Aedes aegypti: characterization of hemocyte polypeptide synthesis during wound healing and immune response to inoculated microfilariae

Hemocytes from adult, female Aedes aegypti, intrathoracically inoculated with microfilariae (mf) of the nematode Dirofilaria immitis, were compared to saline-inoculated and uninoculated controls using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), 125I-labeling, and wheat germ agglutinin (WGA) binding techniques. Activation of wound healing and/or melanotic encapsulation responses by the inoculation of saline or mf into the host hemocoel induced alterations in the hemocyte activity of these mosquitoes. Protein assays of whole hemocyte lysates revealed that hemocytes from saline- and mf-inoculated mosquitoes had higher protein concentrations than uninoculated controls. Many polypeptides were seen within all three hemocytes preparations when stained with silver nitrate, but there was an overall increase in protein synthesis in hemocytes from inoculated mosquitoes. In addition, a 200-kDa polypeptide was uniquely expressed in hemocytes from inoculated mosquitoes. There were several prominent surface proteins labeled with 125I, and several of these increased dramatically in intensity during wound healing and/or a melanotic encapsulation response. Similar results were seen in two-dimensional separations. A set of basic polypeptides comigrated with an acidic polypeptide resulting in a surface protein of approximately 80-90 kDa that increased in inoculated mosquitoes. Hemocytes from inoculated mosquitoes exhibited a group of three acidic polypeptides, whereas hemocytes from uninoculated mosquitoes exhibited only one of these protein fragments. Three surface polypeptides bound 125I-labeled WGA, and binding of WGA to hemocyte surface polypeptides was successfully inhibited by the incubation of cells with the lectin and its competing sugar.     

Quantitative analysis of hemolymph monophenol oxidase activity in immune reactive Aedes aegypti

A hemocyte-mediated melanotic encapsulation reaction is elicited in adult Aedes aegypti in response to intrathoracically inoculated microfilariae of Dirofilaria immitis. The activity of monophenol oxidase in cell-free hemolymph collected from uninoculated, microfilariae-inoculated and saline-inoculated control mosquitoes was investigated using a quantitative radiometric assay that measured the amount of tritiated water formed during the hydroxylation of l-[3,5-³H]tyrosine to dopa. Enzyme activity in immune reactive hosts examined 2 days postinoculation was aproximately twice as high (96–206 nmol/min per mg protein) as in uninoculated or saline inoculated insects (34–80 nmol/min per mg protein). The augmented activity of the enzyme coincides in time with the early development of melanotic capsules around the microfilariae. The possible involvement of hemocytes in the activation and/or generation of monophenol oxidase in response to infection, and the metabolism of catecholamines in relation to insect immune responses are discussed.     

Hemocyte population changes during the immune response of Aedes aegypti to inoculated microfilariae of Dirofilaria immitis

Ultrastructural and lectin-binding studies have established that the melanotic encapsulation reaction of Aedes aegypti Liverpool strain against inoculated Dirofilaria immitis microfilariae (mff) is a hemocyte-mediated reaction. Total hemocyte counts from mff-inoculated (= immune-activated), saline-inoculated, and uninoculated female A. aegypti were determined using a hemocoel perfusion technique. Total hemocyte populations in uninoculated mosquitoes were significantly larger in younger mosquitoes, but no significant change was noted as mosquitoes aged beyond 14 days. Hemocyte populations in immune-activated mosquitoes increased from 1 to 3 days postinoculation (PI) and decreased on days 4 and 5 PI. Hemocyte populations at 1 to 4 days PI were significantly elevated in mff-inoculated A. aegypti as compared with saline-inoculated controls. Saline-inoculated mosquitoes displayed little change in total hemocyte numbers from 1 to 5 days PI, and their hemocyte populations were similar to those seen in uninoculated insects of the same age. Experiments involving the inoculation of [3H]thymidine along with mff or saline alone and studies involving the administration of colchicine suggest that increased hemocyte populations in immune-activated A. aegypti are a result of mitotic division of circulating blood cells.     

中华按蚊和白纹伊蚊接种马来丝虫微丝蚴后酚氧化酶活性的变化(英文)

酚氧化酶在昆虫抵抗外来异物黑化和成囊的免疫反应中起重要作用。中华按蚊和白纹伊蚊分别经胸内接种马来丝虫微丝蚴和生理盐水后,测定蚊血淋巴中酚氧化酶活性。结果表明:未接种的两种蚊,其1日龄蚊的酚氧化酶活性显著高于3、12和16日龄蚊。1日龄中华按蚊接种微丝蚴24h后,其酚氧化酶活性比未接种蚊和接种生理盐水蚊高2—3倍。1日龄白纹伊蚊接种微丝蚴24h后,其酚氧化酶活性是未接种蚊的7倍。白纹伊蚊酚氧化酶活性的免疫激活水平显著高于中华按蚊。讨论了中华按蚊和白纹伊蚊酚氧化酶活性的差异与其免疫力差异的关系。     

Hemocyte monophenol oxidase activity in mosquitoes exposed to microfilariae of Dirofilaria immitis

Monophenol oxidase (MPO) activity in hemocytes collected from Aedes aegypti Liverpool strain and Aedes trivittatus intrathoracically inoculated with saline alone, inoculated with Dirofilaria immitis microfilariae (mff), or from uninoculated mosquitoes was compared using a radiometric tyrosine hydroxylation assay. Hemocyte MPO activity in mff-inoculated (= immune-activated) mosquitoes was significantly increased at 24 hr postinoculation (PI) in A. aegypti and at 6, 12, and 24 hr PI in A. trivittatus as compared with saline-inoculated controls. Baseline and immune-activated levels of hemocyte MPO activity in A. trivittatus were significantly higher compared with those seen in A. aegypti. Baseline hemocyte population levels were similar in both species, but immune activation did not elicit increases in total hemocyte populations in A. trivittatus as has been demonstrated for A. aegypti. Likewise, immune activation by the inoculation of mff did not significantly alter plasma MPO activity in A. trivittatus as compared with uninoculated or saline-inoculated mosquitoes. Plasma MPO activity in A. aegypti, however, appears to constitute a major component of the immune response. The importance of phenol oxidase(s) in the immune response of mosquitoes against mff and the relationship of observed differences in MPO activity to differences in immunological capability between A. aegypti and A. trivittatus are assessed.     

PHENOL OXIDASE ACTIVITY IN ANOPHELES SINENSIS AND AEDES ALBOPZCTUS EXPOSED TO MICROFILARIAE OF BRUGIA MALAYI

Abstract Phenol oxidase (PO activity was investigated in cell-free hemolymph collected from Anopheles sinensis and Aedes albopictlcs female adults intrathoracically inoculated with Brugia dayi microfilariae (mff), inoculated with saline alone, or from uninoculated mosquitoes. The activity of PO from uninoculated 1-day-old mosquitoes was significantly greater than those of mosquitoes on 3, 12 and 16 day. PO activity in mff-inoculated A. sinensis at 24 h postinoculation (PI) was 2–3 times higher as compared with uninoculated or saline-inoculated mosquitoes. Inoculation of B. malayi mff into A. albopictus elicited ?-fold increase in PO activity at 24 h PI as compared with uninoculated mosquitoes. Immune-activated levels of PO activity in A. alboplctus was significantly higher as compared with those seen in A. sinensis. The relationship of observed differences in PO activity to differences in immunological capability between A. sinensis and A. albopictus is discussed.     

High-pressure liquid chromatographic analysis of hemolymph plasma catecholamines in immune-reactive Aedes aegypti

Tyrosine and catecholamines have been implicated as substrates for the encapsulation reactions involved in the immune response of mosquitoes to microfilariae (mff). Identification and quantitation of tyrosine and catecholamines present in Aedes aegypti hemolymph plasma were accomplished by ion-pair high-pressure liquid chromatography with electrochemical detection at either +650 or +850 mV vs Ag/AgCl. Tyrosine, dopamine, and N-beta-alanyldopamine were detected in the hemolymph plasma of naive A. aegypti. Although no differences in these compounds were observed in hemolymph plasma from A. aegypti inoculated with Dirofilaria immitis mff, the chromatogram showed a single major peak (PI) (65 microM, expressed as dopamine equivalents) that was not present in naive hemolymph plasma. Saline-inoculated controls contained only 5% of the PI in immune reactive hemolymph plasma. A high concentration of PI (127 +/- 39 microM) was also detected after treatment of hemolymph plasma with mild alkaline conditions (pH 9.0), indicating that it is normally present as an electrochemically inert form in naive mosquitoes. High concentrations of PI were also detected in the naive hemolymph plasma from three other mosquito species, but no PI was found in A. trivittatus under any conditions. PI did not cochromatograph with any of the catecholamines commonly thought to be involved in immune responses of dipterans against metazoan parasites, suggesting that it may be a unique substrate for these reactions. The biological relevance of PI was evidenced by its appearance in the hemolymph plasma of two strains of D. immitis-inoculated A. aegypti.     

Immune competence of Aedes trivittatus hemocytes as assessed by lectin binding

Hemocytes were perfused from uninoculated, saline-inoculated, and Dirofilaria immitis microfilariae-inoculated Aedes trivittatus and assessed for their capacity to bind wheat germ agglutinin (WGA). Approximately one-fourth of perfused hemocytes were WGA positive in all groups of mosquitoes at all time periods tested. Results suggest A. trivittatus has a larger inherent population of immune-competent hemocytes as compared with Aedes aegypti and, therefore, is faster and more effective in killing microfilariae by melanotic encapsulation reactions.     

Effect of gamma irradiation on the hemocyte-mediated immune response of Aedes aegypti against microfilariae

The effect of gamma irradiation on the melanotic encapsulation response of Aedes aegypti black eye Liverpool strain against inoculated Dirofilaria immitis microfilariae (mff) was assessed at 1, 2, 3, and 6 days postinoculation (PI). Mosquitoes received 6000 rad from a 137Cs source (Shepard Mark I irradiator) at 3 days postemergence and were inoculated with 15-20 mff 24 hr later. These mosquitoes were compared to nonirradiated controls that also were inoculated with 15-20 mff at 3 days postemergence. The immune response was significantly reduced in irradiated mosquitoes as compared with controls at all days PI. Although the response was significantly inhibited compared with controls, irradiated mosquitoes were still capable of eliciting a response against 69% of recovered mff at 6 days PI. External gamma irradiation did not significantly affect the proliferation of hemocytes associated with the melanotic encapsulation response of A. aegypti. The number of circulating hemocytes increased in irradiated mosquitoes in response to inoculated mff in a manner similar to nonirradiated, inoculated controls. Hemocyte monophenol oxidase activity, however, was significantly reduced in gamma-irradiated mosquitoes at 12 hr PI as compared with controls. The reduced immunological capacity of irradiated mosquitoes might be related to an interference with gene activity required for the synthesis or activation of enzymes that are directly or indirectly involved in the biochemical processes associated with the production of melanotic substances that sequester mff.     

The melanization reaction is not required for survival of Anopheles gambiae mosquitoes after bacterial infections

The melanization reaction of insects requires activation of pro-phenoloxidase by a proteolytic cascade leading to melanin production. Studies in adult mosquitoes have shown that bacteria are efficiently melanized in the hemocoel, but the contribution of melanization to survival after bacterial infections has not been established. Here we show that the Anopheles gambiae noncatalytic serine protease CLIPA8, an essential factor for Plasmodium ookinete melanization, is also required for melanization of bacteria in adult mosquitoes. CLIPA8 silencing by RNA interference inhibits pro-phenoloxidase activation and melanization of bacteria in the hemolymph following microbial challenge. However, CLIPA8 is not required for wound melanization nor for melanotic pseudotumor formation in serpin2 knockdown mosquitoes, suggesting a specific role for pathogen melanization. Surprisingly, CLIPA8 knockdown mosquitoes are as resistant to bacterial challenge as controls, indicating that melanization is not essential for defense against bacteria and questions its precise role in mosquito immunity.     

Immune defense mechanisms of Culex quinquefasciatus (Diptera: Culicidae) against Candida albicans infection

Mosquitoes have an efficient defense system against infection. The cellular immune defense mechanism initiated by the mosquito Culex quinquefasciatus infected with the fungus Candida albicans was investigated in this study. Differences in the hemocyte counts in hemolymph perfused from uninoculated, saline-inoculated, and C. albicans-infected mosquitoes were compared using a light microscope. Phagocytosis was also investigated using electron microscopy. Four types of hemocytes were identified in control mosquitoes: prohemocytes (9.8%), plasmatocytes (38.8%), granular cells (44.2%), and oenocytoids (7.3%). Between 3 and 18 h postinoculation the total hemocyte count was significantly higher in infected, compared to uninfected, mosquitoes. Differential hemocyte counts from infected mosquitoes at 3, 6, and 18 h after inoculation showed that the relative proportion of plasmatocytes (48.6, 50.7, 45%) was higher and, concomitantly, the proportion of granular cells was lower (38, 36.8, 35%, respectively). Yeast cells were phagocytosed and limited growth was observed within the plasmatocytes. Melanized nodules were found attached to different insect tissues at 24 to 72 h following infection. These results suggest that phagocytosis, followed by nodule formation, was capable of clearing the hemolymph of yeast cells.     

Further evidence that the genes controlling susceptibility of Aedes aegypti to filarial parasites function independently.

Comparisons were made of in vivo labeled polypeptides from Aedes aegypti strains refractory to either Brugia malayi or Dirofilaria immitis. There does not seem to be a generalized "anti-parasite" polypeptide response that mosquitoes refractory to filarial worm infection produce following bloodfeeding. Instead, it seems that any response produced by these mosquitoes is localized to the tissue in which the filarial parasite develops.     

A synthetic gene increases TGFβ3 accumulation by 75-fold in tobacco chloroplasts enabling rapid purification and folding into a biologically active molecule

Human transforming growth factor-β3 (TGFβ3) is a new therapeutic protein used to reduce scarring during wound healing. The active molecule is a nonglycosylated, homodimer comprised of 13-kDa polypeptide chains linked by disulphide bonds. Expression of recombinant human TGFβ3 in chloroplasts and its subsequent purification would provide a sustainable source of TGFβ3 free of animal pathogens. A synthetic sequence (33% GC) containing frequent chloroplast codons raised accumulation of the 13-kDa TGFβ3 polypeptide by 75-fold compared to the native coding region (56% GC) when expressed in tobacco chloroplasts. The 13-kDa TGFβ3 monomer band was more intense than the RuBisCO 15-kDa small subunit on Coomassie blue-stained SDS-PAGE gels. TGFβ3 accumulated in insoluble aggregates and was stable in leaves of different ages but was not detected in seeds. TGFβ3 represented 12% of leaf protein and appeared as monomer, dimer and trimer bands on Western blots of SDS-PAGE gels. High yield and insolubility facilitated initial purification and refolding of the 13-kDa polypeptide into the TGFβ3 homodimer recognized by a conformation-dependent monoclonal antibody. The TGFβ3 homodimer and trace amounts of monomer were the only bands visible on silver-stained gels following purification by hydrophobic interaction chromatography and cation exchange chromatography. N-terminal sequencing and electronspray ionization mass spectrometry showed the removal of the initiator methionine and physical equivalence of the chloroplast-produced homodimer to standard TGFβ3. Functional equivalence was demonstrated by near-identical dose-response curves showing the inhibition of mink lung epithelial cell proliferation. We conclude that chloroplasts are an attractive production platform for synthesizing recombinant human TGFβ3.     

Relationship of hemolymph phenol oxidase and mosquito age in Aedes aegypti.

Monophenol oxidase (MPO) and diphenol oxidase (DPO) activity in hemocytes and cell-free plasma perfused from 7-, 14-, 21-, and 28-day-old Aedes aegypti mosquitoes were compared. A progressive decrease of enzyme activity was detected as mosquito age increased, and this decrease was significant in both hemocytes and cell-free plasma when mosquitoes were 28 days old as compared with that found in 7-day-old mosquitoes. There was no significant difference in total hemolymph protein as mosquito age increased. Although this decreased MPO and DPO activity might be partially responsible for the reduced immune response against filarial worms previously reported for older mosquitoes, other factors undoubtedly play a significant role.     

Parasite-induced suppression of the immune response in Aedes aegypti by Brugia pahangi

The melanization response against intrathoracically inoculated Brugia pahangi and Dirofilaria immitis microfilariae (mff) isolated from vertebrate host blood was evaluated in both uninfected Aedes aegypti black-eyed Liverpool strain and in mosquitoes harboring a developing B. pahangi infection. The immune response against inoculated mff of either species was significantly reduced by 28-47% in infected as compared with uninfected mosquitoes. Attempts to passively transfer this suppression factor(s) by inoculating naive mosquitoes with 0.1-0.2 microliter of hemolymph from B. pahangi-infected mosquitoes produced equivocal results. The role this parasite-induced immune suppression might play in aiding parasite survival in compatible vectors is discussed.     

The Toll immune-regulated Drosophila protein Fondue is involved in hemolymph clotting and puparium formation

Clotting is critical in limiting hemolymph loss and initiating wound healing in insects as in vertebrates. It is also an important immune defense, quickly forming a secondary barrier to infection, immobilizing bacteria and thereby promoting their killing. However, hemolymph clotting is one of the least understood immune responses in insects. Here, we characterize fondue (fon; CG15825), an immune-responsive gene of Drosophila melanogaster that encodes an abundant hemolymph protein containing multiple repeat blocks. After knockdown of fon by RNAi, bead aggregation activity of larval hemolymph is strongly reduced, and wound closure is affected. fon is thus the second Drosophila gene after hemolectin (hml), for which a knockdown causes a clotting phenotype. In contrast to hml-RNAi larvae, clot fibers are still observed in samples from fon-RNAi larvae. However, clot fibers from fon-RNAi larvae are more ductile and longer than in wt hemolymph samples, indicating that Fondue might be involved in cross-linking of fiber proteins. In addition, fon-RNAi larvae exhibit melanotic tumors and constitutive expression of the antifungal peptide gene Drosomycin (Drs), while fon-RNAi pupae display an aberrant pupal phenotype. Altogether, our studies indicate that Fondue is a major hemolymph protein required for efficient clotting in Drosophila.     

垂体腺苷酸环化酶激活肽衍生多肽RHMP的重组制备及促角膜损伤修复的初步研究

利用基因工程技术高效制备具有促进角膜损伤修复功能的垂体腺苷酸环化酶激活肽(PACAP)衍生多肽RHMP,并在体外研究其生物学效应,为角膜疾病的治疗提供了新思路。采用基因重组技术表达重组肽RHMP,经Chitin-Beads柱纯化、HPLC及SDS-PAGE、质谱鉴定后,研究其对小鼠角膜损伤修复的影响。实验结果表明:利用基因重组技术制备的PACAP衍生多肽RHMP的分子量为3.4kDa,纯度为96%;将重组肽作用于创伤后的小鼠角膜,12h、24h、36h、48h后角膜修复率分别为(49.58±1.74)%、(93.66±3.39)%、(99.6±0.43)%、(99.8±0.14)%,而对照组修复率分别为(9.76±1.58)%、(29.02±1.63)%、(55.10±1.49)%、(78.59±2.52)%,P<0.001,差异具有统计学意义,重组肽RHMP可显著促进小鼠角膜损伤修复。因此,利用以上确立的表达、纯化策略,可实现新型基因重组PACAP衍生多肽RHMP的高效制备,重组多肽RHMP可快速有效地促进损伤角膜的修复,从而有望成为一种新型角膜损伤治疗药物;同时,建立的小鼠角膜上皮损伤模型可用于相关药物的生物学效应研究。