Plant-derived recombinant F1, V, and F1-V fusion antigens of Yersinia pestis activate human cells of the innate and adaptive immune system.

Plague is still endemic in different regions of the world. Current vaccines raise concern for their side effects and limited protection, highlighting the need for an efficacious and rapidly producible vaccine. F1 and V antigens of Yersinia pestis, and F1-V fusion protein produced in Nicotiana benthamiana administered to guinea pigs resulted in immunity and protection against an aerosol challenge of virulent Y. pestis. We examined the effects of plant-derived F1, V, and F1-V on human cells of the innate immunity. F1, V, and F1-V proteins engaged TLR2 signalling and activated IL-6 and CXCL-8 production by monocytes, without affecting the expression of TNF-alpha, IL-12, IL-10, IL-1beta, and CXCL10. Native F1 antigen and recombinant plant-derived F1 (rF1) and rF1-V all induced similar specific T-cell responses, as shown by their recognition by T-cells from subjects who recovered from Y. pestis infection. Native F1 and rF1 were equally well recognized by serum antibodies of Y. pestis-primed donors, whereas serological reactivity to rF1-V hybrid was lower, and that to rV was virtually absent. In conclusion, plant-derived F1, V, and F1-V antigens are weakly reactogenic for human monocytes and elicit cell-mediated and humoral responses similar to those raised by Y. pestis infection.

[French language training course: malaria workshop organized by Institut Pasteur de Madagascar]. / Formation francophone Sud/Sud: l'atelier paludisme de l'Institut Pasteur de Madagascar.

The Malaria Workshop organized by Institut Pasteur de Madagascar is an original course that applies innovative concepts to training of health professionals involved in malaria control in endemic countries. Course objectives are to enhance the skills needed to fight malaria (transversal competencies, critical approach, and position statement), to reinforce project cycle management proficiency, and to demonstrate how the Internet can be used as a source of documentation to compensate for geographical isolation. The Malaria Workshop is a six-consecutive-week full-day course that has been presented once a year since 2003. Seventy-six researchers, physicians or health ministry officials have already benefited from this training. Teaching methods emphasize andragogy that facilitates a learner/mentor relationship promoting exchange rather than transmission of knowledge and problem-based learning that engages learners to take an active part in gathering information. These methods in combination with the diverse backgrounds and experience of course participants foster a positive dynamic environment for learning that is monitored by weekly progress evaluation. Follow-up surveys have confirmed the positive effect of this training on the professional performance of former participants who become more involved in program development and fund-raising efforts. A professional network is growing and learners are starting to their experience. In this report workshop organizers describe the course's origins and concepts and present the conclusions drawn based on the first five yearly sessions.

Monitoring susceptibility to sulfadoxine-pyrimethamine among cases of uncomplicated, Plasmodium falciparum malaria in Saharevo, Madagascar.

Intermittent preventive treatment (IPT) of pregnant women with sulfadoxine-pyrimethamine (SP) is being considered as a routine practice in Madagascar, mainly to decrease the risks of malaria-associated severe anaemia in the women, and of low birthweight in their babies. There is, however, relatively little information available on the efficacy of SP when used, in Madagascar, to treat cases of Plasmodium falciparum malaria. In a preliminary study, carried out in 2003 in the village of Saharevo, 36 uncomplicated cases were each treated with a standard dose of SP and with paracetamol and then followed up for 28 days. No case of therapeutic failure occurred and all the asexual parasitaemias cleared by day 3. It therefore appears that SP is effective against P. falciparum in Saharevo (and probably in the whole, rural district of Moramanga in which the village lies). This is an encouraging observation to make before IPT is initiated throughout the country.

Effects of methotrexate on the oxidative metabolism of cultured rabbit articular chondrocytes.

OBJECTIVE: The mechanism of action of methotrexate (MTX) in inflammatory joint disease is still unclear. We examined the possible interactions of MTX with the oxidative metabolism of rabbit articular chondrocytes. METHODS: Cell cultures of articular chondrocytes enzymatically isolated from juvenile New Zealand white rabbits were incubated 24 h with either MTX (0.22 or 1.1 microM), bacterial lipopolysaccharide (LPS, 50 microg/ml), or both. Cytofluorometry was then performed using 2',7'-dichlorofluorescein diacetate (DCFH-DA), rhodamine 123 (Rh123), or propidium iodide (PI). These fluorochromes allow evaluation of cellular production of H2O2, mitochondrial membrane potential, and cell viability, respectively. In a separate experiment, we used the Griess colorimetric technique to evaluate cellular nitric oxide (NO) production. RESULTS: Addition of MTX alone (0.22 or 1.1 microM) inhibited spontaneous production by chondrocytes of H2O2 (p < 0.01 and p < 0.001, respectively) and NO (p < 0.01 both concentrations). The LPS induced increase in H2O2, production was inhibited by MTX at 0.22 and 1.1 microM (p < 0.01 both concentrations), whereas the LPS induced increase in NO synthesis was not influenced by MTX, even at 1.1 microM. MTX did not significantly modify mitochondrial activity or cell viability. CONCLUSION: MTX at therapeutic concentrations in vitro inhibits the production of H2O2 and NO by unstimulated chondrocytes, and only the H2O2 overproduction by LPS stimulated chondrocytes. These properties may contribute to the therapeutic effect of MTX in RA.

Plasmodium coatneyi: differential clinical and immune responses of two populations of Macaca fascicularis from different origins.

Two species of macaques, including two Macaca fascicularis from the Philippines, two M. fascicularis from Mauritius, and one Macaca mulatta, were experimentally infected with blood stages of Plasmodium coatneyi and followed during their clinical, parasitological, biological, and immunological evolution. Plasma cytokine (TNF-alpha, IL-1beta, IL-6, IFN-gamma) production peaked for all monkeys 11 days after inoculation, concomitantly with peaks of parasitemia. Only the M. fascicularis from the Philippines survived the infection. The main features, which discriminated nonfatal from fatal cases, were the observation in M. fascicularis from the Philippines of a mean CD4+/CD8+ ratio below I and of their ability, as revealed by mitogenic stimulation of whole blood, to produce increasing amounts of IFN-gamma as infection evolved. The contribution of environmental and genetic factors, which may differentiate the three groups of monkeys and therefore explain fatal or nonfatal evolution of the infection among them, is discussed.

Factors influencing resistance to reinfection with Plasmodium falciparum.

A treatment-reinfection study design was used to investigate the relationships between host immunologic and/or genetic factors and resistance to reinfection with Plasmodium falciparum. Sixty-one children in Gabon were enrolled in a cross-sectional study to measure the prevalence of each human plasmodial species. All were given amodiaquine for radical cure of parasites, and 40 were subsequently followed-up for 30 weeks. Successive blood smears were examined to measure the delay of reappearance in blood of asexual stages of P. falciparum parasites. Presence of infection during the cross-sectional survey was associated with male sex, non-deficient glucose-6-phosphate dehydrogenase activity, plasma interleukin-10 level, and anti-LSA-Rep antibody concentration. Resistance to reinfection was related to the presence of anti-LSA-J antibodies, and the absence of anti-LSA-Rep antibodies. Moreover, P. malariae-infected subjects were usually co-infected with P. falciparum, and were also more rapidly reinfected with P. falciparum after treatment, compared with those without P. malariae infection.

In vitro antimalarial activity of a new organometallic analog, ferrocene-chloroquine.

The in vitro activities of new organometallic chloroquine analogs, based on 4-amino-quinoleine compounds bound to a molecule of ferrocene, were evaluated against chloroquine-susceptible, chloroquine-intermediate, and chloroquine-resistant, culture-adapted Plasmodium falciparum lineages by a proliferation test. One of the ferrocene analogs totally restored the activity of chloroquine against chloroquine-resistant parasites. This compound, associated with tartaric acid for better solubility, was highly effective. The role of the ferrocene in reversing chloroquine resistance is discussed, as is its potential use for human therapy.

Lack of correlation between hydrogen peroxide production and nitric oxide production by cultured rabbit articular chondrocytes treated with fluoroquinolone antimicrobial agents.

The arthrotoxicity of fluoroquinolone antibacterial agents so far remains unexplained. Recent experimental data have indicated an early stimulation of the oxidative metabolism within articular chondrocytes. An in vitro model was designed to analyse the production of oxygen-derived reactive species and glutathione by immature rabbit articular chondrocytes, and the influence of different fluoroquinolones on this model was examined. Primary cultures of chondrocytes were exposed to pefloxacin, ofloxacin or ciprofloxacin at 10 mug/ml, for 24 or 48 hr. Flow cytometric analysis used the vital tracer 2',7'-dichlorofluorescein diacetate (DCFH-DA) and evaluated the production of H(2)O(2) and NO by chondrocytes. Separately, NO production and intracellular glutathione levels were evaluated, with the Greiss colorimetric technique and the Tietze method, respectively. With each fluoroquinolone tested, intracellular levels of the fluorescent compound dichlorofluorescein (oxidized form of DCFH-DA) were significantly higher in treated chondrocytes than in control cells. No significant modification of NO or of glutathione cellular levels was noted. Fluoroquinolones stimulate H(2)O(2) production in immature articular chondrocytes, but have no apparent effect on either NO or glutathione production, at least in the early stages of the chondrotoxicity.

Effects of fluoroquinolones on cultured articular chondrocytes flow cytometric analysis of free radical production.

Using flow cytometry, we previously established in an ex vivo model that fluoroquinolones induce a stimulation of the oxidative metabolism in immature chondrocytes. To assess these findings in an in vitro model, primary cultures of immature articular chondrocytes were incubated with four quinolone solutions: ofloxacin, ciprofloxacin, nalidixic acid at 10 micrograms/ml for 24 hr and pefloxacin at 1, 10 and 100 micrograms/ml for various periods of time (2, 4, 6, 12, 24 and 48 hr). Three fluorochromes were used: DCFH-DA, reflecting cellular production of H2O2, rhodamine 123 (Rh123) and 10-N-nonyl-acridine orange (NAO), which are specific for mitochondrial activity and mass, respectively. In immature chondrocyte cultures treated with pefloxacin, ofloxacin and nalidixic acid at 10 micrograms/ml for 24 hr, levels of cellular fluorescent dichlorofluorescein DCF (oxidized form of DCFH-DA) were significantly higher than in control cells. No significant increase could be registered with ciprofloxacin. In the same experimental conditions, incorporation of Rh123 and NAO was not significantly modified. Pefloxacin (10 micrograms/ml, 24 hr) did not induce any significant increase of DCFH-DA processing either in mature chondrocytes or in alveolar macrophages removed from immature rabbits. Quinolones induce in vitro an early stimulation of the oxidative metabolism in immature but not in mature chondrocytes, a phenomenon that could explain juvenile onset of quinolone arthropathy. This in vitro model could be proposed as an easy and reproducible method for screening potential arthrotoxicity of antimicrobial agents, capable of stimulating the formation of H2O2.