Different intrafamilial clinical presentation of FMF mutation carriers

Familial Mediterranean fever (FMF) is a heterogeneous disorder; at present, it is diagnosed using only genetic methods. In the current study, we performed molecular analysis in two families presenting with FMF. In the first family, we report two brothers with a common genotype (M694V/V726A) but with different clinical presentation. In the second family, we identified the M694V and K695R mutations in a presymptomatic carrier.     

Automated analysis of FISH and immunohistochemistry images: a review.

Fluorescent in-situ hybridization (FISH) and immunohistochemistry (IHC) constitute a pair of complimentary techniques for detecting gene amplification and overexpression, respectively. The advantages of IHC include relatively cheap materials and high sample durability, while FISH is the more accurate and reproducible method. Evaluation of FISH and IHC images is still largely performed manually, with automated or semiautomated techniques increasing in popularity. Here, we provide a comprehensive review of a number of (semi-) automated FISH and IHC image processing systems, focusing on the algorithmic aspects of each technique. Our review verifies the increasingly important role of such methods in FISH and IHC; however, manual intervention is still necessary in order to resolve particularly challenging or ambiguous cases. In addition, large-scale validation is required in order for these systems to enter standard clinical practice.     

Parasite nucleotide-metabolizing enzymes and host purinergic signalling

Tissue damage results in a variety of molecular signals that activate elements of the immune system. Recent years have seen a growing awareness that key regulators of these events are extracellular nucleotides that signal through purinergic receptors. Haematophagous insects and ticks secrete enzymes in their saliva that degrade nucleotides, thus inhibiting haemostasis and minimizing the ensuing pain and inflammatory reactions provoked by these mediators. The discovery of an enzymatic cascade of nucleotide-metabolizing enzymes in secreted products of Trichinella spiralis suggests that endoparasites use similar mechanisms to modulate host purinergic signalling.     

The formation of non-bilayer structures in total polar lipid extracts of chloroplast membranes

The structural organisation of aqueous dispersions of total membrane lipid extracts of broad bean (Vicia faba) chloroplasts is dependent on pH and the presence of cations. In the absence of inorganic salts, sonicated dispersions of lipid extract in distilled water form smooth, single-shell vesicles approximately 30–50 nm in diameter. Reducing the pH of the dispersions, to neutralise the acidic lipids present in the extract, or the addition of low concentrations of metal cations, leads to the fusion of the vesicles and a partial phase-separation of the non-bilayer forming lipid monogalactosyldiacylglycerol to form spherical inverted micelles similar to those previously reported for binary mixtures of monogalactosyl and digalactosyldiacylglycerol (Biochim. Biophys. Acta 685, 297–306). Increasing concentrations of polyvalent, but not monovalent, cations lead to further structural rearrangements involving the formation of para-crystalline arrays of tubular and spherical inverted micelles. The factors determining the formation of these different structures, and their possible relevance to the structural organisation of the native chloroplast membrane, are discussed.     

Reconstitution of plastoquinone in the D1/D2/cytochrome b-559 photosystem II reaction centre complex

Reconstitution of plastoquinone in the photosystem II D1/D2/cytochrome b-559 reaction centre complex, in the presence of the detergent Triton X-100, is reported. Illumination of the reconstituted system results in the reduction of cytochrome b-559, the process being partly herbicide-sensitive. In addition, the reconstitution of plastoquinone results in the ability of the isolated reaction centre to catalyse the photoreduction of 2,6-dichlorophenolindophenol in the presence of the exogenous electron donor diphenylcarbazide.     

Characterisation of novel protein families secreted by muscle stage larvae of Trichinella spiralis

Proteins secreted by Trichinella spiralis have a potential role in remodelling host skeletal muscle. However, whilst many parasite-secreted proteins have been identified, it has rarely been demonstrated that these are secreted into the nurse cell. Using an informatics-based analysis, we have searched the T. spiralis expressed sequence tag (EST) datasets for cDNAs encoding potential secreted proteins. Here we describe the characterisation of three of the top candidates isolated from our analysis, termed secreted from muscle stage larvae (SML)-1, -2 and -3. All three proteins were demonstrated to be secreted by muscle stage larvae, and immunohistochemical analysis established that SML-1 and -2 are secreted into developing nurse cells. We also show that SML-2 is processed from a precursor into smaller peptides by a metalloprotease contained within T. spiralis-secreted products. With the identification of these and other secreted proteins, we now have molecules to test in functional assays designed to dissect molecular features of the developing nurse cell.     

Endoscopic Tests for the Diagnosis of Helicobacter pylori Infection in Children: Validation of Rapid Urease Test

Background: Rapid urease test (CLO‐test) is an inexpensive and quick method for diagnosis of Helicobacter pylori infection with controversial results in children. We evaluated the performance of CLO‐test in relation to endoscopic and histological findings in children with H. pylori infection. Materials and methods: We studied the medical records of c hildren with H. pylori infection who were diagnosed between 1989 and 2009. Noninfected children were used as controls. H. pylori infection was defined by positive culture or by two other positive tests (histology and CLO‐test, or urea breath test when a single test was positive). All children had histology together with CLO‐test. Tissue culture was performed whenever possible. Results: Five hundred thirty infected children (10.4 ± 3.0 years) and 1060 controls (7.3 ± 4.4 years) were studied. Sensitivity of CLO‐test was 83.4% (95% CI, 79.9–86.3%), of culture 84.6% (95% CI, 78.7–89.1%), of histology 93.2% (95% CI, 90.7–95.1%), and specificity 99% (95% CI, 98.2–99.4%), 100%, and 100% respectively. CLO‐test positivity was correlated with higher bacterial density (p < .001), activity (p < .001) and severity of gastritis (p < .01), older age (p < .01), and the presence of antral nodularity (p < .001). When CLO‐test was positive, the concordance with histology and culture was high (95.5 and 89.2% respectively), whereas low concordance was observed when CLO‐test was negative (17.05 and 45.83% respectively). Conclusions: CLO‐test had lower sensitivity and comparable specificity with histology. Both tests should be performed concurrently to accurately diagnose H. pylori infection in children.     

Characterisation of the secreted apyrase family of Heligmosomoides polygyrus

Apyrases are a recurrent feature of secretomes from numerous species of parasitic nematodes. Here we characterise the five apyrases secreted by Heligmosomoides polygyrus, a natural parasite of mice and a widely used laboratory model for intestinal nematode infection. All five enzymes are closely related to soluble calcium-activated nucleotidases described in a variety of organisms, and distinct from the CD39 family of ecto-nucleotidases. Expression is maximal in adult worms and restricted to adults and L4s. Recombinant apyrases were produced and purified from Pichia pastoris. The five enzymes showed very similar biochemical properties, with strict calcium dependence and a broad substrate specificity, catalysing the hydrolysis of all nucleoside tri- and diphosphates, with no activity against nucleoside monophosphates. Natural infection of mice provoked very low antibodies to any enzyme, but immunisation with an apyrase cocktail showed partial protection against reinfection, with reduced egg output and parasite recovery. The most likely role for nematode secreted apyrases is hydrolysis of extracellular ATP, which acts as an alarmin for cellular release of IL-33 and initiation of type 2 immunity.     

Activation of Nippostrongylus brasiliensis infective larvae is regulated by a pathway distinct from the hookworm Ancylostoma caninum

Developmentally arrested infective larvae of strongylid nematodes are activated to resume growth by host-derived cues encountered during invasion of the mammalian host. Exposure of Nippostrongylus brasiliensis infective larvae to elevated temperature (37 °C) is sufficient to activate signalling pathways which result in resumption of feeding and protein secretion. This occurs independently of exposure to serum or glutathione, in contrast to the hookworm Ancylostoma caninum, and is not initiated by chemical exsheathment. No qualitative differences in protein secretion were induced by host serum as visualised by two-dimensional SDS–PAGE, although exposure of larvae to an aqueous extract of rat skin did stimulate secretion of a small pre-synthesised bolus of proteins. Infective larvae began feeding after a lag period of 3–4 h at 37 °C, reaching a maximum of 90% of the population feeding by 48 h. Neither a membrane permeant analogue of cyclic GMP nor muscarinic acetylcholine receptor agonists stimulated feeding at 20 °C, and high concentrations of both compounds inhibited temperature-induced activation. LY294002, an inhibitor of phosphatidylinositol 3-kinase, Akt inhibitor IV, an inhibitor of Akt protein kinase, and ketoconazole, an inhibitor of cytochrome P450, all blocked resumption of feeding and protein secretion at 37 °C. Serotonin increased the rate of feeding assessed by uptake of radiolabelled BSA, but could not initiate feeding independently of elevated temperature. Collectively, the data suggest that the early signalling events for larval activation in N. brasiliensis differ substantially from A. caninum, but that they may converge at pathways downstream of phosphatidylinositol 3-kinase involving steroid hormone synthesis.