Steinernema carpocapsae DD136: metabolites limit the non-self adhesion responses of haemocytes of two lepidopteran larvae, Galleria mellonella (F. Pyralidae) and Malacosoma disstria (F. Lasiocampidae)

Live adult and juvenile entomopathogenic Steinernema carpocapsae DD136 (P. Nematoda) were not subjected to adhesion by haemocytes of lepidopteran insect larvae of Galleria mellonella or Malacosoma disstriain vitro or in vivo. In vitro freeze-killed nematodes exhibited haemocyte attachment, the intensity increasing with time. Accumulation of haemocytes on the dead nematodes was associated with host phenoloxidase activity; live nematodes and their exudates did not activate the enzyme whereas dead nematodes but not their exudate did activate phenoloxidase. Live-nematode exudate inhibited granular cell and some plasmatocyte adhesion to slides, increased granular cell but not plasmatocyte dissociation from preformed haemocyte monolayers and in vivo elevated total haemocyte counts and changed the floating haemocyte types while impairing bacterial removal from the haemolymph. Dead-nematode exudate did not affect these parameters thus immunosuppressant activity by live nematodes may represent the release of inhibitors not associated with their cuticle. The third stage juveniles released the inhibitors.     

Immunological characterization of maize starch branching enzymes

Highly purified fractions of three starch branching enzymes from developing maize (Zea mays L.) endosperm were used to prepare antisera in rabbits. In double diffusion experiments, no immunoprecipitate was observed when branching enzyme IIa or IIb was tested against branching enzyme I antiserum. No immunoprecipitate was formed when branching enzyme I was tested against branching enzyme IIa or IIb antiserum. Increasing amounts of antisera in the above combinations also failed to inhibit enzyme activity. Branching enzyme IIa antiserum cross-reacted and formed spurs with branching enzyme IIb when compared with branching enzyme IIa antigen. Comparison of branching enzyme IIb antiserum with branching enzyme IIa also resulted in an immunoprecipitate. Increasing levels of branching enzyme IIa antiserum inhibited branching enzyme IIb as did the reciprocal combination. The data indicated that branching enzymes IIa and IIb are immunologically similar while branching enzyme I is distinct. The data supports the classification of starch branching enzymes based on genetic, kinetic, and chromatographic properties.     

Fibroblast-like cells from embryonic chick cornea, heart, and skin are antigenically distinct.

Populations of fibroblast-like cells from 14 day embryonic chick cornea, heart, and skin were grown in vitro as primary cultures and found to be antigenically distinct from one another. Corneal fibroblasts were obtained by dissection, whereas heart and skin fibroblast-like cells were separated from nonfibroblastic cell types by their rapid adhesion to substrata. Cultured cells were used as antigens in rabbits. Antisera were first absorbed against homogenates of embryonic chicks from which the homologous tissue was removed. Each such 1° absorbed antiserum then was absorbed against homogenates of the two respective heterologous fibroblast-like cell populations (2° and 3° absorptions). Resulting 3° absorbed antisera were tested for specificity by immunodiffusion, immune agglutination, immune cytotoxicity (trypan blue uptake and ⁵¹Cr release), and indirect immunofluorescence. Each 3° antiserum was judged tissue specific when it reacted only with the fibroblast-like cells of its own tissue, i.e., the homologous population. Unabsorbed antisera reacted with both homologous and heterologous fibroblast-like cells, as did 1° absorbed antisera. Absorption of 1° antisera with homogenates of the two heterologous fibroblast-like populations removed antibodies against the heterologous populations without significantly reducing the 3° antiserum titer against the homologous fibroblast cell type. Moreover, absorption of 1° antisera with each of the two heterologous fibroblast-like populations removed antibodies not removed by the other. Thus, the fibroblast-like cells from cornea, heart, and skin are antigenically different from one another in vitro. The stable antigenic differences detected may have arisen during the differentiation of these cells in vivo. Some of the tissue-specific antigens detected must occur on the cell surface.     

A cell adhesion factor from crayfish haemocytes has degranulating activity towards crayfish granular cells

A factor able to mediate cell adhesion of semigranular and granular haemocytes of the crayfish Pacifastacus leniusculus was recently purified from crayfish haemocyte lysate (Johansson and Söderhäll, J. Cell Biol.106, 1795–1803, 1988). It is a protein with a mass of 76 kDa, and its activity seems to be generated concomitantly with the activation of the prophenoloxidase (proPO) activating system. In this paper, we present evidence that this same protein is also responsible for the previously reported degranulating activity of a crayfish haemocyte lysate, in which the proPO system has been activated. First, the 76 kDa band in SDS-polyacrylamide electrophoresis seems to be a single protein, since in isoelectric focusing the purified cell adhesion factor fraction migrated as one band with an isoelectric point of 7.2. Second, this fraction was also able to degranulate crayfish granular cells in vitro, and third, antibodies to this 76 kDa protein, which are known to block cell adhesion, could also inhibit degranulation in vitro.     

Trypanosoma brucei: some properties of the cytotoxic reaction induced by normal human serum.

The trypanocidal activity of normal human serum has been studied in vitro using Trypanosoma brucei as the test organism. The variables affecting the rate and extent of lysis, such as time, temperature, serum concentration, and pleomorphism of trypanosomes, are described. Trypanocidal titers of serum and serum fractions were quantitatively determined under standardized incubation conditions. Inactivation and/or removal of components of both the classical and alternate pathways of complement activation had no effect on the trypanocidal properties of human serum. The active factor was nondialyzable, present in plasma at equivalent levels to that in serum, and not removed by absorption with IgG fractions of antisera against human IgM or α₂-macroglobulin. The trypanocidal factor could be inactivated by heat (65 C), dithiothreitol, urea, and trypsin. Gel filtration studies indicated that the trypanocidal activity eluted as a single protein with a molecular weight of about 500,000.     

Obtention de lignées permanentes d'hémocytes de blatte. Caractéristiques physiologiques et ultrastructurales

Two new cell lines were established from larval and adult cockroach haemolymph. The cells show typical characteristics of plasmatocytes, but under different conditions of cultivation, they modulate from small dividing cells (prohaemocytes) to large storing cells (granulocytes, adipohaemocytes etc.). Consequently, the very confusing haemocyte nomenclature must be reconsidered taking into account the cell physiology. Biochemical analysis of the metabolized culture medium established bacteriolytic activity and chitinase secretion (two enzymes). Moreover, in the same medium we found a macromolecular factor inducing spermatogenesis in cultured germinal cysts of the Cynthia silkworm. All these characteristics of in vitro haemocytes are discussed to explain the involvement of blood cells in insect physiology and especially in immunity, gametogenesis, and ecdysis.     

A study on the possible utilization of immunodiffusion and immunofluorescence techniques as the diagnostic methods for American foulbrood of honeybees (Apis mellifera)

Four types of antisera were obtained from rabbits hyperimmunized with either spores or vegetative rods from two strains of the American foulbrood pathogen, Bacillus larvae. The specificity and sensitivity of these antisera were tested with immunofluorescence and immunodiffusion methods. No cross-reactions were observed between the antisera and other different species of Bacillus or different genera of bacteria. The specificity was not found between the antisera and two strains of B. larvae although stronger fluorescent intensity was observed between the antiserum and its corresponding strain of antigen in the immunofluorescence tests. Eight samples of 1- to 2-day-old larvae, 3- to 4-day-old larvae, decayed tissue, and dry remain, collected from eight infected colonies, were tested against antisera by the immunofluorescence and the immunodiffusion methods. The results indicated that both methods are sensitive and specific for making diagnosis of field samples of American foulbrood of honey bees.     

Toxicity of some insecticides to the haemocytes of giant honeybee,Apis dorsata F. under laboratory conditions

Quantitative studies concerning total and differential haemocyte counts and abnormalities were performed under laboratory conditions for larvae, pupae and adults collected from a wild Apis dorsata colony. Haemolymph samples were observed immediately, thirty and sixty minutes after field recommended concentration exposure of five different insecticides. Total haemocyte counts were significantly higher for larvae and pupae but less for adult bees, however, differential haemocyte counts insignificantly different. Exposure of insecticides showed variable response for tested insecticides with immediate increased change with ethofenprox, diafenthiuron and imidacloprid but decreased for all tested insecticides after sixty minutes. For differential haemocyte counts, plasmatocytes and granulocytes increased with exposure of insecticides. Immune response of haemocytes against insecticides showed different degrees of abnormalities like agglutination, denucleation and cell shape distortion. Such studies may help in possible identification of insect defense mechanisms against their exposure to external hazards for instance insecticide exposure.     

The effects of anti-embryo sera and their localization on the cell surface during mouse preimplantation development.

We have developed two rabbit antisera, one against mouse blastocysts and a second against mouse placentae. After absorption of these antisera with adult mouse tissues and extensive dialysis, results from indirect immunofluorescence, cytotoxicity, and culturing experiments lead us to two major conclusions. First, anti-blastocyst serum detects a group of cell surface molecules whose expression is embryo-specific, stage-specific, and whose unaltered presence is required for preimplantation development in vitro. Second, anti-placenta serum detects a different group of cell surface molecules that are present before fertilization and become segregated to the syncytiotrophoblast, appear unessential for preimplantation development in vitro, and may function in trophoblast differentiation, and/or in protection of the fetus from maternal immunologic attack.     

Purification and Characterization of Chorismate Synthase from Euglena gracilis: Comparison with Chorismate Synthases of Plant and Microbial Origin

Chorismate synthase was purified 1200-fold from Euglena gracilis. The molecular mass of the native enzyme is in the range of 110 to 138 kilodaltons as judged by gel filtration. The molecular mass of the subunit was determined to be 41.7 kilodaltons by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Purified chorismate synthase is associated with an NADPH-dependent flavin mononucleotide reductase that provides in vivo the reduced flavin necessary for catalytic activity. In vitro, flavin reduction can be mediated by either dithionite or light. The enzyme obtained from E. gracilis was compared with chorismate synthases purified from a higher plant (Corydalis sempervirens), a bacterium (Escherichia coli), and a fungus (Neurospora crassa). These four chorismate synthases were found to be very similar in terms of cofactor specificity, kinetic properties, isoelectric points, and pH optima. All four enzymes react with polyclonal antisera directed against chorismate synthases from C. sempervirens and E. coli. The closely associated flavin mononucleotide reductase that is present in chorismate synthase preparations from E. gracilis and N. crassa is the main difference between those synthases and the monofunctional enzymes from C. sempervirens and E. coli.     

Trypanosoma vivax: Absence of host protein on the surface coat

The possible presence of host serum proteins on the surface of Trypanosoma vivax stock Zaria Y486 was studied. Intact washed bloodstream forms from mice were not lysed or neutralized by antisera against mouse serum proteins. Serum against T. vivax prepared in rabbits against an antigen which was a water-soluble trypanosome extract, failed to cross-react with mouse serum when tested by immunoelectrophoresis and immunodiffusion. The T. vivax antigen failed to cross-react with three different anti-mouse sera when tested by the same techniques.Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analyses of ¹²⁵I-surface-labeled parasites showed the presence of a cluster of proteins ranging in molecular weights between 57,000 and 45,000 daltons. None of these proteins was precipitated by anti-mouse serum protein sera. The serum against T. vivax precipitated a protein of 50,000 daltons molecular weight.     

In vitro maturation of ovine fetal adrenal cells adenylate cyclase: corticotropin-dependent and independent development of the response to corticotropin

Polyclonal antibodies against an estrogen induced 52 K protein released by human breast cancer cells have been developed by injecting rabbits with a crude cellular pellet of MCF₇ human breast cancer cells. The rabbit antisera have been tested against [³⁵S]Methionine labelled proteins released by the MCF₇ cells followed by separation of the immune complexes with Protein A Sepharose. In spite of their low specificity and titer, these antisera allowed us to investigate the release of the 52 K protein $\text{in vitro}$ by other mammary cancer or normal cells.     

Immunological Characterization of Plant Ornithine Transcarbamylases

Pea (Pisum sativum L.) ornithine transcarbamylase (OTC) antisera were used to investigate the immunological relatedness of several plant and animal OTC enzymes. The antisera immunoprecipitated OTC activity in all monocot and dicot species tested, and sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis of immunoprecipitated protein revealed monomeric proteins ranging from 35,200 to 36,800 daltons in size. Pea OTC antisera did not recognize mammalian OTC protein. OTC activity and protein levels detected on sodium dodecyl sulfate polyacrylamide gel electrophoresis immunoblots from homogenates of green leaf, etiolated epicotyl and cotyledon, and root tissues of pea were poorly correlated. This might result from differences in amounts of enzymatically active OTC protein in the homogenates. Alternatively, the antisera may fail to recognize different isozyme forms of OTC, which have been reported for some plant species. A putative cytosolic precursor OTC (pOTC) polypeptide exhibiting and M_r = 39,500 to 40,000 daltons was immunoprecipitated from in vitro translation mixtures of total pea leaf poly(A)⁺ RNA. The size of the pOTC polypeptide, as compared with mature OTC monomer (36,000 daltons), suggests that a 4 kilodalton N-terminal leader sequence, like that responsible for mitochondrial targeting of the mammalian enzyme, may be involved in organellar import of the plant enzyme.     

Real-time analysis of Drosophila post-embryonic haemocyte behaviour

Background

The larval stage of the model organism Drosophila is frequently used to study host-pathogen interactions. During embryogenesis the cellular arm of the immune response, consisting of macrophage-like cells known as plasmatocytes, is extremely motile and functions to phagocytise pathogens and apoptotic bodies, as well as produce extracellular matrix. The cellular branch of the larval (post-embryonic) innate immune system consists of three cell types—plasmatocytes, crystal cells and lamellocytes—which are involved in the phagocytosis, encapsulation and melanisation of invading pathogens. Post-embryonic haemocyte motility is poorly understood thus further characterisation is required, for the purpose of standardisation.

Methodology

In order to examine post-embryonic haemocyte cytoskeletal dynamics or migration, the most commonly used system is in vitro cell lines. The current study employs an ex vivo system (an adaptation of in vitro cell incubation using primary cells), in which primary larval or pre-pupal haemocytes are isolated for short term analysis, in order to discover various aspects of their behaviour during events requiring cytoskeleton dynamics.

Significance

The ex vivo method allows for real-time analysis and manipulation of primary post-embryonic haemocytes. This technique was used to characterise, and potentially standardised, larval and pre-pupal haemocyte cytoskeleton dynamics, assayed on different extracellular matrices. Using this method it was determined that, while larval haemocytes are unable to migrate, haemocytes recovered from pre-pupae are capable of migration.     

The induction of an injury reaction in cultured haemocytes from saturniid pupae

The plasmatocytes of diapausing saturniid pupae are round or spindle-shaped cells floating free in the haemolymph. Upon injury to the pupa, these haemocytes become amoeboid and adhesive. A technique is described for the isolation and short-term culture of pupal haemocytes in their inactive state and for their conversion in vitro into the active, ‘injured’ form. The activation of ‘uninjured’ haemocytes was stimulated by fragments of epidermal tissue or by plasma from previously activated blood samples. A fraction capable of stimulating the activation of haemocytes was partially purified from both plasma and epidermal tissue and has been called haemokinin. Haemokinin from either source has a molecular weight in the range of 50,000. The intensity of the haemocyte injury reaction in vitro changes systematically during diapause; the nature and significance of the changes are discussed.     

Characterization of Protective Immune Responses Induced by Pneumococcal Surface Protein A in Fusion with Pneumolysin Derivatives

Pneumococcal surface protein A (PspA) and Pneumolysin derivatives (Pds) are important vaccine candidates, which can confer protection in different models of pneumococcal infection. Furthermore, the combination of these two proteins was able to increase protection against pneumococcal sepsis in mice. The present study investigated the potential of hybrid proteins generated by genetic fusion of PspA fragments to Pds to increase cross-protection against fatal pneumococcal infection. Pneumolisoids were fused to the N-terminus of clade 1 or clade 2 pspA gene fragments. Mouse immunization with the fusion proteins induced high levels of antibodies against PspA and Pds, able to bind to intact pneumococci expressing a homologous PspA with the same intensity as antibodies to rPspA alone or the co-administered proteins. However, when antibody binding to pneumococci with heterologous PspAs was examined, antisera to the PspA-Pds fusion molecules showed stronger antibody binding and C3 deposition than antisera to co-administered proteins. In agreement with these results, antisera against the hybrid proteins were more effective in promoting the phagocytosis of bacteria bearing heterologous PspAs in vitro, leading to a significant reduction in the number of bacteria when compared to co-administered proteins. The respective antisera were also capable of neutralizing the lytic activity of Pneumolysin on sheep red blood cells. Finally, mice immunized with fusion proteins were protected against fatal challenge with pneumococcal strains expressing heterologous PspAs. Taken together, the results suggest that PspA-Pd fusion proteins comprise a promising vaccine strategy, able to increase the immune response mediated by cross-reactive antibodies and complement deposition to heterologous strains, and to confer protection against fatal challenge.     

Effects of the parasitization of a braconid, Apanteles, on the blood of its host, Pieris

Parasitization of a braconid wasp, Apanteles glomeratus, of larvae of a common cabbage butterfly, Pieris rapae crucivora, caused changes in differential haemocyte count (DHC), total haemocyte count (THC), and encapsulative capacity against dead eggs of Apanteles in the fourth and fifth instar host larvae.However, no correlation could be found between the number of Apanteles eggs deposited and THC of the middle fourth instar host larvae or between the number of parasitoid larvae and specific gravity of the haemolymph from the late fifth instar host larvae.From the changes in DHC and in THC of both non-parasitized and parasitized Pieris larvae, an increase in the number of plasmatocytes of non-parasitized Pieris larvae in the early fourth instar period was supposed to be due to transformation of prohaemocytes into plasmatocytes, and a low population of plasmatocytes of parasitized larvae in the comparable period was assumed to be due to a suppression of transformation of prohaemocytes by some factor released from the parasitoid eggs.Failure of the parasitized fourth instar Pieris larvae to encapsulate injected dead eggs of Apanteles indicated that the parasitoid embryos were, in some way, actively inhibiting the encapsulation reactions of the host.The increase in THC of the parasitized fifth instar larvae could not be ascribed to a decrease in the volume of host haemolymph. Rather it could be interpreted by a suppression of adhesive capacity of haemocytes in the host haemocoel to tissue surfaces.Reduced encapsulative capacity of the parasitized fifth instar larvae might be attributed either to a depression of the adhesive activity of plasmatocytes resulting from a depletion of energy source for haemocytes in the host haemolymph by parasitization, or from an active suppression of adhesiveness of the plasmatocytes by secretions from ‘giant cells’ (teratocytes) originated from the parasitoid.     

The role of haemocytes from the crab Carcinus aestuarii (Crustacea,Decapoda) in immune responses: A first survey

For the first time, a functional study of haemocytes from the crab Carcinus aestuarii was performed in order to evaluate their involvement in immune responses. Total haemocyte count (THC), phagocytosis, haemolymph opsonisation properties, hydrolytic and oxidative enzyme activities, and production of intracellular superoxide anion were evaluated. A great variability in THC was recorded among individuals, and haemocyte mean number was 6.4 (×10⁶) cells/ml haemolymph. Although only hyalinocytes were able to phagocytose yeast cells or Zymosan, phagocytic index was low (3%) and did not increase significantly (4%) after pre-incubation of yeast and Zymosan in cell-free haemolymph, suggesting that haemolymph did not have opsonising properties. All haemocyte types produced superoxide anion, whereas only granulocytes were positive to the hydrolytic enzymes assayed. In addition, only granulocytes were positive to phenoloxidase activity. Both Petri dish and spectrophotometric assays revealed a very low lysozyme-like activity in cell-free haemolymph (CFH) and haemocyte lysate (HL), although enzyme activity was higher in CFH than in HL. Interestingly, normalisation of data as to total protein content in CFH and HL resulted in an opposite situation, lysozyme-like activity being higher in HL than in CFH. This demonstrated that haemolymph of C. aestuarii has a high quantity of total proteins, functional properties of which need to be better investigated in future studies. Overall, the results obtained in the present study indicated that C. aestuarii haemocytes are not very active phagocytic cells, but they are more active in terms of both hydrolytic and oxidative enzyme activities and superoxide anion production.     

Changes in haemolymph parameters and insect ability to respond to immune challenge during overwintering

Overwintering is a challenging period in the life of temperate insects. A limited energy budget characteristic of this period can result in reduced investment in immune system. Here, we investigated selected physiological and immunological parameters in laboratory‐reared and field‐collected harlequin ladybirds (Harmonia axyridis). For laboratory‐reared beetles, we focused on the effects of winter temperature regime (cold, average, or warm winter) on total haemocyte concentration aiming to investigate potential effects of ongoing climate change on immune system in overwintering insects. We recorded strong reduction in haemocyte concentration during winter; however, there were only limited effects of winter temperature regime on changes in haemocyte concentration in the course of overwintering. For field‐collected beetles, we measured additional parameters, specifically: total protein concentration, antimicrobial activity against Escherichia coli, and haemocyte concentration before and after overwintering. The field experiment did not investigate effects of winter temperature, but focused on changes in inducibility of insect immune system during overwintering, that is, measured parameters were compared between naïve beetles and those challenged by Escherichia coli. Haemocyte concentration decreased during overwintering, but only in individuals challenged by Escherichia coli. Prior to overwintering, the challenged beetles had a significantly higher haemocyte concentration compared to naïve beetles, whereas no difference was observed after overwintering. A similar pattern was observed also for antimicrobial activity against Escherichia coli as challenged beetles outperformed naïve beetles before overwintering, but not after winter. In both sexes, total protein concentration increased in the course of overwintering, but females had a significantly higher total protein concentration in their hemolymph compared to males. In general, our results revealed that insect’s ability to respond to an immune challenge is significantly reduced in the course of overwintering.     

Localization of vimentin,the nonspecific intermediate filament protein,in embryonal glia and in early differentiating neurons: In vivo and in vitro immunofluorescence study of the rat embryo with vimentin and neurofilament antisera

Antisera raised against vimentin, the protein subunit of nonspecific intermediate-sized filaments (IFs), were used in conjunction with neurofilament (NF) antisera to study the early development of neurons and glia in the rat embryo. Vimentin-positive fibers spanning the entire thickness of the neural tube including the cerebral vesicles were first observed on Day 12, concomitant with the appearance of NF protein in more confined areas (anterolateral regions of spinal cord and brain stem; motor roots emerging from the NF-positive areas). From Day 15 onwards vimentin and NF antisera selectively decorated glia and neurons, respectively, both in vivo and in vitro. Before Day 15 it appeared that NF-positive structures also stained with antivimentin in cryostat sections. In vitro experiments confirmed the presence of vimentin in early differentiating neurons. NF-positive cells were observed which also reacted with antivimentin in cultures obtained from 13- and 14-day embryos, but not later in development. Most neurons in these cultures became vimentin negative after 2–3 days in vitro.