Different Bla-g T cell antigens dominate responses in asthma versus rhinitis subjects.

BACKGROUND AND OBJECTIVE: The allergenicity of several German cockroach (Bla-g) antigens at the level of IgE responses is well established. However, less is known about the specificity of CD4+ TH responses, and whether differences exist in associated magnitude or cytokine profiles as a function of disease severity. METHODS: Proteomic and transcriptomic techniques were used to identify novel antigens recognized by allergen-specific T cells. To characterize different TH functionalities of allergen-specific T cells, ELISPOT assays with sets of overlapping peptides covering the sequences of known allergens and novel antigens were employed to measure release of IL-5, IFNγ, IL-10, IL-17 and IL-21. RESULTS: Using these techniques, we characterized TH responses in a cohort of adult Bla-g-sensitized subjects, either with (n = 55) or without (n = 17) asthma, and nonsensitized controls (n = 20). T cell responses were detected for ten known Bla-g allergens and an additional ten novel Bla-g antigens, representing in total a 5-fold increase in the number of antigens demonstrated to be targeted by allergen-specific T cells. Responses of sensitized individuals regardless of asthma status were predominantly TH 2, but higher in patients with diagnosed asthma. In asthmatic subjects, Bla-g 5, 9 and 11 were immunodominant, while, in contrast, nonasthmatic-sensitized subjects responded mostly to Bla-g 5 and 4 and the novel antigen NBGA5. CONCLUSIONS: Asthmatic and nonasthmatic cockroach-sensitized individuals exhibit similar TH 2-polarized responses. Compared with nonasthmatics, however, asthmatic individuals have responses of higher magnitude and different allergen specificity.

RNAi of ace1 and ace2 in Blattella germanica reveals their differential contribution to acetylcholinesterase activity and sensitivity to insecticides.

Cyclorrhapha insect genomes contain a single acetylcholinesterase (AChE) gene while other insects contain at least two ace genes (ace1 and ace2). In this study we tested the hypothesis that the two ace paralogous from Blattella germanica have different contributions to AChE activity, using RNA interference (RNAi) to knockdown each one individually. Paralogous-specific depletion of Bgace transcripts was evident in ganglia of injected cockroaches, although the effects at the protein level were less pronounced. Using spectrophotometric and zymogram measurements, we obtained evidence that BgAChE1 represents 65-75% of the total AChE activity in nerve tissue demonstrating that ace1 encodes a predominant AChE. A significant increase in sensitivity of Bgace1-interfered cockroaches was observed after 48 h of exposure to chlorpyrifos. In contrast, Bgace2 knockdown had a negligible effect on mortality to this organophosphate. These results point out a key role, qualitative and/or quantitative, of AChE1 as target of organophosphate insecticides in this species. Silencing the expression of Bgace1 but not Bgace2 also produced an increased mortality in insects when synergized with lambda-cyhalothrin, a situation which resembles the synergistic effects observed between organophosphates and pyrethroids. Gene silencing of ace genes by RNAi offers an exciting approach for examining a possible functional differentiation in ace paralogous.

Quantity does matter. Juvenile hormone and the onset of vitellogenesis in the German cockroach.

We aimed to elucidate why cockroaches do not produce vitellogenin in immature stages, by studying the appearance of vitellogenin mRNA in larvae of Blattella germanica. Treatment of female larvae in any of the last three instars with 1 microg of juvenile hormone (JH) III induces vitellogenin gene transcription, which indicates that the fat body is competent to transcribe vitellogenin at least from the antepenultimate instar larvae. In untreated females, vitellogenin production starts on day 1 after the imaginal molt, when corpora allata begin to synthesize JH III at rates doubling the maximal of larval stages. This coincidence suggests that the female reaches the threshold of JH production necessary to induce vitellogenin synthesis on day 1 of adult life. These data lead to postulate that larvae do not synthesize vitellogenin simply because they do not produce enough JH, not because their fat body is incompetent.

The vitellogenin of the honey bee, Apis mellifera: structural analysis of the cDNA and expression studies.

The cDNA of Apis mellifera vitellogenin was cloned and sequenced. It is 5440 bp long and contains an ORF of 1770 amino acids (including a putative signal peptide of 16 residues). The deduced amino acid sequence shows significant similarity with other hymenopteran vitellogenins (58% with Pimpla nipponica and 54% with Athalia rosae). The alignment with 19 insect vitellogenins shows a high number of conserved motifs; for example, close to the C-terminus there is a GL/ICG motif followed by nine cysteines, as occurs in all hymenopteran species, and, as in other insect vitellogenins, a DGXR motif is located 18 residues upstream the GL/ICG motif. Phylogenetic analysis of vitellogenin sequences available in insects gave a tree that is congruent with the currently accepted insect phylogenetic schemes. Using two fragments of the vitellogenin cDNA as probes, we analyzed by Northern blot the sex- and caste-specific patterns of vitellogenin expression in pupae and adults of A. mellifera. In queens, vitellogenin mRNA was first detected in mid-late pupal stage, whereas in workers it was first detected in late pupal stage. Vitellogenin mRNA was also observed in drones, although it was first detected not in pupae but in freshly molted adults.

Ovarian 3-hydroxy-3-methylglutaryl-CoA reductase in Blattella germanica (L.): pattern of expression and critical role in embryogenesis.

In the ovary of adult Blattella germanica, the enzyme 3-hydroxy-3-methylglutaryl-CoA reductase (HMG-CoA reductase) is highly expressed in mid-late vitellogenesis, suggesting a functional link of the mevalonate pathway with choriogenesis. The inhibitor of HMG-CoA reductase, fluvastatin, applied in females in late vitellogenesis, inhibits the activity of the enzyme in the ovary and in the developing embryos within the ootheca. This does not affect choriogenesis or ootheca formation but reduces the number of larvae per ootheca. Our results suggest that fluvastatin is incorporated into the oocytes and has delayed inhibitory effects on the oviposited eggs. HMG-CoA reductase is essential for embryogenesis, but not for chorion formation.

Screening of antifeedant activity in brain extracts led to the identification of sulfakinin as a satiety promoter in the German cockroach. Are arthropod sulfakinins homologous to vertebrate gastrins-cholecystokinins?

The feeding cycle of the adult female cockroach Blattella germanica parallels vitellogenesis. The study of the mechanisms that regulate this cycle led us to look for food-intake inhibitors in brain extracts. The antifeedant activity of brain extracts was tested in vivo by injecting the extract and measuring the carotenoids contained in the gut from carrot ingested after the treatment. By HPLC fractionation and tracking the biological activity with the carrot test, we isolated the sulfakinin EQFDDY(SO3H) GHMRFamide (Pea-SK). A synthetic version of the peptide inhibited food intake when injected at doses of 1 microg (50% inhibition) and 10 microg (60% inhibition). The sulfate group was required for food-intake inhibition. These biological and structural features are similar to those of the gastrin-cholecystokinin (gastrin-CCK) family of vertebrate peptides. However, heterologous feeding assays (human CCK-8 tested on B. germanica, and Pea-SK tested on the goldfish Carassius auratus) were negative. In spite of this, alignment and cluster analysis of these and other structurally similar peptide families suggest that sulfakinins and gastrin-CCKs are homologous, and that mechanisms of feeding regulation involving these regulatory peptides may have been conserved during evolution between insects and vertebrates.

Induction of vitellogenin gene transcription in vitro by juvenile hormone in Blattella germanica.

In the cockroach Blattella germanica, the synthesis of vitellogenin is juvenile hormone III (JH III)-dependent. We have studied the effect of JH III upon vitellogenin gene expression in periovaric fat bodies incubated in vitro. Periovaric fat bodies were obtained from cardioallatectomized females. The response to JH III was measured in terms of vitellogenin and vitellogenin mRNA after 7 h of incubation. A hormonal concentration as low as 1 nM was enough to induce vitellogenin production and its release to the medium, whereas the concentration of 10 nM produced the maximal effects. Although the response of the vitellogenin gene to JH III is fast and efficient, it seems that the action is mediated by protein factors, given that cycloheximide treatment impairs the hormonal effect. The presence in the medium of brain extract (0.5 equivalents), corpora cardiaca (one pair) or hypertrehalosemic hormone (10(-7) or 10(-8) M), partially inhibited the response to JH III.

3-Hydroxy-3-methylglutaryl coenzyme A synthase-1 of Blattella germanica has structural and functional features of an active retrogene.

Blattella germanica has two cytosolic 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) synthase genes, HMG-CoA synthase-1 and -2. HMG-CoA synthase-1 gene shows several features of processed genes (retroposons): it contains no introns but has a short direct-repeat sequence (ATTATTATT) at both ends. An atypical feature is the presence at both ends of the gene of short inverse repeats flanked by direct repeats. There is neither a TATA box nor a CAAT box in the 5' region. Comparative analysis with other species suggests that the HMG-CoA synthase-1 gene derives from HMG-CoA synthase-2. Cultured embryonic B. germanica UM-BGE-1 cells express HMG-CoA synthase-1 but not HMG-CoA synthase-2, suggesting that the intron-less gene is functional. In addition, it can complement MEV-1 cell line, which is auxotrophic for mevalonate. We show that compactin and mevalonate do not significantly affect the mRNA levels of HMG-CoA synthase-1 in UM-BGE-1 cells. Compactin induces a 6.7-fold increase in HMG-CoA reductase activity, which is restored to normal levels by mevalonate. HMG-CoA synthase activity is not modified by either of these effectors, suggesting that the mevalonate pathway in this insect cell line is regulated by post-transcriptional mechanisms affecting HMG-CoA reductase but not HMG-CoA synthase.

Vitellogenin of Blattella germanica (L.) (Dictyoptera, blattellidae): nucleotide sequence of the cDNA and analysis of the protein primary structure.

The cloning and sequencing of a cDNA of the vitellogenin gene from the cockroach Blattella germanica is reported. It is 5,749 nucleotides long and encodes an amino acid sequence of 1,862 residues (including a putative signal peptide of 17 residues). The vitellogenin sequence includes a long serine-rich stretch between amino acids 322 and 349, and two other stretches between amino acids 1691 and 1740. The vitellogenin of B. germanica shows a notable similarity (between 32 and 42%) to those described in other insects, and its alignment shows a high number of motifs conserved in all species, especially in the subdomains I-V. Non-parsimony methods (Neighbor Joining) of phylogenetic analysis of the insect vitellogenin sequences gave a tree showing a topology that is, in general, congruent with the currently accepted insect phylogenetic schemes. Arch.

Molecular cloning and structural analysis of 3-hydroxy-3-methylglutaryl coenzyme A reductase of the moth Agrotis ipsilon.

The enzyme 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, which plays a key role in isoprenoid biosynthesis, catalyses the synthesis of mevalonate from HMG-CoA. Insects do not synthesize cholesterol de novo, rather mevalonate derivatives lead to non-sterol isoprenoids which are essential for development and reproduction. In this paper, we describe an HMG-CoA reductase of the moth Agrotis ipsilon and we report its expression in fat body, ovary, muscle, brain and corpora allata tissues of adult specimens. The analysis of the cDNA reveals that it encodes a polypeptide of 833 amino acids (Mr = 89785). Alignments of this HMG-CoA reductase from A. ipsilon with the homologous sequences of other eukaryotes shows a high degree of conservation in all species studied. Parsimony analysis based on these alignments produced dendrograms congruent with the current systematic schemes. This suggests that, during eukaryote evolution, HMG-CoA reductase diversified in parallel with taxonomic splitting.

Fast induction of vitellogenin gene expression by juvenile hormone III in the cockroach Blattella germanica (L.) (Dictyoptera, Blattellidae).

The present paper describes the effect of juvenile hormone III (JH III) upon vitellogenin (Vg) gene expression in cardioallatectomized females of Blattella germanica. Northern blot analyses of time course studies showed that Vg mRNA can be detected 2 h after the treatment with 1 microgram of JH III. Western blot analyses revealed that Vg protein is detectable 4 h after the same treatment. The study of the influence of the age showed that 48-h-old females seem more sensitive than 24-h-old females, whereas differences were less apparent between 48- and 72-h-old females. Dose-response studies indicated that 0.01 microgram of JH III is ineffective, whereas the doses of 0.1, 1 and 10 micrograms induced the synthesis of Vg in a dose-dependent fashion. Finally, the administration of three successive doses, of 0.01 microgram of JH III each, did not result in detectable Vg production, whereas two doses of 0.01 microgram followed by one of 1 microgram of JH III induced a greater response than that resulting from a sole dose of 1 microgram of JH III, which suggests that sub-effective doses of JH III elicit a priming effect on Vg production.

The molecular evolution of the allatostatin precursor in cockroaches.

Allatostatins (ASTs) of the Tyr/Phe-Xaa-Phe-Gly Leu/Ile-NH2 family are a group of insect neuropeptides that inhibit juvenile hormone biosynthesis by the corpora allata. We have obtained genomic DNA sequences that specify the preproallatostatin precursor for the cockroaches, Blatta orientalis, Blattella germanica, Blaberus (cranufer and Supella longipalpa. The sequences obtained are similar to those of Diploptera punctata and Periplaneta americana reported previously. The precursors of all these cockroach species are similar in size, and the organization of the ASTs that they contain (there are 13 or 14, depending on the species) have been conserved. With the sequences of these precursors, and using the homologous sequence in the orthopteran Schistocera gregari as an outgroup, a phylogenetic analysis using parsimony was carried out. The dendrograms obtained from these analyses. using the amino acid as well as the nucleotide sequences, are comparable with current models for cockroach phylogeny. Parsimony analysis was also used to study the genealogy of the different ASTs within the same precursor. Results suggest that the AST sequences were generated through a process of internal gene duplication which occurred before these species diverged from each other in evolutionary time.

Modulation of cardiac rhythm by allatostatins in the cockroach Blattella germanica (L.) (Dictyoptera, Blattellidae).

Cardiac rhythm was measured in Blattella germanica females during the reproductive cycle. The rate increased from day 0 to 1, remained constant during the vitellogenic period and fell by about 20% during the period of oothecal transport. The effects of allatostatins, allatostatin analogues and corazonin were tested on semi-isolated heart preparations. Allatostatins showed a rapid, reversible and dose-dependent cardioinhibitory activity. Blattella allatostatin 1 (BLAST-1: LYDFGL-NH(2)), was the most active, eliciting 76% inhibition at 10(-7) M and even 19% inhibition at 10(-9) M. BLAST-2 (DRLYSFGL-NH(2)), BLAST-3 (AGSDGRLYSFGL-NH(2)) and BLAST-4 (APSSAQRLYGFGL-NH(2)) were less active. An analogue of BLAST-2 with C-terminus in acid form and a pseudopeptide analogue of BLAST-2 with a methyleneamino Psi[CH(2)NH] peptide bond surrogate between residues L(3) and Y(4) were inactive. Corazonin elicited rapid, reversible and dose-dependent cardioacceleratory activity. When tested together with BLAST-1, corazonin overrode the cardioinhibitory effect of allatostatin. Our previous results had shown that high levels of allatostatin were maintained during the period of oothecal transport. This and the fact that physiological concentrations of allatostatins produce physiological levels of inhibition, suggest that allatostatins are involved in the modulation of cardiac rhythm in this cockroach.

Isolation and sequence of a partial vitellogenin cDNA from the cockroach, Blattella germanica (L.) (Dictyoptera, Blattellidae), and characterization of the vitellogenin gene expression.

A partial cDNA clone of the vitellogenin gene from the cockroach Blattella germanica has been isolated from a cDNA expression library using an anti-vitellin-vitellogenin antiserum probe. The analysis of cDNA inserts gave a sequence of 2,645 nucleotides corresponding to the 3' region. The deduced amino acid sequence is 825 residues long and is similar to the homologous portion of the vitellogenin of other insect species, especially that of the mosquito Aedes aegypti. RNA hybridization studies indicated that the vitellogenin gene expression is limited to the fat body of adult females. The pattern of expression during the first vitellogenic cycle was approximately parallel to that of vitellogenin production by the fat body previously described. The availability of a cDNA probe for the B. germanica vitellogenin gene represents a useful tool to study the molecular action of hormones affecting vitellogenin synthesis in this species.

Localization of allatostatin-immunoreactive material in the central nervous system, stomatogastric nervous system, and gut of the cockroach Blattella germanica.

Immunoreactivity against peptides of the allatostatin family having a typical YXFGL-NH2 C-terminus has been localized in different areas of the central nervous system, stomatogastric nervous system and gut of the cockroach Blattella germanica. In the protocerebrum, the most characteristic immunoreactive perikarya are situated in the lateral and median neurosecretory cell groups. Immunoreactive median neurosecretory cells send their axons around the circumesophageal connectives to form arborizations in the anterior neuropil of the tritocerebrum. A group of cells in the lateral aspect of the tritocerebrum project to the antennal lobes in the deutocerebrum, where immunoreactive arborizations can be seen in the periphery of individual glomeruli. Nerve terminals were shown in the corpora allata. These terminals come from perikarya situated in the lateral neurosecretory cells in the pars lateralis and in the subesophageal ganglion. Immunoreactive axons from median neurosecretory cells and from cells positioned in the anteriormost part of the tritocerebrum enter together in the stomatogastric nervous system and innervate foregut and midgut, especially the crop and the valve between the crop and the midgut. The hindgut is innervated by neurons whose perikarya are located in the last abdominal ganglion. Besides immunoreactivity in neurons, allatostatin-immunoreactive material is present in endocrine cells distributed within the whole midgut epithelium. Possible functions for these peptides according to their localization are discussed.

Ketomethylene and methyleneamino pseudopeptide analogues of insect allatostatins inhibit juvenile hormone and vitellogenin production in the cockroach Blattella germanica.

Metabolic studies on insect allatostatins have suggested that the dipeptide Leu-Tyr may be a target for endopeptidases. In order to increase resistance to degradation, methyleneamino psi [CH2NH] and ketomethylene psi [COCH2] peptide bond surrogates have been introduced at the position Leu3-Tyr4 of the allatostatin Asp-Arg-Leu-Tyr-Ser-Phe-Gly-Leu-amide (BLAST-2), and Leu3-Phe4 of [Phe4]BLAST-2, respectively. Assays of inhibition of juvenile hormone (JH) synthesis in vitro by corpora allata from the cockroach Blattella germanica showed that both analogues were similarly active to the respective model peptides. The methyleneamino analogue was further tested in vivo as an inhibitor of JH synthesis, and in vivo and in vitro as an inhibitor of vitellogenin production by the fat body of B. germanica. The analogue was less active than BLAST-2 when tested in vitro, but more active than it when tested in vivo.

Feeding and activation of corpora allata in the cockroach Blattella germanica (L.) (Dictyoptera, Blattellidae).

Adult females of the cockroach Blattella germanica have clearly-defined feeding cycles related to oogenesis. In the first cycle, food ingestion precedes volumetric increase in the corpora allata, which in turn precedes juvenile hormone production, whereas starved females do not develop the corpora allata and produce very low amounts of juvenile hormone. When the second gonadotropic cycle is provoked by removing the ootheca, the first event observed is an increase in food consumption, followed by an increase in corpora allata volume and activity. However, this increase in corpora allata volume (and activity) does not occur if females are starved, thus indicating that the ootheca in the genital chamber inhibits primarily feeding, and indirectly corpora allata development and activity. Corpora allata volume in isolated heads from starved and decapitated females was able to increase to levels similar to fed controls, but this increase was abolished by allatostatin treatment. We suggest that a factor produced in the thoracico-abdominal compartment, which reaches the head mainly through a nervous pathway, is released during starvation and inhibits corpora allata development. This factor may stimulate allatostatin production or release, or may well be allatostatin itself.

Coordinated expression and activity of 3-hydroxy-3-methylglutaryl coenzyme A synthase and reductase in the fat body of Blattella germanica (L.) during vitellogenesis.

Levels of mRNA for the two 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) synthases, (HMG-S1 and HMG-S2), and for HMG-CoA reductase (HMG-R) of Blattella germanica were analyzed in the fat body during the first gonadotrophic cycle. HMG-S2 and HMG-R showed the highest mRNA levels on day 0 and decreased thereafter, whereas HMG-S1, showed faint expression. Western blot using specific antibodies for HMG-S1 and HMG-S2 showed no detectable levels for HMG-S1 but a clear pattern for HMG-S2. Both results point to a very limited role for HMG-CoA synthase-1 in B. germanica fat body that the functional enzyme in this organ is HMG-CoA synthase-2. HMG-CoA reductase and synthase proteins shared a cyclic pattern (maximum levels at day 4 and minimum levels on days 0 and 8), which was coincident with the pattern of activity. The delay between gene transcription and protein synthesis suggests a finely regulated translation mechanism. Moreover, the pattern of mevalonate synthesis parallels that of vitellogenin production, suggesting a coordinate mechanism between the mevalonate pathway and the production of vitellogenin.

Inhibition of vitellogenin production by allatostatin in the German cockroach.

Allatostatins with a typical YXFGL-amide C-terminus constitute a neuropeptide family, which was discovered because of its inhibitory action on insect juvenile hormone synthesis. In the search for possible new functions for allatostatins we focused our attention on the fat body. Our previous studies on the cockroach Blattella germanica suggested the occurrence of factors terminating vitellogenesis, and the hypothesis here was that allatostatins might be one of these factors. Our experiments have shown that allatostatin impaired vitellogenin release in fat bodies incubated in vitro, and that this effect appears to be mediated by the inhibition of vitellogenin glycosylation. Fluvastatin also inhibited vitellogenin release, and mevalonolactone counteracted the inhibitory effects of allatostatin. These results suggest that allatostatin acts upon the mevalonate pathway and synthesis of dolichol, which would explain the inhibition of vitellogenin glycosylation. We finally conclude that allatostatins may effectively contribute to the termination of the vitellogenic cycle in B. germanica.

Quantification of ecdysteroids by immunoassay: comparison of enzyme immunoassay and radioimmunoassay.

The performance of enzyme immunoassay (EIA) and radioimmunoassay (RIA) in the quantitative analysis of ecdysteroids was compared. The EIA was found to be at least equivalent to the RIA with respect to analytical range and sensitivity and to be more comfortable with respect to safety and time saving. When biological samples were analyzed by both assays a good correlation (r = 0.83) was found. Since the EIA has certain advantages over the RIA, we now recommend the use of the former assay for the quantification of ecdysteroids.