A rhamnose-binding lectin from Rhodnius prolixus and the impact of its silencing on gut bacterial microbiota and Trypanosoma cruzi.

Lectins are ubiquitous proteins involved in the immune defenses of different organisms and mainly responsible for non-self-recognition and agglutination reactions. This work describes molecular and biological characterization of a rhamnose-binding lectin (RBL) from Rhodnius prolixus, which possesses a 21 amino acid signal peptide and a mature protein of 34.6 kDa. The in-silico analysis of the primary and secondary structures of RpLec revealed a lectin domain fully conserved among previous insects studied. The three-dimensional homology model of RpLec was similar to other RBL-lectins. Docking predictions with the monosaccharides showed rhamnose and galactose-binding sites comparable to Latrophilin-1 and N-Acetylgalactosamine-binding in a different site. The effects of RpLec gene silencing on levels of infecting Trypanosoma cruzi Dm 28c and intestinal bacterial populations in the R. prolixus midgut were studied by injecting RpLec dsRNA into the R. prolixus hemocoel. Whereas T. cruzi numbers remained unchanged compared with the controls, numbers of bacteria increased significantly. The silencing also induced the up regulation of the R. prolixus defC (defensin) expression gene. These results with RpLec reveal the potential importance of this little studied molecule in the insect vector immune response and homeostasis of the gut bacterial microbiota.

Rhodnius prolixus: from physiology by Wigglesworth to recent studies of immune system modulation by Trypanosoma cruzi and Trypanosoma rangeli.

This review is dedicated to the memory of Professor Sir Vincent B. Wigglesworth (VW) in recognition of his many pioneering contributions to insect physiology which, even today, form the basis of modern-day research in this field. Insects not only make vital contributions to our everyday lives by their roles in pollination, balancing eco-systems and provision of honey and silk products, but they are also outstanding models for studying the pathogenicity of microorganisms and the functioning of innate immunity in humans. In this overview, the immune system of the triatomine bug, Rhodnius prolixus, is considered which is most appropriate to this dedication as this insect species was the favourite subject of VW's research. Herein are described recent developments in knowledge of the functioning of the R. prolixus immune system. Thus, the roles of the cellular defences, such as phagocytosis and nodule formation, as well as the role of eicosanoids, ecdysone, antimicrobial peptides, reactive oxygen and nitrogen radicals, and the gut microbiota in the immune response of R. prolixus are described. The details of many of these were unknown to VW although his work gives indications of his awareness of the importance to R. prolixus of cellular immunity, antibacterial activity, prophenoloxidase and the gut microbiota. This description of R. prolixus immunity forms a backdrop to studies on the interaction of the parasitic flagellates, Trypanosoma cruzi and Trypanosoma rangeli, with the host defences of this important insect vector. These parasites remarkably utilize different strategies to avoid/modulate the triatomine immune response in order to survive in the extremely hostile host environments present in the vector gut and haemocoel. Much recent information has also been gleaned on the remarkable diversity of the immune system in the R. prolixus gut and its interaction with trypanosome parasites. This new data is reviewed and gaps in our knowledge of R. prolixus immunity are identified as subjects for future endeavours. Finally, the publication of the T. cruzi, T. rangeli and R. prolixus genomes, together with the use of modern molecular techniques, should lead to the enhanced identification of the determinants of infection derived from both the vector and the parasites which, in turn, could form targets for new molecular-based control strategies.

Entomopathogenic Fungi: New Insights into Host-Pathogen Interactions.

Although many insects successfully live in dangerous environments exposed to diverse communities of microbes, they are often exploited and killed by specialist pathogens. Studies of host-pathogen interactions (HPI) provide valuable insights into the dynamics of the highly aggressive coevolutionary arms race between entomopathogenic fungi (EPF) and their arthropod hosts. The host defenses are designed to exclude the pathogen or mitigate the damage inflicted while the pathogen responds with immune evasion and utilization of host resources. EPF neutralize their immediate surroundings on the insect integument and benefit from the physiochemical properties of the cuticle and its compounds that exclude competing microbes. EPF also exhibit adaptations aimed at minimizing trauma that can be deleterious to both host and pathogen (eg, melanization of hemolymph), form narrow penetration pegs that alleviate host dehydration and produce blastospores that lack immunogenic sugars/enzymes but facilitate rapid assimilation of hemolymph nutrients. In response, insects deploy an extensive armory of hemocytes and macromolecules, such as lectins and phenoloxidase, that repel, immobilize, and kill EPF. New evidence suggests that immune bioactives work synergistically (eg, lysozyme with antimicrobial peptides) to combat infections. Some proteins, including transferrin and apolipophorin III, also demonstrate multifunctional properties, participating in metabolism, homeostasis, and pathogen recognition. This review discusses the molecular intricacies of these HPI, highlighting the interplay between immunity, stress management, and metabolism. Increased knowledge in this area could enhance the efficacy of EPF, ensuring their future in integrated pest management programs.

Amino acid derivatives from Lucilia sericata excretions/secretions may contribute to the beneficial effects of maggot therapy via increased angiogenesis.

BACKGROUND: Maggot therapy, utilizing the larvae of Lucilia sericata, has been reported to reduce the bacterial load within wounds and also to enhance wound healing. Maggot excretions/secretions (ES) have been shown to have a role in the success of maggot therapy. While the protein content of ES has been investigated, to date little research has focused on the small metabolites present in ES and their potential contribution to the therapy. Study of the molecular composition of the secretions and the potential bioactivities present will allow for a more detailed evaluation of the efficacy of maggot therapy. OBJECTIVES: We studied the amino acid-like compounds present in ES of L. sericata larvae in order to determine the compounds present and their potential role in the wound healing process. METHODS: These included thin-layer chromatography/mass spectrometric analysis of ES to identify amino acid-like components, a turbidometric assay to investigate their potential antibacterial activity and cell proliferation studies to investigate their potential mitogenic ability. RESULTS: Three prominent compounds were detected and identified as histidine, valinol and 3-guanidinopropionic acid. While these amino acids were not shown to exhibit antibacterial activity, a proliferative effect on the growth of human endothelial cells, but not fibroblasts, was noted. CONCLUSIONS: The demonstrated proliferative effect, selectively on endothelial cells, suggests that the amino acid-like compounds present in maggot ES may have a role in wound healing, by stimulating angiogenesis.

Antiserum against perimicrovillar membranes and midgut tissue reduces the development of Trypanosoma cruzi in the insect vector, Rhodnius prolixus.

Antiserum raised against Rhodnius prolixus perimicrovillar membranes (PMM) and midgut tissue interfered with the midgut structural organization and reduced the development of Trypanosoma cruzi in the R. prolixus insect vector. SDS-PAGE and Western blot analyses confirmed the specific recognition of midgut proteins by the antibody. Feeding, mortality, molt, and oviposition of the insects were unaffected by feeding with the antiserum. However, the eclosion of the eggs were reduced from R. prolixus females treated with antiserum. Additionally, in vivo evaluation showed that after oral treatment with the antiserum, the intensity of infection with the Dm-28c clone of T. cruzi decreased in the digestive tract of fifth-instar nymphs and in the excretions of R. prolixus adults. These results suggest that the changes observed in the PMM organization in the posterior midgut of R. prolixus may not be important for triatomine survival but the antiserum acts as a transmission-reduction vaccine able to induce significant decreases in T. cruzi infection in the vector.

Comparisons of PAH-induced immunomodulation in three bivalve molluscs.

There is growing evidence that contaminants may be partly responsible for the observed increase in disease in marine organisms by adversely affecting their immunity. Bivalve molluscs are common sentinels used in invertebrate immunotoxicology, however, to date, studies have been restricted to a few resilient species. This present study is a comparative investigation into the effects of the polycyclic aromatic hydrocarbon, phenanthrene, on the immunocompetence of three bivalve species. The commonly-studied marine mussel, Mytilus edulis, was compared with two species that have never been studied with respect to immunomodulation, namely, the edible cockle, Cerastoderma edule and the razor shell, Ensis siliqua. Animals were exposed to a range of phenanthrene concentrations (50, 100, 200 or 400 microg l(-1)) and haemocyte immune parameters, including haemocyte counts, phagocytosis, superoxide generation, lysosomal enzymes and lectin-binding, were monitored. Aims were not only to extend existing knowledge of bivalve immunotoxicology, but also to establish whether contaminant-induced immunomodulation in the sentinel species, M. edulis, is comparable to that observed in other bivalves. Results showed that the immune response of the three species was differentially affected by phenanthrene exposure, with immunomodulation in M. edulis not reflecting the immunological changes observed in the other two species. This suggests M. edulis may not be a suitable sentinel bivalve, and that other species, such as C. edule, may more accurately reflect the general immunological response of this group of marine animals.

Bivalve immunity: comparisons between the marine mussel (Mytilus edulis), the edible cockle (Cerastoderma edule) and the razor-shell (Ensis siliqua).

Much of the current knowledge concerning bivalve immunology and immunotoxicology has come from studies on the mussel genus, Mytilus, or from the American oyster, Crassostrea virginica. Following a major oil spill, it was observed that the marine mussel, Mytilus edulis, underwent significant immunosuppression but no oil-induced mortalities, while in contrast, mass mortalities were noted in the edible cockle, Cerastoderma edule, and the razor-shell, Ensis siliqua. A study comparing immune cells and functions in these three species was initiated (i) to assess whether M. edulis was a representative model species and (ii) to provide baseline data on immunity in two common species, which had previously received little or no attention in this respect. While all three species shared similar cell types, their lectin-binding and enzyme cytochemistry differed considerably. M. edulis had significantly different proportions of haemocytes binding with the lectins concanavalin A, wheatgerm agglutinin and Helix pomatia agglutinin and stained positive for eight enzymes, compared with only five in C. edule and three in E. siliqua. In terms of immune function, M. edulis haemocytes were much more active in phagocytosis and superoxide generation than haemocytes of the other two species. The results show that immune cells and functions differed extensively in these three closely related species, with M. edulis showing a much higher level of immunological vigour that may be linked to its considerable resilience to adverse environmental conditions. This suggests that M. edulis may not be particularly representative of the bivalves in terms of immune reactivity and that a wider range of species should be included in studies of molluscan immunotoxicology.

Identification and distribution of carbohydrate moieties on the salivary glands of Rhodnius prolixus and their possible involvement in attachment/invasion by Trypanosoma rangeli.

In the present study, FITC-labelled lectins (WGA, Con A, PNA, HPA, and TPA) were utilized to investigate carbohydrate residues on the surface of Rhodnius prolixus salivary glands. The results revealed that the salivary glands are rich in carbohydrate moieties and the diversity in binding pattern of particular lectins showed the presence of specific carbohydrate residues in the basal lamina, muscle, and cell layers of the glands. Subsequently, the sugars detected on the salivary gland surface were employed to investigate the interaction between Trypanosoma rangeli and the R. prolixus salivary glands. In vitro adhesion inhibition assays using long epimastigote forms (the invasion/adhesion forms) showed that some sugars tested were able to block the receptors on both the surfaces of the salivary glands and on T. rangeli. Among the sugars tested, GlcNAc, GalNAc, and galactose showed the highest overall inhibitory effect, following pre-incubation of either the salivary glands or parasites. These results are discussed in relation to previous work on the role of carbohydrates and lectins in insect vector/parasite interactions.

Calreticulin enriched as an early-stage encapsulation protein in wax moth Galleria mellonella larvae.

To investigate the molecular mechanism of the early-stage encapsulation reaction in insects, we purified a 47kDa protein from injected beads into Galleria mellonella larvae. When a cDNA clone was isolated, the 47kDa protein showed high homology with Drosophila and human calreticulin. Western blotting analysis showed that the 47kDa protein was present in the hemocytes, but not in the plasma. When the early-stage encapsulated beads were coated with 47kDa protein antibody and reinjected into G. mellonella larvae, any further encapsulation reaction was inhibited. These results suggest that calreticulin is involved in non-self recognition in invertebrate cellular defense reactions.

Role of superoxide and reactive nitrogen intermediates in Rhodnius prolixus (Reduviidae)/Trypanosoma rangeli interactions.

This study compares aspects of the superoxide, nitric oxide and prophenoloxidase pathways in Rhodnius prolixus hemolymph, measured in parallel, in response to Trypanosoma rangeli inoculation. Responses to two strains of T. rangeli, and two developmental forms, were studied, and the results obtained were correlated with the ability of the parasites to survive, multiply, and complete their life cycles in the hemolymph of the host. T. rangeli H14 strain parasites, which fail to complete their life cycle in Rhodnius by invading the salivary glands, stimulated high levels of superoxide and prophenoloxidase activity, which peaked 24 h after inoculation. Simultaneously, the concentration of hemolymph nitrites and nitrates increased, indicative of nitric oxide activity, but parasite numbers remained low. T. rangeli Choachi strain parasite inoculation also stimulated superoxide and prophenoloxidase activity, which, though significantly lower than the equivalent responses to the H14 strain, also peaked at 24 h. However, nitrate and nitrite levels in Choachi strain-inoculated hemolymph remained low, and this parasite strain multiplied rapidly, especially following peak superoxide activity, and eventually invaded the salivary glands for transmission to a vertebrate host. In both strains, short form epimastigotes stimulated greater superoxide and prophenoloxidase responses than long form epimastigotes. Injection of the NADPH oxidase inhibitor N-ethylmaleimide or the inducible nitric oxide synthase inhibitor S-methyl isothiourea sulfate caused significantly higher insect mortalities in groups of R. prolixus inoculated with either parasite strain compared with those of uninfected control insects. This indicates that both NADPH oxidase and nitric oxide synthase activity may be involved in the immune response of R. prolixus to infection by T. rangeli. Finally, Western blotting of R. prolixus hemocyte lysates revealed the presence of a protein immunologically related to the human NADPH oxidase complex, the initiator enzyme of the respiratory burst.

Identification, purification and properties of a beta-1,3-glucan-specific lectin from the serum of the cockroach, Blaberus discoidalis which is implicated in immune defence reactions.

A lectin specific for laminarin, a beta-1,3-glucan, agglutinating baker's yeast and enhancing prophenoloxidase activation by laminarin, has been purified from the cockroach, Blaberus discoidalis, serum. Purification involved gel filtration with Bio-gel P300 and affinity chromatography on blue Sepharose CL-6B and laminarin-Sepharose 4B. The purified lectin has a molecular mass estimate of 520 kDa determined by gel filtration, and approximately 80 and 82 kDa by SDS-PAGE, under non-reducing and reducing conditions, respectively. After isoelectric focusing the lectin focused as a single band at pH 4.9. The purified lectin was stained by the periodic acid/Schiff's reagent showing that it is a glycoprotein, and was deglycosylated by endo-beta-N-acetylglu-cosaminidase F. Amino acid composition analysis showed the protein is similar to previously purified beta-1,3-glucan binding proteins from other invertebrates. In electron micrographs by negative staining, the protein formed large aggregates with 'Y'-shaped 'structural units' ca. 79 x 65 nm. Immunological tests confirmed that this lectin is not related to any other lectins previously purified from the same insect. This protein appears to be part of the hexamerin family of proteins. This is one of the first reports of a hexamerin-like molecule with lectin activity.

Studies on a haemolymph lectin isolated from Rhodnius prolixus and its interaction with Trypanosoma rangeli.

We demonstrated that in Rhodnius prolixus haemocyte monolayers, both Trypanosoma cruzi and Trypanosoma rangeli are capable of inducing haemocyte/parasite clump formation. We also purified, by one-step affinity chromatography, a haemolymph galactoside-binding lectin from R. prolixus which we believe could play an important role in the development of T. rangeli in the haemocoel of the insect vector. This lectin markedly enhanced the activation of clump formation by T. rangeli in R. prolixus haemocyte monolayers, with an increase in clump size and haemocyte aggregation. The haemolymph lectin also significantly affected the motilitity and survival of T. rangeli culture short forms, but not the long forms, when they were incubated in vitro. This molecule is also one of the few described in insects with agglutination activity independent of calcium ions. The partial N-terminal amino acid sequence of this lectin demonstrated similarity to a bacterial xylulose kinase and in preliminary experiments the purified haemolymph lectin phosphorylated a tyrosine kinase substrate in a dose-dependent manner. The possible role of this haemolymph lectin in the life cycle of T. rangeli is discussed.

Innate immunity in insects: the role of multiple, endogenous serum lectins in the recognition of foreign invaders in the cockroach, Blaberus discoidalis.

Unlike vertebrates, insects do not have an Ab-based nonself recognition system, and must rely totally on innate immunity to defend themselves from microbial invaders. The most likely candidates for recognizing foreign material in insects are the lectins, which have already been shown to be important in mammalian innate immunity. The hemolymph of the cockroach, Blaberus discoidalis, contains multiple lectins, designated BDL1, BDL2, BDL3, and GSL (beta-1,3-glucan-specific lectin), two of which, namely BDL1 and GSL, have close similarities to acute phase reactants. These endogenous molecules, as well as Con A, wheat germ agglutinin, and Helix pomatia agglutinin, have been shown to induce an enhanced phagocytic response by B. discoidalis plasmatocytes. This effect is related to the carbohydrates presented on the surface of the microorganism and to the sugar specificities of the lectins. Thus, the mannose-specific lectins, BDL1 and Con A, both increase the phagocytosis of baker's yeast and Escherichia coli, whereas the N-acetyl-D-glucosamine/N-acetyl-D-galactosamine-specific lectins, BDL2, wheat germ agglutinin, and H. pomatia agglutinin, induce the phagocytosis of Bacillus cereus and E. coli. GSL, specific for beta-1,3-glucan, and the N-acetyl-D-galactosamine-specific BDL3, only enhance the phagocytosis of yeast and B. cereus, respectively. Phenylthiourea, an inhibitor of the prophenoloxidase system, caused either total, partial, or no inhibition of the lectin-induced increase in phagocytosis, indicating that this immune enhancement results, in some cases, from at least two closely linked mechanisms. These results show that the endogenous lectins in the cockroach hemolymph are capable of acting as nonself recognition molecules for a wide range of microorganisms, and thus obviate the necessity of Abs in these animals.

In vitro superoxide activity in the haemolymph of the West Indian leaf cockroach, Blaberus discoidalis.

The respiratory burst is an NADPH oxidase-driven reduction of molecular oxygen to superoxide, which can occur in phagocytic cells as part of an antimicrobial defence, and is well documented among the vertebrates. This paper describes a process resembling the respiratory burst, which occurs in the haemolymph and haemocytes of the cockroach, Blaberus discoidalis. The in vitro reduction of nitroblue tetrazolium by superoxide to formazan was measured spectrophotometrically in B. discoidalis haemolymph in response to various immune elicitors. Nitroblue tetrazolium reduction was partly impeded in the presence of superoxide dismutase, a specific antioxidant which converts superoxide to hydrogen peroxide, as well as by chemicals known to inhibit the respiratory burst in vertebrates (trifluoperazine, diphenylene iodonium, and N-ethylmaleimide). This suggests the generation of superoxide anions by haemolymph as part of an immune response. Furthermore, formazan staining of elicitor-treated haemocytes was observed microscopically, with less intense staining in the presence of superoxide dismutase. Finally, respiratory burst inhibitors and superoxide dismutase enhanced the growth of E. coli incubated in whole haemolymph, implying a role for haemolymph-derived superoxide in antibacterial defence.

Sub-populations of haemocytes in the adult and developing marine mussel, Mytilus edulis, identified by use of monoclonal antibodies.

Monoclonal antibodies specific for haemocyte sub-populations in the mussel, Mytilus edulis, were raised by use of separated basophilic and eosinophilic cell types as antigens. The antibodies could be broadly divided into 3 groups, reactive with sub-populations of (1) basophilic granular haemocytes, (2) basophilic granular and hyaline cells and (3) eosinophilic granular cells. Non-selective antibodies staining all haemocytes were also generated. The antibodies bound to epitopes of differing molecular masses and, at the ultrastructural level, reacted principally with the granules of the haemocyte sub-populations. The antibodies were used to investigate haemocyte function and ontogeny and to test reactivity with haemocytes from mussels subject to varying degrees of pollution stress. Five antibodies showed reactivity with cells from the trochophore and veliger larvae of M. edulis, indicating that epitopes on adult mussel hae-mocytes are also present at much earlier stages in the life history. Reactivity with the larval stages was most prevalent with non-selective antibodies and those selective for basophilic haemocytes. When mussels from different sites were examined, both immunocytochemistry and ELISA showed reduced expression of a 140 kDa epitope in the haemocytes of mussels subject to greater contaminant loads. These results show that the monoclonal antibodies of the present study are valuable both in tracing immune-cell development and in detecting molecular changes under conditions of stress.

Microcystic adnexal carcinoma.

Microcystic adnexal carcinoma (MAC) is a rare adnexal tumour which has only recently been recognized as a separate clinicopathological entity. It typically affects the face of the middle-aged and often requires extensive surgical excision, due to its locally invasive nature. Its clinical significance is that, despite being locally invasive, MAC is typified by a lack of metastatic spread. We present a case and review of the literature.

Trypanosoma cruzi and erythrocyte agglutinins: a comparative study of occurrence and properties in the gut and hemolymph of Rhodnius prolixus.

The activity of agglutinins found in the gut tissues and hemolymph of Rhodnius prolixus was tested using rabbit erythrocytes, Trypanosoma cruzi, or Trypanosoma rangeli as test particles. In addition, investigations were made of the influence of parasitic infection and insect diet on the agglutination titers. A range of physicochemical tests and carbohydrate-binding studies were performed and inhibitors were subsequently found for both the crop agglutinin (p-nitrophenol-derived sugars) and the hemolymph agglutinin (galactose-type sugars). As a result, affinity chromatography was utilized for an attempted purification of these agglutinins, and a one-step purification protocol for the R. prolixus hemolymph agglutinin has been developed. Preliminary results of some biological and physicochemical characteristics of this pure agglutinin are described. These results represent a starting point for future studies of lectin/parasite interaction in this reduviid-trypanosome model.

Inter- and intra-observer variation in the reporting of cervical smears: specialist cytopathologists versus histopathologists.

One hundred and ten cervical smears were circulated to five specialist consultant cytopathologists and five consultant histopathologists. Of these smears, 100 were randomized and re-circulated. The cytopathologists reported endocervical cells and wart virus infection more frequently than the histopathologists, although neither group showed good inter-observer agreement for either assessment. Apart from smear adequacy and the presence of endocervical cells, both groups showed good intra-observer agreement in all the parameters measured. This suggests that overall individuals were applying their own personal criteria with consistency over time, although a previous study had shown considerable lack of inter-observer agreement among the histopathologists on the grade of dyskaryosis and the management recommendation. The results indicate that specialist cytopathologists bring a different viewpoint to the reporting of cervical smears than histopathologists. They also show a lack of standardization in the reporting of smears despite the guidelines issued by the British Society for Clinical Cytology.

Differential in vitro and in vivo behavior of three strains of Trypanosoma cruzi in the gut and hemolymph of Rhodnius prolixus.

A comparison was made of the agglutination and lysis of three strains of Trypanosoma cruzi in gut extracts and hemolymph of Rhodnius prolixus and the results obtained were correlated with the success or failure of the parasite strain to infect the digestive tube or to survive in the hemocel after inoculation. Both T. cruzi strains Dm28c and Cl urine 35 days after feeding with parasites. Concomitantly, both of these strains were agglutinated but not lysed by the crop extracts. In contrast, T. cruzi Y strain parasites rapidly disappeared from the gut and showed no agglutination, but some lysis, in the crop extract. Following inoculation into the hemocel, only the Cl strain survived at high levels and was also the only strain agglutinated significantly in the hemolymph. Both Dm28c and Y strains rapidly disappeared from the hemocel with the former parasite being removed more slowly than the latter, probably due to clearance by the cellular defenses. The rapid clearance of the Y strain was correlated with the presence of a high titer lysin in the hemolymph. Subsequent experiments using FITC-labeled lectins and FACS to probe the carbohydrates on the parasite surfaces showed significant differences between the three strains. Thus, only Dm28c was stained strongly by Arachis hypogea (PNA) lectin, indicating the presence of galactose/N-acetylgalactosamine residues, and Dm28c and Y strains by Phytolacca lectin for N-acetyl glucosamine moieties. Finally, the fact that, in contrast to Dm28c and Y, the Cl strain strongly interacted with Triticum vulgaris (WGA) but not with Phytolacca lectin may be due to the presence of N-acetyl neuraminic acid residues on these organisms. These surface carbohydrate differences may be correlated both to the behavior and agglutination variations between the three strains recorded in this work.