Study of the effect of Anisakis simplex larval products on the early and late components in the classical complement pathway

In previous studies, we have reported that the larval products (crude extract [CE] and excretory-secretory [ES]) of Anisakis simplex showed a dose-dependent inhibition of the lysis mediated by classical (CP) and alternative pathways (AP) of the human complement system, with the major inhibition on the CP rather than on AP. This inhibition of hemolysis is due to the consumption of complement factors because the assays performed shortening the preincubation period result in a significant decrease of the inhibitory effect on the lysis of the larval products compared with the standard time. Likewise, we found that the larval products reduce the inhibitory percentages in the CP using C3-deficient sera, but not in the AP, which could indicate that other complement components are implicated in the inhibitory effect in the CP. Hence, we have studied the activity of the larval products of A. simplex on individual components in the CP, using different complement-deficient sera. The investigated complement molecules were C1q, C2, C4, C5, C6, C7, C8, and C9. The larval products showed activity at the C2 level but failed to have a significant effect on the other components. Therefore, CE and ES products from A. simplex interact with C3 and C2 complement proteins, which are early components of the complement system, but not with the late complement components.     

Seroprevalence of antibodies against Anisakis simplex larvae among health-examined residents in three hospitals of southern parts of Korea

The present study was performed to estimate the seroprevalence of larval Anisakis simplex infection among the residents health-examined in 3 hospitals in southern parts of Korea. A total of 498 serum samples (1 serum per person) were collected in 3 hospitals in Busan Metropolitan city, Masan city, and Geoje city in Gyeongsangnam-do (Province) and were examined by IgE-ELISA and IgE-western blotting with larval A. simplex crude extract and excretory-secretory products (ESP). The prevalence of antibody positivity was 5.0% and 6.6% with ELISA against crude extracts and ESP, respectively. It was also revealed that infection occurred throughout all age groups and higher in females than in males. A specific protein band of 130 kDa was detected from 10 patients with western blot analysis against crude extract and ESP among those who showed positive results by ELISA. Our study showed for the first time the seroprevalence of anisakiasis in Korea. The allergen of 130 kDa can be a candidate for serologic diagnosis of anisakiasis.     

Excretory-secretory antigen is better than crude antigen for the serodiagnosis of clonorchiasis by ELISA

Although stool examination is the standard diagnostic method of clonorchiasis, serodiagnosis by ELISA using crude antigen is now widely used because of its convenience. However, ELISA diagnosis still suffers from cross-reactions, and therefore there is a need to improve the present conventional ELISA. The present study was undertaken to evaluate the diagnostic value of ELISA using excretory-secretory antigen (ESA) instead of crude antigen (CA) of Clonorchis sinensis. The diagnostic sensitivity of ELISA using excretory-secretory antigen was 92.5%, which was higher than that of ELISA using crude Clonorchis sinensis antigen (88.2%). In addition, the specificity of excretory-secretory antigen was found 93.1% while that of crude antigen was 87.8%. In summary, Clonorchis sinensis ESA was found to be a better serodiagnostic antigen than CA for ELISA.     

饲料中灵芝提取物对异育银鲫生长、饲料利用、免疫应答及抗病的影响

通过生长实验探求饲料中添加灵芝提取物饲料对异育银鲫（6.720.11） g生长、饲料利用及免疫应答的影响,以及停止灵芝提取物饲料后,使用效果的持续性。在实验进行的第21天（D21）、第46天（D46）、第65天（D65）进行取样,结果表明所有的灵芝提取物组的特定生长率在饲喂65d后与对照组无显著差异（P0.05）。摄食率随着灵芝提取物在饲料中含量的增加而增加（P0.05）,饲料效率则有着相反的趋势（P0.05）。在D46和D65时,随着灵芝提取物的含量增高,吞噬活力略有增高,但无显著差异（P0.05）。在D21、D46和D65时,呼吸暴发活力随着灵芝提取物的含量增高而增高（P0.05）,在D21时,（0.6%-21d、0%-44d）处理组和（3%-21d、0%-44d）处理组其呼吸暴发活力高于对照组（P0.05）。在D65时,（0.6%-21d、0%-44d）处理组和（3%-21d、0%-44d）处理组其呼吸暴发活力仍高于对照组（P0.05）,但差异不显著。D21、D46和D65时,替代途径补体溶血活力随着灵芝提取物的增高而降低（P0.05）。所有处理组溶菌酶活力在D21时没有产生显著差异。在D46时,对照组溶菌酶活力比最高添加组低但高于低添加组（P0.05）,（3%-1d、0%-44d）处理组也仍高于对照组（P0.05）。在D65时,所有添加组的溶菌酶活力均低于对照组（P0.05）。在D65时,髓过氧化物酶活力随着灵芝提取物的添加剂量升高而升高（P0.05）,（0.6%-21d、0%-44d）处理组仍高于对照组（P0.05）。灵芝提取物的能提高白细胞呼吸暴发活力,且在在高剂量摄入21d后（D46时）,仍可以保持较高的活力,说明灵芝提取物对免疫应答有一定时间延续效应。随着灵芝提取物添加量的升高,异育银鲫在经爱德华氏菌攻毒后的存活率显著提高。最高存活率组为3%添加组,显著高于对照组（P0.05）。实验周期为65d时,饲料中灵芝提取物推荐添加剂量为3%。       

Immune reactions and allergy in experimental anisakiasis

The third-stage larvae (L3) of the parasitic nematode, Anisakis simplex, have been implicated in the induction of hyperimmune allergic reactions in orally infected humans. In this work, we have conducted a review of an investigation into immune reactions occurring in animals experimentally infected with A. simplex L3. The patterns of serum antibody productions in the experimental animals against excretory-secretory products (ESP) of A. simplex L3 contributed to our current knowledge regarding specific humoral immune reactions in humans. In our review, we were able to determine that L3 infection of experimental animals may constitute a good model system for further exploration of immune mechanisms and allergy in anisakiasis of humans.     

Complement activation by phospholipids: the interplay of factor H and C1q

Complement proteins in blood recognize charged particles. The anionic phospholipid (aPL) cardiolipin binds both complement proteins C1q and factor H. C1q is an activator of the complement classical pathway, while factor H is an inhibitor of the alternative pathway. To examine opposing effects of C1q and factor H on complement activation by aPL, we surveyed C1q and factor H binding, and complement activation by aPL, either coated on microtitre plates or in liposomes. Both C1q and factor H bound to all aPL tested, and competed directly with each other for binding. All the aPL activated the complement classical pathway, but negligibly the alternative pathway, consistent with accepted roles of C1q and factor H. However, in this system, factor H, by competing directly with C1q for binding to aPL, acts as a direct regulator of the complement classical pathway. This regulatory mechanism is distinct from its action on the alternative pathway. Regulation of classical pathway activation by factor H was confirmed by measuring C4 activation by aPL in human sera in which the C1q:factor H molar ratio was adjusted over a wide range. Thus factor H, which is regarded as a down-regulator only of the alternative pathway, has a distinct role in downregulating activation of the classical complement pathway by aPL. A factor H homologue, β2-glycoprotein-1, also strongly inhibits C1q binding to cardiolipin. Recombinant globular domains of C1q A, B and C chains bound aPL similarly to native C1q, confirming that C1q binds aPL via its globular heads.     

Parallel genetic changes and nonparallel gene-environment interactions characterize the evolution of drug resistance in yeast

Beneficial mutations are required for adaptation to novel environments, yet the range of mutational pathways that are available to a population has been poorly characterized, particularly in eukaryotes. We assessed the genetic changes of the first mutations acquired during adaptation to a novel environment (exposure to the fungicide, nystatin) in 35 haploid lines of Saccharomyces cerevisiae. Through whole-genome resequencing we found that the genomic scope for adaptation was narrow; all adapted lines acquired a mutation in one of four late-acting genes in the ergosterol biosynthesis pathway, with very few other mutations found. Lines that acquired different ergosterol mutations in the same gene exhibited very similar tolerance to nystatin. All lines were found to have a cost relative to wild type in an unstressful environment; the level of this cost was also strongly correlated with the ergosterol gene bearing the mutation. Interestingly, we uncovered both positive and negative effects on tolerance to other harsh environments for mutations in the different ergosterol genes, indicating that these beneficial mutations have effects that differ in sign among environmental challenges. These results demonstrate that although the genomic target was narrow, different adaptive mutations can lead populations down different evolutionary pathways, with respect to their ability to tolerate (or succumb to) other environmental challenges.     

Glycyrrhizin inhibits the lytic pathway of complement--possible mechanism of its anti-inflammatory effect on liver cells in viral hepatitis

Glycyrrhizin, an aqueous extract of licorice root, has anti-inflammatory activity and has been used for the treatment of chronic viral hepatitis. In the present study we describe the mechanism by which glycyrrhizin inhibits complement. Glycyrrhizin inhibited the cytolytic activity of complement via the activation of both the classical and alternative pathways, while it had no effect on immune adherence, suggesting that it blocks C5 or a later stage of the complement cascade. Further analysis revealed that glycyrrhizin inhibits the lytic pathway in which the membrane attack complex (MAC) is formed. This mechanism suggests that glycyrrhizin may prevent tissue injury caused by MAC not only in chronic hepatitis but in many autoimmune and inflammatory diseases.     

Analysis of larval antigens of Oestrus ovis for the diagnosis of oestrosis by enzyme-linked immunosorbent assay

A serodiagnostic test for the diagnosis of infestation by the sheep nasal bot fly, Oestrus ovis (Linné) was examined. The enzyme-linked immunosorbent assay (ELISA) technique was used to analyze and compare the production of immunoglobulin G (IgG) antibodies against excretory-secretory products (ESP) and crude extract (CE) antigens from all the different larval stages of O. ovis in the sera of 276 adult sheep sampled in summer (n = 135) and winter (n = 141). ESP from first stage larvae was the most sensitive, coating antigen in winter and ESP from second stage larvae during summer. The most specific values were obtained by ESP against L1 in winter and by CE against L3 in summer. These results show that the stage of larval development has a significant impact on the humoral immune response over the course of a season. A significant correlation (P < 0.001) was found between the number of O. ovis larvae and the serum antibody levels using all differents antigens, except L3 CE. In Spain, where a long favourable period exists for the evolution and development of the different stage larvae between March and November, the ELISA test using L1 ESP antigen during winter and L2 ESP antigen in summer may be used for ovine oestrosis immunodiagnosis.     

MALDI mass sequencing and characterization of filarialglutathione-S-transferase

Glutathione-S-transferase has been detected in the somatic extract and excretory-secretory products of different life stages of Setaria cervi, a bovine filarial parasite. The enzyme was subjected to MALDI-TOF followed by mass spectrometry and the nearest match found was Pleuronectes platessa GST. Molecular mass of the purified enzyme was approximately 26 kDa as determined by SDS-PAGE and MALDI-TOF. Setaria cervi GST exhibited high activity towards 1-chloro-2,4-dinitrobenzene and ethacrynic acid. Kinetic analysis with respect to 1-chloro-2,4-dinitrobenzene and glutathione as substrate revealed a K(m) of 2.22 mM and 0.61 mM, respectively. The activity was inhibited significantly by Cibacron blue and alpha-tocopherol.     

Specific and common antigens of Clonorchis sinensis and Opisthorchis viverrini(Opisthorchidae,Trematoda)

The antigenic characterizations and serological reactions of human liver flukes, Clonorchis sinensis and Opisthorchis viverrini, were analyzed by immunoblot. The antigenic profiles of the crude extract of Clonorchis contained major proteins of 8, 26-28, 34-37, 43, and 70 kDa, and those of Opisthorchis 34-37, 43, 70, and 100 kDa. Of these, the 8, 26-28 and 34-37 kDa bands of Clonorchis and the 100 kDa of Opisthorchis were major components of each excretory-secretory antigen. The 8 and 26-28 kDa bands were specific to Clonorchis but the 100 kDa of Opisthorchis cross-reacted with the sera of clonorchiasis, and the 34-37, 70 and 100 kDa bands cross-reacted with sera of other helminthiases. The frequency and intensity of the immunoblot reactions were positively correlated with the intensity of the liver fluke infection.     

Anisakis simplex: CO(2)-fixing enzymes and development throughout the in vitro cultivation from third larval stage to adult

We studied the effect of CO(2) on the in vitro cultivation of Anisakis simplex, an aquatic parasitic nematode of cetaceans (final hosts) and fish, squid, crustaceans and other invertebrates (intermediate/paratenic hosts), and, occasionally, of man (accidental host). The results showed that a high pCO(2), at a suitable temperature, is vital for the optimum development of these nematodes, at least from the third larval stage (L3) to adult. After 30 days cultivation in air, molting to L4 (fourth larval stage) was reduced to 1/3, while survival was about 1/3 of that when cultivated in air + 5% CO(2). The activity of the CO(2)-fixing enzymes, PEPCK and PEPC, was also studied. Throughout the development of the worms studied, PEPCK activity was much higher than that of PEPC (e.g., 305 vs. 6.8 nmol/min.mg protein, respectively, in L3 collected from the host fish). The activity of these enzymes in the worms cultivated in air + 5% CO(2) was highest during M3, and was also generally higher than that of those cultivated in air only, especially during molting from L3 to L4 (e.g., in recently molted L4, PEPCK activity was 3.7 times greater than that of PEPC 2.9 times greater than when cultivated in air).     

Purification of a phospholipase A2 from Lonomia obliqua caterpillar bristle extract

Lonomia obliqua caterpillar bristle extract induces both direct and indirect hemolytic activity on human and rat washed erythrocytes, and provokes intravascular hemolysis in Wistar rats. Indirect hemolytic activity is assumed to be caused by a phospholipase A(2) (PLA(2)) present in this extract, and this investigation was initiated in order to characterize this enzyme. Phospholipase A(2) activity of crude extract was inhibited by both a PLA(2)-specific inhibitor (pBpb) and the metal ion chelator EDTA. L. obliqua PLA(2) was purified by liquid chromatography from the crude bristle extract and had a molecular mass of 15kDa and a pI of 5.9; its N-terminal sequence showed high homology to a sequence of a putative PLA(2) obtained from a cDNA library of L. obliqua bristles, and it is tentatively placed among Group III phospholipases A(2). This enzyme was stable at 4 degrees C, sensitive to higher temperatures, and its maximum catalytic activity was at pH 8.0. L. obliqua PLA(2) induced hemolysis only when incubated with exogenous lecithin. Thus, the PLA(2) purified herein appears to be responsible for the indirect hemolytic activity of the crude bristle extract.     

Effects of Lonomia obliqua caterpillar venom upon the proliferation and viability of cell lines

Many active principles produced by animals, plants and microorganisms have been employed in the development of new drugs for the treatment of human diseases. Among animals known to produce pharmacologically active molecules that interfere in human cell physiology, the caterpillar Lonomia obliqua has become the focus of toxicological studies due to recent findings about its venom constituents. The objective of this study was to investigate the effects of L. obliqua venom upon the viability and the proliferation of different cell lineages and to propose mechanisms for the herein observed induction of cell proliferation in glioma cell lines. MTT analyses indicate that L. obliqua venom increases the viability of tumor cell lines U138-MG and HT-29; on the other hand, it inhibits the viability of V-79 nontumor cells. Cell count based on the trypan blue exclusion method suggests a proliferating activity of the venom upon U138-MG cells. Exposure of U138-MG to crude venom extract led to a decrease in the production of nitric oxide, and activation of the cAMP signaling pathway inhibited the effects of the venom, indicating that these mechanisms may influence cell proliferation triggered by the venom. Despite the proliferative effects of crude venom on U138-MG and HT-29 cell cultures, a protein purified from L. obliqua hemolymph previously shown to have cytoprotective activity had no effect on U138-MG and HT-29; however, this same protein increased the viability of V-79 cells that had previously been exposed to the cytotoxic activity of the crude venom extract. This study indicates that the venom and the antiapoptotic protein act differently and have different effects on cell cultures, depending on the cell line analyzed. Biomolecules displaying either mitogenic or cytotoxic activities are of great biotechnological interest. Further studies encompassing the purification of active principles from L. obliqua venom are necessary to further elucidate its effects on different cell types.     

A Recombinant Matrix Metalloproteinase Protein from Gnathostoma spinigerum for Serodiagnosis of Neurognathostomiasis

Neurognathostomiasis is a severe form of human gnathostomiasis which can lead to disease and death. Diagnosis of neurognathostomiasis is made presumptively by using clinical manifestations. Immunoblotting, which recognizes antigenic components of molecular mass 21 kDa and 24 kDa in larval extracts of Gnathostoma spinigerum (Gs 21/24), has high sensitivity and specificity for diagnosis of neurognathostomiasis. However, only very small amounts of the Gs 21/24 antigens can be prepared from parasites harvested from natural or experimental animals. To overcome this problem, we recently produced a recombinant matrix metalloproteinase (rMMP) protein from G. spinigerum. In this study, we evaluated this rMMP alongside the Gs 21/24 antigens for serodiagnosis of human neurognathostomiasis. We studied sera from 40 patients from Srinagarind Hospital, Khon Kaen University, Thailand, with clinical criteria consistent with those of neurognathostomiasis, and sera from 30 healthy control adults from Thailand. All sera were tested for specific IgG antibodies against both G. spinigerum crude larval extract and rMMP protein using immunoblot analysis. The sensitivity and specificity for both antigenic preparations were all 100%. These results show that G. spinigerum rMMP protein can be used as an alternative diagnostic antigen, in place of larval extract, for serodiagnosis of neurognathostomiasis.     

Immunoglobulins anti-Anisakis simplex in patients with gastrointestinal diseases

The nematode Anisakis simplex causes anisakidiasis, a disease that often mimics other gastrointestinal diseases. Patients with digestive haemorrhaging, Crohn's disease, digestive cancer and appendicitis were analysed for antibodies to A. simplex. Antibody detection was carried out by enzyme-linked immunosorbent assay (ELISA) and immunoblotting using crude extract (CE) antigen and excretory-secretory (ES) products. Total immunoglobulin (Igs), IgG, IgM, IgA and IgE were studied. The highest percentage was obtained when Igs were tested against CE antigen. A higher percentage of positivity was observed with the appendicitis group. The Crohn's disease group showed the highest levels of IgG against the ES antigen. Using immunoblotting, 24% and 48% of sera from patients with symptoms of Crohn's disease and digestive haemorrhaging, respectively, showed a positive immunorecognition pattern of CE antigen. The prevalence of detectable antibodies against A. simplex is higher in patients with digestive disorders than in the healthy population. A linear correlation was observed between prothrombin activity and Igs-CE, IgA-CE and IgA-ES but not between IgE-CE and the other immunoglobulin levels. Specific IgA is associated with a higher activity index of Crohn's disease. Specific antibodies were observed against A. simplex in patients with appendicitis and gastrointestinal cancer, indicating a higher rate of positivity for IgA.     

Characterization of partially purified 8 kDa antigenic protein of Clonorchis sinensis

The 8 kDa antigenic protein of Clonorchis sinensis was partially purified by ammonium sulfate precipitation and subsequently by a column chromatographic steps. The purified protein was separated into 7 and 8 kDa protein bands through SDS-tricine gel electrophoresis, while the protein was found to migrate to a 8 kDa band in 7.5-15% SDS-PAGE. The molecular weight of the antigen was estimated to be 110 kDa by Superose 6 HR 10/30 gel filtration. The purified antigen strongly reacted with the human sera of clonorchiasis. The hyperimmune sera of BALB/c mice immunized against the 8 kDa protein were reacted with both the crude extract and the excretory-secretory product of adult worms, but not with the metacercarial extract. Immunohistochemical staining demonstrated that the protein was distributed to the tegument and subtegumental cells and also to the seminal receptacle. The present findings suggest that the 8 kDa protein is a partition of the multicomplex protein originating from various organs of adult C. sinensis, and that it is composed of several 7 and 8 kDa proteins.     

Characterization of the mitochondrial respiratory pathways in Candida albicans

Candida albicans is an opportunistic oral pathogen. The flexibility of this microorganism in response to environmental changes includes the expression of a cyanide-resistant alternative respiratory pathway. In the present study, we characterized both conventional and alternative respiratory pathways and determined their ADP/O ratios, inhibitor sensitivity profiles and the impact of the utilization of either pathway on susceptibility to commonly used antimycotics. Oxygen consumption by isolated mitochondria using NADH or malate/pyruvate as respiratory substrates indicated that C. albicans cells express both cytoplasmic and matrix NADH-ubiquinone oxidoreductase activities. The ADP/O ratio was higher for malate/pyruvate (2.2±0.1), which generate NADH in the matrix, than for externally added NADH (1.4±0.2). In addition, malate/pyruvate respiration was rotenone-sensitive, and an enzyme activity assay further confirmed that C. albicans cells express Complex I activity. Cells grown in the presence of antimycin A expressed the cyanide-insensitive respiratory pathway. Determination of the respiratory control ratio (RCR) and ADP/O ratios of mitochondria from these cells indicated that electron transport from ubiquinone to oxygen via the alternative respiratory pathway was not coupled to ATP production; however, an ADP/O ratio of 0.8 was found for substrates that donate electrons at Complex I. Comparison of antifungal susceptibility of C. albicans cells respiring via the conventional or alternative respiratory pathways showed that respiration via the alternative pathway does not reduce the susceptibility of cells to a series of clinically employed antimycotics (using Fungitest®), or to the naturally occurring human salivary antifungal peptide, histatin 5.     

Optimum conditions for the activation of the alternative complement pathway of a cyprinid fish (Tinca tinca L.). Seasonal variations in the titres

The present work describes the existence of a haemolytic activity in the serum of tench, Tinca tinca, against rabbit red blood cells (RRBC) which was identified as belonging to the alternative complement pathway from the following findings: haemolytic activity disappeared when the serum was heated to 45°C for 20 min; 10 mM EDTA, which chelates Ca²⁺ and Mg²⁺, induced a complete loss of haemolysis; Mg²⁺, but not Ca²⁺, was required for the activity, and the use of sheep red blood cells (SRBC), which have a high content of sialic acid, resulted in the serum activity falling to a very low degree of haemolysis. The ACH50 value (units ml^-1 serum) was defined as the reciprocal of the serum dilution necessary to lyse 50% of 4 × 10⁷ RRBC in a buffered medium of normal ionic strength (μ=0·15) containing 10 mM EGTA and optimum concentrations of Mg²⁺. The optimum conditions for the ACH50 assay were: pH 7·2-7-7; reaction temperature, 15°C; concentration of Mg²⁺, 5 mM; and reaction time, 90 min. Under these conditions, the values of ACH50 in spring, summer, autumn and winter for male tench were 69±13, 91±22, 90±36 and 137±41, and for female tench 100±11, 108±13, 82±12 and 145±17. The highest serum activity was found in the winter, suggesting the importance of this pathway during cold periods when the specific immune response is depressed in ectothermic vertebrates.