Schistosoma mansoni: vaccination of mice with cryopreserved irradiated schistosomules.

In our earlier experiments, NIH/Nmri (CV) mice developed protective immunity to a Schistosoma mansoni cercarial challenge when previously exposed percutaneously to highly ⁶⁰Co-irradiated homologous cercariae. Experiments reported here were conducted to assess the immunogenicity of unfrozen and frozen and thawed schistosomules derived from ⁶⁰Co-irradiated cercariae (irradiated schistosomules). Immunization of NIH/Nmri (CV) mice by ⁶⁰Co-irradiated unfrozen schistosomules reduced worm burdens from a subsequent percutaneous challenge with normal cercariae by 41 to 72%. Immunogenicity was not narrowly dependent on irradiation dose rates between 1 and 8 kR/min, or on the total dose of irradiation given the schistosomules between 25 and 50 kR. Comparable protective immunity developed after injection of irradiated schistosomules which had been frozen to ?196 C in liquid nitrogen and thawed. Cryopreservation appears to offer a solution to the problem of storage of attenuated, immunogenic S. mansoni schistosomules.     

Schistosoma mansoni: cryopreservation of schistosomules.

Conditions were established for recovery of active schistosomules of Schistosoma mansoni after cryopreservation and storage in liquid nitrogen (?196 C). Schistosomules prepared from cercariae by a shear pressure technique were subjected to a two-step cooling process consisting of a slow cooling rate to an intermediate temperature, followed by rapid quenching of the sample in liquid nitrogen. Overall averages of 39 and 44% of the schistosomules, with a maximum of 88%, were recovered retaining normal activity with cooling rates of 0.4 C/min to ?32 C or 0.8 C/min to ?35 C, respectively. Methanol at 17.5% in Earle's lactalbumin hydrolysate was the freezing medium. As compared with 24 hr storage in liquid nitrogen, no loss in schistosomule motility was observed after 1 month. Following cryopreservation, attenuated schistosomules derived from ⁶⁰Co-irradiated cercariae (50 kR) exhibited structure and activity equivalent to that of unattenuated schistosomules. Infectivity for mice of unattenuated schistosomules derived from ⁶⁰Co-irradiated cercariae (50 Krad) exhibited structure and activity of unfrozen schistosomules ranged from 0.4 to 15.2%.     

Schistosoma mansoni: defined system for stepwise transformation of cercaria to schistosomule in vitro

Defined conditions are described for the in vitro production of large numbers of tail-free viable schistosomules. These consist of (1) the centrifugation of cold cercarial suspension and the incubation of the packed cercariae in a minimal volume of medium at 30 C for 40 min to effect tail loss and glandular secretion; (2) the isolation of the bodies by resuspension and sedimentation and (3) the induction of surface changes by incubating the bodies in inactivated serum or a defined tissue culture medium for a further 40 min interval at 37 C with mild agitation.The resultant schistosomules are characterized by the depletion of their penetration gland contents, loss of tail, fluoride and water sensitivities, complement insensitivity, negative “Cercarien-hüllen Reaktion,” and loss of the surface coat as demonstrated by periodic acid-Schiff (PAS) staining and electron microscope observations.     

日本血吸虫尾蚴人工方法转变的童虫超微结构的观察

本文报道日本血吸虫尾蚴经注射器推压和血清孵育两种人工方法转变的童虫与载体皮肤型童虫的透射及扫描电镜的观察结果。描述了三种童虫在转变后3小时至12小时其糖膜、外质膜、体被内包含体及腺体的超微结构的变化。     

The ultrastructure of the tegument and the digestive caeca of in vitro cultured metacercariae of Fasciola hepatica

Davies C. 1978. The ultrastructure of the tegument and digestive caeca of in vitro cultured metacercariae of Fasciola hepatica. International Journal for Parasitology8: 197–206. The ultrastructure of the tegument and digestive caeca of metacercariae of Fasciola hepatica grown in vitro in two different media is described and compared with the development of these two systems during maturation in vivo. Although the tegument of metacercariae grown in Medium RC showed no development, that of flukes cultured in Medium CS began to produce T-1 and T-2 granules typical of the liver phase of development in vivo. The gastrodermal cells showed some degree of conversion to an adult-like morphology in vitro with the production of typical secretory granules, a limited amount of orientation of the GER and the development of junctional complexes with adjacent parenchyma cells—this was particularly evident in flukes from Medium CS. The growth achieved in each of the culture media is correlated to the degree of development of the tegument and the digestive caeca.     

Schistosoma mansoni: vaccination of mice with 10-krad-irradiated, cryopreserved schistosomules

Protection against a Schistosoma mansoni cercarial challenge was evaluated in mice immunized with a vaccine composed of 10-krad-irradiated, cryopreserved schistosomules. The level of resistance induced in C57B1/6 or NMRI (CV) mice increased with the number of schistosomules injected. Up to 83% reduction in challenge worm burden was achieved when 5000 schistosomules were injected per mouse. Intramuscular injection of the vaccine was superior to subcutaneous. Multiple immunizations, up to 3 at 4-week intervals, did not increase the resistance induced by a single immunization. A high level of protection developed in as little as 2 weeks and was maintained through at least 12 weeks postimmunization. The vaccine irradiated with 10 krad from either a 60-cobalt or 137-cesium source induced equivalent levels of resistance, and no differences were found in the immunogenicity of vaccines comprised of organisms irradiated as cercariae or as 1- to 3-hr-old schistosomules. These findings are basic to the development of a cryopreserved, live vaccine against schistosomiasis of humans or domestic animals.     

A comparative ultrastructural study of in vivo and in vitro derived adults of Microphallus similis

Davtes C. 1980. A comparative ultrastructural study of in vivo and in vitro derived adults of Microphallus similis. International Journal for Parasitology10: 217–266. The ultrastructure of in vitro cultured adults of Microphallus similis was examined by TEM and SEM and compared to that of metacercariae and in vivo grown adults. In cultured flukes the most conspicuous abnormalities were observed in secretory cells, especially those of the tegument, digestive caecum, forebody glands and vitellaria. In the tegumental cells and the forebody gland cells, the secretory granules appeared to lose some of their contents within the cell bodies suggesting that there may be some defect in the transport and/or packaging of secretory granules in vitro. In the vitellaria of cultured flukes some of the granules lost their characteristic appearance, becoming ragged in outline and very electrondense. The premature tanning of the vitelline secretions within the vitellaria is correlated with abnormal egg production in vitro. The caecal cells of cultured flukes differed from those of the metacercaria and the normal adult in several important respects which suggested that their function was probably impaired.     

日本血吸虫皮肤型童虫透射电镜观察

本文报道应用透射电镜观察并比较0.5、3和12小时龄的日本血吸虫皮肤型童虫的超徽结构特征。结果表明,除了外质膜外,其他的超微结构,如体被、肌层、体被下细胞、胞质桥、头腺、钻腺和食道等结构在尾蚴感染后3小时均未见再有明显的变化。     

Fluorescently tagged pUL47 of Marek's disease virus reveals differential tissue expression of the tegument protein in vivo

Marek''s disease virus (MDV), a lymphotropic alphaherpesvirus, causes Marek''s disease (MD) in chickens. MD is characterized by neurological signs, chronic wasting, and T cell lymphomas that predominate in the visceral organs. MDV replicates in a highly cell-associated manner in vitro and in vivo, with infectious virus particles being released only from feather follicle epithelial (FFE) cells in the skin. Virus produced and shed from FFE cells allows transmission of MDV from infected to naïve chickens, but the mechanisms or roles of differential virus gene expression have remained elusive. Here, we generated recombinant MDV in which we fused enhanced green fluorescent protein (EGFP) to the C terminus of the tegument protein pUL47 (vUL47-EGFP) or pUL49 (vUL49-EGFP). While vUL49-EGFP was highly attenuated in vitro and in vivo, vUL47-EGFP showed unaltered pathogenic potential and stable production of pUL47-EGFP, which facilitated direct analysis of pUL47 expression in cells and tissues. Our studies revealed that pUL47-EGFP is expressed at low levels and localizes to the nucleus during lytic replication in vitro and in lymphocytes in the spleen in vivo, while it is undetectable in tumors. In contrast, pUL47-EGFP is highly abundant and localizes predominantly in the cytoplasm in FFE cells in the skin, where MDV is shed into the environment. We concluded that differential expression and localization of MDV pUL47-EGFP tegument protein is potentially important for the unique cell-associated nature of MDV in vitro and in lymphocytes in vivo, as well as production of free virus in FFE cells.     

The TIP30 protein complex, arachidonic acid and coenzyme A are required for vesicle membrane fusion

Efficient membrane fusion has been successfully mimicked in vitro using artificial membranes and a number of cellular proteins that are currently known to participate in membrane fusion. However, these proteins are not sufficient to promote efficient fusion between biological membranes, indicating that critical fusogenic factors remain unidentified. We have recently identified a TIP30 protein complex containing TIP30, acyl-CoA synthetase long-chain family member 4 (ACSL4) and Endophilin B1 (Endo B1) that promotes the fusion of endocytic vesicles with Rab5a vesicles, which transport endosomal acidification enzymes vacuolar (H⁺)-ATPases (V-ATPases) to the early endosomes in vivo. Here, we demonstrate that the TIP30 protein complex facilitates the fusion of endocytic vesicles with Rab5a vesicles in vitro. Fusion of the two vesicles also depends on arachidonic acid, coenzyme A and the synthesis of arachidonyl-CoA by ACSL4. Moreover, the TIP30 complex is able to transfer arachidonyl groups onto phosphatidic acid (PA), producing a new lipid species that is capable of inducing close contact between membranes. Together, our data suggest that the TIP30 complex facilitates biological membrane fusion through modification of PA on membranes.     

Schistocephalus solidus: pinocytosis by the plerocercoid tegument

The possibility of pinocytosis occurring in the tegument of the plerocercoid of Schistocephalus solidus has been investigated by morphological and experimental methods. Electronmicroscopic study showed that the outer syncytial tegument contained numerous electronlucid vesicles. These vesicles had two gradients, the number of vesicles decreasing from the outer canopy region to the inner canopy and from the apical to the basal plasma membrane for any particular region of tegument. A variety of morphological modifications of the apical plasma membrane very similar to those which have been accepted as evidence of pinocytosis in other tissues were present. In vitro studies using horseradish peroxidase, ruthenium red, and lanthanum nitrate showed that all three tracers are taken up by the tegument into membrane-limited vesicles. Vesicles which contained ruthenium red occurred at the base of the tegument within a 5-min incubation period, and their contents appeared to be released into the underlying interstitial material by exocytosis.     

Schistosoma mansoni: biochemical evidence for morphogenetic change from cercaria to schistosomule

Conditions for obtaining in vitro the transformation of cercariae in larvae similar to schistosomules are described. They consist of either centrifuging cercarial suspension or packed cercariae at room temperature, or incubating at 30 C packed cercariae. The product obtained was a mixture of cercariae, tails, cercarial bodies, and schistosomule-like larvae as revealed by stereomicroscopy. Tails and cercariae were mechanically separated.Analysis of samples of schistosomule-like preparations obtained by centrifuging cercariae at room temperature revealed that many of the organisms were PAS negative and water sensitive, and showed no localized staining with alizarin and no tendency to form Cercarienhüllen Reaktion (envelopes) in immune human sera. Elimination of the contents of the preacetabular glands was followed during centrifugation or incubation by assaying the proteolytic and/or esterolytic activities in the pellets obtained at different times. The specific activity of the secretion collected increased as a function of time. Electrophoresis of extracts of organisms before and after centrifugation at room temperature showed decrease of PAS stained bands.     

The formation of the cyst wael of the metacercaria of Cloacitrema narrabeenensis (Bowell & Bearup, 1967) (digenea: Philophthalmidae)

Dixon K.E. and Colton M. 1978. The formation of the cyst wall of the metacercaria of Cloacitrema narrabeenensis (Howell &; Bearup, 1967) (Digenea: Philophthalmidae). International Journal for Parasitology8: 491–499. The cercaria of Cloacitrema narrabeenensis contains six different types of cystogenic cells which were distinguished on the basis of their position, ultrastructure and chemical composition. Four of the different types secrete carbohydrate-protein complexes and the other two protein granules. Early in development, the cercaria is bounded by a flattened, cellular envelope which contains a few mitochondria but lacks cytoplasmic ground substance and a nucleus. Later in development, this envelope is lost, shortly after a new cellular covering forms at the surface. The nuclei are later lost from this layer and it is suggested that they sink inwards, thus forming a cercarial tegument. The products of the cystogenic cells are gradually discharged through pseudopodial-like connections with the surface layer of the cercarial tegument to form the metacercarial cyst wall. The sequence of secretion is controlled so that the separate layers of the cyst wall are formed in their correct order.     

In vitro synthesis, assembly and function of a photosynthetic membrane protein

Cell-free translation of Chlamydomonas reinhardtii RNA in the presence of photosynthetic membranes resulted in association of the herbicide binding (Qb) protein with membranes. Incubation of recovered membranes with high salt did not extract the polypeptide from membranes. Tryptic digestion of in vivo labeled membranes or membranes recovered from in vitro translation mixtures showed that Qb had similar orientation. In vitro translation in the presence of chloroplast membranes from cells exposed to high light intensity restored the membrane associated kinase activity lost by photoinhibition. Thus, in vitro synthesis resulted in functional integration of the Qb protein within the photosynthetic membrane.     

Schistosoma manonsi: cercarial penetration of host epidermis at the ultrastructural level

Invasion of the outer layers of the epidermis of mouse ear skin by cercariae of Schistosoma mansoni within 7 min of their application to it has been studied with the optical and the scanning and transmission electron microscopes.Entrance of cercariae was under the edges of the dead flattened keratinized cells of the horny layer (squames), and penetration through this layer was by disarticulation of the stacks of squames at their interdigitations. Mucus from the postacetabular glands was recognized with the light and electron microscopes on the skin surface, especially at squame edges and between layers of squames and along the keratogenous zone. The findings suggested that disarticulation of the squames was not effected solely by the muscular probing and pushing of the parasite, but that it might be aided by swelling of the mucus secretion deposited in the area from the postacetabular glands. Loosening of the interlocked edges of the squames by enzymatic action is also a possibility, but was not evaluated for this report.The migration path along the keratongenous zone was marked by extensive damage to the transitional cells of the granular layer subjacent to the squames. Packets of secretion from the cercarial preacetabular glands were identified below the horny layer in the cytoplasm of these cells. It was considered that the host tissue damage in this area was the result not only of tearing of the tissues by passage of the spiny schistosomules, but also of enzymatic activity, the enzyme source being the granules in the packets of preacetabular gland secretion.     

Different incorporation of glucocorticoid hormone into diploid and tetraploid liver nuclei

Tetraploid parenchymal rat liver nuclei incorporate about twice as much (³H)dexamethasone as diploid parenchymal nuclei both in vivo and in vitro. This suggests that the ability of hepatic nuclei to incorporate glucocorticoid hormone is influenced by the number of copies of the genome in these nuclei.     

Antifungal properties of a peptide derived from the signal peptide of the HIV-1 regulatory protein, Rev

Antifungal effects of nuclear entry inhibitory signal peptide of HIV-1 Rev protein (Rev-NIS) were investigated. Rev-NIS contained potent antifungal activities without hemolytic effects. To understand the antifungal mechanism(s), in vivo and in vitro fluorescent studies were conducted. Flow cytometric analysis with bis-(1,3-dibutylbarbituric acid) trimethine oxonol [DiBAC₄(3)] and calcein-leakage measurement from large unilamellar vesicles (LUVs) indicated that Rev-NIS depolarized and disrupted the fungal membranes. These results were further confirmed by using giant unilamellar vesicles (GUVs). The current study suggests that Rev-NIS exerts its antifungal activity with membrane-active mechanism(s).     

PHENOBARBITAL-INDUCED SYNTHESIS OF THE MICROSOMAL DRUG-METABOLIZING ENZYME SYSTEM AND ITS RELATIONSHIP TO THE PROLIFERATION OF ENDOPLASMIC MEMBRANES : A Morphological and Biochemical Study

Liver microsomes, isolated from rats which had been treated with phenobarbital in vivo, were found to exhibit increased activities of oxidative demethylation and TPNH-cytochrome c reductase and an increased amount of CO-binding pigment. Simultaneous administration of actinomycin D or puromycin abolished the phenobarbital-induced enzyme synthesis. Increased rate of P_i³² incorporation into microsomal phospholipid was the first sign of phenobarbital stimulation and appeared 3 hours after a single injection of this drug. Microsomes were divided into smooth-surfaced and rough-surfaced vesicle fractions. The fraction consisting of smooth-surfaced vesicles exhibited the greatest increase in protein content and oxidative demethylation activity after phenobarbital administration in vivo. Ultrastructural studies revealed that drug treatment also gave rise to proliferation of the endoplasmic reticulum in the hepatic parenchymal cells, first noticed after two phenobarbital injections. The phenobarbital-induced synthesis of the metabolizing enzymes is discussed with special reference to the relationship to the stimulated synthesis of the endoplasmic membranes.