Trypanosoma brucei: New radioisotope assay for quantitating cell lysis

A new radioisotope technique for quantitatively determining the extent of cell lysis of trypanosomes is described. Trypanosoma brucei was pulse-labeled with [³H]leucine and then incubated at 37 C in lytic (human serum) or nonlytic (rabbit serum) media. The release into the incubation medium of radioactively labeled intracellular macromolecules was then determined. A linear relationship was found between the release of TCA-precipitable radioactivity and lysis, as determined by conventional hemacytometer counting. This method is used to quantitate the trypanocidal activity in normal human serum.     

Purine requirements for the expression of Cape buffalo serum trypanocidal activity

Cape buffalo serum contains xanthine oxidase which generates trypanocidal H₂O₂ during the catabolism of hypoxanthine and xanthine. The present studies show that xanthine oxidase-dependent trypanocidal activity in Cape buffalo serum was also elicited by purine nucleotides, nucleosides, and bases even though xanthine oxidase did not catabolize those purines. The paradox was explained in part, by the presence in serum of purine nucleoside phosphorylase and adenosine deaminase, that, together with xanthine oxidase, catabolized adenosine, inosine, hypoxanthine, and xanthine to uric acid yielding trypanocidal H₂O₂. In addition, purine catabolism by trypanosomes provided substrates for serum xanthine oxidase and was implicated in the triggering of xanthine oxidase-dependent trypanocidal activity by purines that were not directly catabolized to uric acid in Cape buffalo serum, namely guanosine, guanine, adenine monophosphate, guanosine diphosphate, adenosine 3′:5-cyclic monophosphate, and 1-methylinosine. The concentrations of guanosine and guanine that elicited xanthine oxidase-dependent trypanocidal activity were 30–270-fold lower than those of other purines requiring trypanosome-processing which suggests differential processing by the parasites.     

Effect of serum on prostaglandin production during the co-culture of human thyroid cells and peripheral blood mononuclear cells

Prostaglandin generation by human peripheral blood mononuclear cells is enhanced during co-culture with human thyroid cells. The objective of the present study was to determine the influence of various sera on this process. Human thyroid adenoma cell monolayers were cultured with normal human peripheral blood mononuclear cells for three days in the presence of a variety of sera, or serum fractions. Prostaglandin E (PGE) in the medium was measured by bioassay or by radioimmunoassay. Significantly more PGE was generated in cultures containing fetal calf serum than in those containing human serum. This difference was not abolished by dialysis of the human serum. When the 50% (NH₄) ₂SO₄ precipitate of the serum was used, PGE generation was similar to that in fetal calf serum, indicating the presence of an inhibitory factor in human serum. The degree of this inhibitory activity was similar in autologous and heterologous human serum, as well as in normal subjects and patients with Graves' disease. Gel filtration and ion-exchange chomatography of human serum showed the inhibitor to co-migrate with albumin. Evidence presented suggests that the inhibitor is not albumin itself but is, instead, a factor tightly bound to albumin. Inhibitory activity was also found in rabbit, goat, rat and cow serum. Prostaglandins are potent modulators of immune-cell function. These data indicate that this process may be modulated by a factor in mammalian serum. The relative absence of this factor in fetal serum may have important implications in regard to the profound changes which occur in the immune system after birth.     

Goldfish (Carassius auratus L.) possess natural antibodies with trypanocidal activity towards Trypanosoma carassii in vitro

Natural infection of cyprinids, such as carp, with the extracellular protozoan parasite Trypanosoma carassii can attain up to 100% prevalence and cause significant host morbidity and mortality, particularly in aquaculture settings. Host recovery from T. carassii infection has been shown to be antibody (Immunoglobulin M; IgM)-mediated, conferring long-term immunity in recovered animals upon challenge. To assess the role of IgM in parasite clearance in the goldfish, IgM was purified by PEG-6000 precipitation from goldfish serum collected at 0 (naïve), 21 (peak parasitaemia) and 42 (recovery phase; immune) days post infection (dpi) and used for in vitro assays. Purified IgM from 0, 21, and 42 dpi serum showed dose- and time-dependent trypanocidal activity in vitro. Incubation of T. carassii with 0 dpi IgM showed the greatest reduction in trypanosome numbers after 24 h, followed by 42 dpi IgM, and finally by 21 dpi IgM. The trypanocidal activity of the PEG-purified IgM was abrogated by pre-absorption with parasites in vitro and was affected by temperature. Furthermore, studies using 0 dpi IgM purified using gel permeation chromatography showed increased trypanocidal activity, with complete elimination of parasites after 12 h when incubated with 200 μg of 0 dpi IgM, or by 24 h when incubated with 80 μg or 100 μg of 0 dpi IgM. Lastly, in vivo passive transfer experiments demonstrated that while immune serum or purified IgM from 42 dpi serum conferred protection against a challenge, neither 0 dpi serum or 0 dpi purified IgM conferred protection against challenge with T. carassii.     

Human Transferrin Confers Serum Resistance against Bacillus anthracis

The innate immune system in humans consists of both cellular and humoral components that collaborate to eradicate invading bacteria from the body. Here, we discover that the Gram-positive bacterium Bacillus anthracis, the causative agent of anthrax, does not grow in human serum. Fractionation of serum by gel filtration chromatography led to the identification of human transferrin as the inhibiting factor. Purified transferrin blocks growth of both the fully virulent encapsulated B. anthracis Ames and the non-encapsulated Sterne strain. Growth inhibition was also observed in serum of wild-type mice but not of hypotransferrinemic mice that only have ∼1% circulating transferrin levels. We were able to definitely assign the bacteriostatic activity of transferrin to its iron-binding function: neither iron-saturated transferrin nor a recombinant transferrin mutant unable to bind iron could inhibit growth of B. anthracis. Additional iron could restore bacterial growth in human serum. The observation that other important Gram-positive pathogens are not inhibited by transferrin suggests they have evolved effective mechanisms to circumvent serum iron deprivation. These findings provide a better understanding of human host defense mechanisms against anthrax and provide a mechanistic basis for the antimicrobial activity of human transferrin.     

Rat Serum Factors Inhibiting the G1-S Transition In Hepatocytes

An attempt was made to detect the serum factors inhibiting the G₁-S transition in synchronized, baby rat hepatocytes. In untreated adult rat serum, this inhibitory activity was always linked to high molecular weight (HMW) compounds. Incubation of serum with trypsin or chymotrypsin resulted in the formation of a low molecular weight (LMW) G₁-S inhibitory factor. the same result was obtained with fractions from adult rat liver but not with kidney or spleen fractions. Separation of the LMW factor by ultrafiltration increased its specific activity by about 10³. the active period in the cell cycle of both the LMW and HMW factors was the same: the late G₁ phase. However, the activity of the LMW factor was not blocked by the Kunitz factor. an enzymatic transformation of the HMW factor might be induced by liver cell membrane-bound proteases and constitute a mechanism regulating hepatocyte proliferation.     

Schistosomicidal and trypanocidal structure-activity relationships for (±)-licarin A and its (-)- and (+)-enantiomers

(±)-Licarin A (1) was obtained by oxidative coupling, and its enantiomers, (?)-licarin A (2) and (+)-licarin A (3), were resolved by chiral HPLC. Schistosomicidal and trypanocidal activities of these compounds were evaluated in vitro against Schistosoma mansoni adult worms and trypomastigote forms of Trypanosoma cruzi. The racemic mixture (1) displayed significant schistosomicidal activity with an LC₅₀ value of 53.57 μM and moderate trypanocidal activity with an IC₅₀ value of 127.17 μM. On the other hand, the (?)-enantiomer (2), displaying a LC₅₀ value of 91.71 μM, was more active against S. mansoni than the (+)-enantiomer (3), which did not show activity. For the trypanocidal assay, enantiomer 2 showed more significant activity (IC₅₀ of 23.46 μM) than enantiomer 3, which showed an IC₅₀ value of 87.73 μM. Therefore, these results suggest that (±)-licarin A (1) and (?)-licarin A (2) are promising compounds that could be used for the development of schistosomicidal and trypanocidal agents.     

Hautriwaic acid as one of the hepatoprotective constituent of Dodonaea viscosa

It is widely known that hepatitis and its complications such as cirrhosis or hepatocellular carcinoma are one of the major health problems of the world especially since no specific treatment is available. In the present study we investigated the hepatoprotective potential of the methanolic extract of the whole plant of Dodonaea viscosa and its ethyl acetate, aqueous, butanol and n-hexane fractions against carbon tetrachloride (CCl₄) induced hepatoxicity in rats. Hepatoprotection was assessed in terms of reduction in serum enzymes (ALT, AST, and ALP) that occur after CCl₄ injury, and by histopathology and immunohistochemistry. The methanolic extract reduced the serum enzyme level (ALT, AST, and ALP) down to control levels despite CCl₄ treatment. It also reduced the CCl₄-induced damaged area to 0% as assessed by histopathology. The CD68+ macrophages were also reduced in number around the central vein area by the methanolic extract. These hepatoprotective effects were better than the positive control silymarin. Similar hepatoprotective activities were found with the ethyl acetate, and aqueous fractions of the methanolic extract. The butanol and n-hexane fractions showed elevated levels of ALT, AST and ALP as compared to the positive control silymarin. Histopathology showed ∼30% damage to the liver cells with the butanol and n-hexane fractions which still showed some protective activity compared to the CCl₄ treated control. HPLC fingerprinting suggested that hautriwaic acid present in the methanolic extract and its ethyl acetate, and aqueous fractions may be responsible for this hepatoprotective activity of Dodonaea viscosa which was confirmed by in vivo experiments.     

Immunological studies of the anticryptococcal factor of normal human serum

Preliminary studies have shown a very high inhibitory activity in the alpha₂ and gamma zone of human serum towards the growth of Cryptococcus neoformans. These findings are now corroborated by single radial immunodiffusion tests, which showed the some loss of IgA and IgM globulins and of the other three globulin fractions (ceruloplasmin, alpha₂ macroglobulin and alpha₂ HS glycoprotein) which migrate in the alpha₂ zone. The data was obtained by single radial immunodiffusion tests. The losses were not statistically significant however. No change in the immunoglobulin content of the sera kept for 6 days in contact with a heat-killed suspension of C. neoformans was noted. These findings suggest, that the inhibitory activity of the normal human serum on the in-vitro growth of C. neoformans is due to the above mentioned globulin fractions and not to a single specific factor.     

An assay for osteoclast-activating factor (OAF) in biological fluids: Detection of OAF in the serum of myeloma patients

A procedure is described for the partial purification and biological assay of a lymphokine, osteoclast-activating factor, in samples of serum. This procedure involves two steps of ultra-filtration of samples, followed by polyacrylamide gel electrophoresis and bioassay of fractions eluted from the gels. The procedure effectively discriminates between osteoclast-activating factor and other agents with similar biological activities (parathyroid hormone, prostaglandin E₂, and 1, 25-dihydroxyvitamin D₃), when each agent is added to normal human serum. Using this procedure as a specific assay for osteoclast-activating factor, serum from normal individuals and myeloma patients was examined. The activity was detected in 11 of 23 myeloma sera but in none of 12 normal sera.     

Synthesis of 5-enamine-4-thiazolidinone derivatives with trypanocidal and anticancer activity

A series of novel 2-(5-aminomethylene-4-oxo-2-thioxothiazolidin-3-yl)-3-phenylpropionic acid ethyl esters has been synthesized. Target compounds were evaluated for their trypanocidal activity towards Trypanosoma brucei brucei and Trypanosoma brucei gambiense. Several hit-compounds (8, 10, 12) inhibited growth of the parasites at sub-micromolar concentrations (IC₅₀ 0.027–1.936 µM) and showed significant selectivity indices (SI = 108–1396.2) being non-toxic towards the human primary fibroblasts. The screening of anticancer activity in vitro within NCI DTP protocol allowed to identify active 2-(5-{[5-(2,4-dichlorobenzyl)-thiazol-2-ylamino]-methylene}-4-oxo-2-thioxothiazolidin-3-yl)-3-phenylpropionic acid ethyl ester 14 that demonstrated inhibition against all 59 human tumor cell lines with the average GI₅₀ value of 2.57 μM. It was established that the activity type (antitrypanosomal or anticancer) as well as its level depends on the character of enamine fragment in the C5 position of thiazolidinone core.     

2-Phenylaminonaphthoquinones and related compounds: Synthesis,trypanocidal and cytotoxic activities

A series of new 2-aminonaphthoquinones and related compounds were synthesized and evaluated in vitro as trypanocidal and cytotoxic agents. Some tested compounds inhibited epimastigote growth and trypomastigote viability. Several compounds showed similar or higher activity and selectivity as compared with current trypanocidal drug, nifurtimox. Compound 4l exhibit higher selectivity than nifurtimox against Trypanosoma cruzi in comparison with Vero cells. Some of the synthesized quinones were tested against cancer cells and normal fibroblasts, showing that certain chemical modifications on the naphthoquinone moiety induce and excellent increase the selectivity index of the cytotoxicity (4g and 10). The results presented here show that the anti-T. cruzi activity of 2-aminonaphthoquinones derivatives can be improved by the replacement of the benzene ring by a pyridine moiety. Interestingly, the presence of a chlorine atom at C-3 and a highly lipophilic alkyl group or aromatic ring are newly observed elements that should lead to the discovery of more selective cytotoxic and trypanocidal compounds.     

Synthesis and tripanocidal activity of ferrocenyl and benzyl diamines against Trypanosoma brucei and Trypanosoma cruzi

Trypanosoma brucei and Trypanosoma cruzi are the etiologic agents of sleeping sickness and Chagas disease, respectively, two of the 17 preventable tropical infectious diseases (NTD) which have been neglected by governments and organizations working in the health sector, as well as pharmaceutical industries. High toxicity and resistance are problems of the conventional drugs employed against trypanosomiasis, hence the need for the development of new drugs with trypanocidal activity. In this work we have evaluated the trypanocidal activity of a series of N1,N2-dibenzylethane-1,2-diamine hydrochlorides (benzyl diamines) and N1-benzyl,N2-methyferrocenylethane-1,2-diamine hydrochlorides (ferrocenyl diamines) against T. brucei and T. cruzi parasite strains. We show that incorporation of the ferrocenyl group into the benzyl diamines increases the trypanocidal activity. The molecules exhibit potential trypanocidal activity in vitro against all parasite strains. Cytotoxicity assay was also carried out to evaluate the toxicity in HepG2 cells.     

Trypanosoma cruzi: in vitro activity of Epoxy-α-Lap, a derivative of α-lapachone, on trypomastigote and amastigote forms

Chagas disease is an endemic parasitic infection caused by Trypanosomacruzi that affects 18-20 million people in Central and South America. Recently we described the Epoxy-α-Lap, an oxyran derivative of α-lapachone, which presents a low toxicity profile and a high inhibitory activity against T.cruzi epimastigotes forms, the non-infective form of this parasite. In this work we described the trypanocidal effects of Epoxy-α-Lap on extracellular (trypomastigote) and intracellular (amastigote) infective forms of two T. cruzi strains (Y and Colombian) known by their different infective profile. Our results showed that Epoxy-α-Lap is lethal to trypomastigote Y and Colombian strains (97% and 84%, respectively). Interestingly, Epoxy-α-Lap also showed a trypanocidal effect in human macrophage infected with T. cruzi Y (85.6%) and Colombian (71.9%) strains amastigote forms. Similar effects were observed on T. cruzi amastigote infected Vero cells (96.4% and 95.0%, respectively). Our results pointed Epoxy-α-Lap as a potential candidate for Chagas disease chemotherapy since it presents trypanocidal activity on all T. cruzi forms with low) toxicity profile.     

Gonadotropin and prostaglandins binding sites in rough endoplasmic reticulum and Golgi fractions of bovine corpora lutea.

Highly purified rough endoplasmic reticulum and three subfractions of golgi were prepared from 105,000g pellet of the homogenate by centrifugation in floatation and sedimentation discontinuous sucrose gradients. Highly purified plasma membranes were also prepared from 9,000g pellet of the same homogenates for assessment under the same experimental conditions. Although 5′-nucleotidase, a marker for plasma membranes, was markedly enriched in plasma membranes, very little or none of this enzyme activity was found in other fractions. Very little or no NADH cytochrome c reductase activity, a marker for rough endoplasmic reticulum, was found in fractions other than rough endoplasmic reticulum. Galactosyl transferase, a marker for golgi, was found and enriched in all the fractions; however, enrichment in golgi fractions was higher than in other fractions. Very little or no lysosomal marker activity, i.e., acid phosphatase, was found in rough endoplasmic reticulum or golgi fractions as compared to lysosomes. These marker enzyme data suggested that rough endoplasmic reticulum and golgi fractions were relatively pure with little or no cross contamination with other organelles. The [¹²⁵I]human choriogonadotropin ([¹²⁵I]hCG), [³H]prostaglandin (PG)E₁, and [³H]PGF_2a specifically bound to rough endoplasmic reticulum and golgi fractions in addition to plasma membranes. The enrichments of binding in the former two fractions, in some cases, were as high as plasma membranes itself. The specific binding of some of the ligands was found to be partially latent in rough endoplasmic reticulum and golgi fractions but not in plasma membranes. Marker enzyme data, ratio between bindings and marker enzyme activities (an index of organelle contamination), and partial latency of binding suggest that rough endoplasmic reticulum and golgi fractions intrinsically contain gonadotropin and PGs binding sites.     

Ability of dialysates containing transfer factor to induce lymphocyte activating factor by human mononuclear cells.

Dialysates of human leukocyte lysates containing transfer factor (TF_d) stimulated human mononuclear cells (MNL) to produce lymphocyte activating factor (LAF). Both unfractionated and adherent MNL cultures were stimulated by TF_d to produce a factor which was mitogenic for murine thymocytes and had the biochemical characteristics of LAF as determined by Bio-Gel P-100, DEAE cellulose, and hydroxylapatite chromatography. Fractionation of TF_d on Sephadex G-25 showed that the specific transfer factor activity of converting in vivo skin tests was present in the major uv-absorbing peak, whereas the substance(s) that induced LAF activity was present in a number of the other fractions. Therefore, the capacity of TF_d to induce monocytes to produce LAF is not a measure of classical transfer factor activity. However, this effect of TF_d may instead participate in the nonspecific immunoenhancing effects of TF_d.     

Serum factors required for arginase induction in macrophages

Two nondialyzable factors in the sera of several species were required for the induction of arginase in resident peritoneal macrophages in vitro. One factor stimulated PGE₂ production by macrophages, was inhibited by indomethacin, and could be replaced by the addition of PGE₂ or DBcAMP. Thirty percent serum was required for maximal activity of this factor. DBcAMP reduced the requirement for serum by 10-fold, suggesting the requirement of a second factor which required less than 3% serum for maximal activity. A factor with similar activity was found in the supernatant of tumor cells grown in vitro.     

Effect of serum on organogenesis of the rat testis in vitro

Summary It was observed previously that primordia of fetal rat testes when explanted in vitro in a synthetic medium at the outset of sexual differentiation differentiate seminiferous cords during the following days, but that the addition of 15% fetal bovine serum prevents this morphogenesis. In the present study, human, horse, bovine calf, and rat sera were shown to exert the same effect. Very low concentrations of human or fetal bovine serum (0.5 or 1%) were sufficient to produce the serum effect, which was only slightly reduced when the serum was heated. The serum activity was not removed by dialysis (membrane cut-off 15 000), but it disappeared after treatment with trichloroacetic or perchloric acids or after trypsin digestion. Partial purification of the active factor(s) from human serum was achieved by successive gel filtration, affinity chromatography, and ion exchange chromatography. Analysis of the active fractions by electrofocusing and immunoelectrophoresis placed the activity within the α globulin group. Among a series of purified serum proteins tested, α₂-HS-glycoprotein was found to exhibit the serum effect, though this activity was heat labile.