Transcriptomic and Coexpression Network Analyses Revealed Pine Chalcone Synthase Genes Associated with Pine Wood Nematode Infection.

Pine wood nematode (PWN) causes serious diseases in conifers, especially pine species. To investigate the transcriptomic profiles of genes involved in pine-PWN interactions, two different pine species, namely, Pinus thunbergii and P. massoniana, were selected for this study. Weighted gene coexpression network analysis (WGCNA) was used to determine the relationship between changes in gene expression and the PWN population after PWN infection. PWN infection negatively affects the expression of most genes in pine trees, including plant defense-related genes such as genes related to plant hormone signal transduction, plant-pathogen interactions, and the MAPK signaling pathway in plants. However, the expression of chalcone synthase genes and their related genes were proportional to the changes in nematode populations, and chalcone synthase genes were dominant within the coexpression module enriched by genes highly correlated with the nematode population. Many genes that were closely related to chalcone synthase genes in the module were related to flavonoid biosynthesis, flavone and flavonol biosynthesis, and phenylpropanoid biosynthesis. Pine trees could actively adjust their defense strategies in response to changes in the number of invasive PWNs, but the sustained expression of chalcone synthase genes should play an important role in the inhibition of PWN infection.

Apicomplexa, trypanosoma and parasitic nematode protein kinases as antiparasitic therapeutic targets.

Parasitic infections caused by Plasmodium, Trypanosoma, Leishmania, Toxoplasma and parasitic nematodes affect hundreds of millions of individuals worldwide and are the cause of significant mortality and morbidity, particularly in developing countries. These diseases also have an impact on individuals from developed countries; for example, some US troops in Iraq and Afghanistan have been infected with Leishmania. The annual mortality associated with parasitic infections is estimated to be 1.5 million deaths. The socioeconomic impact of the morbidity associated with parasitic infections is significant, and the development of new drugs, aimed at novel targets, is urgently needed to develop effective treatments for these diseases. The small-molecule inhibitors discussed in this review constitute useful tools with which to explore the relevance of kinase inhibition in inducing antiparasitic activity. The aim of recent target-based approaches used in the development of parasite kinase inhibitors is to identify novel antiparasitic agents with therapeutic potential.

Cloning and characterisation of a Heterodera glycines aminopeptidase cDNA.

An aminopeptidase full-length cDNA (Hg-amp-1) was cloned from the adult female soybean cyst nematode Heterodera glycines by heterologous screening of a cDNA library with a Caenorhabditis elegans EST sequence. The predicted open reading frame encoded an 882-amino acid protein containing the conserved zinc-binding domain and GAMEN motif that are characteristic of M1 family aminopeptidases. The putative protein lacks any subcellular targeting signals and displays strong similarity to puromycin-sensitive aminopeptidases from C. elegans, Drosophila and mammals. Hg-amp-1 is expressed in juvenile nematodes and both male and female adults, with highest expression in gravid females. In situ mRNA hybridisation localised the Hg-amp-1 transcript to the genital primordium of pre-parasitic juvenile nematodes and the reproductive tract of adult females. Suppression of Hg-amp-1 transcript level by RNA-interference led to a 61% reduction in the number of female nematodes parasitising soybean roots 21 days post infection with infective juvenile nematodes that had been exposed to double-stranded RNA.

Tissue-specific expression of mast cell granule serine proteinases and their role in inflammation in the lung and gut.

Serine proteinases with trypsin-like (tryptase) and chymotrypsin-like (chymase) properties are major constituents of mast cell granules. Several tetrameric tryptases with differing specificities have been characterized in humans, but only a single chymase. In other species there are larger families of chymases with distinct and narrow proteolytic specificities. Expression of chymases and tryptases varies between tissues. Human pulmonary and gastrointestinal mast cells express chymase at lower levels than tryptase, whereas rodent and ruminant gastrointestinal mast cells express uniquely mucosa-specific chymases. Local and systemic release of chymases and tryptases can be quantified by immunoassay, providing highly specific markers of mast cell activation. The expression and constitutive extracellular secretion of the mucosa-specific chymase, mouse mast cell proteinase-1 (mMCP-1), is regulated by transforming growth factor-beta1 (TGF-beta1) in vitro, but it is not clear how the differential expression of chymases and tryptases is regulated in other species. Few native inhibitors have been identified for tryptases but the tetramers dissociate into inactive subunits in the absence of heparin. Chymases are variably inhibited by plasma proteinase inhibitors and by secretory leucocyte protease inhibitor (SLPI) that is expressed in the airways. Tryptases and chymases promote vascular permeability via indirect and possibly direct mechanisms. They contribute to tissue remodelling through selective proteolysis of matrix proteins and through activation of proteinase-activated receptors and of matrix metalloproteinases. Chymase may modulate vascular tissues through its ability to process angiotensin-I to angiotensin-II. Mucosa-specific chymases promote epithelial permeability and are involved in the immune expulsion of intestinal nematodes. Importantly, granule proteinases released extracellularly contribute to the recruitment of inflammatory cells and may thus be involved in innate responses to infection.

Ecto and endoparasites in two primary schools in Qualyob City, Egypt.

Ecto and endoparasites are still one of the public health problems in Egypt. This is particularly true among school students who are exposed to the parasitic infections or infestations by autoinfection or by contagious. In this paper, two primary schools were selected in Qualyob City, Qualyobia Governorate (in the Nile Delta). Examination of 486 school children (6-12 years old) revealed pediculosis (16.04), schistosomiasis (8.8%), amoebiasis (7.81%), giardiasis (9.05%), taeniasis saginata (0.41%), ascariasis (9.05%), enterobiasis (0.9%) and hymenolepiasis nana (9.87%). It was found that ectoparasites (lice) represented 17.8% of the total parasites detected in the children. Endoparasites transmitted by autoinfection represented 43.02%, those transmitted by skin penetration represented 9.84%, those transmitted by meat consumption represented 0.45% and by other modes of infection represented 28.8%. It was concluded that school children are the group of individuals at risk. They spend long time outside their homes in a crowd area. Besides, they convey the parasites, particularly those transmitted by contagious and autoinfection to their family members.

Identification of the secreted neutral proteases from Anisakis simplex.

Ingestion of larval nematodes (family: Anisakidae) can cause the human disease known as anisakiasis. After ingestion, Anisakis larvae can be invasive, penetrating host stomach or intestinal wall. Observation of larvae penetrating the tissue layers of human stomach in vitro by SEM showed tunnels and burrows were formed in the mucosa and submucosa. Based on these observations, we hypothesized that secreted proteases may be involved in the degradation of host tissue macromolecules to allow tunnel formation. Using a model of connective tissue extracellular matrix (ECM), we found that as few as 5 Anisakis simplex larvae could degrade approximately 25% of the ECM in a 16-mm culture well in 24 hr. Further characterization of the secreted proteases using synthetic peptide substrates and inhibitors revealed that there were 2 classes of proteases present: a metallo aminopeptidase and a trypsinlike serine protease. Extracts of Anisakis larvae contained a 25-kDa protease that was recognized by rabbit anti-rat trypsin antibody on western blots. This suggests that there is structural as well as functional similarity between the Anisakis trypsin and vertebrate trypsins.

Histamine metabolism in lungs of rats infected with Nippostrongylus brasiliensis.

Several parameters connected to histamine metabolism and mast cell number were examined in the lungs of rats infected with the nematode Nippostrongylus brasiliensis. Histamine levels as well as mast cell numbers were found to be increased on day 14 after infection and were elevated during the whole time of the experiment. Histidine decarboxylase activity also reached a peak on day 14. There was no measurable activity of diamine oxidase in the lungs of parasitized and normal rats. It is postulated that the increase in histidine decarboxylase activity and histamine concentration observed in the present study is related to the process of mastocytosis.

Antigen-induced mucosal damage and restitution in the small intestine of the immunized rat.

Intestinal mucosal damage and restitution were examined following antigen-induced systemic anaphylaxis in Nippostrongylus brasiliensis immunized rats. The rats were injected intravenously with N. brasiliensis antigen or saline. At 60 min, morphological and biochemical parameters were determined in jejunum and ileum, and the epithelial permeability was assessed by measuring recovery of 51Cr-ethylenediaminetetraacetic acid in the blood after injecting it into a ligated segment. Antigen challenge resulted in significant abnormalities: (1) villus damage with sloughing of enterocytes; (2) decreased activities of brush border enzymes; (3) decreased levels of mucosal histamine and rat mast cell protease II (mast cell mediators), and (4) increased uptake of 51Cr-ethylenediaminetetraacetic acid. Progression of the injury was examined by taking consecutive biopsies at 15-min intervals for 60 min and then at 5 h. At 15 min, an abnormality was present in all sections which ranged from minor oedema and enterocyte detachment at villus tips to virtual complete destruction of the apical region. Restitution occurred by villus contraction with migration of the epithelium over the damaged regions. At 5 h, the epithelium had resealed, but the villi were significantly reduced in height.

Possible lymphocyte effects on lactase expression by mouse jejunal enterocytes.

Jejunal intraepithelial lymphocyte numbers fall and lactase activity increases during early infection with the intestinal parasite Nematospiroides dubius. Both these variables later return to values found in control mice. These results support the view that local immune reactions, suppressed by the presence of N. dubius, normally inhibit lactase expression by mouse enterocytes.

Intestinal infection with Nematospiroides dubius selectively increases lactase expression by mouse jejunal enterocytes.

1. Intestinal structure, lactase (beta-galactosidase; EC 3.2.1.23) activity and alkaline phosphatase activity have been determined in mouse jejunal and ileal tissues before and during infection with the intestinal parasite Nematospiroides dubius. 2. Oral infection with small numbers of N. dubius larvae caused villus height, crypt depth and enterocyte migration rate to increase in the mouse jejunum. None of these effects occurred in ileal tissue. 3. Lactase activity also increased in jejunal, but not ileal, tissue of infected mice. This increase was associated with a doubling of the rate at which activity appeared in the brush-border membrane of enterocytes during migration over the basal regions of jejunal villi. Alkaline phosphatase activity in jejunal tissue remained unchanged in infected mice. 4. Attention is drawn to the fact that this is the first occasion when crypt cell hyperplasia has been found to be positively correlated with an increase in lactase activity and a decrease in cytotoxic/suppressor T-cells. Further work is needed to establish the primary cause of these effects.

Fluctuations in rat liver alanine-amino-transferase activity during experimental nippostrongylosis.

The activity of the gluconeogenic enzyme, alanine-amino-transferase (ALT), in a preparation from the liver of rats was studied by means of an in vitro assay throughout the course of a primary infection of Nippostrongylus brasiliensis, established by a subcutaneous injection of approximately 4000 3rd-stage larvae. The activity was measured on days 1-14 p.i. in both uninfected and infected rats and a marked pattern in the enzyme's activity was observed. In infected rats, the activity increased from 1.46 +/- 0.19 U/g liver on day 1 p.i. to a peak on day 4 p.i. of 10.75 +/- 1.62 U/g liver, then decreased to a trough of 0.44 +/- 0.18 U/g liver on day 10 p.i. before returning to original levels by day 14 p.i., by which time the infection had been largely eliminated. In uninfected rats the activity of the liver enzyme remained constant throughout this period with a value of 2.54 +/- 0.12 U/g liver. The activity of the enzyme in vitro was found to be related to the size of the inoculum on days 4 and 10 p.i. It was proposed that these observations could be due to either (1) a direct effect of the parasite, or (2) a consequence of the host immune response to the infection. In order to investigate the second proposition more fully, liver ALT activity was investigated by in vitro assay on selected days p.i. in rats experiencing a secondary N. brasiliensis infection.(ABSTRACT TRUNCATED AT 250 WORDS)

Testing the hypothesis that crypt cell hyperplasia inhibits lactase expression by mouse jejunal enterocytes.

Lactase activity and crypt cell proliferation both increased significantly in mouse jejunal villi in the presence of the intestinal parasite Nematospiroides dubius. Comparisons are made between this result and others showing lactase activity to decline whenever crypt cell proliferation is increased.

Suppression of Nippostrongylus brasiliensis (Nematoda)-induced lysophospholipase activity and peripheral eosinophilia by Eimeria nieschulzi (Apicomplexa).

Interspecific interactions between Nippostrongylus brasiliensis and Eimeria nieschulzi were studied by measuring fecal lysophospholipase (LYPH) activity and relative numbers of peripheral eosinophils in rats singly or concurrently infected with one or both parasite species. Three groups of 10 rats each were inoculated with 2 X 10(3) N. brasiliensis L3 larvae and/or 5 X 10(5) E. nieschulzi sporulated oocysts. Groups 1 and 2 were infected with E. nieschulzi or N. brasiliensis, respectively. Group 3 rats were infected first with N. brasiliensis, followed on day 8 postinoculation (PI) with E. nieschulzi. Each rat served as its own control. Results revealed LYPH levels rose steadily in Group 2 rats, reaching significant peaks on days 10 and 12 PI before decreasing to control levels. Lysophospholipase activity in Groups 1 and 3, however, did not differ from control values. Group 2 rats also demonstrated peripheral eosinophilia, with peak values occurring on days 10, 12, 14, and 16 PI, while rats in Groups 1 and 3 exhibited no eosinophilia. These results demonstrate that E. nieschulzi suppressed intestinal LYPH activity and relative peripheral eosinophilia and demonstrate that a host's immune response to a single parasite may be significantly altered when a second parasite species is present.

Peroxidase and gamma-glutamyl transpeptidase activities during Eimeria nieschulzi (Apicomplexa) and/or Nippostrongylus brasiliensis (Nematoda) infections in the rat.

Results suggest that malabsorption of amino acids which occurs during Eimeria nieschulzi and Nippostrongylus brasiliensis infections in rats is not due to impairment by intestinal inflammation of gamma-glutamyl transpeptidase activity.

Activation of alveolar macrophages following infection with the parasitic nematode Nippostrongylus brasiliensis.

Alveolar macrophages (AM) of rats infected with 3000 Nippostrongylus brasiliensis infective larvae for 2, 8 or 32 days (D2, D8 or D32 AM) quantitatively surpassed AM from uninfected rats in one or more of IgG- or C3-dependent phagocytosis indices, beta-D-glucuronidase release, or spontaneous release of thymocyte activating factor (interleukin-1, IL-1) and hepatocyte stimulation factor (HSF). These observations suggest that N. brasiliensis infection results in the activation of AM. We have reported previously that a greater proportion of AM from infected rats expressed C3 receptors and were helminthocidal in vitro in the presence of complement than normal AM which were not helminthocidal. The acquisition of the activated state by AM during infection may play a role in vivo lung resistance against migrating helminth parasites.

Anaphylactic release of mucosal mast cell protease and its relationship to gut permeability in Nippostrongylus-primed rats.

The systemic secretion of rat mucosal mast cell protease (RMCPII) was examined in Nippostrongylus-primed rats injected intravenously with N. brasiliensis whole worm antigen. The secretory response in primed rats was both time- and dose-dependent whereas no RMCPII was present in the sera of naive rats challenged with antigen. RMCPII was detected in the sol and gel phases of intestinal perfusates, the release of enzyme into the gut lumen occurring very rapidly after challenge in immune rats. Mucosal permeability, assessed by measuring the passage of Evan's blue from the blood into the gut lumen was both time- and dose-dependent and reflected the combined capillary and epithelial permeability. Although the release of RMCPII into the gut lumen occurred more rapidly than the intraluminal accumulation of Evan's blue, these two events were highly correlated. There were, in addition significant correlations between the systemic and enteric secretion of RMCPII and the enteric accumulation of Evan's blue. These results indicate that RMCPII may have a role in altering intestinal mucosal permeability during systemic anaphylaxis in the rat.