Highly Differentiated,Resting Gn-Specific Memory CD8+ T Cells Persist Years after Infection by Andes Hantavirus

In man, infection with South American Andes virus (ANDV) causes hantavirus cardiopulmonary syndrome (HCPS). HCPS due to ANDV is endemic in Southern Chile and much of Argentina and increasing numbers of cases are reported all over South America. A case-fatality rate of about 36% together with the absence of successful antiviral therapies urge the development of a vaccine. Although T-cell responses were shown to be critically involved in immunity to hantaviruses in mouse models, no data are available on the magnitude, specificity and longevity of ANDV-specific memory T-cell responses in patients. Using sets of overlapping peptides in IFN-γ ELISPOT assays, we herein show in 78 Chilean convalescent patients that Gn-derived epitopes were immunodominant as compared to those from the N- and Gc-proteins. Furthermore, while the relative contribution of the N-specific response significantly declined over time, Gn-specific responses remained readily detectable ex vivo up to 13 years after the acute infection. Tetramer analysis further showed that up to 16.8% of all circulating CD3⁺CD8⁺ T cells were specific for the single HLA-B*3501-restricted epitope Gn_465–473 years after the acute infection. Remarkably, Gn_465–473–specific cells readily secreted IFN-γ, granzyme B and TNF-α but not IL-2 upon stimulation and showed a ‘revertant’ CD45RA⁺CD27⁻CD28⁻CCR7⁻CD127⁻ effector memory phenotype, thereby resembling a phenotype seen in other latent virus infections. Most intriguingly, titers of neutralizing antibodies increased over time in 10/17 individuals months to years after the acute infection and independently of whether they were residents of endemic areas or not. Thus, our data suggest intrinsic, latent antigenic stimulation of Gn-specific T-cells. However, it remains a major task for future studies to proof this hypothesis by determination of viral antigen in convalescent patients. Furthermore, it remains to be seen whether Gn-specific T cells are critical for viral control and protective immunity. If so, Gn-derived immunodominant epitopes could be of high value for future ANDV vaccines.     

Influence of Cross-linked Waxy Maize Starch on the Aggregation Behavior of Casein Micelles During Acid-induced Gelation

The influence of cross-linked waxy maize starch on the aggregation behavior of casein micelles was investigated using a combination of physico-chemical techniques. Milk was homogenized at two different temperatures (55 and 65 °C) and then heated at 95 °C for 5 min in a pilot scale system. The possible interactions between modified starch and milk proteins during lactic acid fermentation were evaluated. While 1% starch did not show differences in the whey protein complexes formed during heating compared to milk with no starch (as measured by size exclusion chromatography), a higher (2.5%) concentration of starch clearly showed an increased amount of heat-induced whey protein aggregates. The gelation pH also increased significantly with 2.5% starch compared to that of the control samples. The storage modulus (G′) increased with increasing levels of starch, and confocal microscopy confirmed that the microstructure of the casein gels was altered by the presence of modified starch. Milk-starch mixtures preheated and homogenized at 55 or 65 °C exhibited similar physico-chemical behavior during acidification. The results suggested a lack of interaction between starch granules and casein micelles during acidification, and scanning electron microscopy images collected with a self-assembled monolayer technique also confirmed that starch granules were not attached to milk caseins but only embedded in the protein gel matrix.     

Phase Separation Behavior of Caseins in Milk Containing Flaxseed Gum and κ-Carrageenan: A Light-Scattering and Ultrasonic Spectroscopy Study

The physico-chemical properties of skim milk containing κ-carrageenan (in the concentration range 0–0.06% w/v), flaxseed gum (in the concentration range 0–0.40% w/v), or a mixture of both polysaccharides were studied using dynamic light scattering, under diluted conditions, as well as in situ, undiluted, using diffusing wave spectroscopy (DWS) and ultrasonic spectroscopy. Flaxseed gum causes phase separation in milk mixtures, because of thermodynamic incompatibility between the casein micelles and the polysaccharide chains. Confocal microscopy and ultrasonic spectroscopy showed that while the addition of 0.01% κ-carrageenan was not sufficient to hinder phase separation, when 0.03% was added, the helix–helix interactions between κ-carrageenan molecules were sufficient to form a network and stabilize the system. DWS clearly demonstrated that clusters of casein micelles still form even at very low concentrations of polysaccharides (below the visible phase separation threshold) and that κ-carrageenan hinders visible phase separation by decreasing the mobility of the casein micelles.     

Factors influencing the production of stilbenes by the knotweed, <Emphasis Type="Italic">Reynoutria</Emphasis> × <Emphasis Type="Italic">bohemica</Emphasis>

Background  

Japanese knotweed, Reynoutria japonica, is known for its high growth rate, even on adverse substrates, and for containing organic substances that are beneficial to human health. Its hybrid, Reynoutria × bohemica, was described in the Czech Republic in 1983 and has been widespread ever since. We examined whether Reynoutria × bohemica as a medicinal plant providing stilbenes and emodin, can be cultivated in spoil bank substrates and hence in the coalmine spoil banks changed into arable fields. We designed a pot experiment and a field experiment to assess the effects of various factors on the growth efficiency of Reynoutria × bohemica on clayish substrates and on the production of stilbenes and emodin in this plant.     

aroA and aroD mutations influence biofilm formation in Salmonella Enteritidis

aro mutants of Salmonella enterica are frequently used as live vaccines for the oral vaccination of domestic animals. Interestingly, besides their auxotrophy, they appear to be of reduced resistance to the components of innate immune response due to a defect in outer membrane and/or cell wall integrity. Because different extracellular structures associated with the cell wall or outer membrane are involved in biofilm formation, we were interested in the ability of aroA and aroD mutants of S . Enteritidis to adhere to solid surfaces. We found that aroA and aroD mutants did not adhere to solid surfaces although they bind Congo red and produced d -mannose and d -glucose capsular polysaccharides in the same amounts as the wild-type strain. However, the aro mutants exhibited a decreased production of cellulose, N -acetyl- d -glucosamine or N -acetylneuraminic acid containing capsular polysaccharide and fimbriae which explains their inability to form biofilms. aroA and aroD containing plasmids complemented all the defects of the aro mutants. Beside its attenuation for different hosts, the loss of ability to form biofilm is an additional interesting characteristic of aro mutants.     

Involvement of oxidative stress in the impairment in biliary secretory function induced by intraperitoneal administration of aluminum to rats

We have shown that aluminum (Al) induces cholestasis associated with multiple alterations in hepatocellular transporters involved in bile secretory function, like Mrp2. This work aims to investigate whether these harmful effects are mediated by the oxidative stress caused by the metal. For this purpose, the capability of the antioxidant agent, vitamin E, to counteract these alterations was studied in male Wistar rats. Aluminum hydroxide (or saline in controls) was administered ip (27 mg/kg body weight, three times a week, for 90 d). Vitamin E (600 mg/kg body weight) was coadministered, sc. Al increased lipid peroxidation (+50%) and decreased hepatic glutation levels (-43%) and the activity of glutation peroxidase (-50%) and catalase (-88%). Vitamin E counteracted these effects total or partially. Both plasma and hepatic Al levels reached at the end of the treatment were significantly reduced by vitamin E (-40% and -44%, respectively;p< 0.05). Al increased 4 times the hepatic apoptotic index, and this effect was fully counteracted by vitamin E. Bile flow was decreased in Al-treated rats (-37%) and restored to normality by vitamin E. The antioxidant normalized the hepatic handling of the Mrp2 substrates, rose bengal, and dinitrophenyl-S-glutathione, which was causally associated with restoration of Mrp2 expression. Our data indicate that oxidative stress has a crucial role in cholestasis, apoptotic/necrotic hepatocellular damage, and the impairment in liver transport function induced by Al and that vitamin E counteracts these harmful effects not only by preventing free-radical formation but also by favoring Al disposal.     

Neutrophils activate macrophages for intracellular killing of Leishmania major through recruitment of TLR4 by neutrophil elastase

We investigated the role of neutrophil elastase (NE) in interactions between murine inflammatory neutrophils and macrophages infected with the parasite Leishmania major. A blocker peptide specific for NE prevented the neutrophils from inducing microbicidal activity in macrophages. Inflammatory neutrophils from mutant pallid mice were defective in the spontaneous release of NE, failed to induce microbicidal activity in wild-type macrophages, and failed to reduce parasite loads upon transfer in vivo. Conversely, purified NE activated macrophages and induced microbicidal activity dependent on secretion of TNF-alpha. Induction of macrophage microbicidal activity by either neutrophils or purified NE required TLR4 expression by macrophages. Injection of purified NE shortly after infection in vivo reduced the burden of L. major in draining lymph nodes of TLR4-sufficient, but not TLR4-deficient mice. These results indicate that NE plays a previously unrecognized protective role in host responses to L. major infection.     

Low seroprevalence of Neospora caninum infection associated with the limousin breed in cow-calf herds in Andorra, Europe

Neospora caninum seroprevalence and risk factors affecting seroprevalence in beef cattle in Andorra were investigated. Antibodies to N. caninum were evaluated by enzyme-linked immunosorbent assay performed on a yearly basis in 1,758 animals older than 6 mo, belonging to 26 herds. Mean seroprevalence of antibodies to N. caninum for the herds was 7.4 +/- 1.2% (130/1,758). Logistic regression analyses were performed on data from each animal, considering N. caninum seropositivity as the dependent variable, and herd, grazing area, year of sampling, repeat-test animal (animals sampled twice or more), sex, breed, age (animals <4 yr old or > or =5 yr old), and country of birth as possible risk factors. Based on the odds ratio, the prevalence of infection was 2.1 times higher (P < 0.01) in animals from the Ordino grazing area, 1.64 times higher in animals older than 5 yr (P < 0.01), and 6.7 times (1/0.15) lower in Limousin-mixed Limousin cattle (P < 0.002). The results suggest that the particular grazing location could promote the horizontal transmission of this parasite and that certain breeds are less susceptible to N. caninum infection than others.