Segmental pairs of dermal secretory cells release proteins into the hemolymph at the larval-pupal molt.

At each molt of Manduca, the large dermal secretory cells expel the protein contents of their vacuoles into the hemocoel. The constellation of proteins expelled at the last larval-pupal molt, however, differs qualitatively from those proteins released at earlier larval-larval molts. Secretory cells at the two stages not only have different lectin staining properties but also have different proteins that separate on two-dimensional gels. Numerous physiological changes accompany the termination of the last larval instar, including increased chitin synthesis, diminished oxygen delivery, and reduced humoral immunity. Secretion of trehalase that is essential for chitin synthesis and the release of hypoxia up-regulated protein to ameliorate oxygen deprivation help ensure normal transition from larva to pupa. Proteins released by dermal secretory cells at this last molt could supplement the diminished immune defenses mediated by fat body and hemocytes at the end of larval life. Additional immune defenses provided by dermal secretory cells could help ensure a safe transition during a period of increased vulnerability for the newly molted pupa with its soft, thin cuticle and reduced mobility.

Dual role of the serine protease homolog BmSPH-1 in the development and immunity of the silkworm Bombyx mori.

Serine proteases and serine protease homologs are involved in the prophenoloxidase (proPO)-activating system leading to melanization. The Bombyx mori serine protease homolog BmSPH-1 regulates nodule melanization. Here, we show the dual role of BmSPH-1 in the development and immunity of B. mori. BmSPH-1 was expressed in hemocytes after molting and during the larval-pupal transformation in normal development. In contrast, following infection, BmSPH-1 was expressed in hemocytes and cleaved in the hemolymph, which resulted in the induction of PO activity. Moreover, BmSPH-1 was cleaved in the cuticle during the larval-pupal transformation and early pupal stages. In BmSPH-1 RNAi-treated silkworms, the reduced BmSPH-1 mRNA levels during the spinning stage or the prepupal stage resulted in the arrest of pupation or pupal cuticular melanization, respectively. The binding assays revealed that BmSPH-1 interacts with B. mori immulectin, proPO, and proPO-activating enzyme. Our findings demonstrate that BmSPH-1 paticipates larval-pupal transformation, pupal cuticular melanization and innate immunity of silkworms, illustrating the dual role of BmSPH-1 in development and immunity.

An MBL-like protein may interfere with the activation of the proPO-system, an important innate immune reaction in invertebrates.

An important characteristic of the innate immune systems of crayfish and other arthropods is the activation of a serine proteinase cascade in the hemolymph, which results in the activation of prophenoloxidase and subsequently leading to the formation of toxic quinones and melanin. Although no true complement homologues have been detected in crayfish or crustaceans, several proteins with similarities to vertebrate pattern recognition receptors (PRRs), which are involved in the lectin pathway of complement activation in vertebrates, are present. One is a C-type lectin, a mannose-binding lectin (Pl-MBL), which is secreted from granular hemocytes. Here we report that Pl-MBL has LPS-binding capacity and is dependent upon high Ca(2+) for its solubility and Pl-MBL interferes with proPO activation in vitro when HLS is prepared at high Ca(2+). The proPO-activating system is efficiently activated by microbial polysaccharides and it has to be neatly regulated to avoid activation in places where it is inappropriate and the active enzyme PO should be prevented from spreading throughout the body of the animal. This may be particularly important during molting when proPO is involved in hardening of a new cuticle and the animal is vulnerable to microbes. The presence of high amount of Pl-MBL in the granular hemocytes may play a role in this process. Since a hemocyte lysate supernatant (HLS) prepared at 100 mM Ca(2+) could become activated when the concentration of LPS was increased up to 3 mg/ml, this may indicate that Pl-MBL acts as a scavenger for LPS to prevent spreading of LPS in the hemolymph to avoid further activation of the proPO-system.

A second proPO present in white shrimp Litopenaeus vannamei and expression of the proPOs during a Vibrio alginolyticus injection, molt stage, and oral sodium alginate ingestion.

Prophenoloxidase (proPO) is a melanin-synthesising enzyme that plays important roles in immune responses by crustaceans. Previously, we cloned and characterized proPO-I from white shrimp, Litopenaeus vannamei. In the present study, a novel prophenoloxidase-II (proPO-II) cDNA was also cloned from haemocytes of L. vannamei using oligonucleotide primers and reverse-transcriptase polymerase chain reaction (RT-PCR). Both 3'- and 5'-regions were isolated by the rapid amplification of complementary (c)DNA end (RACE) method. The 2504-bp cDNA contained an open reading frame (ORF) of 2073 bp, an 84-bp 5'-untranslated region, and a 347-bp 3'-untranslated region containing the poly A tail. The molecular mass of the deduced amino acid sequence (691 amino acids) was 78.8 kDa with an estimated pI of 6.07. It contains two putative tyrosinase copper-binding motifs and a conserved C-terminal region common to all known proPOs. Comparisons of the amino acid sequences showed that white shrimp proPO-II is more closely related to the proPO of other penaeids than to that of crayfish, lobsters, crab, or a freshwater prawn, and is the ancestor type of known penaeid proPOs. proPO-I and proPO-II messenger (m)RNAs of shrimp were located on different loci, and were constitutively expressed mainly in haemocytes. The transcriptional regulation of these two proPOs in shrimp at different molt stages, those administered dietary sodium alginate, and those challenged with Vibrio alginolyticus were surveyed. The results showed that the proPOs may be directly involved in the acute-phase immune defence, and proPO-II may contribute earlier to immune defence in shrimp injected with V. alginolyticus, and it may be regulated by ecdysone. However, a similar effect was found by stimulating proPO-I and proPO-II mRNA expression in shrimp fed a sodium alginate-containing diet. Results of this study provide a basis for developing a comprehensive understanding of expression/function relationships of individual proPOs in shrimp.

Dynamics of innate immune response in Gallus domesticus using two methods of induced molting.

The aim of this study was to compare the ability of laying hen abdominal macrophages during the second production cycle by using two different methods of induced molting. Two groups of Single Comb White Leghorn hens were induced to molt at the end of their first production cycle using feed restriction and ZnO supplementation. Macrophages were isolated from the abdomen and in vitro cytotoxic ability, at which point macrophage bactericidal moiety nitric oxide (NO) was recorded. Serum IgM and IgG titers against sheep red blood cells (SRBC) were determined at various stages: before molting (BM), 5% production level (5P), peak production stage (PP) and at the end of production (EP) level after fast and Zn-induced molt. Macrophages adherence percentage remained unaffected (p< or =0.05) during all production cycles, whereas the macrophage engulfment percentage and engulfment/cell was significantly higher (p< or =0.05) at PP in both fast and Zn-induced molted groups, as compared to all other studied stages. Macrophage NO production was increased (p< or =0.05) at PP and after SRBC and lipopolysaccrides (LPS) stimulus, when molted with ZnO supplementation. Serum total antibody titer against SRBC increased serum IgG and IgM titers during the second production cycle by Zn-induced molt. However, molting stress greatly reduced IgG and IgM production at the 5P stage. Serum Zn concentration increased with the onset of production but decreased at the EP stage irrespective of their molting regimes. Our results validate the strengthened innate and acquired immune response during the second production cycle after Zn-induced molting instead of fasting.

Cytokines in reproductive remodeling of molting White Leghorn hens.

The role of cytokines in regression of the ovary and oviduct during induced molting in chickens was investigated by evaluating the expressions of IL-1beta, IL-6, IFN-gamma, IL-2, TGF-beta2, MIP-1beta and IL-8 in the regressing ovary and oviduct by semi-quantitative RT-PCR. In addition, serum hormonal profiles (estrogen, progesterone and corticosterone), along with the gross regression and histological changes of the ovary and oviduct, were investigated. The correlation between expression of cytokines and hormonal changes during the induced molting was also studied. The expression of IL-6, IL-8, MIP-1beta and IFN-gamma mRNAs in the ovary, and IL-1beta, IL-6, IL-8, MIP-1beta, IFN-gamma and TGF-beta2 mRNAs in the oviduct, were up-regulated significantly during induced molting, suggesting their role in tissue regression. However, histological findings suggested no significant increase in immune cells in the regressing oviduct and ovary. Significant up-regulation of TGF-beta2 in the regressing oviduct might have suppressed leukocyte recruitment thereby preventing the inflammatory response and tissue damage. The down-regulation of estrogen and progesterone and up-regulation of corticosterone is well correlated with increased expression of cytokines. It appears that cytokines released during the process of induced molting may have a role in decreasing ovarian steroids and increasing the corticosterone levels in chicken. From this study, it may be concluded that cytokines play a major role in regression of the ovary and oviduct during induced molting in chickens.

Brugian infections in the peritoneal cavities of laboratory mice: kinetics of infection and cellular responses.

Standard, immunocompetent, inbred strains of mice are non-permissive for infection with the human filarial nematode, Brugia malayi or the closely related Brugia pahangi. This non-permissiveness allows one to address the mechanism(s) that might be used by mammalian hosts to eliminate large, multicellular, metazoan, extracellular invertebrate pathogens. We describe here the time course of intraperitoneal Brugian infections in naïve and primed +/+ mice from two commonly used, inbred laboratory strains (C57BL/6J and BALB/cByJ). We believe that this documentation of the course of infection in normal mice will serve as a reference for future studies using mice with gene-targeted immunological deficits or which have been pharmacologically or immunologically manipulated to manifest such deficits. Our data show that even though both strains of mice eliminate the parasite before the onset of patency, there are significant differences in the time course of infection and in the fractions of input larvae that can be recovered at any time after infection. In a secondary infection, the time course of elimination is accelerated. We examined the cells in the peritoneal cavity, the site of infection, by flow microfluorimetry using forward and side scatter properties and cell surface antigen expression using fluorescent antibodies. These studies reveal a complex cellular pattern, predominated by B lymphocytes, macrophages, and eosinophils. The most notable gross morphological findings at necropsy during the phase of elimination of the parasite are nodules of tissue containing larvae, which appear viable in some cases and undergoing various stages of disintegration in others. These nodules, which are histologically granulomas, are primarily composed of macrophages and eosinophils, with few if any lymphocytes. Transmission electron micrographs reveal that eosinophils can penetrate under the cuticles of the larvae and be seen in close approximation with internal structures. These granulomas may represent an important mechanism by which worms are eliminated.

Antibodies to tyvelose exhibit multiple modes of interference with the epithelial niche of Trichinella spiralis.

Infection with the parasitic nematode Trichinella spiralis is initiated when the L1 larva invades host intestinal epithelial cells. Monoclonal antibodies specific for glycans on the larval surface and secreted glycoproteins protect the intestine against infection. Protective antibodies recognize tyvelose which caps the target glycan. In this study, we used an in vitro model of invasion to further examine the mechanism(s) by which tyvelose-specific antibodies protect epithelial cells against T. spiralis. Using cell lines that vary in susceptibility to invasion, we confirmed and clarified the results of our in vivo studies by documenting three modes of interference: exclusion of larvae from cells, encumbrance of larvae as they migrated within epithelial monolayers, and inhibition of parasite development. Excluded larvae bear cephalic caps (C. S. McVay et al., Infect. Immun. 66:1941-1945, 1998) of immune complexes that may physically block invasion or may interfere with sensory reception. Monovalent Fab fragments prepared from a tyvelose-specific antibody also excluded larvae from cells, demonstrating that antibody binding can inhibit the parasite in the absence of antigen aggregation and cap formation. In contrast, encumbered larvae caused extensive damage to the monolayer yet were not successful in establishing a niche, as reflected by their failure to molt. These results show that antibodies to tyvelose exhibit multiple modes of inhibitory activity, further implicating tyvelose-bearing glycoproteins as mediators of invasion and niche establishment by T. spiralis.