Effect of Holothurin on Trypanosoma musculi infection in Three Strains of Mice1

The effect of holothurin (a marine biotoxin) on the resistance of mice to Trypanosoma musculi was measured by studying changes in the parasite population in vivo. Swiss Webster (SW), Beige (BG), and Black (BL) mice treated with holothurin prior to and simultaneously with infection of trypanosomes had lower parasitemias than controls. Higher levels of parasitemia were observed in mice treated after infection with trypanosomes. The timing of administration of holothurin appeared to be an important factor in the observed effect. The minor variations in the parasitemia seemed to be related to the mouse strain.     

Effects of heparin administration on Trypanosoma brucei gambiense infection in rats

We examined whether heparin administration influences in vivo trypanosome proliferation in infected rats. Administration of heparin every 8 hr via cardiac catheter inhibited growth of Trypanosoma brucei gambiense and prolonged survival of treated rats. Heparin administration increased lipoprotein lipase activity, high-density lipoprotein (HDL) concentration in the blood, and haptoglobin messenger RNA content of the liver. The presence of heparin in culture media did not directly affect proliferation of trypanosomes in vitro. However, the addition of plasma from infected rats treated with heparin to culture media decreased the number of trypanosomes. This effect was decreased by incubating the trypanosomes with benzyl alcohol, a known inhibitor of receptor-mediated endocytosis of lipoprotein. These data suggested that heparin administration reduced the number of trypanosomes in infected rats. Trypanosome lytic factor, a HDL and haptoglobin-related protein, protects humans and some animals from infection by Trypanosoma brucei brucei. In rats, increases in HDL and haptoglobin may affect the proliferation of T. b. gambiense.     

Trypanosoma lewisi Infections in Normal Rats and in Rats Treated with Dexamethasone

SYNOPSIS. Administration of dexamethasone to rats infected with Trypanosoma lewisi resulted in the development of exceedingly large populations of trypanosomes which were fatal to their hosts. The elevated levels of parasitemia in treated rats early in infections were thought not to be a result of an increased reproductive rate. However, trypanosomes in treated rats 2 days postinfection did have a higher coefficient of variation in total length and a greater percentage of dividing forms than those observed from infected rats which were not given the drug. The course of infection may be markedly altered not only in intensity but also in length by this corticosteroid. It is suggested that dexamethasone administered at the levels recorded to rats infected with T. lewisi inhibits the production of ablastin and trypanocidal antibodies.     

Trypanosoma brucei: anti-tubulin antibodies specifically inhibit trypanosome growth in culture

We previously observed that trypanosome tubulin immunizes mice against infection by this parasite. Here we describe the direct effect of anti-tubulin antibodies on trypanosomes, using rabbit antibodies to renatured (nTbTub) or SDS-PAGE denatured (dTbTub) Trypanosoma brucei tubulin. We also evaluate antibodies to synthetic tubulin peptides (STP) and rat brain tubulin (RbTub). The anti-nTbTub serum strongly inhibited trypanosome proliferation in culture, and immunoagglutinated trypanosomes even after heat inactivation of complement. The anti-dTbTub and the anti-STP sera also inhibited trypanosome growth and immunoagglutinated trypanosomes, but to a lesser extent than the anti-nTbTub, whereas the anti-RbTub serum had no effect. In Western blots these antibodies were species specific. Immunofluorescence showed that the surface of intact trypanosomes was not uniformly stained by any of these antibodies, but cells that had been permeabilised were labeled throughout the cytoplasm. This suggests that the variant surface glycoproteins (VSG) played no part in the generation of these inhibitory antibodies.     

Formation of the complex of the triterpene glycoside holothurin A with cholesterol in liposomal membranes

Interaction between holothurin A triterpene glycoside and lipid-cholesterol liposomes was studied by differential scanning microcalorimetry. Partial restoration of the peak of basic phase transition of dipalmitoyl phosphatidyl choline was shown to be related to the formation of holothurin A (in the membrane)-cholesterol complex. The data obtained are in favor of "sterol" hypothesis of the mechanism of membrane-tropic action of holothurin A.     

Lymphocyte function in experimental African trypanosomiasis. V. Role of antibody and the mononuclear phagocyte system in variant-specific immunity

The role of parasite-specific antibody and the mononuclear phagocyte system (MPS) in immunity to the African trypanosomes was examined. For this study C57BL/10SnJ mice were infected with Trypanosoma rhodesiense clone LouTat 1.0. Infected mice were injected with 75Se-labeled LouTat 1.0 trypanosomes, and clearance from the blood upon reexposure was measured throughout the course of infection. Clearance of labeled organisms occurred only on or after day 5, which was the day of natural elimination of LouTat 1.0 from the blood. Clearance was dependent on a functional immune system and correlated with the appearance of antibody to the variant-specific surface antigen (VSSA) of the trypanosomes. The ability to clear trypanosomes was transferred to normal, uninfected mice by immune serum. Both the IgM and IgG fractions of immune serum mediated the clearance, and VSSA-specific IgM fractions were as efficient in clearing LouTat 1.0 as the IgG fractions. Normal levels of complement (C3) were not required for clearance. The liver was the primary organ of clearance, and the ability of the liver to sequester radiolabeled trypanosomes was not impaired in the terminal phase of the disease or by large numbers of circulating trypanosomes present representing different variant antigenic types (VAT). We conclude that in African trypanosomiasis the MPS is not depressed in its ability to clear trypanosomes of the infecting VAT at any time during the course of infection. The observed clearance function requires parasite-specific antibody but normal levels of C3.     

Macrophage cytostatic effect on Trypanosoma musculi involves an L-arginine-dependent mechanism

Peritoneal macrophages from mice infected with an extracellular parasite, Trypanosoma musculi were effective in inhibiting parasite proliferation in vitro. This trypanostatic activity could be suppressed by NG monomethyl-L-arginine (NGMMA), a specific inhibitor of a biochemical pathway synthesizing L-citrulline and inorganic nitrogen oxides from L-arginine. Macrophages exerted this in vitro antiproliferative effect from the 10th day of infection on and this activity was maximum around 14th day of infection. Nitrite production paralleled development of macrophage trypanostatic activity. Macrophages collected from BCG-infected mice or treated with IFN-gamma in vitro also exerted a trypanostatic activity which was suppressed by NGMMA. A trypanostatic activity suppressed by NGMMA was also exerted by splenic macrophages from T. musculi-infected mice. Trypanostatic activity of IFN-gamma-treated macrophages was reduced by addition of anti-TNF-alpha showing the participation of TNF-alpha in IFN-gamma-mediated macrophage trypanostatic activity. Nitric oxide (NO) gas inhibited T. musculi proliferation. Addition of excess iron reversed the trypanostatic effect of both macrophages and NO gas. All these data showed that, as reported for a broad spectrum of microorganisms, activated macrophages displayed an antimicrobial effect on trypanosomes through the L-arginine: NO pathway that could participate in controlling infection in T. musculi-infected mice before appearance of antibody-dependent mechanisms. NO production by activated macrophages could trigger iron loss from critical target enzymes in trypanosomes.     

Beta-interferon inhibits cell infection by Trypanosoma cruzi

Preparations containing alpha/beta-interferon produced by L-929 cells were found to inhibit the capacity of bloodstream forms of Trypanosoma cruzi to associate with and infect mouse peritoneal macrophages or rat heart myoblasts. Marked reductions in the number of parasites per cell as well as in the percentage of cells associated with the trypanosomes were systematically observed in cultures of these cells that contained interferon. The inhibitory effect was abrogated in the presence of specific antibodies against alpha/beta-interferon, and purified beta-interferon induced a similar inhibitory effect, indicating that the active principle in the preparation was indeed interferon. Pretreatment of the parasites with alpha/beta-interferon reduced their infectivity for untreated host cells, whereas pretreatment of either type of host cell had no consequence on the interaction. The effect of interferon on the trypanosomes was reversible; the extent of the inhibitory effect was significantly reduced after 20 min, and was undetectable after 60 min when macrophages were used as host cells. Longer periods of time were required for the inhibitory effect to begin to subside (60 min) and to become undetectable or insignificant (120 min) when rat heart myoblasts were used. The results of additional studies performed with purified preparations of alpha- or beta-interferon revealed that only the latter was inhibitory of cell-parasite association. Because interferon is known to be produced shortly after T. cruzi infection and its administration has been shown to have a marked protective effect against this infection, our results suggest that the latter may involve inhibition of cell infection by interferon.     

The in vitro and in vivo effects of mefloquine on Trypanosoma brucei brucei.

Blood stream forms of Trypanosoma brucei brucei were grown over mouse kidney (MK) cells in minimum essential medium with various concentrations of mefloquine. The drug was observed to inhibit multiplication of the parasites in vitro. Groups of male albino mice were treated with mefloquine at 24, 48 and hours after T. b. brucei infection. Mefloquine at 0.03 mg/kg body weight administered for 4 consecutive days cleared the infection. No trypanosomes were detected in the blood of these mice for 90 days and over after the clearance of parasite from the blood. The doses for both the in vitro and in vivo therapy, were well below those prescribed for humans.     

The Biology of Trypanosoma diemyctyli, Tobey. III. Factors influencing the cycle of Trypanosoma diemyctyli in the vertebrate host Triturus v. viridescens.

SYNOPSIS. The effects of some environmental influences on the cycle of Trypanosoma diemyctyli in Triturus v. viridescens are described. Bleeding of the host produced a reduction in the number of trypanosomes but did not affect their growth rate. The temperature at which the host was maintained affected the cycle of the trypanosomes. The length of the post-inoculation latent stage increased from 24 hours at 25°C. to an indefinitely long time at 5°C. The trypanosomes were found to be dimorphic. Adult parasites of the short form had a range of 45–75 μ and those of the long form of 76–116 μ. Growth rate of the trypanosomes was inhibited or greatly retarded at temperatures of 10°C. or lower and was greatest at 25°C. The size attained by the parasites and the number of parasites were greatest at 15°C. At this temperature the infection was pathogenic and the dimorphic parasites were in their long form. At the higher temperatures (20–25°C.) the infection was non-pathogenic with the trypanosomes in their short form.
The infection is primarily one of adult newts. Experiments indicated that the larvae were resistant to the trypanosomes at all temperatures while the red efts were not. The latter are usually free from the trypanosomes because they are not exposed to them. Attempts to infect other newts and to locate any cryptic stages by the injection of blood and tissues from infected newts gave negative results.
Starvation, sodium salicylate, and treatments used to control fungus infection of the newts had no detectable effects on the trypanosomes.     

The effect of BCG on the course of experimental Chagas' disease in mice

Experiments were done to determine the effect of BCG treatment on longevity, development of parasitemia, and in vivo distribution of ⁵¹Cr-labelled trypanosomes in C3H(He) female mice infected with a Brazil strain of Trypanosoma cruzi. BCG sensitization of mice was accomplished by a single IV injection of 3·0 mg (wet weight) of BCG. Twenty-one days after BCG injection mice were infected with 5 × 10⁴ blood-form trypomastigotes. Parasitemia determinations were made on alternate days during the experiment while in vivo distribution of exogenously supplied ⁵¹Cr-epimastigotes was made in groups of BCG or PBS stimulated mice on day 15 of the T. cruzi infection.It was found that BCG sensitization had no effect on longevity or parasitemia development in T. cruzi infected C3H(He) female mice. There were, however, some differences in the in vivo distribution of parasites between BCG treated and control mice. BCG stimulated mice accumulated greater numbers of radiolabelled trypanosomes in the kidneys and small intestines while PBS treated mice were found to have greater numbers of labelled parasites in the liver. Although no significant differences were observed in longevity of BCG or PBS treated mice, it was noted that BCG treated animals which were bled for parasitemia determinations lived significantly longer than those which were merely observed for longevity.     

Nitric oxide-mediated cytostatic activity on Trypanosoma brucei gambiense and Trypanosoma brucei brucei.

Macrophages collected from BCG-infected mice or exposed in vitro to interferon-gamma plus lipopolysaccharide developed a cytostatic activity on Trypanosoma brucei gambiense and Trypanosoma brucei brucei. This trypanostatic activity of activated macrophages was inhibited by addition of N-monomethyl-L-arginine, an inhibitor of the L-arginine-nitric oxide (NO) metabolic pathway, indicating a role for NO as the effector molecule. Contrary to trypanosomes treated with N2gas, trypanosomes treated with NO gas did not proliferate in vitro on normal macrophages. Compared to mice infected with control parasites, mice infected with NO-treated parasites had decreased parasitemias in the first days postinfection and had a prolonged survival. Addition of excess iron reversed the trypanostatic effect of both activated macrophages and NO gas. These data show that activated macrophages exert an antimicrobial effect on T.b. gambiense and T.b. brucei through the L-arginine-NO metabolic pathway. In trypanosomes, NO could trigger iron loss from critical targets involved in parasite division. The participation of this effector mechanism among the other immune elements involved in the control of African trypanosomes (antibodies, complement, phagocytic events) remains to be defined.     

The Effects of Bacterial Endotoxin on the Infection of Mice with Trypanosoma cruzi*

Mice (Rockland strain) infected with Trypanosoma cruzi strain Tulahuén were treated with Escherichia coli endotoxin before, simultaneously with, and after inoculation of the parasites. The peak parasitemias of endotoxin-treated mice were higher than those of nontreated infected animals, regardless of the time of endotoxin administration. Peak parasitemias occurred at the same time in infected nontreated mice as in animals given endotoxin before or simultaneously with the trypanosomes. If endotoxin was administered 24 hr after the infection, a delay in the peak parasitemia was noted. Changes in the survival time were not observed unless endotoxin was given 24 hr postinfection. Infected mice had an increasing susceptibility to the lethal effect of endotoxin. The LD₅₀ of endotoxin decreased from 675 μg for normal mice to 230, 92, and 18 μg for infected animals 1, 3, and 8 days after the infection, respectively. In the infected mice, the endotoxin-detoxifying ability of the spleen was found to be impaired.     

Immunologic-mediated Protection of Trypanosoma congolense-Infected Mice by Polyribonucleotides

SYNOPSIS. When the synthetic polyribonucleotides polyinosinic acid-polycytidylic acid (poly I poly C) and polyadenylic acid-polyuridylic acid (poly A poly U) were tested against mice infected with varying numbers of Trypanosoma congolense the results varied with the method of passage of trypanosomes in mice. Thus, when 100 flagellates were passaged every 7th day and experiments were initiated with these trypanosomes from mice on the 7th day of their infection, the protective effects of poly I poly C and poly A poly U apparently varied independently of each other as assayed by the mean parasitemias and cumulative mortalities of infected mice. Poly I poly C-resistant and poly I poly C-susceptible variants (“R” and “S”, respectively) were isolated and maintained in mice by passage of 10⁶ trypanosomes every 4th day. Mice infected with these variants responded consistently to poly I poly C and poly A poly U injections in that mice infected with the “R” variant showed no response to either polyribonucleotide but those infected with the “S” variant consistently had a decrease in mean parasitemias and cumulative mortality when treated with poly I poly C, but not with poly A poly U. Using mice infected with the “S” variant, the protective effect of poly I poly C was dose-dependent and best protection was afforded when 1st injections of poly I poly C were given around the time of infection of the mice. The protective effects of the synthetic polyribonucleotides used in these experiments are probably due to their immunologic enhancing capacities and not to interferon. To support this, injections of Newcastle disease virus, a strong inducer of interferon in mice, did not protect against T. congolense in mice. It was not possible to determine whether serum from poly I poly C-treated mice had a greater neutralizing effect upon trypanosomes in vitro than serum from saline-treated mice since any effect of antibody was masked by a naturally occurring inhibitor in normal mouse serum which was reduced during infection. The protective effects of poly I poly C in T. congolense-infected mice were reversed by treatment with cyclophosphamide. This strong immunosuppressant, however, could not reverse the protective effects of poly I poly C against mice infected with Semliki Forest virus, strongly suggesting that the protective mechanisms stimulated by poly I poly C in these 2 infections were different. The response of mice infected with the “R” and “S” variants of T. congolense to synthetic polyribonucleotides is discussed in relation to antigenic variation of trypanosomes.     

Antifungal activity in triterpene glycosides from the sea cucumber Actinopyga lecanora

Bioassay-guided fractionation of methanol extract of sea cucumber Actinopyga lecanora led to the isolation of a new triterpene glycoside (1), along with two known glycosides holothurin B (3) and holothurin A (4). The structure has been elucidated on the basis of extensive 2D NMR spectroscopic analysis. The saponin (3) showed in vitro antifungal activity against all the twenty fungal test isolates including ATCC strain and was found to be most effective against Trychophyton mentagrophytes and Sporothrix schenckii, MIC range of 1.56 microg/ml.     

African trypanosomes and brain infection – the unsolved question

African trypanosomes induce sleeping sickness. The parasites are transmitted during the blood meal of a tsetse fly and appear primarily in blood and lymph vessels, before they enter the central nervous system. During the latter stage, trypanosomes induce a deregulation of sleep–wake cycles and some additional neurological disorders. Historically, it was assumed that trypanosomes cross the blood–brain barrier and settle somewhere between the brain cells. The brain, however, is a strictly controlled and immune‐privileged area that is completely surrounded by a dense barrier that covers the blood vessels: this is the blood–brain barrier. It is known that some immune cells are able to cross this barrier, but this requires a sophisticated mechanism and highly specific cell–cell interactions that have not been observed for trypanosomes within the mammalian host. Interestingly, trypanosomes injected directly into the brain parenchyma did not induce an infection. Likewise, after an intraperitoneal infection of rats, Trypanosoma brucei brucei was not observed within the brain, but appeared readily within the cerebrospinal fluid (CSF) and the meninges. Therefore, the parasite did not cross the blood–brain barrier, but the blood–CSF barrier, which is formed by the choroid plexus, i.e. the part of the ventricles where CSF is produced from blood. While there is no question that trypanosomes are able to invade the brain to induce a deadly encephalopathy, controversy exists about the pathway involved. This review lists experimental results that support crossing of the blood–brain barrier and of the blood–CSF barrier and discuss the implications that either pathway would have on infection progress and on the survival strategy of the parasite. For reasons discussed below, we prefer the latter pathway and suggest the existence of an additional distinct meningeal stage, from which trypanosomes could invade the brain via the Virchow–Robin space thereby bypassing the blood–brain barrier. We also consider healthy carriers, i.e. people living symptomless with the disease for up to several decades, and discuss implications the proposed meningeal stage would have for new anti‐trypanosomal drug development. Considering the re‐infection of blood, a process called relapse, we discuss the likely involvement of the newly described glymphatic connection between the meningeal space and the lymphatic system, that seems also be important for other infectious diseases.     

Late stage infection in sleeping sickness

At the turn of the 19(th) century, trypanosomes were identified as the causative agent of sleeping sickness and their presence within the cerebrospinal fluid of late stage sleeping sickness patients was described. However, no definitive proof of how the parasites reach the brain has been presented so far. Analyzing electron micrographs prepared from rodent brains more than 20 days after infection, we present here conclusive evidence that the parasites first enter the brain via the choroid plexus from where they penetrate the epithelial cell layer to reach the ventricular system. Adversely, no trypanosomes were observed within the parenchyma outside blood vessels. We also show that brain infection depends on the formation of long slender trypanosomes and that the cerebrospinal fluid as well as the stroma of the choroid plexus is a hostile environment for the survival of trypanosomes, which enter the pial space including the Virchow-Robin space via the subarachnoid space to escape degradation. Our data suggest that trypanosomes do not intend to colonize the brain but reside near or within the glia limitans, from where they can re-populate blood vessels and disrupt the sleep wake cycles.     

Contribution of the complement system to antibody-mediated binding of Trypanosoma gambiense to macrophages

The role of complement in the process of binding of trypanosomes to macrophages in the presence of specific antibody was studied. The aggregation of trypanosomes observed at the optimal antigen-antibody ratio or in the presence of excess antigen inhibited the binding. Complement caused clumped trypanosomes to dissociate, and the free trypanosomes, which were presumed to be coated with antibody that had fixed complement, readily attached to surfaces of phagocytes. Thus, complement was shown to contribute at the site of the antigen-antibody reaction to the creation of an environment suitable for the binding. It seems likely that the trypanosomes dissociated by complement adhered to C3 receptors of the macrophage. However, in the absence of complement and in regions of antibody excess, free trypanosomes also attached to phagocytes. Thus phagocytes may also have receptors for the Fc portion of aggregated antibody. Complement activated by the alternate pathway also enhanced attachment of trypanosomes to phagocytes, but the effect was not as rapid as it was when complement was activated by classical means.     

Anti-Trypanosoma brucei activity in Cape buffalo serum during the cryptic phase of parasitemia is mediated by antibodies

Cape buffalo are reservoir hosts of African trypanosomes. They rapidly suppress population growth of the highly antigenically variable extracellular haemoprotozoa and subsequently maintain a cryptic infection. Here we use in vitro cultures of trypanosomes cloned from Cape buffalo blood during cryptic infection, as well as related and unrelated trypanosomes, to identify anti-trypanosome components present in cryptic-phase infection serum. Trypanosome clone-specific complement-dependent trypanolytic IgM and IgG arose after appearance of target trypanosomes during cryptic infection. Serum collected late in the cryptic phase of infection contained complement-independent growth-inhibitory IgG which varied in activity among target trypanosomes. Removal of protein A/G-binding IgG from the serum restored its capacity to support trypanosome growth in vitro. Recovered growth-inhibitory IgG reacted with the variable surface glycoprotein (VSG) of parasites most affected by it, and reacted with trypanosome common antigens, notably the endosome-restricted tomato lectin-binding glycoproteins (TL-antigens). The inclusion of purified TL-antigens in culture medium did not affect the trypanosome growth-inhibitory activity of immune Cape buffalo serum. In addition, hyperimmune rabbit IgG against TL-antigens showed little or no binding to intact trypanosomes and did not affect trypanosome growth in vitro although it did react strongly with TL-antigens and trypanosome endosomes. We conclude that antibodies, particularly clone-specific (putatively VSG-specific) antibodies are responsible for the anti-trypanosome activity of cryptic phase infection serum consistent with a dominant role in parasite control in Cape buffalo.     

Trypanosoma congolense: Specific transformation in vitro of leukocytes from infected or immunized cattle

An assay to measure the specific proliferation in vitro of peripheral blood leukocytes (PBL) in response to ultrasonicated trypanosomes was adapted for use in cattle. The kinetics of mitosis exhibited by PBL from cattle which had been treated following infection with Trypanosoma congolense paralleled the development of a delayed-type skin reaction elicited with ultrasonicated and Formalin-fixed T. congolense. Responses in both tests were maximal on the fourth day after initiation. Specific proliferation of PBL harvested from cattle which had been immunized with intact, nonviable trypanosomes was also elicited in vitro by trypanosomal antigen. Peripheral blood leukocytes taken from cattle immunized with 50 μg of variant-specific surface antigen (VSSA) from T. brucei and from cattle infected with T. congolense were not stimulated to divide in vitro by ultrasonicated trypanosomes.