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This study is an attempt to reach some understanding of how insects recognize intruding microorganisms and foreign entities while executing an immune response. We used lipopolysaccharide (LPS) from Escherichia coli, bound to a radiolabeled iodinated crosslinker, to identify hemolymph proteins from the Hyalophora cecropia moth that have the capacity to bind LPS. High amounts of radioactivity were conferred to hemolin, an immunoglobulin and NCAM-related protein, the concentration of which increases in insect hemolymph upon bacterial infection. We could demonstrate a concentration-dependent binding of hemolin to LPS. In addition we could show that Lipid A can compete for this binding, whereas KDO has no effect, indicating that hemolin interacts specifically with the Lipid A moiety of LPS.

The aim of the study was to investigate the involvement of the actin binding protein tropomyosin in the allergic sensitization of patients to household arthropods, as well as to study its panallergenic character in relation to other invertebrate extracts. Three arthropod extracts were prepared, namely fly (Musca domestica), moth (Ephestia spp.) and spider (Tegenaria spp.), and used to evaluate by cutaneous and RAST tests a population of 100 household arthropod allergic patients. Twenty-nine sera were selected for the subsequent SDS-PAGE Immunoblotting assays. IgE binding bands at 36, 34, 31, 27 and 17 kDa were detected in the fly extract by more than 50% of tested sera. In moth and spider extracts, the more relevant allergens were found at 34, 31, 24 and 110, 38, 35, 26, 19 kDa, respectively. A commercial polyclonal antiserum anti-chicken muscle tropomyosin was used for tropomyosin identification. Cross-reactivity studies performed by SDS-PAGE Immunoblotting, using a pool of household arthropod allergic patients and tropomyosin antiserum, preliminarily demonstrated the presence of such protein as a cross-reacting allergen in a large variety of extracts obtained from insects, mites, crustaceans, mollusks and parasites.

Gallysin-1, an inducible effector protein in the protective response of Galleria mellonella larvae is a 75 kDa component of hemolytically active material (HAM) isolated from immune cell-free hemolymph. The sequence of the first 20 N-terminal amino acids of the antibacterial protein Gallysin-1 is identical to the predicted sequence of the first 20 amino acids of the Galleria arylphorin Lhp76 (larval hemolymph protein 76). A murine monoclonal antibody to the 20 amino acid N-terminal peptide of Gallysin-1 (GYPQYHYDVETRKLDPSLVN) provides additional evidence for a link between Gallysin-1 and Lhp76, and is used to characterize HAM further. HAM, initially characterized as a mixture of two proteins, Gallysin-1 and a 69 kDa component is now identified as a 450-500 kDa heteromultimer, designated Gallysin. In vivo levels of Gallysin rise during the effector phase of an induced immune response. The monoclonal antibody inhibits the hemolytic activity of Gallysin. In addition to a hemolytic activity for mammalian erythrocytes, Gallysin possesses a cytotoxic activity for the human tumor cell line, K562. Lipopolysaccharides (LPS) and a Pseudomonas aeruginosa vaccine induce a cytotoxic activity which reaches its maximum levels in the hemolymph early (2 hours post-vaccination) in the protective response. The partially purified cytotoxic material (Cyt-M) obtained from cell-free hemolymph collected 2 hours after vaccination has hemolytic activity and shows structural similarities to Gallysin and Lhp76. The previously established role of Gallysin-1 as an effector protein in the protective response of Galleria mellonella indicates that arylphorins may play a role in insect immune responses.

The nasal bot fly, Oestrus ovis, was investigated to establish which specific cuticular component is most immunogenic to infested sheep and how larval cuticle attains a protective role, if any, against the host immune system. To accomplish these goals, larval cuticle was extracted by a variety of agents and tested against immune sera from infested sheep and experimentally immunized rabbits. The cuticle substructure remaining after extraction was examined to localize various immunogenic components. O. ovis larval integument comprises an inner cellular layer, the epidermis, and an overlying cuticle layer. In 3rd instar larvae, the cuticle comprises 2 additional layers: the procuticle with numerous pore canals and the epicuticle which includes the wax canals. Three additional layers, altogether comprising the cuticulin layer, are present external to the epicuticle. The epicuticle is completed by apposition of an amorphous electrondense material extending for up to 1 micron in thickness. When fixed with ruthenium red, cuticle becomes heavily stained all along the epicuticular surface in larvae of all developmental stages. However, in 3rd instar larvae, ruthenium red deposits are restricted to the cuticulin layer alone. By gel electrophoresis, 3rd instar larval cuticle is shown to contain a number of polypeptides ranging in molecular weight from 180 to 4.5 kDa. The number and relative concentration of low molecular weight polypeptides was shown to vary in relation to the extraction media employed. Cuticular fragments examined after extraction exhibit an altered ultrastructure. When tested by immunoblotting, the cuticular polypeptides most reactive against sheep antisera are in the range of 180-56 kDa. A similar reaction was also detected with sera from rabbits infested experimentally with O. ovis larvae. Results are interpreted in relation to differential polypeptide distribution within the larval cuticle and to accessibility of the host immune system.

In a previous study, electrospray ionization mass spectrometry was used to analyze the structure of the O-glycopeptide diptericin, an antibacterial peptide from the fleshfly Phormia terranovae. Several glycoforms of diptericin differing in the length of their oligosaccharide chains were present at the final stage of purification. In order to determine the origin of this glycan heterogeneity, we analyzed by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) the relative abundance of the different diptericin glycoforms in fractions obtained after each purification step, and directly in the hemolymph and in the fat body which produces diptericin. MALDI-MS clearly shows that the purification procedure had no effect on the O-linked oligosaccharide chains of diptericin, suggesting that diptericin is synthesized as a family of heterogeneous glycopeptides. In addition, in these experiments, differential mapping by MALDI-MS of the hemolymph and fat body tissue from bacteria-challenged and naive larvae allowed us to detect induced or repressed molecules which may be involved in the immune response of P. terranovae.

During Musca domestica vitellogenesis a protein is preferentially synthesized by the female fat body and accumulates in the haemolymph but not in the ovaries. This protein, designated nonvitellogenic female protein (NVFP), was purified and shown to be a hexamer with an M(r) = 430 kDa, and subunits of M(r) = 70 kDa. The hexamer dissociates into subunits when the pH is elevated from 7.0 to 9.0. Two cDNA clones, F0 and F2, were isolated and analysed. The 2.2 kb F2 clone has an open reading frame that encodes a conceptual translation product that has similarity to the Drosophila melanogaster LSP-2 hexamerin. Recombinant protein from the F2-cDNA is recognized by a specific anti-NVFP serum. The temporal pattern of mRNA expression of the gene represented by the F2 clone follows that determined for the synthesis of NVFP. The data support the conclusion that NVFP is an hexamerin specific to the adult stage of Musca domestica.

Background: DE-cadherin is an epithelial cadherin in Drosophila, and forms adherens junctions by associating with Armadillo (beta-catenin). To investigate its role in oogenesis, we generated germ-line clones homozygous for a null mutation in shotgun (shg) encoding this molecule, and examined their phenotypes, comparing with those of armadillo (arm) mutants., Results: In the wild-type ovaries, DE-cadherin was expressed by both the germ-line and somatic derivatives, colocalizing with Armadillo. In the shg mutant ovaries in which the mutation was restricted to the germ line, germ cells were rounded, and generated gaps between themselves, suggesting that their surface adhesiveness was reduced or lost. However, the positioning of germ cells in the egg chamber was normal. Two groups of somatic follicle cells--the border cells and centripetal follicle cells--frequently migrated along incorrect pathways, indicating that DE-cadherin is required for their appropriate migration. Notably, the shg phenotypes were distinct from those of arm null mutants. Intercellular adhesion appeared to be less severely affected by arm than by the shg mutation, and the actin-based cytoskeleton and cell arrangement were disorganized only in the arm mutants., Conclusions: These findings suggest that DE-cadherin is critical for cell-cell adhesion, and functional to a certain extent without Armadillo, whereas Armadillo is required for cytoskeletal organization and for the control of cell positioning. We therefore propose that the molecular complex of DE-cadherin and Armadillo which is present in normal cells is endowed with multiple functions derived from each molecule.

Manduca sexta larvae infected per os with a sublethal dose of Erinnyis ello granulosis virus (EeGV) contain an induced plasma protein of ca 33-36 kDa [Finnerty et al. (1994) J. Invertebr. Pathol. 63, 140-144]. This virus-induced protein shares characteristics with scolexin, a bacteria-induced plasma protein from M. sexta, so in this study we compared the two proteins. Two-dimensional gel electrophoresis reveals the induction of polypeptides corresponding to the subunits of scolexin in the plasma from EeGV-infected, as well as bacteria-injected, larvae. Immunoblots of these two-dimensional polyacrylamide gels show that antiserum against the virus-induced protein cross-reacts with the putative scolexin polypeptides induced by either EeGV infection or bacteria injection. Amino acid analysis of the virus-induced protein shows it to be very similar to scolexin, and the N-terminal sequences of the two proteins are nearly identical. From these data, we conclude that the virus-induced protein and scolexin are identical, or at least isoforms of the same protein. Using immunoblot, we also demonstrate that scolexin is induced in M. sexta plasma by injection with yeast or lipopolysaccharide from Serratia marcescens.

Two additional members of the American cockroach (Periplaneta americana) Per a 3 (Cr-PI) allergen, C13 and C28, were isolated and sequenced. They encoded proteins of 470 and 393 amino acids with two and no potential N-glycosylation sites, respectively. The molecular weights for C13 and C28 cloned proteins are 56,200 and 46,7000, with PI values of 7.06 and 6.54. C13 and C28 display 95.4% identity with several overlapping predicted central antigenic determinants. Both allergens were also found to have a 95% sequence homology with previously cloned C20 and share similar antigenic determinants, as defined by the structural prediction and ELISA analysis. However, the recombinant C13 and C28 allergens showed 26.3 and 94.7% skin reactivities on asthmatic patients while C20 elicited 47.4%. While no sequence similarity was found to other known allergens, these two aromatic amino acid-rich allergens were highly related to insect hemolymph proteins (28.7-36.5%), as with C20 cloned protein. Results suggest that these two are isoallergenic variants of C20. Sequence variations among isoforms, resulting a significant difference in skin reactivities, will be useful in elucidating the allergenic determinants.

Background: Commercial extracts are available for the diagnosis and treatment of mosquito allergy, but their antigen content has never been analyzed., Objective: We wanted to analyze commercially available mosquito extracts and to compare these extracts with different laboratory preparations., Methods: Seven commercially available mosquito whole body extracts from six companies and four laboratory mosquito preparations including saliva extract were studied. Epicutaneous tests and measurement of protein concentration were performed. Protein components were identified by sodium sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and silver stain. IgE and IgG binding antigens were analyzed by SDS-PAGE and immunoblotting with sera from mosquito-allergic subjects., Results: The seven commercial materials produced wheals and papules ranging from 0 to 36 mm2. Their protein concentrations varied from 0.1 to 4.9 mg/mL. There were significant differences in their protein and antigen components. Some extracts contained multiple highly immunoreactive proteins and IgE- and IgG-binding antigens that are not present in mosquito saliva, but few actual salivary antigens. In the four laboratory preparations, rank ordered from whole body, head and thorax, salivary gland to saliva extracts, the amount of salivary antigens significantly increased, while non-salivary proteins and antigens significantly decreased., Conclusions: Commercial mosquito extracts should be standardized. Purer mosquito extracts should be used in diagnosis and immunotherapy of mosquito allergy.

This paper reviews recent advances in our understanding of changes in local cellular traffic and cytokine synthesis that occur as a result of infection of sheep with the ectoparasite Lucilia cuprina. Changes in the cellular composition and cytokine profile of infected skin and draining afferent and efferent lymph were assessed using standard approaches and, in addition, a variety of techniques that have only recently become available as a result of advances in ruminant cytokine biology. These include cytokine-specific immunoassay, reverse transcription PCR (RT-PCR) and immunohistology. The initial acute inflammatory response was characterised by the infiltration of polymorphonuclear cells followed by selected lymphocyte subsets into discrete areas adjacent to the site of infection. Analysis of cytokine expression in skin prior to and following infection provided a molecular basis for the observed cellular events. Both cellular and molecular events within the skin were reflected within draining afferent lymph providing a basis for the conclusion that events within the skin (other than antigen uptake and transport) may influence events within the draining node and thus the outcome of the immune response to the parasite. Analysis of cellular and molecular changes in efferent lymph during infection suggested initiation of antigen-specific immunity.

Purified lipophorin, metabolically labelled with 32P exclusively in the phospholipid moiety, was used to study the process of phospholipid delivery to the oocyte. The kinetics of phospholipid transfer "in vitro," from lipophorin to the oocytes, was linear at least up to 4 h and was impaired by low temperature. A net transfer of phospholipids from lipophorin particles to the oocytes was observed. The rate of phospholipid uptake was dependent on the concentration of lipophorin in the medium and was shown to be a saturable process. The addition of a molar excess of purified unlabelled lipophorin to the culture medium resulted in a substantial decrease in the transfer of [32P]phospholipids, but no reduction occurred in the presence of a molar excess of albumin. The lipophorin binding sites were localized in the oocytes by immunogold techniques using two different protocols for oocyte fixation. Strong labelling was observed especially at the microvilli. No labelling was detected in the yolk granules.

The distribution of integument peptides in relation to chitin and structural features has been studied in the surface epidermis of the caterpillar of Calpodes ethlius by immunoblotting and immunogold labelling using antibodies prepared to peptides isolated from lamellate endocuticle or from hemolymph. The intermoult cuticle consists of an epicuticle, an endocuticle of many chitin containing lamellae, and a chitin containing assembly zone directly above the apical epidermal microvilli and the perimicrovillar space. During the intermoult, the epidermis secretes peptides constitutively, that is, secretory vesicles containing peptides exocytose without accumulating, traverse the perimicrovillar space and form lamellae in the assembly zone. At moulting, the epidermis deposits ecdysial droplets in addition. These interrupt the last few lamellae which later go on to become the perforated ecdysial membrane. The integument is involved with four routing classes of peptide. Secretion is apical into the cuticle (C), basal into the hemolymph (H), bidirectional (BD), or transported to the cuticle across the epidermis from the hemolymph (T). Some peptides change their routing at moulting. There are several patterns of localization. (1) C and BD cuticular peptides occur mainly in chitin containing lamellate cuticle. (2) Some are also present in epicuticle, and are therefore not obligatorily linked to chitin or matrix between chitin fibers. Cuticular peptides that also occur in the hemolymph are glycosylated, whereas most that are only secreted apically into the cuticle are not. All BD but few C peptides carry alpha-D-glucose/alpha-D-mannose. Some C and BD peptides carry N-acetyl glucosamine. (3) C36 extracted from cuticle has most N-acetyl glucosamine and colocalizes with chitin rather than the protein matrix. It is therefore probably the main link between chitin fibers and the matrix. (4) H235 is barely detectable at the apical cell surface during the intermoult but is abundant at moulting around and below the ecdysial droplets. (5) T66 occurs in intermoult lamellate cuticle. At moulting, alone among the peptides examined, it is in ecdysial droplets. Intermoult C and BD peptides are not in ecdysial droplets but continue to be present in the ecdysial membrane, suggesting that constitutive secretion is independent from the exocytosis of transported moult peptides. T66 differs from most hemolymph peptides in that it does not carry N-acetyl glucosamine or alpha-D-glucose/alpha-D-mannose. (6) Weakly reacting BD peptides (and some H peptides barely detectable in cuticle) localize near the apical surface. Their distribution therefore favours apical secretion and retrieval as a mechanism for basal secretion.