (SE) or a synthetic Toll-like receptor 4 (TLR4) agonist (glucopyranosyl lipid A [GLA]) in SE (GLA-SE). Sera and peripheral blood mononuclear cells (PBMCs) were tested for the presence of antigen-specific humoral and cellular responses, respectively. All groups of monkeys generated high titers of anti-P. vivax IgG antibodies, as detected by enzyme-linked immunosorbent assays (ELISAs) and immunofluorescence assays. In addition, all groups generated a cellular immune response characterized by antigen-specific CD4(+) T cells secreting predominantly interleukin-2 (IL-2) and lesser amounts of tumor necrosis factor (TNF). We conclude that the combination of VMP001 and GLA-SE is safe and immunogenic in monkeys and may serve as a potential second-generation vaccine candidate against P. vivax malaria.     

Major Surface Protein LipL32 Is Not Required for Either Acute or Chronic Infection with Leptospira interrogans

Leptospira interrogans is responsible for leptospirosis, a zoonosis of worldwide distribution. LipL32 is the major outer membrane protein of pathogenic leptospires, accounting for up to 75% of total outer membrane protein. In recent times LipL32 has become the focus of intense study because of its surface location, dominance in the host immune response, and conservation among pathogenic species. In this study, an lipL32 mutant was constructed in L. interrogans using transposon mutagenesis. The lipL32 mutant had normal morphology and growth rate compared to the wild type and was equally adherent to extracellular matrix. Protein composition of the cell membranes was found to be largely unaffected by the loss of LipL32, with no obvious compensatory increase in other proteins. Microarray studies found no obvious stress response or upregulation of genes that may compensate for the loss of LipL32 but did suggest an association between LipL32 and the synthesis of heme and vitamin B₁₂. When hamsters were inoculated by systemic and mucosal routes, the mutant caused acute severe disease manifestations that were indistinguishable from wild-type L. interrogans infection. In the rat model of chronic infection, the LipL32 mutant colonized the renal tubules as efficiently as the wild-type strain. In conclusion, this study showed that LipL32 does not play a role in either the acute or chronic models of infection. Considering the abundance and conservation of LipL32 among all pathogenic Leptospira spp. and its absence in saprophytic Leptospira, this finding is remarkable. The role of this protein in leptospiral biology and pathogenesis thus remains elusive.Leptospira interrogans is a zoonotic spirochete with a worldwide distribution. In the chronic carrier state, host animals such as rats do not exhibit overt disease but are colonized by Leptospira in their renal tubules and shed bacteria in their urine. Humans are incidental hosts that become infected through exposure to contaminated water, soil, or urine. In the acute form of leptospirosis, disease severity ranges from asymptomatic infection to multiple organ failure, pulmonary hemorrhage, and death (13).The cellular and molecular mechanisms of leptospiral pathogenesis remain unclear. One of the major sites of interaction with the host is the bacterial outer membrane (OM). Analysis of the L. interrogans OM has identified a number of proteins (11, 12), the most abundant of which is LipL32, a 32-kDa lipoprotein estimated to account for a remarkable 75% of the OM proteome (11). LipL32 is also the most abundant surface-exposed protein (12).LipL32 is found in all pathogenic species tested to date and is highly conserved, with average amino acid identity over 98%, but it is not found in saprophytic species (16, 17). LipL32 is expressed during both chronic and acute infection and is highly immunogenic (15, 16, 27). These features have generated interest in LipL32 as a potential diagnostic reagent in both PCRs (21) and enzyme-linked immunosorbent assays (14). There has also been much interest in the potential of LipL32 to generate heterologous immunity, overcoming the limitations of serovar-specific immunity. However, to date LipL32-based vaccines have met with limited success (5, 6).The abundance, conservation, unique presence in pathogenic species, and immunogenicity of LipL32 are consistent with an important role in pathogenesis. Available microarray data provide little insight into the role of LipL32 since gene expression is unchanged under conditions of different temperatures (22). Recent studies have shown that LipL32 may act as an adhesin binding to collagen, laminin, and fibronectin (18, 19) while LipL32 has also been associated with hemolysis (20). However, the precise role of LipL32 in pathogenesis remains unknown.In this study an lipL32 mutant was constructed by transposon mutagenesis. To our surprise, analysis of this mutant in the hamster model of acute infection and rat model of chronic infection showed that LipL32 is not required for causing either acute leptospirosis or renal colonization.     

Single amino acid substitution in Plasmodium yoelii erythrocyte ligand determines its localization and controls parasite virulence

The major virulence determinant of the rodent malaria parasite, Plasmodium yoelii, has remained unresolved since the discovery of the lethal line in the 1970s. Because virulence in this parasite correlates with the ability to invade different types of erythrocytes, we evaluated the potential role of the parasite erythrocyte binding ligand, PyEBL. We found 1 amino acid substitution in a domain responsible for intracellular trafficking between the lethal and nonlethal parasite lines and, furthermore, that the intracellular localization of PyEBL was distinct between these lines. Genetic modification showed that this substitution was responsible not only for PyEBL localization but also the erythrocyte-type invasion preference of the parasite and subsequently its virulence in mice. This previously unrecognized mechanism for altering an invasion phenotype indicates that subtle alterations of a malaria parasite ligand can dramatically affect host–pathogen interactions and malaria virulence.     

Use of Luminescent Leptospira interrogans for Enumeration in Biological Assays

Rapid and reliable in vitro methods for the detection of pathogenic leptospires, such as Leptospira interrogans, are lacking. The present study investigated the use of luminescence to replace the existing enumeration techniques. Transposon TnSC189 was modified to incorporate the luxCDABE cassette from Photorhabdus luminescens and was used to construct luminescent Leptospira spp. There was a linear relationship between luminescence and cell number, with the theoretical detection limit being less than 10⁴ leptospires. A comparison of enumeration by a standard method (counting by dark-field microscopy) and enumeration by luminescence was conducted with luminescent L. interrogans. There was a good correlation between the two methods of enumeration (R² = 0.766), although variation in the luminescence early and late in growth phase reduced the degree of correlation. To demonstrate the utility of luminescence as a viability and cell number reporter, in vitro assays, including MIC determination, an extracellular matrix binding experiment, and a complement killing experiment, were conducted. In each case, the results obtained by luminescence matched those obtained by traditional means with high correlations (binding assay R² = 0.916, complement killing assay R² = 0.988). A strain expressing the luxCDABE transposon retained virulence in the hamster model of infection. Despite some variation in luminescence as a result of the growth phase or the particular assay conditions, enumeration by luminescence was found to be a quick, reliable, and highly sensitive method for the in vitro detection of leptospires that has the potential to replace more time-consuming methods of enumeration.The spirochete Leptospira interrogans causes widespread zoonosis, contracted through contact with contaminated water, soil, or animal urine. Disease syndromes can range from no symptoms and a mild influenza-like illness through to multiple-organ failure and death (3).Despite intensive research into the pathogenic mechanisms of L. interrogans, progress has been limited due to the practicalities of working with these bacteria. One significant technical difficulty is the quantitative detection of leptospires in vitro. Conventional bacterial enumeration methods are unsuitable due to the unusual features of Leptospira spp. A very slow rate of growth (Leptospira spp. take up to 4 weeks to form colonies on plates [1]) makes growth-dependent outputs such as CFU counts and minimal dilution slow and prone to overgrowth by contaminants. The unusual morphology of leptospires makes identification by light microscopy difficult, while a low maximum culture density makes estimation of the concentration by measurement of the optical density (OD) of limited use. Quantitative real-time PCR may be used to estimate the leptospiral cell number through the detection of chromosomal DNA (10), but this technique is time-consuming and expensive and does not distinguish live cells from dead cells. The current standard for the enumeration of leptospires involves counting by use of a Petroff-Hausser chamber under dark-field illumination (3). This process requires a trained operator, is time-consuming, and puts the operator at risk of eye strain. Furthermore, this technique is difficult in the presence of other diffracting material that may be present in the output of an experimental assay (e.g., cell debris) and is compromised by the autoagglutination of leptospires and the high degree of motility of some strains. New methods of enumeration of spirochetes are required, such as the recently described flow cytometry method for the enumeration of Treponema denticola, an oral spirochete that shares many of the difficult features found in Leptospira (16).To further complicate the study of L. interrogans, this pathogen has no replicating plasmids and genetic manipulation through mutagenesis is very difficult. Recently, an L. interrogans mutagenesis method was developed with a mariner transposon, TnSC189 (1, 12). This is currently the only efficient means for constructing defined leptospiral mutants. This method has been applied for widespread random mutagenesis of the bacterial chromosome (13), facilitating the identification of two virulence factors (14, 17) and the unexpected finding that the major outer membrane protein (LipL32) is not required for pathogenesis (15).Luminescent bacteria have previously been used for in vitro enumeration and viability reporting and for monitoring the progress of infection in vivo (8, 9). In the study described here, in the absence of replicating plasmids for L. interrogans, transposon TnSC189 was modified to incorporate the luxCDABE cassette from Photorhabdus luminescens (20). This construct was used to stably integrate luxCDABE into the chromosome, producing luminescent Leptospira interrogans isolates. Luminescence was found to be a highly efficient means for determining bacterial numbers and population viability.