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.     

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.     

An electron microscope study of the tegument of Hunterella nodulosa Mackiewicz and McCrae, 1962 (cestoidea: Caryophyllidea)

Ultrastructural observations using transmission and scanning electron microscopy reveal the tegument to be basically similar to that of other cestodes. The syncytial distal cytoplasm is devoid of organelles except for rod-shaped bodies, believed to be secretory vesicles, and lamellated bodies which probably contribute the raw material for new microtriches. There is evidence that these vesicles originate from the Golgi found in the sub-cuticular cells.Three types of microtriches are described: typical ones with well-developed spines, ones with short filaments instead of spines, and ones with no spines. Microtriches with spines are found only on the anterior part of the worm and may serve to anchor the worm. Microtriches on the posterior have no spines and are believed to be primarily involved in the absorption of nutrients. Between these two regions there is a transitional zone where all three types of microtriches can be found. In general the microtriches are quite uniformly distributed throughout the surface of the worm. The presence of cestodarian-like microtriches raises interesting evolutionary questions.Histochemical tests localized acid and alkaline phosphatase activity on various parts of the tegument, as well as on host intestine, while acian blue tests showed that acid mucopolysaccharide levels correspond with the concentration of the tegument vesicles.     

Synthesis of macromolecules by the epithelial surfaces of Schistosoma mansoni: an autoradiographic study.

The use of tritiated leucine as a marker for protein synthesis and of tritiated glucosamine as a marker for polysaccharide/glycoprotein synthesis, is described. Adult worms were pulse-labelled by incubation in medium containing the substrate. Labelled worms were then incubated in chase medium, without labelled substrate, for varying lengths of time before fixation. The distribution of label which had been incorporated into macromolecules in the worm tissues, was examined by light and electron microscope autoradiography. It was estimated that the tegument and tegument cell bodies were the source of 67--80%, and the gut epithelium of 20--30%, of exportable leucine-containing protein. Conversely, the gut epithelium was the source of 72%, and the tegument cells 28%, of exportable glucosamine-containing polysaccharide. The specific activity of labelled protein reached a peak in the tegument cytoplasm after 1.5 h of chase incubation. Half of the labelled protein was secreted into the worm's environment by 3 h of chase incubation. The half-life of secretory protein in gut cells appears to be around 2 h. Labelled protein disappears from the gut lumen relatively rapidly but labelled polysaccharide remains in the lumen at high specific activity for at least 24 h. The major carbohydrate labelled may be the glycocalyx on the luminal surface of the gut epithelial cells. The results suggest that the bulk of worm secretions have a rapid turnover with a half-life of a few hours. Against this background of rapid mass secretion a slower process of membrane turnover would be difficult to detect and quantitatively small.     

Hymenolepis diminuta: ultrastructural observations on complement-mediated tegumental lysis and destrobilation of 4-day-old worms in vitro

Changes in the ultrastructure of the tegument and subtegumental cells of 4-day-old Hymenolepis diminuta were studied in vitro in 50% fresh normal rat serum over a 5-h period and compared with heat-inactivated serum and saline controls. First, membrane-bound vesicles accumulate above the microthrix-border. After 30–40 min large vacuoles, which may contain membranous elements, appear in the tegument at a time when the surface of the young strobila is virtually denuded of the microthrix-border. With prolonged incubations there are subtegumental secretory inclusions with dark, enveloping cytoplasm in the tegument and finally the apical plasma membrane, together with the majority of the matrix, is lost. The disrupted portion of the worm is abruptly demarcated from the comparatively intact scolex/anterior neck region by a constriction. Even after 5 h incubation there is no evidence of loss of tegumental matrix components from regions anterior to the constriction but the neck region shows a significant denudation of the microthrix layer and the tegument contains numerous inclusions. The scolex tegument only showed little evidence of loss of membrane from the surface. Possible mechanisms for the avoidance of complement-mediated lysis in the anterior region are discussed.     

Exposed proteins of the Schistosoma japonicum tegument

The ability of the mammalian blood fluke Schistosoma japonicum to survive in the inhospitable environment of the mammalian bloodstream can be attributed, at least in part, to its host-exposed outer surface, called the tegument. The tegument is a dynamic organ and is involved in nutrition, immune evasion and modulation, excretion, osmoregulation and signal transduction. Given its importance for parasite survival, proteins exposed to the host at the surface of the tegument are ideal targets for the development of vaccines and drugs. By biotinylating live adult worms and using a combination of OFFGEL electrophoresis and tandem mass spectrometry 54 proteins were identified as putatively host-exposed in S. japonicum. These included glucose transport proteins, an amino permease, a leucine aminopeptidase and a range of transporters, heat shock proteins and novel immune-active proteins. Members of the tetraspanin protein family and a homologue of Sm 29, a tegument membrane protein from Schistosoma mansoni, both effective vaccine antigens in S. mansoni, were also identified. The fate of labelled surface proteins was monitored over time using electron microscopy and revealed that biotinylated proteins were rapidly internalised from the surface of the tegument and trafficked into the cytoplasmic bridges that connect the distal cytoplasm of the tegument to the underlying cell bodies. The results reported herein dramatically increase the number of S. japonicum proteins known to be exposed to the host and, hence, those of interest as therapeutic targets. The ability of the parasite to rapidly internalise proteins at its surface has implications for the development of vaccines and may explain how these parasites are able to avoid the host immune system for long periods of time.     

Schistosoma mansoni: antigen preparations which induce antibodies to schistosomula surface antigens

To determine the easiest method of raising antibodies to antigens exposed on the surface of schistosomula of Schistosoma mansoni, several crude preparations of the parasite were used to immunize mice. Schistosomula released products, whole worm homogenate, and parasite eggs all raised antibodies which bound to the surface of live schistosomula, although the anti-egg antiserum did so less strongly. Anti-schistosomula released products antiserum recognized three schistosomula surface antigens of Mr 15,000, 20,000, and 32,000, anti-whole worm homogenate recognized 20,000, 32,000, and 38,000 Mr surface antigens, and anti-egg recognized a less than 200,000 Mr surface antigen. None of these antigens was recognized when the labeled preparation was immunoprecipitated with its homologous antiserum. When these antisera were used to immunoprecipitate cell free translation products of adult worm RNA, the antischistosomula released products and anti-whole worm homogenate recognized an 11,000 Mr doublet while the anti-egg precipitated 14,000 and 44,000 Mr antigens. Other crude preparations were used to immunize rabbits; Formalin-fixed schistosomula, denuded adult worms, and purified worm tegument all induced antibodies which recognized the 20,000, 32,000, and 38,000 Mr schistosomula surface antigens.     

The formation and turnover of the membranocalyx on the tegument of Schistosoma mansoni.

The multilaminate vesicles present in the tegument cytoplasm appear to fuse with side channels projecting out into the cytoplasm from the base of the surface pits. Their lamellate contents then unroll and spread out to form a trilaminate membranocalyx lining the pits and covering the tegument surface. The plant lectin concanavalin A appears to stabilize the process of vesicle fusion leading to an aggregation of multilaminate vesicles trapped in the lumen of the surface pits. The membranocalyx can be labelled with cationized ferritin. Chase incubations in normal medium indicate that by 4 h most of the label and the membranocalyx to which it is bound have been lost to the medium. This suggests that under the conditions of these experiments the membranocalyx has a half-life of 2-3 h.     

Reproductive development of female Schistosoma mansoni (Digenea: Schistosomatidae) following bisexual pairing of worms and worm segments

Maturation and maintenance of normal reproductive function in female Schistosoma mansoni require a permanent association with the male, but the nature of this relationship is not well understood. The regional localization of a stimulatory factor in the male and its target in the female were investigated. Unisexual female and mature male worms were transected into segments of various lengths. Various combinations of transected male and female segments and intact worms were transferred to the mesenteric veins of recipient hamsters and were also maintained in vitro. In hamsters and in vitro, pairing took place between intact worms of each sex and segments of the other, and between segments of both sexes. The majority of female worms and segments so paired showed some reproductive development, as assessed by vitelline gland differentiation. In intact unisexual females paired with small male segments, vitelline gland development was limited to that portion of the worm that had been held by the male. Worm segments continued to display normal body contractions throughout 24 days of in vitro maintenance and morphological integrity was retained. It is concluded that 1) in the absence of a functioning gut, worm segments can survive for prolonged periods on nutrients absorbed through the tegument; 2) worm pairing, male stimulation, and the female developmental response are independent of central nervous control by the cerebral ganglia; 3) males have no centralized localization for the female-stimulating factor; 4) vitelline gland differentiation in the female requires local stimulation through male contact, and this is not propagated throughout the worm.     

Formation of the plasma membrane during regeneration ofParamecium caudatum

Summary Fragments ofParamecium caudatum cells obtained by merotomy were fixed in 1% OsO₄ within 5 seconds after cutting. The ultrastructure of the damaged area of the fragment was studied in oriented ultrathin sections and by scanning electron microscopy. The cytoplasm exposed by merotomy was covered during a few seconds with a new membrane. This was a typical trilaminar membrane continuous with the plasma membrane covering the undamaged surface of the cell. The surface over the wound was wrinkled into irregular grooves and ridges. The cytoplasm, mitochondria and trichocysts in the injured region were electron translucent. The cytoplasm under the new membrane contained an unusually high amount of small membrane vesicles, 20–90 nm in diameter. These were probably the remnants of subpellicular alveoli and the plasma membrane destroyed by microsectioning. The possibility that the exposed cytoplasm would be covered by mere shifting of the existing plasma membrane can be excluded. The complex structure of the cortex with its subpellicular alveoli and regularly distributed cilia provide a strong argument against this notion. It seems probable that the new membrane was built up from the available molecular material,e.g., phospholipids and proteins present in the cytoplasm. Fragments of the membrane and alveolar membranes in the form of small vesicles may have also been included into the new membrane.     

鱊头槽绦虫原尾蚴皮层的超微结构观察

利用透射电镜观察了头槽绦虫 (Bothriocephalusacheilognathi)原尾蚴皮层的超微结构。头槽绦虫原尾蚴的皮层为典型的合胞体结构。皮层表面有浓密的微毛与少量的突起。与成虫相比 ,原尾蚴的微毛长而且粗 ,呈现单态性 ,推测在进入终末宿主的过程中有脱落和再生现象。突起分布在头部与体侧 ,含有较少的电子致密颗粒。在原尾蚴皮层细胞质中观察到三种腺细胞的分泌过程 ,即顶分泌、外分泌和微分泌过程。原尾蚴身体后部观察到尾钩和穿刺腺管道的开口。在核周区和纤维层下可见发育较好的环肌与纵肌。本文还对原尾蚴皮层突起的功能进行了探讨。     

扩张莫尼茨绦虫(圆叶目:裸头科)节间腺的超微结构及组织化学

用光学显微镜和透射电子显微镜观察了扩张莫尼茨绦虫节间腺形成过程的精细结构及一些组化变化。结果表明：节间腺是扩张莫尼茨绦虫皮层的特化部分,由节片后缘的皮层及其邻近细胞体向绦虫实质组织中陷入开始其形成过程,随着虫体发育的进行,新的陷入不断形成,原陷入的部分不断脱离皮层形成簇状腺体结构。节间腺的数目随着体节的发育不断增加,幼节中仅有少数几个(6～9个),而远端的孕节中多于100个。电镜下可见腺细胞体由细胞质管与腺皮层相联,簇状腺体结构为一合胞体形态,腺细胞体围绕并开口于椭球体或不规则形状的皮层腔中。离腺皮层远的腺细胞体电子密度高并含有与腺皮层相应的典型分泌颗粒,而靠近腺皮层的腺细胞体电子密度低,所含分泌颗粒较少。扩张莫尼茨绦虫节间腺的组化性质尚不完全清楚。糖与蛋白质等组化结果不稳定,随染液pH值及染色时间的变化等多种因素而改变。基于我们的研究及其他研究者的观察表明,节间腺可能参与外源基质形成虫卵的转运,同时他们可能在虫体节片脱落及虫卵溢出时起作用。     

Cyton II: A subtegumental cell type in the cercaria of Schistosoma mansoni

In Schistosoma mansoni cercaria, an aggregate of subtegumental cells is found in a small, dorsoanterior area of the body (middivision). These cells are nestled between two laterally positioned flame cells and the muscle that delimits the anterior end of the body, and the anterior end of the central ganglion. This highly amorphous cell type, designated as cyton II, has a heterochromatic nucleus and a cytoplasm that is elaborated into coarse, tortuous processes. Its cytoplasm contains ribosomes, mitochondria, sparse amounts of endoplasmic reticulum, and two types of circular-to-oval concentric membranous bodies. One type has an electron-dense core and measures 200–250 nm on the short axis, and the other is completely membranous and measures 100–125 nm on the short axis. The cell body of cyton II communicates with the tegument that covers a small, dorsoposterior area of the anterior organ (oral sucker); however, we could not confirm a tegumental connection with the body division. When cercariae transform into schistosomules, the concentric membranous bodies of cyton II migrate into the anterior organ's tegument via cytoplasmic processes of the cell. The major function of previously described cells that have similar membranous bodies is to supply additional membranes to the outer tegument during development into an adult worm. A multilaminated outer membrane is an adaptation to the survival of the schistosomule and adult worm in the bloodstream of the vertebrate host (Hockley amd McLaren [′73]). The presence of membranous bodies from cyton II in the tegument does not confirm that this cell type participates in the formation of multilaminated membranes. Its precise function remains to be determined. © 1995 Wiley-Liss, Inc.     

Early post-larval development of the endoparasitic platyhelminth Mesocestoides corti: trypsin provokes reversible tegumental damage leading to serum-induced cell proliferation and growth

Mesocestoides corti is a suitable in vitro model for studying the development of human endoparasitic platyhelminthes. Treatment with trypsin, supplemented with fetal bovine serum (FBS), induces M. corti development from larvae (tetrathyridia) to segmented adult worm; however, the role of this protease and of FBS in post-larval development induction remains unknown. To characterize the participation of trypsin enzymatic activity and of FBS in the induction of tetrathyridia growth and development, both stimuli were added to the larvae either together or sequentially. Additionally, specific inhibition of trypsin activity was also monitored. Finally, the effect of the enzyme on the parasite tegument as well as the proliferative activity and location of proliferating cells after induction of tetrathyridia development were also studied. We conclude that trypsin-induced tetrathyridia development to adult worm is FBS-dependent and that the effect of serum factors is dependent upon a previous trypsin-induced reversible damage to the larva tegument. In dividing and non-dividing tetrathyridia, proliferative activity of cells is mainly located within the apical massif in the anterior region and nerve cords of larvae, respectively. In tetrathyridia stimulated to develop to adult worms, an intense proliferative activity is evident along the nerve cords. Our results suggest that in natural infections the tetrathyridia tegument is temporally made permeable to growth factors by proteolytic enzyme activity in the intestine juice of the definitive host, thus leading to development to adult worms.     

Interferon-gamma inhibits T84 epithelial cell migration by redirecting transcytosis of beta1 integrin from the migrating leading edge

Intestinal inflammation is associated with epithelial damage and formation of mucosal wounds. Epithelial cells migration is required for wound closure. In inflammatory status, migrating epithelial cells are exposed to proinflammatory cytokines such as IFN-gamma. However, influence of such cytokines on intestinal epithelial wound closure remains unknown. The present study was designed to investigate the effect of IFN-gamma on migration of model T84 intestinal epithelial cells and recovery of epithelial wounds. IFN-gamma significantly inhibited rate of T84 cell migration and closure of epithelial wounds. This effect was accompanied by the formation of large aberrant lamellipodia at the leading edge as well as significant decrease in the number of beta(1) integrin containing focal adhesions. IFN-gamma exposure increased endocytosis of beta(1) integrin and shifted its accumulation from early/recycling endosomes at the leading edge to a yet unidentified compartment at the cell base. This redirection in beta(1) integrin transcytosis was inhibited by depolymerization of microtubules with nocodazole and was unaffected by stabilization of microtubules with docetaxel. These results suggest that IFN-gamma attenuates epithelial wound closure by microtubule-dependent redirection of beta(1) integrin transcytosis from the leading edge of migrating cells thereby inhibiting adequate turnover of focal adhesion complexes and cell migration.     

Immunolocalization of Taenia solium gap junction innexins

In previous studies, ultrastructural observations revealed a large number of gap junctions (GJs) in the neck and immature proglottid tissues of Taenia solium tapeworms. In these helminths, cytoplasmic glycogen sacs are connected by numerous discrete GJs to other cells throughout the maturing strobilar tissue. Discontinuous sucrose gradients were used to purify membrane fractions containing GJs, which were identified by ultrastructural analysis. A trans-membrane peptide sequence from a highly conserved innexin region was used to construct a 20-amino acid synthetic peptide and used to raise polyclonal antibodies in rabbits that recognized both a 55 and a 67 kDa protein in a Western blot of the GJ-enriched pellet. Immunohistochemistry of larval and adult worm sections incubated with antiserum to the synthetic peptide and a secondary anti-rabbit IgG bound to fluorescein, revealed strong binding to the tegumentary surface of the worm, as well as patchy fluorescent areas in the parenchyma. The results indicate that both the tegument of cysticerci and adult T. solium contain innexin-rich membranes, which may function as a tegumentary transport system for small molecules.     

Ultrastructural studies on migrating epidermal cells during the wound healing stage of regeneration in the adult newt, Notophthalmus viridescens

The ultrastructure of the epidermal cells which migrate over the wound surface of the amputated limb of the adult newt, Notophthalmus viridescens, was observed with transmission (TEM) and scanning (SEM) electron microscopy. In order to aid in the visualization of polyanionic surface materials on the wound epithelium and wound surface with TEM, the basic dye, ruthenium red, was introduced into the fixatives and buffer. Control limbs were processed without ruthenium red. Shortly after amputation, basal cells at the wound margin possessed elongated, flattened profiles with long pseudopodial projections (lamellipodia and filopodia) that appeared to make contact with the fibrin exudate covering the stump tissues. Epidermal cells proximal to the site of amputation were also in a state of mobilization. Large intercellular spaces and a reduction in the number of desmosomes were observed in the migrating cells. Epidermal cell nuclei became characteristically euchromatic with well-developed nucleoli. Microfilaments were seen within the cytoplasm, extending toward the plasma membrane of cellular processes. Phagocytosed material was also present in the migrating cells. By approximately 9 hours post-amputation, wound closure was complete, and the wound epithelium consisted of three to four cell layers of a non-cornified epidermis. Generally, the amount of extracellular material present on the surface and in the enlarged intercellular spaces of migrating epidermal cells remained the same throughout the period of wound closure. A layer of polyanionic material was observed consistently over the fibrin meshwork covering the wound surface with TEM.     

Antigenic localities in the tissues of Metagonimus yokogawai in the period of growth]

In order to observe the antigenic localization in the tissues of Metagonimus yokogawai in growth stages, immunogoldlabeling method was applied to using serum of the cat which infected with isolated metacercariae from Plecoglossus altivelis. The sectioned worm tissues from each growth stages 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 worm tissues of all experimental groups, the gold particles were specifically concentrated on the tegumental syncytium and cytoplasm of the tegumental cell as well as the secretory granules in the parenchymal tissue. In the 16th and 20th week grown worm tissues, the gold particles were specifically concentrated on the vesicles in the tegumental syncytium and cytoplasm of the tegumental cell. The gold particles were specifically concentrated on the caecal epithelia of the 4th, 8th and 12th week growth groups but slightly concentrated on those of the 16th and 20th week.