Onchocerca volvulus. Monoclonal anti-idiotype antibody as antigen signal for the microfilaricidal cytotoxicity of diethylcarbamazine-treated platelets

Over the past 35 yr, diethylcarbamazine (DEC) has been the most widely used agent for the treatment of filarial diseases, particularly in onchocerciasis. The microfilaricidal action of DEC has been recently shown to be mediated by blood platelets with the additional triggering of a filarial excretory Ag (FEA). This FEA could be detected by using mAb in the serum of infected patients. By using one mAb (IA2(23] directed against Onchocerca volvulus and recognizing circulating Ag (Ab1), we purified by affinity chromatography the target molecule of IA2(23) (an O. volvulus glycoprotein recognized by IA2(23) mAb). This compound had a dose-dependent effect on the cytotoxic action of DEC-treated platelets. We subsequently produced an anti-idiotype mAb to Ab1 (Ab2), and considered the possibility of replacing the O. volvulus glycoprotein recognized by IA2(23) mAb by Ab2. Ab2 was selected according to its ability to inhibit the binding of radioiodinated Ab1 to the filarial target Ag. It induced the production of anti-O. volvulus antibodies (Ab3) in rats. At a constant concentration of DEC platelets, the addition of increasing amounts of Ab2 led to a dose-dependent cytotoxic effect against parasite larvae. Experiments performed with Ab2 on detergent solubilized surface proteins of platelets identified four bands of Mr 18, 26, 43.5, and 100 kDa, supporting the idea of the presence of binding sites on the platelets for a FEA required for the microfilaricidal cytotoxicity of DEC-treated platelets.     

Protection of mice and nude rats against toxoplasmosis by a multiple antigenic peptide construction derived from Toxoplasma gondii P30 antigen.

The first part of this work presents the sequence of the first 20 NH2 terminus residues obtained from P30, the major surface Ag of Toxoplasma gondii, purified by HPLC. A synthetic peptide (P30 48-67) has been prepared both in linear form and as a multiple antigenic peptide (MAP) construct. Immunization of mice and rats with the P30 48-67 MAP in the presence of IFA induces high levels of IgG antibodies that recognize both the linear peptide and the MAP construct in ELISA, and P30 in Western blots of NP-40-extracted tachyzoite Ag. Because these sera are negative in immunofluorescence assays with whole tachyzoites, it seems that IgG antibodies induced by P30 48-67 MAP, although recognizing the denatured structure, are unable to recognize the native protein. However, the protective effect of both constructs has then been studied in mice and nude rats. Whereas immunization of mice with the monomeric peptide does not confer any protection against oral infection with 1200 cysts of T. gondii 76K strain (mortality within 11 days), 40% of mice immunized with the MAP construct survived up to 75 days after infection. Nude rats were passively transferred with 5 x 10(4) T lymphocytes from P30 48-67 MAP-immunized Fischer rats before infection with 5 x 10(4) RH strain tachyzoites. They survived up to 40 days after infection and raised an intense IgG antibody response against P30, whereas nude rats transferred with control lymphocytes died within 21 days. This shows that immunization with P30 48-67 MAP also induces an efficient T cell immune response. The present work confirms the recently demonstrated role of P30 in protective immunity and shows the interest of peptide octameric constructions as inducers of partially protective immune responses in toxoplasmosis, as already demonstrated in schistosomiasis.     

Repeated oral administration of capsaicin increases anxiety-like behaviours with prolonged stress-response in rats

This study was conducted to examine the psycho-emotional effects of repeated oral exposure to capsaicin, the principal active component of chili peppers. Each rat received 1 mL of 0.02% capsaicin into its oral cavity daily, and was subjected to behavioural tests following 10 daily administrations of capsaicin. Stereotypy counts and rostral grooming were significantly increased, and caudal grooming decreased, in capsaicin-treated rats during the ambulatory activity test. In elevated plus maze test, not only the time spent in open arms but also the percent arm entry into open arms was reduced in capsaicin-treated rats compared with control rats. In forced swim test, although swimming duration was decreased, struggling increased in the capsaicin group, immobility duration did not differ between the groups. Repeated oral capsaicin did not affect the basal levels of plasma corticosterone; however, the stress-induced elevation of plasma corticosterone was prolonged in capsaicin treated rats. Oral capsaicin exposure significantly increased c-Fos expression not only in the nucleus tractus of solitarius but also in the paraventricular nucleus. Results suggest that repeated oral exposure to capsaicin increases anxiety-like behaviours in rats, and dysfunction of the hypothalamic-pituitary-adrenal axis may play a role in its pathophysiology.     

TAT and HA2 Facilitate Cellular Uptake of Gold Nanoparticles but Do Not Lead to Cytosolic Localisation

The methods currently available to deliver functional labels and drugs to the cell cytosol are inefficient and this constitutes a major obstacle to cell biology (delivery of sensors and imaging probes) and therapy (drug access to the cell internal machinery). As cell membranes are impermeable to most molecular cargos, viral peptides have been used to bolster their internalisation through endocytosis and help their release to the cytosol by bursting the endosomal vesicles. However, conflicting results have been reported on the extent of the cytosolic delivery achieved. To evaluate their potential, we used gold nanoparticles as model cargos and systematically assessed how the functionalisation of their surface by either or both of the viral peptides TAT and HA2 influenced their intracellular delivery. We evaluated the number of gold nanoparticles present in cells after internalisation using photothermal microscopy and their subcellular localisation by electron microscopy. While their uptake increased when the TAT and/or HA2 viral peptides were present on their surface, we did not observe a significant cytosolic delivery of the gold nanoparticles.     

Long-term treadmill exercise improves spatial memory of male APPswe/PS1dE9 mice by regulation of BDNF expression and microglia activation

Increasing evidence suggests that physical activity could delay or attenuate the symptoms of Alzheimer''s disease (AD). But the underlying mechanisms are still not fully understood. To investigate the effect of long-term treadmill exercise on the spatial memory of AD mice and the possible role of β-amyloid, brain-derived neurotrophic factor (BDNF) and microglia in the effect, male APPswe/PS1dE9 AD mice aged 4 months were subjected to treadmill exercise for 5 months with 6 sessions per week and gradually increased load. A Morris water maze was used to evaluate the spatial memory. Expression levels of β-amyloid, BDNF and Iba-1 (a microglia marker) in brain tissue were detected by immunohistochemistry. Sedentary AD mice and wildtype C57BL/6J mice served as controls. The results showed that 5-month treadmill exercise significantly decreased the escape latencies (P < 0.01 on the 4th day) and improved the spatial memory of the AD mice in the water maze test. Meanwhile, treadmill exercise significantly increased the number of BDNF-positive cells and decreased the ratios of activated microglia in both the cerebral cortex and the hippocampus. However, treadmill exercise did not significantly alleviate the accumulation of β-amyloid in either the cerebral cortex or the hippocampus of the AD mice (P > 0.05). The study suggested that long-term treadmill exercise could improve the spatial memory of the male APPswe/PS1dE9 AD mice. The increase in BDNF-positive cells and decrease in activated microglia might underpin the beneficial effect.     

Optimization of fecal sample preparation for untargeted LC-HRMS based metabolomics

Introduction

Collecting feces is easy. It offers direct outcome to endogenous and microbial metabolites.

Objectives

In a context of lack of consensus about fecal sample preparation, especially in animal species, we developed a robust protocol allowing untargeted LC-HRMS fingerprinting.

Methods

The conditions of extraction (quantity, preparation, solvents, dilutions) were investigated in bovine feces.

Results

A rapid and simple protocol involving feces extraction with methanol (1/3, M/V) followed by centrifugation and a step filtration (10 kDa) was developed.

Conclusion

The workflow generated repeatable and informative fingerprints for robust metabolome characterization.

     

A suppressive lymphokine of platelet cytotoxic functions

The in vitro stimulation of mononuclear cells from human peripheral blood with mitogens is known to induce the release of factors (monokines and lymphokines) that possess distinct biologic activities. The present data describe the presence in Con A- and antigen-stimulated T cell supernatants (of man or rat) of a factor able to inhibit, in a dose-dependent manner, the platelet cytotoxicity toward the young larvae of Schistosoma mansoni. The production of oxygen metabolites by IgE-coated platelets, stimulated by anti-IgE or the specific antigen, was, likewise, strongly inhibited by this lymphokine. The producing T lymphocyte subpopulation was identified as OKT 8+. This suppressive lymphokine of platelet functions had an m.w. of 15,000 to 20,000 and a pI of 4.6. It was heat- and acid-stable and sensitive to trypsin and proteinase K, but neuraminidase had no effect on its activity. This platelet suppressive activity was specifically absorbed by platelet membrane, suggesting its action through the binding to a receptor.