Schistosoma mekongi: a prominent neutrophil chemotactic activity of egg antigen with reference to that of Schistosoma japonicum

Schistosoma mekongi causes granulomatous lesions around eggs deposited in the liver with neutrophil-rich inflammatory reactions in the early stage of the egg laying. To define the aspects of the typical pathogenesis of S. mekongi infection, we determined the difference between soluble egg antigen (SEA) from S. mekongi and S. japonicum with a focus on chemotactic factors for neutrophils or eosinophils. Mean volume and protein amount of S. mekongi eggs was 71 and 58% of those of Schistosoma japonicum eggs, respectively. Neutrophil chemotactic activity of S. mekongi SEA was about two times higher than that of S. japonicum. In contrast, eosinophil chemotactic activity of S. mekongi SEA was about half of that of S. japonicum SEA. Molecular analysis revealed that S. mekongi SEA contains higher molecular-weight components with a lower level of glycosylation, and this is likely to be related to the intense neutrophil chemotactic activity in comparison with S. japonicum SEA. The prominent chemotactic reactivity for neutrophils is likely to be involved in the typical pathogenesis of mekongi schistosomiasis.     

Schistosoma japonicum: identification and characterization of neutrophil chemotactic factors from egg antigen

Two neutrophil chemotactic factors were identified in soluble egg antigen preparations of Schistosoma japonicum. The higher-molecular-weight neutrophil chemotactic factor was not separable from eosinophil chemotactic factor by means of gel filtration, anion-exchange chromatography, isoelectric focusing, or affinity chromatography; this neutrophil chemotactic factor is apparently identical to the higher-molecular-weight eosinophil chemotactic factor which we purified previously from the soluble egg antigen. The chemotactic activity of the eosinophil chemotactic factor for neutrophils was stable to periodate oxidation but was notably affected by heating or Pronase digestion, suggesting that the determinant for neutrophil chemotaxis exists on the peptide moiety of the eosinophil chemotactic factor. The lower-molecular-weight neutrophil chemotactic factor was separable from the higher-molecular-weight eosinophil chemotactic factor by gel filtration or anion-exchange chromatography. This neutrophil chemotactic factor was rather hydrophobic and heat-stable, but was sensitive to Pronase or carboxypeptidase A digestion. These results suggest that the receptors on the surfaces of neutrophils and eosinophils for those chemoattractants would be different from each other. We suppose that neutrophil chemotactic factors and eosinophil chemotactic factors from the eggs are responsible for neutrophil and eosinophil accumulation around the eggs in schistosomiasis japonica.     

Eosinophil and neutrophil chemotactic activities of adult worm extracts of Schistosoma japonicum in vivo and in vitro

Large numbers of eosinophils and neutrophils attracted to the soluble extract of Schistosoma japonicum adult worms (SjAW-ext) were detected at the injection site of normal guinea pig skin. Eosinophil and neutrophil chemotactic activities were also confirmed in in vitro assay by using blind-well chambers with Millipore filters in dose-dependent fashion. Two components of SjAW-ext showed eosinophil chemotactic activity; one was in the high molecular weight fraction (JAE-H), estimated to be more than 440,000 daltons, the other in the low molecular weight fraction (JAE-L) obtained by Sephadex G-200 gel filtration. High neutrophil chemotactic activity was detected in the JAE-L. These eosinophil and neutrophil chemotactic activities were also detected in culture fluid of S. japonicum adult worms. Eosinophil chemotactic factor (ECF) of JAE-H was stable to heating (100 C, 30 min) and pronase digestion, but completely destroyed by periodate oxidation. It is suggested that the ECF of JAE-H is a glycoprotein. JAE-L was also stable to heating (56 and 100 C, 30 min) and pronase digestion for eosinophil chemotaxis. Possible roles of those activities in schistosome infections are discussed.     

Leukocyte accumulation in sparganosis: further characterization of an eosinophil chemotactic factor of the plerocercoid of Spirometra erinacei

An eosinophil chemotactic (ECF) was partially purified from plerocercoids of Spirometra erinacei by a combination of anion-exchange chromatography on DE52 and gel filtration on Sephacryl S-200. The molecular weight of ECF was estimated to be 25,000-45,000 by high-pressure liquid chromatography. The ECF was bound with concanavalin A-Sepharose. The ECF was sensitive to periodate oxidation and to heating (56 degrees C, 30 min). On isoelectric focusing, eosinophil chemotactic activity was clearly revealed at pI 4.1. These results suggest that ECF of S. erinacei plerocercoid is an acidic glycoprotein. An intradermal injection of ECF eosinophil attractions in the normal guinea pig skin.     

Purification and characterization of a neutrophil chemotactic factor from Dirofilaria immitis

A neutrophil chemotactic factor (NCF-Di) was purified from a crude extract of Dirofilaria immitis adult worm by a combination of anion-exchange chromatography on DE52 and gel filtration on Sephacryl S-200. NCF-Di showed a single protein band by both polyacrylamide gel electrophoresis (PAGE) and sodium dodecyl sulfate (SDS) PAGE. The molecular weight of NCF-Di was estimated to be 17,000 by gel filtration on Sephadex G-150, and 14,000 by SDS-PAGE. NCF-Di was an acidic protein with isoelectric point of 4.5. NCF-Di was absorbed neither to lentil lectin-Sepharose nor to concanavalin A-Sepharose. The chemotactic activity of NCF-Di was heat labile (56 C, 1 hr), but was resistant to periodate oxidation. These results suggest that NCF-Di is a simple peptide which has few or no sugar chains. These physicochemical properties of NCF-Di were compared to previously reported parasite-derived chemoattractants or purified allergen of D. immitis.     

Generation and functional characterization of T cell lines and clones specific for Schistosoma japonicum egg antigen in humans

T cell lines specific for Schistosoma japonicum egg Ag were established in vitro from patients with chronic schistosomiasis japonica, and investigated their possible immunopathologic roles by testing lymphokines production and in vitro granuloma formation assay. All lines tested had surface phenotypes of CD3+ CD4+ CD8-, and showed S. japonicum soluble egg Ag (SEA)-specific proliferation requiring HLA-DR-restricted Ag presentation. Of these fractions of SEA separated by gel filtration, Fraction II (m.w. 7,000 to 18,000) and III (m.w. 7,000) induced strong proliferation of T cell lines, whereas fraction I (m.w. 18,000+) failed to induce detectable proliferation to any T cell lines tested. One of the T cell lines was cloned by micromanipulation: two of eight clones responded only to fraction II, and six to both fractions II and III. We observed that four of eight clones tested produced IL-2 in response to SEA, and three of them were able to transfer S. japonicum egg-specific granulomatous hypersensitivity in vitro to an HLA haplo-identical individual without previous schistosome infection. These immunopathologic functions of T cell clones seemed to be activated by at least two distinct epitopes of SEA. Our present observations suggest that at least two distinct CD4+ human T cells, both of which recognize epitopes expressed on SEA molecules of less than 18 kDa, might have critical roles in granulomatous hypersensitivity to eggs of S. japonicum in humans.     

亲和素结合蛋白性质的研究

前曾发现在日本血吸虫虫卵中,存在一种新的能与亲和素专一性结合的蛋白质,称为亲和素结合蛋白（ＡＢＰ）。在分离纯化ＡＢＰ的基础上,用ＳＤＳ－ＰＡＧＥ及ＳｅｐｈａｄｅｘＧ－１５０分别测定了ＡＢＰ的分子量,并做了糖蛋白染色及等电,大测定。实验结果表明ＡＢＰ是一个分子量为６５ｋＤ的碱性糖蛋白,由数个相同的分子量为１２．７ｋＤ的亚单位组成。ＳＤＳ、β－巯基乙醇处理的ＡＢＰ仍能与亲和素结合,提示ＡＢＰ的亚单位能与亲和素结合。氨基酸修饰剂ＮＡＩ、ＤＴＮＢ、ＮＥＭ及ＮＢＳ对ＡＢＰ与亲和素的结合有不同程度的影响,而ＨＮＢＢ、ＴＮＢＳ及ＩＡＡ对ＡＢＰ与亲和素的结合无影响,提示ＡＢＰ分子中氨基酸残基Ｔｙｒ、Ｃｙｒ是ＡＢＰ与亲和素结合所必需的,可能参与结合位点的形成。另外,测得ＡＢＰ与亲和素结合的结合常数为１．４×１０~（－７）ｍｏｌ／Ｌ。     

Eosinophils and immune mechanisms. IV. Culture conditions, antigen requirements, production kinetics and immunologic specificity of the lymphokine eosinophil stimulation promoter.

The culture conditions which contribute to the production of the lymphokine, eosinophil stimulation promoter (ESP), have been investigated. Spleen or lymph node cells from mice infected for 8–10 weeks with the helminth Schistosoma mansoni respond to challenge with a soluble egg antigenic preparation (SEA) from S. mansoni eggs by the elaboration of ESP into the culture medium. Exposure to as little as 0.1 μg of SEA elicits this response. Furthermore, 30 min of exposure to antigen is sufficient to stimulate subsequent ESP production. Production begins within 4 hr, is rapid for 12 hr, and continues for at least another 8 hr. The use of this information allows the standardization of ESP production to be such that the concentration of SEA is insufficient to interfere with the indirect assay of ESP upon eosinophil-rich peritoneal exudates from S. mansoni-infected mice. The specificity of the direct assay elicited by SEA was confirmed, and the ability of the lymphokine to stimulate eosinophil migration from eosinophil-rich peritoneal exudates from either S. mansoni-or Trichinella spiralis-infected mice was demonstrated.     

Immune responses during human schistosomiasis mansoni. VI. In vitro nonspecific suppression of phytohemagglutinin responsiveness induced by exposure to certain schistosomal preparations.

Simultaneous in vitro exposure of human peripheral blood mononuclear cells to phytohemagglutinin-P (PHA) and either soluble schistosomal egg antigenic preparation (SEA) or soluble cercarial antigenic preparation (CAP) obtained from Schistosoma mansoni resulted in decreased responsiveness as compared to exposure to PHA alone. The addition of a soluble adult worm antigenic preparation (SWAP) did not predictably alter PHA responses in this system. The suppression due to in vitro exposure to either SEA or CAP was expressed whether the lymphocyte donors were S. mansoni patients (early infection, chronic, or treated), or uninfected subjects. The degree of suppression was related to the concentration of SEA used, and the timing of exposure. Preexposure to SEA for 3 days before the addition of PHA resulted in more potent suppression. However, a delay in the time of the addition of SEA of 6 and 24 hr after PHA exposure decreased and eliminated, respectively, its suppressive capacity. SEA and CAP were not directly toxic to responding cells, and appeared to exert their nonspecific suppressive influences through T lymphocyte-related mechanisms. It was observed that although these suppressive events could be induced and observed in vitro, the responsiveness of S. mansoni patient lymphocytes to PHA was equal with that of uninfected controls.     

Low molecular weight eosinophil chemotactic factor in granulomatous liver of murine schistosomiasis.

An acidic peptide, preferentially chemotactic for eosinophils, was extracted from livers of mice infected with Schistosoma mansoni. Sephadex G-25 column chromatography showed that the majority of the eosinophil chemotactic activity was detected in the fractions just after elution of the molecular marker vitamin B12 (m.w. 1355.4). This activity began to appear in the livers of some mice 5 weeks after infection. Peak activity was detected at 8 to 12 weeks after infection and persisted at least until 16 weeks. It was sensitive to carboxypeptidase-A. By Dowex-1 anion exchange chromatography, the activity eluted as a narrow peak at pH 3.1 TO 2.6 as shown for eosinophil chemotactic factor of anaphylaxis (ECF-A). The activity was also detected in a broad peak at pH 6.3 to 3.7. Unlike ECF-A, the activity was stable to boiling in both acid and alkali. These findings suggest that granulomatous liver of murine schistosomiasis-derived eosinophil chemotactic factor (ECF-G) may play a specific role in eosinophil accumulation in this chronic inflammation.     

An eosinophil chemotactic factor present in blister fluids of bullous pemphigoid patients.

Tissue eosinophilia is often found in the inflammatory lesions of bullous perphigoid. A study made on naturally occurring eosinophil chemotactic activity in the blister fluids of four bullous pemphigoid patients revealed the existence of this activity in all of them. Sephadex G-25 column chromatography showed that the greater part of this eosinophil chemotactic activity was composed of low molecular substance of which the weight was close to that of vitamin B12 (m.w. 1357). The blister fluids and the sera of these patients contained elevated levels of IgE. An IgE anti-skin basement, membrane antibody was found in two if the four sera, and deposits of IgE were detected along the basement membrane zone of the involved skin in one of the patients. On the basis of these findings, we have reason to believe that an eosinophil chemotactic factor of anaphylaxis (ECF-A) participates in the accumulation of eosinophils in the lesions of bullous pemphigoid.     

Characterization of a membrane surface glycoprotein associated with T-cell activation

A cell surface antigen (gp140) was previously shown to exist on T cell subsets as well as on monocytes and macrophages in normal peripheral blood. Elevated expression of this antigen was associated with immune system disorders, acute lymphocytic leukemias, and in vitro activation of T cells. The antigen could be identified with monoclonal antibody (MAb) T305. Gp140 was a biosynthetic product of T cells because it could be labeled with [3H]leucine or [3H] glucosamine. Biochemical studies of gp140 used high performance liquid chromatography with nitrocellulose blotting to isolate aliquots suitable for 125I radiolabeling and immunoprecipitation to demonstrate: a) a reduction in m.w. of gp140 KD to 90 KD after deglycosylation by trifluoromethanesulfonic acid, b) alteration of isoelectric point from 4.1 to 5.7 after neuraminidase treatments, c) absence of N-linked sugars based on resistance to endoglycosidase F, d) resistance to trypsin and chymotrypsin digestion but susceptibility to pronase, and e) presence of sialic acid and lactosaminoglycan as O-linked sugars. Gp140 could be labeled with the periodate/NaB[3H]4 technique, indicating its similarity to a class of sialoglycoproteins previously described on activated T-cells in mouse and man. The antigenic epitope recognized by MAb T305 contains sialic acid linked (2----3) to galactose; however, periodate oxidation of the exocyclic ring of sialic acid did not affect binding by MAb T305. In an attempt to determine the functional role of gp140, we tested the ability of MAb T305 to block: a) proliferation of peripheral blood lymphocytes to mitogens, b) response to interleukin 2 (IL 2) of an IL 2 dependent T cell line, and c) growth of a T-ALL derived cell line. No inhibition of proliferation or growth was noted. Although the function of gp140 remains unknown, its association with lymphocyte activation and certain disease states suggests that it may provide a target for modulation of the immune response. These studies characterize the structural features of gp140 and further define the epitope recognized by MAb T305.     

Characterization of developmentally regulated epitopes of Schistosoma mansoni egg glycoprotein antigens

The chemical and antigenic composition of a major group of concanavalin A-binding glycoprotein antigens of Schistosoma mansoni eggs was examined by the use of monoclonal antibodies. The individual glycoproteins of this group each displayed a very wide range of apparent m.w. and had isoelectric points of less than 5 when analyzed by two-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis. These glycoproteins could be chemically labeled with 125Iodine by two different methods and biosynthetically labeled with 35S-methionine during in vitro synthesis by isolated eggs. By using five monoclonal antibodies, the individual egg glycoproteins were shown to share several antigenic determinants when analyzed by radioimmunoprecipitation and solid-phase binding assays. These epitopes were present in a high level, and the degree of expression appeared to be developmentally regulated. In addition, Schistosoma haematobium and Schistosoma japonicum eggs contained antigenically related glycoprotein antigens. Preliminary evidence suggests that the epitopes involve carbohydrate moieties.     

Immunoaffinity purification of Schistosoma mansoni soluble egg antigens

Schistosoma mansoni egg antigens were purified from a heterogeneous mixture of soluble egg antigens (crude SEA) with an immunoaffinity column that consisted of the specific anti-SEA antibodies contained in 16-week S. mansoni-infected mouse serum bound to Sepharose 4B. On sodium dodecyl sulfate (SDS) gel electrophoresis, the purified antigen fraction yielded at least eight bands staining with Coomassie blue and at least five bands staining with Coomaisse blue and at least five bands reacting with periodic acid-Schiff (PAS). All of the proteins in the antigenic fraction appear to contain carbohydrate residues. Upon immunoelectrophoresis the antigen yielded four precipitin arcs. The antigenic fraction isolated by means of the immunoaffinity column was then compared to various fractions obtained from concanavalin A (Con A) chromatography of SEA. The results of Ouchterlony immunodiffusion and immunoelectrophoresis indicate that the antigenic fraction isolated by immunoaffinity purification of SEA contains the major antigens found in the fractions obtained from Con A chromatography of SEA. The results of SDS gel electrophoresis indicate that the major PAS-reacting bands of the antigenic fraction isolated by immunoaffinity purification are found in the 3rd peak (bound fraction) resulting from Con A chromatography of SEA, whereas the major Coomaisse blue-staining band in the isolated antigenic fraction is found in the 2nd peak (unbound fraction) from Con A chromatography of SEA.     

The effect of removal of D-fructose on the antigenicity of the lipopolysaccharide from a rough mutant of Vibrio cholerae Ogawa

The lipopolysaccharide (LPS) of a rough mutant (95R) of Vibrio cholerae Ogawa has been investigated chemically and serologically. D-Fructose was released from LPS under conditions (10mM trifluoroacetic acid, 60 degrees, 1 h) that liberated no other sugar constituent of the LPS (2-amino-2-deoxy-D-glucose, D-glucose, L-glycero-D-manno-heptose). Upon periodate oxidation, D-fructose and D-glucose were oxidised quantitatively, whereas approximately 50% of heptose was periodate-resistant. The data indicate that D-fructose does not link the polysaccharide and lipid A portion as proposed earlier, and suggest that D-fructose is present as a branch. By passive hemolysis inhibition, it was shown that the release of D-fructose paralleled the exposure of an antigenic determinant cryptic in LPS.     

A murine IgM monoclonal antibody binds an antigenic determinant in outer surface protein A, an immunodominant basic protein of the Lyme disease spirochete

A hybridoma cell line formed by the fusion of the P3x63-Ag8.653 myeloma cell line with splenocytes from BALB/c mice immunized with Borrelia burgdorferi produced an IgM monoclonal antibody (mAb-11G1) with kappa-light chains which detected an antigenic determinant in a major spirochetal protein of m.w. approximately 31,000, also known as outer surface protein A (OSP-A). Apparent saturation was reached in approximately 35 min with 34 ng of mAb-11G1 binding to 5 X 10(7) spirochetes giving an estimated 4.8 X 10(2) IgM molecules per spirochete and thus a minimum of 480 binding sites per organism. Enzymatic digestion studies suggest that the antigenic determinant to mAb-11G1 is contained within the peptide chain of OSP-A as binding could be eliminated by treatment of the spirochetes with proteinase K, Pronase and pepsin (100 to 200 micrograms/ml of enzyme) but not by trypsin or bromelain treatment. Periodate oxidation as well as mixed and endoglycosidase treatment of the spirochetes did not alter the binding of mAb-11G1. Two-dimensional gel electrophoresis of whole spirochetal cell lysates disclosed that OSP-A is a heterogeneously charged basic protein with an apparent isoelectric point range from 8.5 to 9.0. Amino acid analysis of OSP-A showed a 10% lysine component which could provide the basic nature to the protein. OSP-A with the intact antigenic determinant for mAb-11G1 can be found in the urine of hamsters experimentally infected with B. burgdorferi.     

Schistosoma mansoni: detection and characterization of antigens and antigenemia by inhibition enzyme-linked immunosorbent assay (IELISA)

An inhibition enzyme-linked immunosorbent assay (IELISA) was used to detect the presence of schistosome antigens obtained from cercariae, adult worms, and eggs of the parasite. Using appropriate titers of Schistosoma mansoni infected mouse serum (IMS), it was possible to detect less than 10 ng/ml of schistosome antigen when added to phosphate-buffered saline (PBS, pH 7.2) or normal human serum (NHS). The sensitivity of the test was highly contingent on the number of experimental variables including antibody titer and antigenic source. The results of specificity studies were complicated. Although there was no cross-reactivity detected with other unrelated antigen preparations, extensive cross-reactivity between various schistosome species and "stage-specific" antigens was observed. The IELISA, utilizing IMS, can quantitate the degree of antigenic cross-reactivity, i.e., genus-specific and cross-reacting antigenic determinants. Soluble egg antigen (SEA) preparations obtained from S. mansoni and S. japonicum actually "cross-reacted" more than cercarial- and egg-derived antigens obtained from the same species (S. mansoni). This test also showed a 32-fold increase in specificity for the quantitative detection of specific antigenic determinants when monoclonal antibodies were used to restrict the heterogeneity of the measured response. The technique proved satisfactory for the quantification of parasitic burden in mice and the detection of active infections in humans. Circulating antigen disappeared with a t 1/2 of 72-96 hr after successful treatment.     

A Model System for Convenient Fluorescent Labeling of Sugar Chain in Taka-amylase A

A convenient detection of sugar chains in Taka-amylase A (TAA) was done by using 40 μg of enzyme, where a decrease in the UV absorption of NaIO₄ during the periodate oxidation reaction was monitored. The periodate-oxidized sugar chain was labeled with a fluorescent reagent, N-1-ethylenediaminonaphthalene (EDAN), by incubation at pH 9.5 and 30°C for 1 h. The excess EDAN was removed by either quenching with o-phthaladehyde or Bio-Gel P-2 gel adsorption. Among the peptide fragments prepared from the EDAN-labeled TAA, a fluorescent peptide corresponding to the sugar chain was distinguished by the ODS column. These results suggest that periodate oxidation and subsequent fluorescent labeling were useful for the sensitive analysis of various glycoprotein samples.     

日本血吸虫凋亡基因Sjcaspase3的克隆、真核表达 及其功能分析

为深入研究日本血吸虫细胞凋亡机制。利用PCR技术扩增得到Sjcaspase3的全长序列,其ORF含900 bp,编码299个氨基酸,理论分子量为33 509.7 Da,理论等电点为6.39。Real-time PCR分析表明该基因在日本血吸虫生长发育的各个时期均有表达,其中21 d表达量最高,42 d雌虫表达量高于42 d雄虫。成功构建了p XJ40-FLAG-Sjcaspase3重组质粒并转染到Hela细胞内,荧光定量PCR和Western blotting分析表明Sjcaspase3成功在Hela细胞中表达。酶活分析提示重组Sjcaspase3具有切割特异性底物天冬氨酸-谷氨酸-缬氨酸-天冬氨酸(DEVD)的活性。流式细胞术检测了Sjcaspase3可诱导Hela细胞发生早期细胞凋亡。研究结果为深入探讨Sjcaspase3的生物学功能及日本血吸虫细胞凋亡机制奠定了基础。     

Isolation and partial characterization of an eosinophil chemotactic factor from metacestodes of Taenia taeniaeformis (ECF-Tt)

Eosinophil chemotactic activity associated with protein extracts of Taenia taeniaeformis metacestodes was investigated. Chemotactic activity was associated with the nonbound protein after QAE cellulose chromatography of a 3 M KCl extract of homogenized larvae. When this material was precipitated with ammonium sulfate, activity was present in the 40 to 80% precipitate. Upon rechromatography on QAE cellulose equilibrated in a low ionic strength buffer, eosinophil chemotactic activity was retained by the gel and eluted after application of the NaCl gradient. Gel filtration of Sephacryl S-300 yielded an estimated m.w. of 91,000. Chromatofocusing revealed a broad peak of activity with a pI of 4.5 to 5.0. SDS-PAGE showed the active fraction migrated as a protein with a m.w. of 10,400. ECF-Tt had chemotactic and chemokinetic activity for equine eosinophils and murine eosinophils, but not for equine and murine neutrophils.