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F Levi-Schaffer

Publications and source records attributed to F Levi-Schaffer.

107 records · Page 6Linked to original sources
PubMed1986

Schistosoma mansoni: killing of transformed schistosomula by the alternative pathway of human complement.

The interaction of mechanically transformed schistosomula of Schistosoma mansoni with the alternative pathway of human complement was studied in vitro. To detect early changes in transformation, the schistosomula were prepared at a low temperature and used immediately. As shown previously, freshly transformed schistosomula were highly susceptible to killing by normal human serum and by C4-depleted normal human serum. This serum activity was concentration dependent and was markedly reduced on a twofold serum dilution. Upon incubation at 37 C in defined synthetic medium, schistosomula rapidly became refractory to killing by the alternative pathway of complement. After 1 hr of incubation at 37 C, the percentage of schistosomula which were resistant to killing increased from 16 to 85. This conversion was accompanied by a fivefold decrease in deposition of C3b on schistosomula which had been exposed to 37 C for 1 hr and then further incubated with C4-depleted normal human serum. The following events occurred concomitantly during incubation of freshly transformed schistosomula at 37 C with a half-life of 30-60 min: (1) Decrease in activation and consumption of the alternative pathway of complement by schistosomula; (2) appearance of a strong complement consuming activity in the supernatant of incubating schistosomula; and (3) shedding of protein- and carbohydrate-containing substances from the surface of schistosomula into the supernatant. Isolated external membranes of freshly transformed schistosomula consumed the alternative pathway of complement to a greater extent than membranes of schistosomula preincubated in medium at 37 C. The results demonstrate that transformed schistosomula acquire resistance to complement killing via the alternative pathway by shedding complement-activating substances.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1986

Coculture of interleukin 3-dependent mouse mast cells with fibroblasts results in a phenotypic change of the mast cells.

The heparin-containing mast cells that reside in the connective tissue of the mouse, but not the chondroitin sulfate-containing mast cells in the gastrointestinal mucosa, stain with safranin when exposed to alcian blue/safranin. Mouse bone marrow-derived mast cells (BMMC), the probable in vitro counterparts of in vivo mucosal mast cells, were cultured for 14 days with mouse skin-derived 3T3 fibroblasts in RPMI 1640 medium containing 10% fetal calf serum and 50% WEHI-3 conditioned medium. Although the BMMC adhered to the fibroblast monolayer, they continued to divide, probably due to the presence of interleukin 3 in the conditioned medium. The mast cells remained viable throughout the period of coculture, since they failed to release lactate dehydrogenase and because they increased their histamine content approximately 15-fold. After 12-14 days of coculture, greater than 50% of the BMMC changed histochemically to become safranin+; 30-40% of the 35S-labeled glycosaminoglycans on the proteoglycans synthesized by these cocultured mast cells were heparin, whereas heparin was not detected in the initial BMMC. In the absence of WEHI-3 conditioned medium, BMMC adhered to the fibroblast monolayer, and after 8 days of coculture, the number of mast cells did not change and their histamine content remained the same. However, these mast cells also became safranin+ and synthesized 40% heparin glycosaminoglycans. Thus, coculture of BMMC with fibroblasts induces a phenotypic change so that the resulting mast cells stain safranin+ and synthesize heparin proteoglycans, whereas the presence of WEHI-3 conditioned medium stimulates proliferation and an increase in histamine content.

Animals↗
PubMed1986

A novel lipid-conjugated marker for the direct labeling of the schistosomulum membrane.

Introduction of the fluorescent group lissamine rhodamine B into the surface of schistosomula of S. mansoni was achieved by brief incubation of the worms with liposomes carrying the lipid bound fluorophore in their bilayers. The liposomes were made of egg lecithin (PC) and lissamine-phosphatidylethanolamine (lissamine-PE). The lissamine groups could be directly detected on the parasite membrane by observation of the single worms under a fluorescent microscope. The fluorescent marker was employed to measure the lateral diffusion coefficient (D) of lipids in the schistosomular membrane and as a surface marker during the isolation of the schistosomula surface membrane.

Animals↗
PubMed1985

Fibroblasts maintain the phenotype and viability of the rat heparin-containing mast cell in vitro.

Rat serosal heparin-containing mast cells (HP-MC) were maintained in vitro for as long as 30 days when co-cultured with mouse skin-derived 3T3 fibroblasts. In contrast, when the mast cells were cultured alone, on fibronectin-, gelatin-, or dermal-collagen-coated dishes, on acid and heat-killed fibroblasts in the presence or absence of 24 hr fibroblast-conditioned medium, or on a monolayer of mouse serosal macrophages, they failed to adhere to the dishes, released significant amounts of their histamine and lactate dehydrogenase, and stained with trypan blue, indicating a loss of viability. The rat serosal HP-MC cultured with the 3T3 fibroblasts became so adherent to the fibroblasts that the two cell types could be separated from one another only by trypsinization. The cultured HP-MC stained with both alcian blue and safranin and continued to synthesize proteoglycan at a rate comparable to that of freshly isolated cells. The 35S-labeled proteoglycan synthesized by these cultured cells, like that produced by freshly isolated rat serosal HP-MC, was a 750,000 to 1,000,000 m.w. proteoglycan containing only heparin glycosaminoglycans of 50,000 to 100,000 m.w. When HP-MC were cultured for 1 wk with the fibroblasts and were then incubated for 5 min with a 1/20 dilution of rabbit anti-rat IgE, they generated and released an average of 22 +/- 10 ng (mean +/- SD, n = 5) of prostaglandin D2 per 10(6) cells and exocytosed a higher net percentage of their total histamine content (44 +/- 11% [mean +/- SD, n = 8]) than did cells just isolated from the animal (6 +/- 4% [mean +/- SD, n = 4]). As assessed by electron microscopy, many of the cultured HP-MC resembled freshly isolated cells except that some secretory granules had fused with one another in some cells. Morphologically, after activation the cultured HP-MC underwent compound exocytosis like freshly isolated cells. These results demonstrate that the in vivo differentiated rat HP-MC maintain their histology, morphology, immunologic responsiveness, histamine content, and ability to synthesize heparin proteoglycan when co-cultured with living fibroblasts.

Animals↗
PubMed1985

Protective monoclonal antibody against Schistosoma mansoni: antigen isolation, characterization, and suitability for active immunization.

Monoclonal antibodies that bind to the surface of developing schistosomula were generated from the spleens of chronically infected mice that were boosted with cercarial glycoproteins. The two most reactive monoclonal antibodies, denoted 152-66-9B and 152-66-1C, were used for identification of surface antigens. The antigen detected by these monoclonal antibodies persisted on the surface of the developing larva for 72 hr posttransformation. This monoclonal antibody effected complement-mediated killing of schistosomula in vitro as efficiently as infected mouse sera. It was also very efficient in inhibiting the infectivity of both cercariae and schistosomula. The antigen reactive with the 152-66-9B monoclonal antibody contains two major polypeptides (45 and 30 KD). These polypeptide chains might have originated from the same protein, because they have the same isoelectric point in two-dimensional gel electrophoresis. Moreover, the affinity-purified antigen migrated as only one protein band of approximately 200 KD in SDS-PAGE in nonreducing conditions. The 9B antigen was isolated, purified, and used for immunization, resulting in an antigen dose-dependent partial protection against S. mansoni infection.

Animals↗
PubMed1984

Acetylcholinesterase of Schistosoma mansoni. Molecular forms of the solubilized enzyme.

Several molecular forms of acetylcholinesterase were obtained from Schistosoma mansoni homogenates by extraction in either low-salt buffer, high-salt buffer or detergent buffer. The low-salt soluble form amounts to 25% of the total activity. By contrast, the extract obtained in the presence of Triton X-100 possessed almost almost 3-fold higher enzymatic activity, most of it (86%) being retained in the soluble extract (100 000 X g). High-salt concentration (1 M NaCl) also has a solubilizing effect, but to a lesser extent (50%). Acetylcholinesterase can also be solubilized by treatment with a solution of 1% methylmannoside (40%). In the presence of non-ionic detergents, the enzyme behaves as monodisperse 8 S form. In the absence of detergent the low-salt soluble extract is polydisperse: it contains a 10 S and a 32 S component, the latter could represent high polymers. The molecular form released from tissue homogenate by treatment with alpha-methylmannoside is polydisperse: it contains a major 10 S and a minor 32 S component. Differences in sedimentation coefficient were observed among the enzymes extracted with detergent from the various life cycle stages of the parasite. The enzyme from the cercarial stage sediments as a single 8 S peak. The adult worm exhibits an additional acetylcholinesterase peak of 18 S representing approx. 30% of the total enzymatic activity. The molecular weight of the major 8 S species, as determined by gel filtration, is 450 000.

Acetylcholinesterase↗
PubMed1984

Acetylcholinesterase of Schistosoma mansoni: antigenic cross-reactivity with Electrophorus electricus and its functional implications.

Acetylcholinesterase (AChE) of the parasite Schistosoma mansoni cross-reacts with rabbit antibodies against AChE from the electric eel. Cross-reactivity was demonstrated by radioimmunoassay with sonicate, as well as soluble, preparations from various stages of the parasite life cycle. On sucrose density gradient, the parasite enzyme migrates as an 7.5 S form, whereas an 9.5 S peak is observed with the enzyme-antibody complex. Immunofluorescence microscopy shows specific staining of intact schistosomula as well as of adult worms. Moreover, the interaction of these antibodies resulted in a marked complement-dependent cytotoxicity towards intact schistosomula, indicating that AChE is an antigen of significance on the surface of the parasite.

Acetylcholinesterase↗
PubMed1984

Isolation and partial characterization of the tegumental outer membrane of schistosomula of Schistosoma mansoni.

Separation of the external membranes from freshly converted mechanical schistosomula of Schistosoma mansoni was achieved by osmotic shock under hypertonic conditions, followed by mechanical shearing and ultracentrifugation. Prior to treatment, the schistosomula were surface labeled by introduction of N-DNP-epsilon-aminocaproylphosphatidylethanolamine molecules into their lipid bilayer followed by anti-DNP antibodies and stained with either 125I-protein-A or ferritin labeled secondary anti-DNP antibodies. This label provided a membrane marker by which the purity of the preparation could be assessed at each stage. Fluorescence staining with FITC-conjugated secondary antibodies prior to treatment revealed that the homogeneously stained membrane of the intact schistosomula became swollen and ruptured after the osmotic shock. The isolated membrane pellet was intensely fluorescent. Electron microscopical examination revealed mostly vesicles, some of them with organized multilayer assembly. The vesicles were ferritin labeled, indicating that they originated from the outer surface membrane of the schistosomula. A 100 fold enrichment in the alkaline phosphatase activity and about 300 fold enrichment in acetylcholinesterase activity in the membrane preparations, as compared to the intact schistosomula, was found. The isolated tegument was analyzed by SDS-polyacrylamide gel electrophoresis. The pattern obtained showed three major bands, of molecular weights 69 000, 45 000 and 12 000 alongside with a large number of minor bands. Immunoprecipitation of the isolated 125I-labeled membrane antigens with antisera from chronically infected mice revealed these three major bands together with three other bands of molecular weight 38 000, 23 000 and 16 000.

Acetylcholinesterase↗
PubMed1984

Effect of phosphonium salts and phosphoranes on the acetylcholinesterase activity and on the viability of Schistosoma mansoni parasites.

In this study we investigated the effects of a series of phosphonium salts and phosphoranes on the catalytic activity of acetylcholinesterase and on the viability of the various life stages of Schistosoma mansoni worms. All the tested compounds showed an inhibitory effect towards the S. mansoni acetylcholinesterase (AChE) activity. The most effective compound, p-xylylene bis(triphenylphosphonium) dibromide (No. 16) displayed approximately 100% inhibition at concentration of 10(-5)-10(-6)M. No significant difference was found in the sensitivity of the enzyme obtained from the various stages of the parasite life cycle to the effect of the drugs. Each compound was also tested for its toxicity towards 3 h old schistosomula and 7-9 week adult worms under in vitro culture conditions. In the case of the larvae, after 2 days in culture, only three compounds (Nos. 4, 11 and 12) out of sixteen tested exhibited efficient killing of the schistosomula while the others had a very slight toxic activity or no toxicity at all. On the other hand, all the compounds showed a significant toxicity towards the adult worms and the most effective one, allytriphenylphosphonium bromide (No. 11), retained its toxic effect even at an extremely high dilution (10(-8)M). However, the cumulative results in this paper do not demonstrate a significant correlation between the inhibitory effect of the phosphonium salts and phosphoranes on the AChE activity of the schistosomes and their toxicity towards the worms. The LD50 value (i.v.) of the compound which showed the highest toxic effect in vitro (No. 11) was found to be 30 +/- 1.7 mg/kg in mice.(ABSTRACT TRUNCATED AT 250 WORDS)

Acetylcholinesterase↗
PubMed1983

A method of introducing lipid-conjugated antigens into the surface of schistosomula.

Introduction of synthetic antigens into the surface of schistosomula of Schistosoma mansoni was achieved by brief incubation of the worms with liposomes carrying the lipid-bound antigens in their bilayers. Three-hour-old schistosomula were surface-labeled with lipid-conjugated dinitrophenyl (DNP) groups by using liposomes made of egg lecithin-N-dinitrophenil-epsilon-aminocaproyl-phosphatidylethanolamine (5:1). The DNP groups incorporated in this way could be detected for more than 21 hours in vitro by using rabbit anti-DNP antibodies stained with fluorescein isothiocyanate-conjugated goat anti-rabbit IgG. Immunofluorescence microscopy showed the lipid antigen to be uniformly distributed over the entire surface of the worms. Electron microscope studies, performed with purified rabbit anti-DNP antibodies followed by ferritin-conjugated goat anti-rabbit IgG, showed that the DNP groups were evenly and densely distributed over the entire outer membrane of the schistosomula, including spines. The distance between the ferritin molecules and the parasite's surface was 24 +/- 5 nm, indicating that the lipid antigen had been incorporated into the outer membrane of the schistosomula.

Animals↗
PubMed1982

Reduced toxicity of daunorubicin by conjugation to dextran.

Daunorubicin-dextran conjugate (dau-dex) was compared to free daunorubicin for acute and subacute toxicity and efficacy in tumor chemotherapy. LD50 and LD2 values of dau-dex are about threefold higher than those of the free drug. The therapeutic index of dau-dex is also higher, manifesting a "safe-region" in which no mortality occurs either from YAC lymphoma or from drug toxicity. Dau-dex caused almost no damage to heart tissue during the 2 months following four injections of the therapeutic dose and caused no change in the differential count of bone marrow cells. Altogether, the subacute toxicity of dau-dex is much lower than that of free daunorubicin, as expressed by negligible histologic damage to all organs examined and compared to the massive atrophy of spleen and bone marrow effected by the free drug.

Animals↗
PubMedDate not supplied

Nedocromil sodium modulates the function of long-term rat peritoneal mast cell cultures.

Long-term rat peritoneal mast cell (MC) cultures consisting of MC co-cultured with 3T3 fibroblasts (MC/3T3) were employed to study the effects of repeated and prolonged incubation with nedocromil sodium (10(-3) and 10(-5) M) on MC activation. When nedocromil sodium was added simultaneously with compound 48/80 to MC/3T3 on the first day of the experiment, it inhibited histamine release to the same extent as when added to the cultures a week later upon rechallenge. In other experiments activated MC/3T3 were incubated with nedocromil sodium for a week and on the last day of the experiment fresh nedocromil sodium was added simultaneously with compound 48/80. Under these conditions the drug was as potent in inhibiting histamine release from the activated MC as it was in cultures incubated for a week with medium alone. In addition, preincubation of naive MC with nedocromil sodium for two days inhibited histamine release from these cells when activated for the first time. Salbutamol (10(-3) and 10(-5) M) exhibited a similar inhibitory effect to nedocromil sodium upon simultaneous incubation with MC/3T3. However, prolonged incubation of salbutamol with MC/3T3 cultures resulted in tachyphylaxis. We conclude that nedocromil sodium is an effective MC stabilizing drug since it similarly inhibits histamine release from both primarily and secondarily challenged MC. Moreover this drug does not lose its efficacy upon prolonged incubation with MC.

3T3 Cells↗
PubMedDate not supplied

Nerve growth factor: an important molecule in allergic inflammation and tissue remodelling.

BACKGROUND: Several studies provide evidence that nerve growth factor (NGF) has an expanding role in neuroimmune interactions. METHODS: We review our data on circulation levels of NGF in allergic diseases as well as on the relationships between this neurotrophin and primary and secondary effector cells of allergic inflammation. RESULTS: In vernal keratoconjunctivitis, a close relationship exists between the increased NGF plasma values and the number of mast cells infiltrating the conjunctiva. NGF serum values are also increased in other allergic diseases and asthma, and are related to the severity of the inflammatory process and disease. Human CD4+ T cell clones (preferentially of activated Th2 type) produce and release NGF, and express high-affinity NGF receptors. NGF is preformed in and can act on human peripheral blood eosinophils to preferentially release inflammatory mediators. Immunoreactivity for high affinity NGF receptors is present both in basal epithelial cells and in the inflamed stroma of the allergic conjunctiva. Topical administration of NGF results in a complete healing of neurotophic corneal ulcers in man, thus suggesting a profound effect of NGF on human fibroblasts and extracellular matrix. CONCLUSION: Data presented suggest that NGF is an important molecule in allergic inflammation and tissue remodelling occurring in allergic diseases.

Animals↗