Toxoplasma gondii profilin does not stimulate an innate immune response through bovine or human TLR5

Toxoplasma gondii is responsible for one of the most prevalent infections in people. T. gondii profilin (TgPr) is a protein integral to parasite movement and cellular invasion. Murine TLR has been described to bind TgPr. Furthermore, more recently, human TLR5 has been described to recognise recombinant TgPr, as well as bacterial flagellin. In addition to infections in humans, T. gondii infects farm animals, but little information is available about its innate recognition. We aimed to investigate whether, similarly to their human orthologue, bovine and porcine TLR5 could also be stimulated by TgPr by using a combination of reporter cell lines expressing full length TLR5 from each species as well as primary cells. Although human and bovine TLR5-transfected cells responded to flagellin, no response was detected upon stimulation with profilin. Furthermore, TgPr failed to elicit IL-6 secretion in human peripheral blood mononuclear cells and CD14þ monocytes. In contrast, exposure of RAW cells, known to express TLR11 to TgPr, slightly increased the IL-6 response. Our data cast doubts on the possibility that profilin is a specific ligand for human TLR5 and bovine TLR5. This leaves the immunogenic properties of this potential target antigen uncharacterised outside of the murine system

Herpesviruses carrying a Brainbow cassette reveal replication and expression of limited numbers of incoming genomes

Whether all the infectious herpesvirus particles entering a cell are able to replicate and/or express their genomes is not known. Here, we developed a general method to determine the number of viral genomes expressed in an infected cell. We constructed and analysed fluorophore expression from a recombinant pseudorabies virus (PRV263) carrying a Brainbow cassette (Cre-conditional expression of different fluorophores). Using three isogenic strains derived from PRV263, each expressing a single fluorophore, we analysed the colour composition of cells infected with these three viruses at different multiplicities. We estimate that fewer than seven incoming genomes are expressed per cell. In addition, those templates that are expressed are the genomes selected for replication and packaging into virions. This finite limit on the number of viral genomes that can be expressed is an intrinsic property of the infected cell and may be influenced by viral and cellular factors

Effect of pH and ionic strength on the interaction of humic acid with aluminium oxide

The effect of pH and neutral electrolyte on the interaction between humic acid/humate and gamma-AlOOH (boehmite) was investigated. The quantitative characterization of surface charging for both partners was performed by means of potentiometric acid-base titration. The intrinsic equilibrium constants for surface charge formation were log K-a,1(int) = 6.7 +/- 0.2 and log K-a,2(int) = 10.6 +/- 0.2 and the point of zero charge was 8.7 +/- 0.1 for aluminium oxide. The pH-dependent solubility and the speciation of dissolved aluminium was calculated (MINT-EQA2). The fitted (FITEQL) pK values for dissociation of acidic groups of humic acid were pK(1) = 3.7 +/- 0.1 and pK(2) = 6.6 +/- 0.1 and the total acidity was 4.56 mmol g(-1) The pH range for the adsorption study was limited to between pH 5 and 10, where the amount of the aluminium species in the aqueous phase is negligible (less than 10(-5) mol dm(-3)) and the complicating side equilibria can be neglected. Adsorption isotherms were determined at pH similar to 5.5, similar to 8.5 and similar to 9.5, where the surface of adsorbent is positive, neutral and negative, respectively, and at 0.001, 0.1, 0.25 and 0.50 mol dm(-3) NaNO3. The isotherms are of the Langmuir type, except that measured at pH similar to 5.5 in the presence of 0.25 and 0.5 mol dm-3 salt. The interaction between humic acid/humate and aluminium oxide is mainly a ligand-exchange reaction with humic macroions with changing conformation under the influence of the charged interface. With increasing ionic strength the surface complexation takes place with more and more compressed humic macroions. The contribution of Coulombic interaction of oppositely charged partners is significant at acidic pH. We suppose heterocoagulation of humic acid and aluminium oxide particles at pH similar to 5.5 and higher salt content to explain the unusual increase in the apparent amount of humic acid adsorbed