Clinical and virological study of dengue cases and the members of their households: the multinational DENFRAME Project

Background

Dengue has emerged as the most important vector-borne viral disease in tropical areas. Evaluations of the burden and severity of dengue disease have been hindered by the frequent lack of laboratory confirmation and strong selection bias toward more severe cases.

Methodology

A multinational, prospective clinical study was carried out in South-East Asia (SEA) and Latin America (LA), to ascertain the proportion of inapparent dengue infections in households of febrile dengue cases, and to compare clinical data and biological markers from subjects with various dengue disease patterns. Dengue infection was laboratory-confirmed during the acute phase, by virus isolation and detection of the genome. The four participating reference laboratories used standardized methods.

Principal Findings

Among 215 febrile dengue subjects—114 in SEA and 101 in LA—28 (13.0%) were diagnosed with severe dengue (from SEA only) using the WHO definition. Household investigations were carried out for 177 febrile subjects. Among household members at the time of the first home visit, 39 acute dengue infections were detected of which 29 were inapparent. A further 62 dengue cases were classified at early convalescent phase. Therefore, 101 dengue infections were found among the 408 household members. Adding these together with the 177 Dengue Index Cases, the overall proportion of dengue infections among the study participants was estimated at 47.5% (278/585; 95% CI 43.5–51.6). Lymphocyte counts and detection of the NS1 antigen differed significantly between inapparent and symptomatic dengue subjects; among inapparent cases lymphocyte counts were normal and only 20% were positive for NS1 antigen. Primary dengue infection and a specific dengue virus serotype were not associated with symptomatic dengue infection.

Conclusion

Household investigation demonstrated a high proportion of household members positive for dengue infection, including a number of inapparent cases, the frequency of which was higher in SEA than in LA.     

Associations of the IL2Rα, IL4Rα, IL10Rα, and IFN γ R1 cytokine receptor genes with AIDS progression in a French AIDS cohort

We have performed an extensive analysis of Th1/Th2 cytokine receptors IL2Rα, IL4Rα, IL10Rα, and IFNγR1 gene polymorphisms to evaluate their impact on AIDS progression. The coding regions and promoters of these genes were sequenced in the genetics of resistance to immunodeficiency virus cohort, composed of 327 HIV-1-positive patients with extreme progression phenotypes, slow and rapid progressors, and of 446 healthy control subjects, all of them of Caucasian descent. Overall, 104 single nucleotide polymorphisms and four insertions/deletions with a minor allelic frequency higher than 1% were identified, 21 of them being newly characterized. We observed weak associations for 13 polymorphisms of IL2Rα, IL4Rα, IL10Rα, and IFNγR1, and 11 haplotypes of IL2Rα, IL4Rα, and IFNγR1. However, we could not relate these positive signals to any relevant biological information on the gene function. To affirm these putative associations in AIDS, further confirmation on other AIDS cohorts will be needed. This complete catalog of polymorphisms in IL2Rα, IL4Rα, IL10Rα, and IFNγR1 cytokine receptor genes should also be useful for investigating associations in other immune-related diseases.Electronic Supplementary Material Supplementary material is available for this article at     

Contemporary variations of immune responsiveness during range expansion of two invasive rodents in Senegal

Biological invasions provide unique opportunities for studying life history trait changes over contemporary time scales. As spatial spread may be related to changes in parasite communities, several hypotheses (such as the evolution of increased competitive ability (EICA) or EICA‐refined hypotheses) suggest immune changes in invasive species along invasion gradients. Although native hosts may be subject to similar changes in parasite selection pressures, their immune responses have been rarely investigated in invasion contexts. In this study, we evaluated immune variations for invasive house mice Mus musculus domesticus, invasive black rats Rattus rattus and native rodents Mastomys erythroleucus and Mastomys natalensis along well‐characterised invasion gradients in Senegal. We focused on antibody‐mediated (natural antibodies and complement) and inflammatory (haptoglobin) responses. One invasion route was considered for each invasive species, and environmental conditions were recorded. Natural‐antibody mediated responses increased between sites of long‐established invasion and recently invaded sites only in house mice. Both invasive species exhibited higher inflammatory responses at the invasion front than in sites of long‐established invasion. The immune responses of native species did not change with the presence of invasive species. These patterns of immune variations do not support the EICA and EICA refined hypotheses, and they rather suggest a higher risk of exposure to parasites on the invasion front. Altogether, these results provide a first basis to further assess the role of immune changes in invasion success.     

Unexpected high circulation of <Emphasis Type="Italic">Plasmodium vivax</Emphasis> in asymptomatic children from Kédougou,southeastern Senegal

Background

Malaria in Senegal is due essentially to infections by Plasmodium falciparum and, to a lesser extent to Plasmodium malariae and Plasmodium ovale. By the use of molecular methods, detection of Plasmodium vivax has been recently reported in the region of Kedougou, raising the question of appraisal of its potential prevalence in this setting.

Methods

A retrospective serological study was carried out using 188 samples taken from 2010 to 2011 in a longitudinal school survey during which 48 asymptomatic children (9–11 years) were recruited. Four collections of samples collected during two successive dry and rainy seasons were analysed for antibody responses to P. vivax and P. falciparum. Recombinant P. falciparum and P. vivax MSP1 antigens and total P. falciparum schizont lysate from African 07/03 strain (adapted to culture) were used for ELISA. Nested PCR amplification was used for molecular detection of P. vivax.

Results

A surprising high prevalence of IgG responses against P. vivax MSP1 was evidenced with 53% of positive samples and 58% of the individuals that were found positive to this antigen. There was 77% of responders to P. falciparum outlined by 63% of positive samples. Prevalence of responders did not differ as function of seasons. Levels of antibodies to P. falciparum fluctuated with significant increasing between dry and rainy season (P < 0.05), contrary to responses to P. vivax. There was a significant reciprocal relationship (P < 10^?3) between antibody responses to the different antigens, but with weak coefficient of correlation (Rho around 0.3) underlining a variable profile at the individual level. Clear molecular signature was found in positive IgG to P. vivax msp1 samples by PCR.

Conclusion

This cross-sectional longitudinal study highlights the unexpected high circulation of P. vivax in this endemic area. Sero-immunology and molecular methods are powerful additive tools to identify endemic sites where relevant control measures have to be settled and monitored.

     

Stereospecific binding of a new benzazepine, [3H]SCH23390, in cortex and neostriatum

The binding of the D1 antagonist SCH23390 to membrane preparations from rat cerebral cortex was examined using enantiomers of dopamine agonists and antagonists to compete with the bound [3H]SCH23390 at its Kd value. The competition curves were compared with those obtained with preparations from the neostriatum. The results demonstrate that specific [3H]SCH23390 binding in the cerebral cortex has the same pharmacological profile as in the neostriatum, so that this radioligand can be used to label dopamine D1 receptors in brain regions with a sparse dopaminergic innervation.     

DC-SIGN from African green monkeys is expressed in lymph nodes and mediates infection in trans of simian immunodeficiency virus SIVagm

African green monkeys (AGMs) infected by simian immunodeficiency virus (SIV) SIVagm are resistant to AIDS. SIVagm-infected AGMs exhibit levels of viremia similar to those described during pathogenic human immunodeficiency virus type 1 (HIV-1) and SIVmac infections in humans and macaques, respectively, but contain lower viral loads in their lymph nodes. We addressed the potential role of dendritic cell-specific intercellular adhesion molecule 3-grabbing nonintegrin (DC-SIGN; CD209) in viral dissemination. In previous studies, it has been shown that human DC-SIGN and macaque DC-SIGN allow transmission of HIV and SIVmac to T cells. Here, we looked at the ability of DC-SIGN derived from AGM lymph nodes to interact with SIVagm. We show that DC-SIGN-expressing cells are present mainly in the medulla and often within the cortex and/or paracortex of AGM lymph nodes. We describe the isolation and characterization of at least three isoforms of dc-sign mRNA in lymph nodes of AGMs. The predicted amino acid sequence from the predominant mRNA isoform, DC-SIGNagm1, is 92 and 99% identical to the corresponding human and rhesus macaque DC-SIGN amino acid sequences, respectively. DC-SIGNagm1 is characterized by the lack of the fourth motif in the repeat domain. This deletion was also detected in the dc-sign gene derived from thirteen animals belonging to five other African monkey species and from four macaques (Macaca fascicularis and M. mulatta). Despite three- to seven-amino-acid modifications compared to DC-SIGNmac, DC-SIGNagm1 allows transmission of SIVagm to T cells. Furthermore, AGM monocyte-derived dendritic cells (MDDC) expressed at least 100,000 DC-SIGN molecules and were able to transmit SIVagm to T cells. At a low multiplicity of infection (10(-5) 50% tissue culture infective doses/cell), viral transmission by AGM MDDC was mainly DC-SIGN dependent. The present study reveals that DC-SIGN from a natural host species of SIV has the ability to act as an efficient attachment and transmission factor for SIVagm and suggests the absence of a direct link between this ability and viral load levels in lymph nodes.     

Viral load in tissues during the early and chronic phase of non-pathogenic SIVagm infection

African green monkeys (AGMs) persistently infected with SIVagm do not develop AIDS, although their plasma viremia levels can reach those reported for pathogenic HIV-1 and SIVmac infections. In contrast, the viral burden in lymph nodes in SIVagm-infected AGMs is generally lower in comparison with HIV/SIVmac pathogenic infections, at least during the chronic phase of SIVagm infection. We searched for the primary targets of viral replication, which might account for the high viremias in SIVagm-infected AGMs. We evaluated for the first time during primary infection SIVagm dissemination in various lymphoid and non-lymphoid tissues. Sixteen distinct organs at a time point corresponding to maximal virus production were analyzed for viral RNA and DNA load. At days 8 and 9 p.i., viral RNA could be detected in a wide range of tissues, such as jejunum, spleen, mesenteric lymph nodes, thymus and lung. Quantification of viral DNA and RNA as well as of productively infected cells revealed that viral replication during this early phase takes place mainly in secondary lymphoid organs and in the gut (5 x 10(4)-5 x 10(8) RNA copies/10(6) cells). By 4 years p.i., RNA copy numbers were below detection level in thymus and lung. Secondary lymphoid organs displayed 6 x 10(2)-2 x 10(6) RNA copies/10(6) cells, while some tissue fragments of ileum and jejunum still showed high viral loads (up to 10(9) copies/10(6) cells). Altogether, these results indicate a rapid dissemination of SIVagm into lymphoid tissues, including the small intestine. The latter, despite showing marked regional variations, most likely contributes significantly to the high levels of viremia observed during SIVagm infection.     

Dual axenic culture of sheared-root inocula of vesicular-arbuscular mycorrhizal fungi associated with tomato roots

Surface-sterilized sheared-root inocula of two vesicular-arbuscular mycorrhizal (VAM) fungi (Glomus intraradices and G. versiforme) from pot cultures associated with excised tomato roots showed significant sporulation and the production of an extensive hyphal biomass. As many as 10²–10³ axenic mature spores were recovered in Petri dishes during 3 months incubation in the dark. Propagules of both species were able to complete their vegetative life cycle in vitro and efficiently colonize Acacia albida roots after 1 month under greenhouse conditions. The effectiveness of 0.5 cm pieces of VAM roots as starter inocula indicates the high inoculum potential of intravesicle propagules.     

A dual role for integrin‐linked kinase and β1‐integrin in modulating cardiac aging

Cardiac performance decreases with age, which is a major risk factor for cardiovascular disease and mortality in the aging human population, but the molecular mechanisms underlying cardiac aging are still poorly understood. Investigating the role of integrin‐linked kinase (ilk) and β1‐integrin (myospheroid, mys) in Drosophila, which colocalize near cardiomyocyte contacts and Z‐bands, we find that reduced ilk or mys function prevents the typical changes of cardiac aging seen in wildtype, such as arrhythmias. In particular, the characteristic increase in cardiac arrhythmias with age is prevented in ilk and mys heterozygous flies with nearly identical genetic background, and they live longer, in line with previous findings in Caenorhabditis elegans for ilk and in Drosophila for mys. Consistent with these findings, we observed elevated β1‐integrin protein levels in old compared with young wild‐type flies, and cardiac‐specific overexpression of mys in young flies causes aging‐like heart dysfunction. Moreover, moderate cardiac‐specific knockdown of integrin‐linked kinase (ILK)/integrin pathway‐associated genes also prevented the decline in cardiac performance with age. In contrast, strong cardiac knockdown of ilk or ILK‐associated genes can severely compromise cardiac integrity, including cardiomyocyte adhesion and overall heart function. These data suggest that ilk/mys function is necessary for establishing and maintaining normal heart structure and function, and appropriate fine‐tuning of this pathway can retard the age‐dependent decline in cardiac performance and extend lifespan. Thus, ILK/integrin‐associated signaling emerges as an important and conserved genetic mechanism in longevity, and as a new means to improve age‐dependent cardiac performance, in addition to its vital role in maintaining cardiac integrity.     

Mutations in Domain I Interhelical Loops Affect the Rate of Pore Formation by the Bacillus thuringiensis Cry1Aa Toxin in Insect Midgut Brush Border Membrane Vesicles

Pore formation in the apical membrane of the midgut epithelial cells of susceptible insects constitutes a key step in the mode of action of Bacillus thuringiensis insecticidal toxins. In order to study the mechanism of toxin insertion into the membrane, at least one residue in each of the pore-forming-domain (domain I) interhelical loops of Cry1Aa was replaced individually by cysteine, an amino acid which is normally absent from the activated Cry1Aa toxin, using site-directed mutagenesis. The toxicity of most mutants to Manduca sexta neonate larvae was comparable to that of Cry1Aa. The ability of each of the activated mutant toxins to permeabilize M. sexta midgut brush border membrane vesicles was examined with an osmotic swelling assay. Following a 1-h preincubation, all mutants except the V150C mutant were able to form pores at pH 7.5, although the W182C mutant had a weaker activity than the other toxins. Increasing the pH to 10.5, a procedure which introduces a negative charge on the thiol group of the cysteine residues, caused a significant reduction in the pore-forming abilities of most mutants without affecting those of Cry1Aa or the I88C, T122C, Y153C, or S252C mutant. The rate of pore formation was significantly lower for the F50C, Q151C, Y153C, W182C, and S252C mutants than for Cry1Aa at pH 7.5. At the higher pH, all mutants formed pores significantly more slowly than Cry1Aa, except the I88C mutant, which formed pores significantly faster, and the T122C mutant. These results indicate that domain I interhelical loop residues play an important role in the conformational changes leading to toxin insertion and pore formation.Once ingested by susceptible insect larvae, the insecticidal crystal proteins of Bacillus thuringiensis are solubilized and converted to their toxic form by midgut proteases. The activated toxins bind to specific receptors on the surface of the luminal membrane of midgut columnar cells, insert into the membrane, and form pores that abolish transmembrane ionic gradients and osmotic balance, leading to the disruption of the epithelium and death of the insect (47, 51). Members of the B. thuringiensis Cry toxin family for which the atomic structure has been reported share a similar three-domain organization in which domain I is composed of a bundle of six amphipathic α-helices surrounding a hydrophobic helix (α5), and domains II and III are formed mostly of β-sheets (7, 8, 18, 26, 37, 38, 43). While domains II and III are thought to be involved in receptor binding and toxin specificity (47), domain I is believed to play a major role in membrane insertion and pore formation (51). Toxin fragments corresponding to domain I of Cry1Ac (62), Cry3Aa (53), and Cry3Ba (61) or to the first five α-helices of Cry4B (48) have been shown to form pores in model membranes. Pore formation in artificial membranes has also been demonstrated with synthetic peptides corresponding to α5 of Cry1Ac (13) and Cry3Aa (19, 21) and to the α4-loop-α5 segment of Cry3Aa (23). Spectroscopic studies have also revealed that while synthetic peptides corresponding to α4 and α5 can coassemble within a lipid bilayer, those corresponding to α2, α3, α6, and α7 adopt a membrane surface orientation (20, 22). In agreement with these findings, α4 was shown to line the lumen of the pores (42). On the other hand, convincing evidence supporting previous suggestions that most of the toxin molecule may become imbedded in the membrane (3, 39, 60) has recently been reported (44, 45).Thus, several models have been proposed for the mechanism of toxin insertion and pore formation (4, 9, 28, 32, 39, 44, 52, 56). Although these models differ in the identities of the toxin segments that are suggested to insert into the membrane, they all imply that the toxin undergoes conformational changes following binding to the membrane surface. Even though such changes imply rotations about the polypeptide backbone in domain I interhelical loops, little attention has been devoted so far to the role of domain I loop residues in pore formation.In the present study, amino acid residues strategically located within each of these loops in Cry1Aa were replaced by a cysteine using site-directed mutagenesis. The resulting mutant toxins were assayed with Manduca sexta midgut brush border membrane vesicles using a light-scattering technique. Mutations mapping within several of these loops altered the functional properties of Cry1Aa, suggesting the involvement of most domain I α-helices in the pore-forming process.