Diagnostic epitope variability within Taenia solium 8 kDa antigen family: implications for cysticercosis immunodetection

To study diagnostic epitopes within the Taenia solium 8 kDa antigen family, six overlapping synthetic peptides from an 8 kDa family member (Ts8B2) were synthesized and evaluated by ELISA and MABA with sera from patients with neurocysticercosis (NCC), from infected pigs and from rabbits immunized with recombinant Ts8B2 protein. The pre-immune rabbit sera and the Ts8B2 recombinant protein served as negative and positive controls, respectively. A similar analysis was done with the already described antigenic peptides from another member of the 8 kDa family, highly similar to Ts8B2, the CyDA antigen. Surprisingly, neither the Ts8B2 peptides nor the CyDA peptides were recognized by infected human and porcine sera. However, the entire Ts8B2 recombinant, as well as amino and carboxy-terminal halves were recognized by the positive serum samples. The observed lack of recognition of linear Ts8B2 peptides suggests that the principal serological response to the Ts8B2 family is focused on conformational epitopes in contrast to the previously observed antigenicity of the CyDA peptides. This differential antigenicity of 8 kDa family peptides could be related with parasite antigenic variability. The fact that rabbits experimentally immunized with Ts8B2 did make anti-peptide antibodies to peptides Ts8B2-6 and CyDA-6, located in the carboxy-terminal region demonstrated that the Ts8B2 peptides are not intrinsically non-immunogenic.     

Salinity tolerance of Saprolegnia parasitica coker

Experiments on two strains ofS. parasitica have shown that this species together with other species ofSaprolegnia have a high tolerance to salinity when vegetative growth alone is considered. The effect of temperature and salinity on asexual sporulation shows that at temperatures of up to 20°C good sporulation occurs in salinities of 3.5%.. At temperatures above this, sporulation is repressed and gammae tend to be formed. Temperature appears to have a marked effect on the initial germination of zoospores, but after 12 hours this effect is minimized.

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Zusammenfassung Untersuchungen an zwei Stämmen vonS. parasitica haben gezeigt, daß diese Art, zusammen mit anderen Arten vonSaprolegnia, eine hohe Toleranz gegen Salinität hat, wenn das vegetative Wachstum allein in Betracht gezogen wird. Die Wirkung von Temperatur und Salinität an asexuellen Sporulation zeigt, daß bei Temperaturen bis zu 20°C eine gute Sporulation in einer Salinität von 3.5%. vorherrscht. Bei höheren Temperaturen wird Sporulation unterdrückt und bilden sich Gammae. Temperatur scheint eine bedeutende Wirkung an der initialen Germination von Zoosporen zu haben, jedoch ist diese Wirkung nach 12 Stunden minimal.

     

Echinostome infection in green frogs (Rana clamitans) is stage and age dependent

The increasing threat of emerging infectious diseases in both wildlife and humans has spurred interest in the causes of disease emergence, including the role of anthropogenic change. A prior field study of infection patterns in amphibians suggests that echinostome infection may be an emerging disease of green frogs Rana clamitans living in urbanized environments. We examined the impact of echinostome infection on green frog tadpoles at a wide range of developmental stages (Gosner stage 25–39). Echinostome infection was associated with green frog mortality rates of up to 40% in an early developmental stage, and none in later developmental stages. Tadpoles exposed to higher echinostome doses exhibited higher edema rates, a potential sign of compromised renal function. Histopathological analysis further supported the hypothesis that echinostome-induced tadpole mortality resulted from compromised renal function. Given that the timing of highest cercarial shedding can coincide with the most vulnerable stages of green frog tadpole development, echinostomes could significantly impact green frog survival in nature.     

A chimeric protein of simian immunodeficiency virus envelope glycoprotein gp140 and Escherichia coli aspartate transcarbamoylase

The envelope glycoproteins of the human immunodeficiency virus and the related simian immunodeficiency virus (SIV) mediate viral entry into host cells by fusing viral and target cell membranes. We have reported expression, purification, and characterization of gp140 (also called gp160e), the soluble, trimeric ectodomain of the SIV envelope glycoprotein, gp160 (B. Chen et al., J. Biol. Chem. 275:34946-34953, 2000). We have now expressed and purified chimeric proteins of SIV gp140 and its variants with the catalytic subunit (C) of Escherichia coli aspartate transcarbamoylase (ATCase). The fusion proteins (SIV gp140-ATC) bind viral receptor CD4 and a number of monoclonal antibodies specific for SIV gp140. The chimeric molecule also has ATCase activity, which requires trimerization of the ATCase C chains. Thus, the fusion protein is trimeric. When ATCase regulatory subunit dimers (R(2)) are added, the fusion protein assembles into dimers of trimers as expected from the structure of C(6)R(6) ATCase. Negative-stain electron microscopy reveals spikey features of both SIV gp140 and SIV gp140-ATC. The production of the fusion proteins may enhance the possibilities for structure determination of the envelope glycoprotein either by electron cryomicroscopy or X-ray crystallography.     

Characterization and functional activity of murine monoclonal antibodies specific for α1,6-glucan chain of Helicobacter pylori lipopolysaccharide

Background: The outer core region of H. pylori lipopolysaccharide (LPS) contains α1,6‐glucan previously shown to contribute to colonizing efficiency of a mouse stomach. The aim of the present study was to generate monoclonal antibodies (mAbs) specific for α1,6‐glucan and characterize their binding properties and functional activity. Materials and Methods: BALB/c mice were injected intraperitoneally with 10⁸ formalin‐fixed H. pylori O:3 0826::Kan cells 3× over 56 days to achieve significant titer. Anti‐α1,6‐glucan‐producing hybridomas were screened by indirect ELISA using purified H. pylori O:3 0826::Kan LPS. One clone, 1C4F9, was selected for further characterization. The specificities of mAbs were determined by indirect and inhibition ELISA using structurally defined H. pylori LPS and synthetic oligosaccharides, and whole‐cell indirect ELISA (WCE) of clinical isolates. They were further characterized by indirect immunofluorescent (IF) microscopy and their functional activity in vitro determined by serum bactericidal assays against wild‐type and mutant strains of H. pylori. Results: The generated anti‐α1,6‐glucan IgM, 1C4F9, has demonstrated an excellent specificity for the glucan chain containing 5 to 6 α1,6‐linked glucose residues and showed surface accessibility by IF microscopy with H. pylori cells adherent to gastric adenocarcinoma cells monolayers. Of 38 isolates from Chile, 17 strains reacted with antiglucan mAbs in WCE (OD₄₅₀ ≥ 0.2). Bactericidal activity was observed against selective wild‐type and mutant H. pylori strains exhibiting OD₄₅₀ values of ≥0.45 in WCE. Conclusions: Anti‐α1,6‐glucan mAbs could have potential application in typing and surveillance of H. pylori isolates as well as offer insights into structural requirements for the development of LPS‐based vaccine against H. pylori infections.     

Single particle reconstructions of the transferrin-transferrin receptor complex obtained with different specimen preparation techniques

The outcome of three-dimensional (3D) reconstructions in single particle electron microscopy (EM) depends on a number of parameters. We have used the well-characterized structure of the transferrin (Tf)-transferrin receptor (TfR) complex to study how specimen preparation techniques influence the outcome of single particle EM reconstructions. The Tf-TfR complex is small (290kDa) and of low symmetry (2-fold). Angular reconstitution from images of vitrified specimens does not reliably converge on the correct structure. Random conical tilt reconstructions from negatively stained specimens are reliable, but show variable degrees of artifacts depending on the negative staining protocol. Alignment of class averages from vitrified specimens to a 3D negative stain reference model using FREALIGN largely eliminated artifacts in the resulting 3D maps, but not completely. Our results stress the need for critical evaluation of structures determined by single particle EM.     

Multicenter evaluation of reverse line blot assay for detection of drug resistance in Mycobacterium tuberculosis clinical isolates

A multicenter study was conducted with the objective to evaluate a reverse line blot (RLB) assay to detect resistance to rifampin (RIF), isoniazid (INH), streptomycin (STR), and ethambutol (EMB) in clinical isolates of Mycobacterium tuberculosis. Oligonucleotides specific for wild type and mutant (drug resistance linked) alleles of the selected codons in the genes rpoB, inhA, ahpC, rpsL, rrs, embB, were immobilized on a nylon membrane. The RLB assay conditions were optimized following analysis of DNA samples with known sequences of the targeted genes. For validation of the method at different geographical locations, the membranes were sent to seven laboratories in six countries representing the regions with high burdens of multudrug-resistant tuberculosis. The reproducibility of the assay for detection of rpoB genotypes was initially evaluated on a blinded set of twenty reference DNA samples with known allele types and overall concordant results were obtained. Further mutation analysis was performed by each laboratory on the local strains. Upon RLB analysis of 315 clinical isolates from different countries, 132 (85.2%) of 155 RIF-resistant and 28 (51.0%) of 55 EMB-resistant isolates were correctly identified, showing applicability of the assay when targeting the rpoB hot-spot region and embB306. Mutations in the inhA and ahpC promoter regions, conferring resistance to INH, were successfully identified in respectively 16.9% and 13.2% of INH-resistant strains. Likewise, mutations in rrs513 and rpsL88 that confer resistance to STR were identified in respectively 15.1% and 10.7% of STR-resistant strains. It should be mentioned that mutation analysis of the above targets usually requires rather costly DNA sequencing to which the proposed RLB assay presents rapid and inexpensive alternative. Furthermore, the proposed method requires the same simple equipment as that used for spoligotyping and permits simultaneous analysis of up to 40 samples. This technique is a first attempt to combine different targets in a single assay for prediction of antituberculosis drugs resistance. It is open to further development as it allows easy incorporation of new probes for detection of mutations in other genes associated with resistance to second-line (e.g., fluoroquinolones) and new antituberculosis compounds.