Ex vivo and in vitro impairment of CD36 expression and tumor necrosis factor-alpha production in human monocytes in response to Plasmodium falciparum-parasitized erythrocytes.

Severe malaria is associated with the failure of host defenses to control parasite replication, with the excessive secretion of proinflammatory cytokines such as tumor necrosis factor-alpha (TNF-alpha), and with the sequestration of parasitized erythrocytes (PEs) in the microcirculation of vital organs. The scavenger receptor CD36, known as a major sequestration receptor, has also been identified as an important factor in mediating nonopsonic phagocytosis of PEs by monocytes and macrophages. The specific consequence of this phagocytosis is a decrease in parasite-induced TNF-alpha secretion. We evaluated the variations in CD36 level and in lipopolysaccharide (LPS)-induced TNF-alpha production in monocytes from Plasmodium falciparum-infected patients and in vitro in the presence of PEs. Both the monocytes from infected patients and from in vitro culture showed a decrease of CD36 expression and a reduced production of TNF-alpha induced by LPS. Using incubation assays with no contact between monocytes and PEs, or in the presence of a soluble supernatant obtained from the incubation of monocytes and PEs, this study shows that decreased CD36 expression was posttranscriptional and not directly related to PEs phagocytosis. In addition, these culture models suggest that the reduced capacity of TNF-alpha production occurred in 2 phases. The early phase (24 hr) appeared to be CD36 dependent and the second phase (48 hr) was due to a soluble factor produced by PEs. These observations suggest that the control of the TNF-alpha production in malaria by monocytes was not entirely dependent on the phagocytosis of PEs by CD36 and that soluble factors produced by PEs could play a role in this process.

Neonatal screening for congenital toxoplasmosis in a cohort of 165 women infected during pregnancy and influence of in utero treatment on the results of neonatal tests.

The aim of this study was to determine the performances of methods used for the neonatal diagnosis of congenital toxoplasmosis. We included 165 pregnant women infected during pregnancy over a 10-year period. Fifty-seven cases of congenital toxoplasmosis were demonstrated (34.5%). Neonatal diagnosis gave positive results in 50 cases (88%). Parasites were isolated from placenta or cord blood in 61% of the infected newborns, more frequently from placenta (60%) than from cord blood (43%). This method was the only criterion of infection in 18% of these infected infants. The detection of specific IgM and IgA antibodies performed on 42 sera of infected infants allowed the diagnosis of congenital infection in 34 cases (81%). IgA antibodies were more frequently detected (60%) than specific IgM (50%). Neonatal and prenatal screening were carried out for 143 pregnant women. This combination diagnosed 39 of 40 infected infants (98%). Prenatal diagnosis identified 30 of 40 cases (75%). Nine cases were diagnosed through neonatal screening and one case with the postnatal follow-up. When prenatal diagnosis was positive, pyrimethamine and sulfadoxine were administered to the mothers (25 cases) in addition to spiramycin. Toxoplasma gondii was less frequently isolated in the placenta and the cord blood of these women (32% and 19%, respectively) than in women treated by spiramycin alone (83% and 63%) proving the antiparasitic action of these drugs. In conclusion, neonatal screening combining parasite detection in placenta and immunological methods on cord blood is essential particularly when prenatal diagnosis is negative. Therefore, when this diagnosis is positive, a treatment with pyrimethamine and sulfamide can be started in the first month of life.

Toxoplasma gondii secretes a calcium-independent phospholipase A(2).

Phospholipases A(2) (PLA(2)) play an important role in Toxoplasma gondii host cell penetration. They are also key enzymes in the host cell response to the parasite invasion. PLA(2) hydrolyse cellular phospholipids, releasing multiple inflammatory lipidic mediators. We have investigated the biochemical characterisation of T. gondii PLA(2) activity in a mouse-cultured tachyzoite homogenate and in the peritoneal exudate from infected mice, using the hydrolysis of a fluorescent phosphatidylglycerol labelled at the sn-2 position. Spectrofluorimetry and thin-layer chromatography showed a PLA(2) activity (about 0.5-2 nmol/min per mg), calcium-independent, secreted into infected mice peritoneal exudate, with a broad pH activity ranging between 6.5 and 9.5 and resistant to a great number of potential PLA(2) inhibitors except dithio-nitrobenzoic acid (1 mM). An associated phospholipase A(1) activity was also displayed. These results suggest that Toxoplasma gondii displays specific phospholipases different from host enzymes and probably involved at critical steps of infectious cycle.

Inhibitory activity of anti-interleukin-4 and anti-interleukin-10 antibodies on Toxoplasma gondii proliferation in mouse peritoneal macrophages cocultured with splenocytes from infected mice.

Cocultures of splenocytes from Toxoplasma gondii-immunized mice or from naive mice, separated by a transwell membrane from naive macrophage layers, induced a decrease in T. gondii proliferation in macrophages in comparison with cultures without splenocytes or cocultures with splenocytes from infected mice. Interleukin 4 (IL-4) and IL-10 levels increased in cocultures of splenocytes from infected mice with naive macrophages. In contrast, the levels of these cytokines decreased in cocultures with splenocytes from immunized mice. No correlation was found between the release of interferon-gamma (IFN-gamma) and the inhibition of parasite multiplication. Cocultures with splenocytes from immunized mice induced an increase in tumor necrosis factor-alpha (TNF-alpha) levels. In contrast, in cocultures with splenocytes from infected mice, TNF-alpha production decreased. In cocultures with splenocytes from infected mice, T. gondii proliferation in macrophages was neutralized by anti-IL4 or anti-IL10 antibodies and was associated with increased TNF-alpha production. Moreover, this study demonstrates the significant combined effect of IL-4 and IL-10 on the down-regulation of macrophage-effector functions. A soluble positive signal was given by splenocytes to induce the production of TNF-alpha by macrophages. This signal was inhibited by IL4 and IL10. This process is biologically relevant in the regulation of T. gondii proliferation.

Prevalence of drugs of abuse in urine of drivers involved in road accidents in France: a collaborative study.

The collaborative, anonymous, case-control study was intended to determine the prevalence of opiates, cocaine metabolites, cannabinoids and amphetamines in the urine of drivers injured in road accidents and to compare these values with those of non-accident subjects ("patients") in France. Recruitment was performed nationwide in the emergency departments of five hospitals and comprised 296 "drivers" aged 18 to 35 and 278 non-traumatic "patients" in the same age range. Females represented 28.4% of "drivers" and 44.2% of "patients." Screening for drugs in urine was performed by fluorescence polarization immunoassays in each center. Each positive result was verified using gas chromatography-mass spectrometry (GC-MS), in a single laboratory. Statistical analysis comprised single-step logistic regression and simultaneously took account of confounding factors and the final differences in prevalence values between the two populations or different subgroups. Cannabinoids were found in 13.9% of drivers (16.0% of males and 8.3% of females, p < 0.05) and 7.5% of patients (12.3% of males, 1.6% of females, p < 0.0001); only in females was this prevalence higher in injured drivers than in patients (p < 0.05). Opiates were present in 10.5% of drivers' and 10.4% of patients' urine samples (NS), and were more frequent in urine samples positive for cannabinoids, in drivers (p < 0.01) as well as in patients (p < 0.001). The prevalence of cocaine metabolites in drivers and patients was 1.0 and 1.1% and that of amphetamines 1.4 and 2.5%, respectively. No causal relationship between drugs and accidents should be inferred from this retrospective study. Nevertheless, the high prevalence of cannabis and opiate (licit or illicit) use in young people, whether injured drivers or patients, has potential implications for road traffic safety in France. Cocaine and amphetamines did not appear to be a major problem, unlike the experience in other countries.

Parasitemia and parasitic loads in acute infection and after anti-gamma-interferon treatment in a toxoplasmic mouse model.

Toxoplasma gondii infection was induced in Swiss-Webster mice by intraperitoneal inoculation of avirulent Beverley strain cysts. We studied parasitemia and parasitic loads first in acute toxoplasmosis, then in the chronic stage of the disease. In the latter stage a group of mice received weekly administration of a rabbit antiserum directed against mouse gamma-interferon. Parasitemia, sequentially determined by amplification of the B1 gene using polymerase chain reaction, persisted for more than 1 month in acute toxoplasmosis. Brain and lung parasitic loads, assessed by a tissue-culture method, were significantly increased in mice treated with anti-interferon. Moreover, this increase was prevented by concomitant administration of pyrimethamine and sulfadiazine, suggesting that early prophylaxis would be suitable. Surprisingly, the anti-interferon treatment induced neither abnormal clinical signs nor a significant rise in the parasitemia level. Such a model seems to be particularly appropriate for the comparison of different strains of Toxoplasma gondii in a moderately immunodeficient state.

The evolution of ocular toxoplasmosis in anti-interferon gamma treated mice.

PURPOSE: A clinico-histopathological cross correlation was made to study the mechanism of tissue damage in toxoplasmic retino-choroiditis during an experimental reactivation of chronic toxoplasmosis and to compare the influence of treatment by sulfadiazine on the retinal lesions. METHODS: Chronically infected Swiss-Webster mice were treated, six weeks after infection, with an avirulent strain of Toxoplasma gondii (Beverley strain) with polyclonal rabbit antibody directed against murine interferon gamma. RESULTS: Mice treated by anti-interferon gamma developed clinical lesions between day 5 and day 30 (lesions including single foci of retinochoroiditis, multifocal lesions or diffuse areas of retinal necrosis). These lesions did not arise from borders of pre-existing scars. The retina was photographed with an operating microscope fitted with a 90 diopter lens. Biological study showed a significant rise of parasitic loads in the eye and brain. Histological examination is in favour of free organism dissemination via retinal vessels; the lesions are restricted to the inner retina and ciliary body, the parasites migrated from extra-ocular cysts via the vasculature. No cysts were seen at the beginning of the study; they were found at the scar phase and appeared in mice treated with sulfadiazine. The clinical lesions were not caused by cysts but by coagulated necrosis in the retinal tissue. Parasite migration may have played a trigger role. CONCLUSIONS: The retinal damage was constituted either as a result of a toxic effect of the organisms or as a hypertensive reaction to the toxoplasma organism. The results of this study showed that the treatment with anti interferon gamma was sufficient to reactivate chronic infection.

[Efficacy and tolerance of praziquantel (Biltricide) in the treatment of distomatosis caused by Fasciola hepatica]. / Efficacité et tolérance du praziquantel (Biltricide) dans le traitement de la distomatose à Fasciola hepatica.

OBJECTIVES: Due to the side-effects of dehydroemetine, we have chosen praziquantel, a broad-spectrum antihelmintic, as a treatment for distomatosis secondary to Fasciola hepatica in humans. The aim of this retrospective study was to evaluate the efficacy and tolerance to praziquantel in patients with this disease. METHODS: Twenty-five patients (12 men) with a definite diagnosis of distomatosis and no previous treatment were followed-up between 8 months and 3 years (> 18 months in 76% of cases). The follow-up was based on clinical, biochemical and serological criteria. All patients received praziquantel (75 mg/kg/day orally) for 5 days. Treatment was started after endoscopic or surgical removal of parasites locolized in the biliary tract, in two patients. A similar therapeutic course was administered twice in four patients with persistent clinical symptoms, hypereosinophilia or arch 2 on immunoelectrophoresis. RESULTS: Cumulative rates of patients with normalized eosinophilia and seronegativation at 6, 9 and 12 months were 55, 65, 75% and 55, 70, 100%, respectively. Complete recovery occurred in 18 patients (72%) whereas hypereosinophilia persisted for more than one year in 5 patients. No side-effects, except transient nausea in a few cases, were observed. CONCLUSION: Since praziquantel seems to be both effective and well tolerated in a large proportion of patients, this drug can be recommended as a first choice for distomatosis due to Fasciola hepatica in human.

New in vitro assay based on glucose consumption for determining intraconazole and amphotericin B activities against Aspergillus fumigatus.

We developed a new in vitro method of evaluating antifungal molecules. Fungal growth was determined by measuring glucose consumption, the only carbon source in a synthetic medium. First, the growth of 12 Aspergillus fumigatus strains was studied. Glucose consumption was an excellent indicator of fungal growth. Second, the partial inhibition of growth was calculated in terms of the 90% or 50% inhibitory concentration for the 12 strains after treatment with itraconazole and amphotericin B. With a 3-day incubation time, the calculated 90% and 50% inhibitory concentrations agreed with those obtained by a macromethod and with those reported in previous publications. In each case the high degrees of efficacy of itraconazole and amphotericin B against A. fumigatus were confirmed. Partial inhibition induced by low concentrations of antifungal agents was quantifiable by this new method.

Failure of mouse peritoneal macrophage activation by the purified excreted/secreted antigens of Toxoplasma gondii.

This study was carried out to evaluate the effect of excreted/secreted antigens on macrophages infected by Toxoplasma gondii. Six proteins 28, 30, 45, 58, 63 and 145 kDa were separated by different chromatographic techniques. Mouse peritoneal macrophages were treated in vitro and in vivo with these purified fractions. Penetration and proliferation assays of T. gondii in the macrophages were performed in vitro. The different antigens used did not change the rate of penetration and proliferation of the parasites. Therefore, the secreted products, which are capable of provoking an immune response, could not directly activate the macrophages. Furthermore, the secreted products were not cytotoxic and neither did they possess a visible phospholipasic activity which would have increased penetration.

Kinetics study and characterisation of target excreted/secreted antigens of immunoglobulin G, M, A and E antibodies from mice infected with different strains of Toxoplasma gondii.

A kinetics study and characterisation of target excreted/secreted antigens of immunoglobulin (Ig) G, M, A and E antibodies were realised by Western blotting with immune sera of mice inoculated with three strains of Toxoplasma gondii: RH, C56 and S3. IgG antibodies of the immune sera recognised the major proteins of the three excreted/secreted antigen preparations with molecular masses of 30, 45, 63 and 77 kDa. IgM antibodies recognised proteins revealed by IgG antibodies but with variable intensities; some proteins were revealed during a short period. IgA antibodies did not recognise the 35-kDa antigen or the antigens inferior to 28 kDa. The RH excreted/secreted antigens were revealed with the highest intensity. The IgE antibodies were briefly detected in trace amounts during period from the 20th to the 35th day. The RH strain with its excreted/secreted antigens had the best antigenicity and is a good model for immunoprotection studies.

A method to obtain large quantities of Toxoplasma gondii tachyzoites with extreme purity.

In order to extract genetic material from the intracellular parasite Toxoplasma gondii, large numbers of organisms free of host cells are necessary. A method is described in which the RH strain of T. gondii was cultivated in nonadherent U937 cells. The purification was done by countercurrent elutriation. The resulting population of 10(10) T. gondii contained only 0.17% host cell material. The method is simple, rapid, and minimizes the use of animals.

Eicosanoid production by mouse peritoneal macrophages during Toxoplasma gondii penetration: role of parasite and host cell phospholipases.

The metabolism of endogenous arachidonic acid by mouse resident peritoneal macrophages infected in vitro with Toxoplasma gondii was studied. Prelabeling of macrophages with [5,6,8,9,11,12,14,15-3H]arachidonic acid and challenge with tachyzoites for 15 min resulted in a high mobilization of free labeled arachidonic acid (178%) in the culture medium. The parasites also triggered the synthesis of 6-keto-prostaglandin F1 alpha (47%), prostaglandin E2 (44%), leukotrienes C4 and D4 (33%) and 5-, 12-hydroxyeicosatetraenoic acids (155%). The study indicated that during the intracellular development phase of the parasites, 6-keto-prostaglandin F1 alpha (38%), prostaglandin E2 (31%) leukotrienes C4 and D4 (15%), hydroxyeicosatetraenoic acids (43%), and free arachidonic acid (110%) were secreted into the culture medium. Pretreatment of tachyzoites with phospholipase A2 inhibitors (4-p-bromophenacyl bromide and quinacrine) and no calcium in the culture medium resulted in inhibition of tachyzoite penetration into the macrophages and a decrease of the arachidonic acid metabolism. The triggering of the arachidonic acid cascade by T. gondii was dependent on the active penetration of the parasites into the macrophages, whereas preincubation of the macrophages with phospholipase A2 inhibitors did not affect penetration or free arachidonic acid release, thereby supporting a role for parasite phospholipase in the penetration process and in arachidonic acid mobilization from macrophage membrane phospholipids. Moreover, treatment of macrophages with phospholipase A2 inhibitors decreased the activities of the cyclooxygenase and lipoxygenase pathways, also suggesting an activation of host cell phospholipase A2 by the parasite.

IgA antibody response during acquired and congenital toxoplasmosis.

Toxoplasma gondii specific IgA and IgM antibodies were quantitated by an antibody capture agglutination assay in 260 patients with acquired toxoplasmosis and from 94 fetuses suspected of congenital toxoplasmosis and 30 infected children. In acquired toxoplasmosis, IgA antibodies to T gondii were found in 95% of the cases. In congenital toxoplasmosis IgA antibodies were more frequently detected (75%) in cord blood than IgM antibodies (61%). They persisted after birth, in some cases for up to 24 months. IgA antibodies were also detected in fetuses whose mothers had toxoplasmosis during their pregnancy. In infected fetuses IgM and IgA antibodies were detected in fetal blood as early as week 24 of pregnancy. Detection of IgA T gondii antibodies may be useful for the diagnosis of some recently acquired infection and for the diagnosis and follow up of the infection in the fetus and neonate.

Analysis by immunoblotting of Toxoplasma gondii exo-antigens and comparison with somatic antigens.

Two soluble Toxoplasma gondii antigen preparations were compared using the immunoblotting technique. The first preparation, which is commonly employed in ELISA tests, corresponds to a lytic extract of the parasite. The second known as exo-antigen, has yielded good results in cell immunity research and is obtained from Toxoplasma gondii culture supernatants. In both preparations, components exhibiting same molecular weight of 57-52 (doublet), 43, 38, 35, 30 and 20 kDa were revealed. In addition, two major 27- and 78-kDa components were detected in exo-antigens. The 30 kDa protein was intensely recognised by all sera, confirming the advantage of its use as an antigen in serological reactions. Exo-antigens could be employed as a soluble antigenic solution because they are easy to obtain, they display good antigenicity and their antigenic composition has been defined. Moreover their preparation does not require mouse inoculation. An evaluation of the enzyme-linked immunosorbent assays (ELISAs) involving these antigens has yet to be performed.

[Anatomopathologic diagnosis of parasitosis in Congo]. / Le diagnostic anatomopathologique des parasitoses au Congo.

In the part ten years, 9,650 anatomopathologic examinations have been carried out in the National Laboratory of Histopathology for Public Health in Brazzaville: 250 cases of parasitic infestation were diagnosed. In order of frequency were noted; 144 cases of bilharzia due to Schistosoma haematobium with 75 genito-urinary localisations and 51 appendicular cases, 31 cases of onchocercosis with a majority of subcutaneous nodules (17), 17 cases of digestive helminthiasis all locate in the ileo-caecal-appendix and discovered during systematic appendicular exploration, 13 cases of colitic or recto-sigmoidal amebiasis. These results point out 68 cases of appendicular parasitism. This should be in favour of more systematic anatomopathological examination of the ileo-caecal appendix both in tropical and in temperate regions.