Recombinant dissection of myosin heavy chain of Toxocara canis shows strong clustering of antigenic regions

Myosins from nematode parasites elicit strong humoral and cellular immune responses and have been investigated as vaccine candidates. In this study we cloned and sequenced a cDNA coding for myosin heavy chain from Toxocara canis, a nematode parasite of canids which may also infect humans and cause various unspecific symptoms. To determine the major antigenic regions the myosin heavy chain was systematically dissected into ten overlapping recombinant fusion polypeptides which were purified by metal chelate chromatography. Single fragments were then tested for their IgG reactivity in sera from toxocarosis patients and healthy probands. Two regions, one region at the mid to carboxy-terminal end of the head domain and one region in the rod domain, were identified as major antigens, which in combination were positive with 86% of the sera. The other domains were less reactive. This shows that the patients' IgG reactivity was not directed evenly against all parts of the molecule, but was rather clustered in few regions.     

Characterisation of recombinant human IgE-Fc fragments expressed in baculovirus-infected insect cells

IgE mediates its effector functions through the Fc region and it has been demonstrated that structures in the Cvarepsilon3-domain are crucial for FcvarepsilonR-binding. In order to further study structures of importance for the function of IgE, such as the carbohydrates, fragments with unmodified amino acid sequence were blunt-end cloned and expressed in baculovirus-infected Sf9 cells. Two fragments of human IgE, one encompassing the entire Fc-region (rCvarepsilon2-4) and a smaller one comprising the second and third domain (rCvarepsilon2-3), were produced and characterised with respect to epitope expression, glycosylation and FcvarepsilonR-binding. N-terminal analysis showed the expected VCSRDF-sequence of the Cvarepsilon2-domain, confirming correct cleavage of the secretion signal. Immunoblotting and gel permeation chromatography demonstrated that rCvarepsilon2-4 mainly formed a dimer, whereas rCvarepsilon2-3 also existed as monomers and oligomers. Endoglycosidase-treatment revealed that both fragments were N-glycosylated. In inhibition ELISA, rCvarepsilon2-4 and myeloma protein IgE(DES) reacted in a near equimolar way with monoclonal antibodies against the Cvarepsilon2-, Cvarepsilon3- and Cvarepsilon4-domains, whereas rCvarepsilon2-3 only reacted with anti-Cvarepsilon2 mAbs. Moreover, in FACS analysis rCvarepsilon2-4 interacted with two cell-lines constitutively expressing FcvarepsilonRI or FcvarepsilonRII, whereas rCvarepsilon2-3 lacked reactivity. A substantial reduction in the ability of rCvarepsilon2-4, following endoglycosidase treatment, to react with recombinant alpha-chain of the high affinity receptor for IgE in sandwich ELISA, indicated a role of N-linked oligosaccharides in stabilising receptor binding structures. Taken together, our results show that rCvarepsilon2-4, but not rCvarepsilon2-3, will be useful in studies of structure-function relationships of IgE, including the role of N-glycosylation, since it demonstrated appropriate epitope expression, conformation and ability to bind Fcvarepsilon-receptors.     

Detection of Herpes simplex virus DNA by real-time PCR

Molecular detection of herpes simplex virus (HSV) DNA is recognized as the reference standard assay method for the sensitive and specific diagnosis of central nervous system infections caused by HSV. In this study, a molecular assay based on real-time PCR on the LightCycler (LC) instrument was evaluated and compared with a home-brew molecular assay. The detection limit of the LC assay was determined with 10-fold dilutions of plasmid pS4 with the SalI restriction fragment of the DNA polymerase gene and with the First European Union Concerted Action HSV Proficiency Panel. A total of 59 cerebrospinal fluid (CSF) specimens were investigated for the comparative study. With plasmid pS4, the detection limit of the LC assay was found to be 10(4) copies per ml, i.e., 12.5 copies per run. When samples of the First European Union Concerted Action HSV Proficiency Panel were tested, 2x10(3) to 5x10(3) HSV type 1 genome equivalents (GE) per ml, i.e., 2.5 to 6.3 GE per run, could consistently be detected. There was a correlation between the LC assay and the home-brew assay in 55 of 59 specimens. In conclusion, the LC assay allows very rapid detection of HSV DNA in CSF. It was found to be laborsaving and showed sufficient sensitivity.     

Binding of heparan sulfate to Staphylococcus aureus.

Heparan sulfate binds to proteins present on the surface of Staphylococcus aureus cells. Binding of 125I-heparan sulfate to S. aureus was time dependent, saturable, and influenced by pH and ionic strength, and cell-bound 125I-heparan sulfate was displaced by unlabelled heparan sulfate or heparin. Other glycosaminoglycans of comparable size (chondroitin sulfate and dermatan sulfate), highly glycosylated glycoprotein (hog gastric mucin), and some anionic polysaccharides (dextran sulfate and RNA) inhibited heparan sulfate binding to various extents. Heat treatment (80 degrees C for 10 min) and treatment of the bacteria with pronase E, proteinase K, pepsin, and chymotrypsin considerably reduced their ability to bind 125I-heparan sulfate, but treatment with trypsin and neuraminidase did not affect binding. Scatchard plot analysis indicated the presence of cell surface components with low affinity (Kd = 3 x 10(-5) M) for heparan sulfate. Cell surface components were released by stirring bacteria with 1 M LiCl at 37 degrees C for 2 h. Proteins of this extract that competitively inhibited binding of 125I-heparan sulfate to S. aureus were isolated by affinity chromatography on heparin-Sepharose. Two proteins having molecular masses of approximately 66 and 60 kDa and the ability to bind 125I-heparan sulfate were obtained. The first 9 amino-terminal amino acid residues of the 66-kDa protein are Asp-Trp-Thr-Gly-Trp-Leu-Ala-Ala-Ala, and the first 4 amino-terminal amino acid residues of the 60-kDa protein are Met-Leu-Val-Thr.     

Contraluminal p-aminohippurate transport in the proximal tubule of the rat kidney

Using the stop-flow peritubular capillary microperfusion method the inhibitory potency (apparent Ki values) of cyclic nucleotides and prostanoids against contraluminal p-aminohippurate (PAH), dicarboxylate and sulphate transport was evaluated. Conversely the contraluminal transport rate of labelled cAMP, cGMP, prostaglandin E2, and prostaglandin D2 was measured and the inhibition by different substrates was tested. Cyclic AMP and its 8-bromo and dibutyryl analogues inhibited contraluminal PAH transport with an app. Ki,PAH of 3.4, 0.63 and 0.52 mmol/l. The respective app. Ki,PAH values of cGMP and its analogues are with 0.27, 0.04 and 0.05 mmol/l, considerably lower. None of the cyclic nucleotides tested interacted with contraluminal dicarboxylate, sulphate and N1-methylnicotinamide transport. ATP, ADP, AMP, adenosine and adenine as well as GTP, GDP, GMP, guanosine and guanine did not inhibit PAH transport while most of the phosphodiesterase inhibitors tested did. Time-dependent contraluminal uptake of [3H]cAMP and [3H]cGMP was measured at different starting concentrations and showed facilitated diffusion kinetics with the following parameters for cAMP: Km = 1.5 mmol/l, Jmax = 0.34 pmol S-1 cm-1, r (extracellular/intracellular amount at steady state) = 0.91; for cGMP: Km = 0.29 mmol/l, Jmax = 0.31 pmol S-1 cm-1, r = 0.55. Comparison of app. Ki,cGMP with app. Ki,PAH of ten substrates gave a linear relation with a ratio of 1.83 +/- 0.5. All prostanoids applied inhibited the contraluminal PAH transport; the prostaglandins E1, F1 alpha, A1, B1, E2, F2 alpha, D2, A2 and B2 with an app. Ki,PAH between 0.08 and 0.18 mmol/l. The app. Ki of the prostacyclins 6,15-diketo-13,14-dihydroxy-F1 alpha (0.22 mmol/l) and Iloprost (0.17 mmol/l) as well as that of leukotrienes B4 (0.2 mmol/l) was in the same range, while the app. Ki,PAH of the prostacyclins PGI2 (0.55 mmol/l), 6-keto-PGF1 alpha (0.77 mmol/l) and 2,3-dinor-6-keto-PGF1 alpha (0.57 mmol/l) as well as that of thromboxane B2 (0.36 mmol/l) was somewhat higher. None of these prostanoids inhibited contraluminal dicarboxylate transport and only PGB1, E2 and D2 inhibited contraluminal sulphate transport (app. Ki,SO4(2-) 5.4, 11.0, 17.9 mmol/l respectively). Contraluminal influx of labelled PGE2 showed complex transport kinetics with a mixed Km = 0.61 mmol/l and Jmax of 4.26 pmol S-1 cm-1. It was inhibited by probenecid, sulphate and indomethacin. Contraluminal influx of PGD2, however, was only inhibited by probenecid. The data indicate that cyclic nucleotides as well as prostanoids are transported by the contraluminal PAH transporter. For prostaglandin E2 a significant uptake through the sulphate transporter occurs in addition.(ABSTRACT TRUNCATED AT 400 WORDS)     

Surface-expressed invariant chain (CD74) is required for internalization of human leucocyte antigen-DR molecules to early endosomal compartments

Transport of major histocompatibility complex (MHC) class II molecules to the endocytic route is directed by the associated invariant chain (Ii). In the endocytic pathway, Ii is proteolytically cleaved and, upon removal of residual Ii fragments, class II alpha beta dimers are charged with antigenic peptide and recognized by CD4+ T cells. Although distinct peptide-loading compartments such as MIIC (MHC class II loading compartment) and CIIV (MHC class II vesicles) have been characterized in different cells, there is growing evidence of a multitude of subcellular compartments in which antigenic peptide loading takes place. We employed a physiological cellular system in which surface Ii (CD74) and surface human leucocyte antigen (HLA)-DR were induced either alone or in combination. This was achieved by transient exposure of HT-29 cells to recombinant interferon-gamma (rIFN-gamma). Using distinct cellular variants, we showed that: (i) the majority of Ii molecules physically associate on the cell membrane with class II dimers to form DR alpha beta:Ii complexes; (ii) the presence of surface Ii is a prerequisite for the rapid uptake of HLA-DR-specific monoclonal antibodies into early endosomes because only the surface DR+/Ii+ phenotype, and not the DR+/Ii- variant, efficiently internalizes; and (iii) the HLA-DR:Ii complexes are targeted to early endosomes, as indicated by co-localization with the GTPase, Rab5, and endocytosed bovine serum albumin. Internalization of HLA-DR:Ii complexes, accommodation of peptides by DR alphabeta heterodimers in early endosomes and recycling to the cell surface may be a mechanism used to increase the peptide repertoire that antigen-presenting cells display to MHC class II-restricted T cells.     

Characterization of the major secreted zinc metalloprotease- dependent glycerophospholipid:cholesterol acyltransferase, PlaC, of Legionella pneumophila

Legionella pneumophila, an intracellular pathogen causing a severe pneumonia, possesses distinct lipolytic activities which have not been completely assigned to specific enzymes so far. We cloned and characterized a gene, plaC, encoding a protein with high homology to PlaA, the major secreted lysophospholipase A of L. pneumophila and to other hydrolytic enzymes belonging to the GDSL family. Here we show that L. pneumophila plaC mutants possessed reduced phospholipase A and lysophospholipase A activities and lacked glycerophospholipid:cholesterol acyltransferase activity in their culture supernatants. The mutants' reduced phospholipase A and acyltransferase activities were complemented by reintroduction of an intact copy of plaC. Additionally, plaC conferred increased lysophospholipase A and glycerophospholipid:cholesterol acytransferase activities to recombinant Escherichia coli. Furthermore, PlaC was shown to be another candidate exported by the L. pneumophila type II secretion system and was activated by a factor present in the bacterial culture supernatant dependent on the zinc metalloprotease. Finally, the role of plaC in intracellular infection of Acanthamoeba castellanii and U937 macrophages with L. pneumophila was assessed, and plaC was found to be dispensable. Thus, L. pneumophila possesses another secreted lipolytic enzyme, a protein with acyltransferase, phospholipase A, and lysophospholipase A activities. This enzyme is distinguished from the previously characterized phospholipases A and lysophospholipases A by its capacity not only to cleave fatty acids from lipids but to transfer them to cholesterol. Cholesterol is an important compound of eukaryotic membranes, and an acyltransferase might be a tool for host cell modification to fit the needs of the bacterium.     

Dietary antigen-specific T-cell responses: switch from an interleukin-10-dominated response in normal mice to a T-helper 1 cytokine profile in Galphai2-deficient mice prior to colitis

We investigated dietary antigen-specific T-cell responses in mesenteric lymph nodes (MLN) and Peyer's patches (PP) in noncolitic control mice as well as in colitis-prone mice prior to onset of histological active colitis. T cells were restimulated in vitro with constituents isolated from the mouse diet. Interestingly, MLN T cells of littermate G(alpha)i2+/- control mice responded to soya with high production of interleukin (IL)-10, but did not produce proinflammatory T-helper 1 (Th1) cytokines. Recall dietary antigen stimulation of G(alpha)i2+/- PP T cells did not result in increased IL-10 production above the spontaneous production in the absence of antigenic stimulation. In strong contrast, MLN T cells from precolitic G(alpha)i2-/- mice produced high levels of interferon-gamma (IFN-gamma) upon restimulation with soya, which could be abolished using a major histocompatibility complex class II-blocking antibody. In conclusion, the present study demonstrates that MLN T lymphocytes in normal healthy mice respond with a significantly increased production of the regulatory cytokine IL-10 on re-encounter with dietary proteins in vitro. In marked contrast precolitic G(alpha)i2-/- mice respond to dietary antigens with a Th1-dominated cytokine response in the mucosa, prior to onset of colitis, with excessive IFN-gamma production. These results suggest that aberrant immune responses to dietary antigens could contribute as a potential pathogenic mechanism in the onset of colitis in G(alpha)i2-deficient mice.     

Multilocus sequence typing of methicillin-resistant Staphylococcus aureus from an area of low endemicity by real-time PCR

A protocol for multilocus sequence typing (MLST) of methicillin-resistant Staphylococcus aureus (MRSA) was adapted to real-time LightCycler System PCR for efficient and rapid amplification of seven housekeeping genes in the same PCR run and real-time detection of the products. The method was evaluated on a representative and well-characterized collection of clinical MRSA isolates (n = 57) obtained from an area of low endemicity. Twenty sequence types (STs) and nine clonal complexes were identified. Combining STs and the staphylococcal cassette chromosome mec (SCCmec) type identified 27 different genotypes, and type IV SCCmec was present in 11 different STs. The presence of the Panton Valentine leukocidin (PVL) genes was found in isolates of four different STs. Eleven different STs were found among the community-acquired as well as among the hospital-acquired MRSA. The genetic heterogeneity was also denoted by pulsed-field gel electrophoresis analysis that showed 24 different pulsotypes among the 57 MRSA isolates. The presence of more than one different type of SCCmec in the same ST indicates that the MRSA clones have arisen at several occasions in the same genetic background by independent acquisition of SCCmec into methicillin-sensitive strains. This circumstance shows the importance of combining MLST data with SCCmec-typing results when investigating the origins of MRSA.     

Hfq-dependent regulation of OmpA synthesis is mediated by an antisense RNA

This paper shows that the small RNA MicA (previously SraD) is an antisense regulator of ompA in Escherichia coli. MicA accumulates upon entry into stationary phase and down-regulates the level of ompA mRNA. Regulation of ompA (outer membrane protein A), previously attributed to Hfq/mRNA binding, is lost upon deletion of the micA gene, whereas overexpression of MicA inhibits the synthesis of OmpA. In vitro, MicA binds to the ompA mRNA leader. Enzymatic and chemical probing was used to map the structures of MicA, the ompA mRNA leader, and the complex formed upon binding. MicA binding generates a footprint across the ompA Shine-Dalgarno sequence, consistent with a 12 + 4 base-pair interaction, which is additionally supported by the effect of mutations in vivo and by bioinformatics analysis of enterobacterial micA/ompA homolog sequences. MicA is conserved in many enterobacteria, as is its ompA target site. In vitro toeprinting confirmed that binding of MicA specifically interferes with ribosome binding. We propose that MicA, when present at high levels, blocks ribosome binding at the ompA translation start site, which-in line with previous work-secondarily facilitates RNase E cleavage and subsequent mRNA decay. MicA requires the presence of the Hfq protein, although the mechanistic basis for this remains unclear.