Study of the stress proteins secreted by Leishmania donovani after treatment with edelfosine, mitelfosine and ilmofosine, and morphological alterations analyzed by electronic microscopy.

We studied the stress proteins induced in protozoa Leishmania donovani after treatment with edelfosine, miltefosine and ilmofosine. We studied the morphological and structural modifications caused in the promastigote forms of the parasite after treatment with the three alkyl-lysophospholipids (ALPs). A resistant strain of L. donovani to miltefosine was obtained and the morphological modifications were observed. The stress proteins induction was studied in promastigote forms and also in amastigote-like forms obtained in vitro. The proteins synthesized with the three alkyl-lysophospholipids were compared to those obtained by heat shock. The axenic amastigote forms synthesized a pattern of different proteins for those observed in the promastigote forms. The morphological alterations were observed under electronic microscopy. The membrane and mitochondria were the organs most affected by the three ALPs. We noted an apparition of vacuoles and vesicles in the treated promastigotes. In the resistant strain, we noted myelin bodies in the treated and untreated parasites.

Effect of alkyl-lysophospholipids on some aspects of the metabolism of Leishmania donovani.

Alkyl-lysophospholipids (ALPs), developed initially to be antitumor agents, have proved highly effective in the treatment of visceral leishmaniasis, a disease caused by the species making up the protozoan complex Leishmania donovani. Although their effectiveness is known, the mode of action against this parasite is not completely understood. In the present work, we have studied the effect of 3 derivatives, edelfosine, miltefosine, and ilmofosine. Using nuclear magnetic resonance spectroscopy ('H-NMR), we have examined the excreted catabolites from glucose metabolism in the promastigote forms treated with these compounds. The ALPs at concentrations of 19 and 38 microM inhibit the excretion of acetate, succinate, and pyruvate. The effect of edelfosine, miltefosine, and ilmofosine on the activity of the enzymes hexokinase, glycerolkinase 3-PD, phosphoglucose isomerase, superoxide dismutase, and phospholipase C were also examined. Glycerolkinase 3-PD and phosphoglucose isomerase are generally insensitive to the compounds, whereas hexokinase and superoxide dismutase are inhibited by miltefosine and ilmofosine. The ALPs exhibited an activated effect against the phospholipase C activity. Alkyl-lysophospholipids were shown to have a significant effect on several enzymes in important biochemical pathways indispensable for the survival of L. donovani promasigotes.

Aspects of the cytological activity of edelfosine, miltefosine, and ilmofosine in Leishmania donovani.

To discover the mode of action of alkyl-lysophospholipids in Leishmania donovani, we studied the effects of edelfosine, miltefosine, and ilmofosine on intracellular pH, the parasite's cell cycle, and the induction of apoptosis. The effect of the alkyl-lysophospholipids was combined with that of inhibitors of some pumps and exchange regulators of intracellular pH (Na+/ H+; Cl-/CO- 3; and the Na+/K+ ATPase). The effect of the 3 alkyl-lysophospholipids on intracellular pH was indirect; the primary action occurred in the parasite's cell membrane. To determine intracellular pH, we used flow cytometry for the macrophages and axenic amastigotes and spectrofluorometry for the promastigote forms. Apoptosis and the cell cycle were studied by flow cytometry. Treatment of the extracellular promastigote form of L. donovani with the 3 alkyl-lysophospholipids induced death by apoptosis, whereas in the infected cell they caused necrosis rather than apoptosis. Miltefosine and ilmofosine at doses of 38 microM caused G2/M cell cycle inhibition in L. donovani promastigotes.

Host humoral immune response to Leishmania lipid-binding protein.

SUMMARY We report on the use of Leishmania donovani lipid-binding proteins (LBPs) as antigens capable of being recognized by serum from immunocompetent patients from southern Spain suffering from visceral leishmaniasis and from Peruvian patients with localized cutaneous leishmaniasis caused by Leishmania braziliensis. The absorbance found by immunoenzymatic techniques gave significantly different results for the serum samples from patients with and without leishmaniasis. Specificity by ELISA testing was 93.2% and sensibility 100%. Dot blots from human patient serum samples or naturally infected dogs from Spain gave similarly significant results. All the human serum samples from individuals with visceral leishmaniasis and the Leishmania-positive canine samples recognized two bands, with molecular weights of 8 and 57 kDa. The serum from individuals with cutaneous leishmaniasis caused by L. braziliensis recognized an additional band of 16 kDa. We discuss the role of Leishmania FABP and compare the immunological reactions found with serum samples from other protozoan infections such as toxoplasma and Chagas as well as bacterial infections such as tuberculosis and syphilis.

Immunogenicity of two Echinococcus granulosus antigens EgA31 and EgTrp in mice.

Here, we investigate in mice the immunogenicity of two antigens EgA31 and EgTrp which are expressed by the larval stage of Echinococcus granulosus. These recombinant proteins were used alone or as a mixture (EgA31-EgTrp) to immunize BALB/c mice. By flow cytometry, we have shown that the ratio CD4+/CD8+ of splenocytes were significantly higher in the antigen-immunized groups. The specific antibody in the sera and cytokine producing splenocytes was evaluated by enzyme-linked immunosorbent assay. EgA31, EgTrp or EgA31-EgTrp elicited high antibody titer of IgG and IgA. Among IgG isotypes, IgG1 was predominant for each antigen tested alone or combined. The production of IL-12, IFN-gamma, IL-10 and IL-6 cytokines was significantly higher in mice immunized with recombinant proteins. Our results suggest that, in BALB/c mice, a mixed Th1/Th2, response to EgA31, EgTrp and EgA31-EgTrp is obtained. The use of both antigens separately or in combination as candidate vaccine proteins is discussed.

An in vitro model to evaluate the cytokine response in Echinococcus infections.

With the aim of proposing an alternative model to animal experimentation, we investigated cytokine production in response to antigens in an in vitro system. This is a co-culture system of healthy human leukocytes and enterocyte-like Caco-2 cells. The antigens tested, EgA31, EgTrp, and FABP1, are candidates for vaccines in infections caused by Echinococcus spp. in the gut. All three have previously been described in the protoscolex stage and belong to protein families which confer protective immunity against several helminths. In this study, we evaluate the Th1/Th2 profile (Th1: IL-12, IFN-gamma; Th2: IL-6, IL-10) in response to protoscoleces, EgA31 and the mixture of EgA31, EgTrp and FABP1. No cytokine production was detected in response to protoscoleces. Neither IFN-gamma nor IL-6, but a significant IL-10 and IL-12 concentration was detected in response to both types of antigens. These findings suggest that EgA31 and the mixture EgA31/EgTrp/FABP1 generated an immunogenic response associated with a mixed Th1/Th2 cytokine.

Ion regulation in the different life stages of Trypanosoma cruzi.

Different ion and pH regulation mechanisms have been detected in the three main life stages of Trypanosoma cruzi: epimastigote, metacyclic trypomastigote and amastigote. Treatment with amiloride showed that the Na(+)/H(+) exchanger participated in all three forms. The Na(+)/K(+) ATPase exchanger appeared to be more active in the epimastigote than in the other forms. V-H(+)-ATPase inhibitors revealed the activity of this regulatory mechanism in the amastigote and epimastigote forms, while treatment with oligomycin only affected the amastigotes. The HCO(-)(3)/Cl(-) exchanger was found in all stages as well as in the intracellular pH-regulatory mechanism after abrupt basification. We deduce that ion regulation in T. cruzi is a complex process and depends upon the precise stage of the cell cycle of the parasite. It would seem to be an important mechanism, allowing the parasite to adapt to the changing environmental conditions within which it develops.

Course of Modic 1 six months after lumbar posterior osteosynthesis.

STUDY DESIGN: A prospective study was conducted to investigate the outcome of the Modic Type 1 inflammatory signal in magnetic resonance imaging (MRI) in 17 patients with chronic low back pain 6 months after instrumented posterior lumbar arthrodesis. OBJECTIVE: To assess the course of the inflammatory signal after stabilization of a painful intervertebral segment by posterior instrumentation alone visualized on MRI systematically performed 6 months after the operation. SUMMARY OF BACKGROUND DATA: In 1988, Modic and colleagues described three degenerative stages of vertebral endplates and subchondral bone. The inflammatory stage, or Stage 1, is correlated with substantial functional disability. According to these authors, Stage 1 lesions naturally transform into Stage 2, the fatty stage. In the literature, patients with Modic 1 signal tend to have good results after arthrodesis, better than those with Modic 2 lesions. METHODS: This study included 17 patients (average age, 46 years) who had experienced chronic low back pain more than 1 year and showed Modic 1 changes in MRI and disc narrowing on plain radiographs. Every patient underwent posterior screw-rod osteosynthesis and posterolateral arthrodesis. Disc disease had occurred subsequently to discectomy (n = 7), rapidly destructive disc disease (n = 5), or spondylolisthesis resulting from spondylolysis (n = 5). Clinical results were assessed according to a visual analog scale for pain, a functional disability score for the evaluation of patients with low back pain (Eiffel), and the validated French version of the self-administered Dallas quality-of-life test (DRAD). RESULTS: Systematic MRI at 6 months showed transformation from Modic 1 to Modic 0 (normal endplate signal) in 4 patients and transformation from Modic 1 to Modic 2 in the remaining 13 patients. Clinical evaluation was performed at 6 months (at the same time as the MRI) and at 1 year. In every patient, there was improvement in the visual analog score and the functional score, which remained stable at 1 year. CONCLUSIONS: According to the literature, most Modic 1 lesions change to become Stage 2 lesions in 18 to 24 months. In this study, 17 patients with Modic Type 1 signal had changes after 6 months. It appears that posterior osteosynthesis combined with posterolateral arthrodesis accelerates the course of Modic 1 lesions, probably by correcting mechanical instability.

Seroprevalence of anti-cysticercus antibodies among the children living in the urban environs of Maputo, Mozambique.

Blood and faecal samples were collected from 269 children (aged 0-15 years) who lived in the urban environs of Maputo, the capital city of Mozambique. Antibodies against Cysticercus cellulosae were detected, at a titre of at least 1:100, in 56 (20.8%) of the blood samples. When the stool samples were checked for Taenia solium and other helminths, both as direct smears and after formalin-ether concentration, 180 (67.0%) were found to contain at least one helminth species. The parasites most commonly detected in the faecal samples were Trichurus trichiura (36.0%) and Ascaris lumbricoides (35.7%). Only in one sample (0.4%) were gravid proglottids of Ta. solium detected, but Hymenolepis nana (1.1%) and H. diminuta (0.4%) were also found. A positive correlation between seropositivity for anti-cysticercus antibodies and subject age, and positive associations between such seropositivity and infection with A. lumbricoides and infection with Tr. trichiura were observed. None of the other demographic and environmental factors investigated--the child's sex, religion and access to toilets and/or piped water, the type of house in which he or she lived, the number of individuals in the household to which he or she belonged, and whether that household had pets or raised livestock--showed any apparent association with either the seroprevalence of anti-cysticercus antibodies or infection with any intestinal helminth. The use of water from the common sewage-drainage system for agricultural irrigation in the study area probably causes most of the contamination with intestinal parasites.

In vitro and in vivo activity of new rhodium (III) complexes against Leishmania donovani.

The activities of 17 new rhodium drug complexes were determined against Leishmania donovani promastigotes. The five most active salts were selected: [Rh(III)(2-amino-6-ethoxybenzothiazole)(4)Br(2)](+)Br(-); [Rh(III)(2-bromothiazole)(4)(Br)(2)](+)Br(-); [Rh(III)(mefloquine)(4)(Cl)(2)](+)Cl(-); [Rh(III)(2-mepacrine)(4)(Cl)(2)](+)Cl(-), and [Rh(III)(oxamniquine)(4)(Cl)(2)](+)Cl(-), which induced growth-inhibition rates of more than 50% at 24 h of treatment and at the maximum dosage tested. The cytotoxicity assays on the macrophage cell line J-774 showed high cytotoxicity for the salts [Rh(III) (mefloquine)(4)(Cl)(2)](+)Cl(-), [Rh(III)(2-mepacrine)(4)(Cl)(2)](+)Cl(-) and [Rh(III)(oxaminquine)(4)(Cl)(2)](+)Cl(-) with a percentage of specific (15)Cr release of 49.3, 64.8 and 53.2% at 24 h of incubation and 100 microg/ml. Meanwhile, assays of the other compounds showed practically no cytotoxicity. The ultrastructural studies in the flagellates treated with the salt [Rh(III)(2-amino-6-ethoxybenzothiazole)(4)Br(2)](+)Br(-) showed some alterations in the nucleus of the parasites with a very condensed chromatin and an electrodense endosome. This compound showed a high in vivo activity in parasitized Wistar rats.