Antigenic localities in the tissues of Paragonimus westermani by developmental stages using immunogold labeling method]

In order to observe the antigenic localization in the tissues of Paragonimus westermani of developmental stages, immunogold labeling method was applied using serum of the cats which were infected with isolated metacercariae from Cambaroides similis. The sectioned worm tissues from each developmental stage were embedded in Lowicryl HM 20 medium, stained with infected serum IgG and protein A gold complex (particle size: 12 nm) and observed by electron microscopy. In the young adult worm tissue of 4 weeks after infection with metacercariae, the gold particles were specifically concentrated on the tegumental syncytium and cytoplasm of the tegumental cells as well as the secretory granules in the parenchymal tissue. The antigenic materials in the adult worm tissue were specifically concentrated on the secretory granules in the parenchymal tissue, the cytoplasm between granules in the vitelline gland and the epithelial lamella in the lumen of the caecum.     

Antigenic localities in the tissues of Metagonimus yokogawai observed by immunogoldlabeling method]

In order to determine the antigenic localization in the tissues of the adult Metagonimus yokogawai, immunogoldlabeling method was applied using serum immunoglobulins(IgG) of cats which were infected with isolated metacercariae from Plecoglossus altivelis. The sectioned worm tissue was embedded in Lowicryl HM 20 medium and stained with infected serum IgG and protein A gold complex(particle size: 12 nm). It was observed by electron microscopy at each tissue of the worm. The gold particles were observed on the tegumental syncytium as well as cytoplasm of tegumental cells and epithelial lamella of the caecum. The gold particles were not observed on the basal lamina of the tegument, interstitial matrix of the parenchyma, the muscle tissue and mitochondria of the tegument. The gold particles were specifically labeled in the secretory granules in the vitelline cells. They were also labeled on the lumen of bladder and egg shell. The above findings showed that antigenic materials in the tissue of adult worms were specifically concentrated on the tegumental syncytium as well as cytoplasm of tegumental cells and epithelial lamella of the caecum.     

Antigenic localities in the tissues of the young adult worm of Paragonimus westermani using immunogold labeling method

In order to observe the antigenic localization in the tissues of the young adult Paragonimus westermani, immunogold labeling method was applied using serum immunoglobulins(IgG) of the dog which infected with isolated metacercariae from Cambaroides similis. The sectioned worm tissue was embedded in Lowicryl HM 20 medium and stained with infected serum IgG and protein A gold complex (particle size; 12 nm). It was observed by electron microscopy at each tissues of the worm. The gold particles were not observed on the basal lamina of the tegument, interstitial matrix of the parenchyma, the muscle tissue and mitochondria of the tegument. The gold particles were specifically labeled in the secretory granules in the vitelline cells. They were predominantly labeling on the epithelial lamela and lumen of caecum. The above finding showed that antigenic materials in young adult worm tissue were specifically concentrated on the tegumental syncytium as well as cytoplasm of tegumental cells.     

Immunological damage to Hymenolepis diminuta following a challenge infection in C57 mice

Immunological damage to Hymenolepis diminuta following a challenge infection in C57 mice. International journal for Parasitology17: 795–803. Ultra-structural observations were made on the scpleces of Hymenolepis diminuta from 10-worm 2° infections in C57 mice to determine whether the immune stress causing reduced establishment, stunting of growth and early rejection was accompanied by significant damage to the worm fine structure. In almost all of the worms recovered there was extensive damage to tissues throughout the scolex, even in those examined 24 h post-secondary-infection (p-2°-i). Many of the muscle, tegumental and parenchyma cells were completely disrupted and dispersed, leaving only isolated strands of cytoplasm and cell debris. A number of excretory ducts were breached, and large and apparently empty spaces were evident throughout the scolex tissues. There was an increase in lipid accumulation in the tegumental syncytium, underlying cytons and in the suckers. Extensive vacuolation also occurred in the sucker tegument. Although some scoleces showed certain regions to be apparently undamaged, close examination revealed a fine structure consistent with enhanced metabolic activity. The structural abnormalities observed are discussed in relation to host immunity.     

Fasciola hepatica: Glycocalyx replacement in the juvenile as a possible mechanism for protection against host immunity

The response of newly excysted juvenile Fasciola hepatica to immune sheep serum under in vitro conditions was examined using indirect fluorescent antibody labeling and electron microscopy. Flukes acquired a continuous layer of host IgG over the surface during incubation in the presence of antiserum, but when transferred to a medium lacking antiserum they actively sloughed this layer and replaced the former glycocalyx, by a new antigenically similar surface coat. Electron microscope examination of juvenile flukes verified than an immune complex formed at and sloughed from the tegumental surface of those which were incubated in immune serum. T0 secretory bodies produced by the GER/Golgi system of the tegumental cells and stored in the metacercariae were discharged at the apical surface of the tegument, possibly in response to antibody binding. When cycloheximide was included with immune serum in the incubation medium the tegumental cells were unable to synthesize new T0 bodies to replace losses and the number of T0 bodies decreased so that the cytoplasm of the tegumental cells and surface syncytium became virtually devoid of T0 bodies within 48 hr.     

A study on the body fluid antigen of Clonorchis sinensis using immunogold labeling method

In order to observe the antigenic localization in the tissues of the adult Clonorchis sinensis, immunogold labeling method was applied using serum immunoglobulins (IgG) of either worm-infected rabbits (group I) or antigen-immunized rabbits (group II) (by the body fluid obtained from the adult worms). The electron micrographs of the sectioned worm tissue antigens, embedded in Lowicryl HM 20 medium and stained with protein A-gold complex (particle size: 12 nm), were compared between the group I and group II. The gold particles were observed in the interstitial matrix of the worm parenchyma, the epithelial lamellae of the cecum, and the cecal lumen both in group I and II. But the particles were in general more concentrated in group II. The gold particles were not observed on the basal lamina of the tegument or on vitelline glands in group I, while they were highly concentrated on those areas in group II. There were also differences in the antigenicity of interstitial matrix(reacted with group I IgG) and head part(reacted with group II IgG) of the sperm cells in the seminal receptacle. Conclusively, it is suggested that the substances comprising the basal lamina of the tegument or vitelline glands act as specific antigens reacting with antigen(body fluid) immunized rabbit IgG. On the other hand, the substances in the cecal lumen and cecal epithelial lamellae are thought to be the specific antigen that react with the worm-infected rabbit IgG.     

Wound healing in the trematode Schistosoma

The ability of adult Schistosoma mansoni to effect wound healing over an exposed surface has been demonstrated. In transected worm segments a new external plasma membrane formed over the exposed tegumental cytoplasm. An elevated leading edge of tegument developed around the margin of the wound; the surface of this region was highly convoluted and there was a proliferation of membranous bodies within its cytoplasm. Inward migration of the leading edge over the exposed internal tissues took place. The resulting new tegument lacked spines and sensory endings. There was no regeneration of basal lamina or tegumentary cytons. In vitro maintenance of worm segments for 3 weeks did not give rise to any major ultrastructural changes in the tissues away from the wound.     

Fasciola hepatica: development of monoclonal antibodies against somatic antigens and their characterization by ultrastructural localization of antibody binding

A series of monoclonal antibodies was prepared against tegumental and internal antigens of Fasciola hepatica by immunizing mice with whole adult-fluke homogenates prior to harvesting the splenic lymphocytes for fusion. Preliminary screening by the Indirect Fluorescent Antibody technique indicated the occurrence of discrete groups of monoclonals differing from one another in tissue-specificity but within which IFA labelling patterns were fairly consistent. Representative hybridomas for 5 of these groups were stabilized and used to produce ascites fluid in mice. By application of an immunogold labelling technique it was possible to map the distribution of antigens for which each monoclonal antibody had affinity throughout the tissues of 4-week and 12-week flukes. Several monoclonals specifically labelled antigenic determinants on the important tegumental antigen T1. However the distribution of gold colloid labelling suggested that epitopes other than that normally exposed to the infected host were recognized; and several monoclonals specifically attached to T1 antigen in the tegument of juvenile worms only. The glycocalyx of the gut and excretory system of flukes shared T1 antigenicity with the tegument. Monoclonal antibodies were produced against an internal immunogen associated with ribosomes and heterochromatin in active protein-producing cells, and against interstitial material of adult flukes. Monoclonals against antigens in parenchymal cell cytoplasm and in mature vitelline cells were recognized but the corresponding hybridomas were not stabilized.     

Fasciola gigantica: Immunolocalization of 28.5 kDa antigen in the tegument of metacercaria and juvenile fluke

Specific monoclonal antibody (MoAb) to 28.5 kDa tegumental antigen (TA) was used to localize this antigen in the tissues of metacercariae, newly excysted juvenile (NEJ), 1, 3, 5, and 7-week-old juveniles of Fasciola gigantica by using indirect immunofluorescence, immunoperoxidase and immunogold techniques. Both indirect immunofluorescence and immunoperoxidase detections showed that this antigen was concentrated in the tegument particularly in its outer rim, tegumental cells and their processes as well as epithelial linings of the oral sucker. Unlike adult F. gigantica, it was not detected in spermatogenic cells in the testes, cells of Mehlis’gland, oocytes within the ovary, and ovum within the egg of parasites. At the ultrastructural level, the immunogold labeling showed deposit of gold particles specifically in G₂ tegumental granules and on the surface membrane. Thus, this 28.5 kDa antigen is expressed in the tegument and associated structures of juvenile parasites, and it could be a major component of the G₂ granules which are shown to fuse with the surface membrane and contribute material to replace the casted-off membrane. This process is the replenishment and turnover of the surface membrane to prevent the attachment of the host immune effector cells.     

Ultrastructural observations on the redial tegument of Paramphistomum epiclitum from the planorbid snail, Indoplanorbis exustus.

The morphology of the tegument in the redia of Paramphistomum epiclitum (Digenea: Paramphistomidae) resembles that shown by most larval and adult digeneans; an outer surface syncytium is in continuity with the cytoplasm of in-sunken, nucleated cytons. Although tegumental cytons usually contain a single nucleus, some display up to six nuclei. The tegumental syncytium lining the pharynx of P. epiclitum rediae lack underlying cytons. The apical membrane of the tegument is elaborated by folds and microvilli, which presumably facilitate uptake of nutrients and/or exchange of ions involved in osmoregulation. A single type of secretory body, resulting from the fusion of smaller vesicles produced at Golgi complexes in the cytons, occurs throughout the tegument. Uniciliate sensory receptors occur in the surface syncytium particularly around the oral opening.     

Scanning electron microscopy of in vitro serum-mediated destruction of juvenile Fasciola hepatica

The tegumental surface of immature Fasciola hepatica was damaged when incubated in vitro with serum collected from an experimentally infected calf. Degeneration of the tegumental surface was observed by scanning electron microscopy (SEM) at 4 hr. after incubation. Decomposition was observed 8 to 12 hr after incubation and complete destruction of the tegument occurred by 16 hr. The flukes became inactive after 8 to 12 hr of incubation. None of the above findings were observed for the tegument of flukes incubated in tissue culture media or in media containing normal calf serum and the trematodes remained motile throughout the incubation period. Latex particles were used as an immunological marker for SEM studies to determine if gamma globulin could be responsible for the observed changes and, if so, the site of antibody attachment. The coated latex particles covered the entire surface of flukes recovered from mice 5 days after infection with metacercariae. In contrast, latex particles coated with either normal gamma globulin or gamma globulin from serum of the experimentally infected calf that had been adsorbed with disrupted adult flukes were not attached to the surface of the flukes. Absorption of the serum with disrupted, adult flukes decreased the concentrations of immunoglobulins (Ig)G1 and G2 whereas IgA and IgM were apparently not affected.     

Fasciola hepatica: Autoradiography of protein synthesis,transport, and secretion by the tegument

Transverse slices of Fasciola hepatica adults were incubated for up to 3 hr in the presence of [³H]leucine. The incorporation of this tracer molecule into macromolecules and its subsequent movement through the tegument was visualized by electron microscope autoradiography. It appeared that protein synthesis in the Type 1 tegumental cells occurred by a GER/Golgi-mediated mechanism similar to that in vertebrate tissues. Mature T1 secretory bodies entered the surface syncytium via the connecting tubules and accumulated in the basal cytoplasm prior to rapid transit to the surface and discharge at the apical plasma membrane. In adult flukes, which are partly protected from immune attack by virtue of their location, glycocalyx replacement and T1 synthesis may be retarded. The Type 1 cells also appear to manufacture proteins which are not membrane bound. These move with the T1 secretory bodies into the surface syncytium and may have structural or enzymic functions within the cytoplasm.     

Fasciola hepatica: development of tegument during migration in mouse.

T-0 granules of the type 0 tegumental cells of newly excysted juveniles appear in the syncytium of juveniles recovered from the abdominal cavity of mice 12 hr postinfection (p.i.). They undergo exocytosis and/or add their contents to the glycocalyx of the syncytial apical plasma membrane. While in the abdominal cavity the syncytial ground cytoplasm has an increased electron density. After arrival in the liver the type 0 cells metamorphose into type 1 cells of the adult and begin to synthesize T-1 granules. The type 2 cells of the adult arise by differentiation of embryonic cells in the parenchyma, 2–3 days p.i., and subsequently form protoplasmic tubular connections with the syncytium. On arrival in the bile ducts, 4 wk p.i., T-2 granules, formed in the type 2 cells, congregate in the apical cytoplasm of the thickened syncytium and the apical plasma membrane becomes much invaginated. The discussion correlates the development of the tegument with the changes in environment and a mechanism of spine growth is proposed.     

Ultrastructure and cytochemistry of the tegument of Orthocoelium scoliocoelium and Paramphistomum cervi (Trematoda: Digenea)

The tegument of Orthocoelium scoliocoelium and Paramphistomum cervi was examined using histochemical techniques and electron microscopy. On the basis of the distribution of acid and alkaline phosphatase (E.C. 3.1.3.2, E.C. 3.1.3.1), non-specific esterase (E.C. 3.1.1.1), cholinesterase (E.C. 3.1.1.7) and succinate dehydrogenase (E.C. 1.3.99.1) at light microscope level two distinct regions were recognized, an outer and an inner zone. Electron microscopy revealed that the tegument comprises an outer surface syncytium underlain by a thick subsyncytial zone and musculature. Deeper still occur the nucleated "tegumental cells". The latter are in cytoplasmic continuity with the surface syncytium via vacuolated cytoplasmic trabeculae which traverse the muscle layers and the subsyncytial zone. Three types of tegumental cells each lacking mitochondria were observed. The T1 cells synthesize discoid and electron dense T1 bodies while T2 cells produce oval and electron lucent T2 bodies. The third type of tegumental cells apparently produce no secretory bodies and may represent an embryonic cell type. The surface syncytium contains T1 and T2 secretory bodies and is bounded apically by a plasma membrane invested externally by a fuzzy and filamentous glycocalyx. The surface syncytium lacks mitochondria and is traversed by infoldings of the basal plasma membrane. Beneath the surface syncytium the subsyncytial zone is largely comprised of fibrous interstitial material. This zone, which is particularly thick in the amphistomes, is traversed by trabeculae and extensions of underlying parenchymal cells which usually contain mitochondria and lysosomes. The subsyncytial zone overlies numerous circular and longitudinal muscle fibres. The absence of mitochondria and enzymes associated with active transport suggests that the amphistome tegument may be mainly specialized for protection of the worm against mechanical and chemical conditions prevailing in the rumen. Active uptake of nutrients is probably not a primary function.     

利用人脐带血干细胞制备人鼠肝组织嵌合体模型

目的利用人脐带血干细胞研究制备HBV感染人鼠嵌合小鼠模型。方法将人脐血干细胞,经尾静脉分两次注射到裸鼠体内。分别于第7,14,21天取肝组织,进行AFP免疫组织化学检测,分析人肝细胞在裸鼠体内嵌合生长情况,并进行乙肝病毒感染实验,定量检测感染后小鼠血清中AFP、ALB。结果实验组小鼠在第7、14、21天肝脏内AFP持续阳性表达,AFP表达于细胞质内。HBV感染后小鼠肝脏HBsAg组化显示密集成片的阳性细胞。实验组和对照组表达差异显著（P〈0.05）。结论将人脐血干细胞移植裸鼠肝脏内可以存活并分化成人肝细胞形成嵌合鼠,并能被HBV感染。     

Ultrastructural localization of 28 kDa glutathione S-transferase in adult Clonorchis sinensis

Glutathione S-transferase (28GST) with molecular mass of 28 kDa is an antioxidant enzyme abundant in Clonorchis sinensis. In adult C. sinensis, 28GST was localized in tegumental syncytium, cytons, parenchyma, and sperm tails examined by immunoelectron microscopy. C. sinensis 28GST was earlier found to neutralize bioreactive compounds and to be rich in eggs. Accordingly, it is suggested that 28GST plays important roles in phase II defense system and physiological roles in worm fecundity of C. sinensis.     

Tegumental alterations in juvenile Schistosoma haematobium harboured in hamsters following artemether treatment

We report the findings of a detailed temporal study on tegumental alterations in juvenile Schistosoma haematobium, induced by artemether, using scanning electron microscopy. Hamsters infected with S. haematobium cercariae for 28 days were treated intragastrically with a single dose of 300 mg/kg artemether. Groups of two hamsters were killed 24 h, 72 h and 7 days after treatment, and schistosomula were recovered from livers by perfusion and subsequent systematic examination of the tissue, before routinely processing for scanning electron microscopic examination. Most schistosomula collected 24 h after artemether administration showed severe tegumental damage, usually including swelling, fusion, vesiculation, peeling and collapse of enlarged sensory structures. After 72 h, tegumental damage had increased and schistosomula generally showed contraction with extensive swelling, erosion and peeling of the tegument. Seven days post-treatment, severe tegumental damage was only seen in a single male specimen with swelling of the worm body and destruction of the oral sucker. The other schistosomula showed only light to moderate damage, suggesting that schistosomula surviving the treatment began to recover. Our findings of tegumental damage following artemether treatment correlate with the efficacy of this novel antischistosomal drug in killing the juvenile stages of S. haematobium and complement recent findings with S. japonicum and S. mansoni.     

Ultrastructure of the tegument of Metamicrocotyla macracantha (Alexander, 1954) Koratha, 1955 (Monogenea, Microcotylidae).

The ultrastructure of the body tegument of Metamicrocotyla macracantha (Alexander, 1954) Koratha, 1955, parasite of Mugil liza from Brazil, was studied by transmission electron microscopy. The body tegument is composed of an external syncytial layer, musculature, and an inner layer containing tegumental cells. The syncytium consists of a matrix containing three types of body inclusions and mitochondria. The musculature is constituted of several layers of longitudinal and circular muscle fibers. The tegumental cells present a well-developed nucleus, cytoplasm filled with ribosomes, rough endoplasmatic reticulum and mitochondria, and characteristic organelles of tegumental cells.     

Distribution of glycine receptors at central synapses: an immunoelectron microscopy study

The distribution of receptors for a neurotransmitter was investigated cytochemically for the first time in the central nervous system, at synapses established on cells of the ventral horn of the rat cervical spinal cord. Three monoclonal antibodies (mAb's) raised against glycine receptors were used. Immunofluorescent staining already showed discontinuous labeling at the surface of neurons, and immunoenzymatic electron microscopy further revealed that the antigenic determinants were confined to the postsynaptic membrane and concentrated at the level of the synaptic complex. More specifically, one mAb directed against the receptive subunit of the oligomeric receptor recognized an epitope on the extracellular side of the plasma membrane, whereas two other mAb's bound to the cytoplasmic face. Epitopes for the last two mAb's were more accurately localized with protein A-colloidal gold, using an intermediate rabbit anti-mouse immunoglobulin serum. (a) In addition to the presence of gold particles in areas facing the presynaptic active zone (visualized with ethanolic phosphotungstic acid), the labeling extended beyond this zone for approximately 50-60 nm, which corresponds to the width of one presynaptic dense projection. (b) The distances between the mid membrane and the gold particles were different for the two mAb's (with means of 21.7 +/- 8.5 nm and 29.8 +/- 10.4 nm, respectively). The data suggest that one of the recognized epitopes is close to the plasma membrane, whereas the second protrudes into the cytoplasm. Our results indicate that the receptor is a transmembrane protein which has a restricted spatial distribution on the postsynaptic neuronal surface.