Complete tumour regressions induced by vaccination with IL-12 gene-transduced tumour cells in combination with IL-15 in a melanoma model in mice

In the present study, IL-12 gene-transduced B78-H1 melanoma cells (B78/IL-12) were used in combination with IL-15 to treat melanoma-bearing mice. Genetically modified B78/IL-12 cells, when injected subcutaneously, induced strong activation of antitumour mechanisms resulting in complete loss of tumourigenicity. In a therapeutic model, intratumoural injection of irradiated B78/IL-12 cells significantly delayed tumour growth and led to the regression of melanoma in one case. Similarly, consecutive daily injections of IL-15 markedly inhibited tumour progression with occasional curative effects. When used in combination, vaccination with B78/IL-12 cells and treatment with IL-15 caused eradication of established tumours in all treated mice. The combined treatment with B78/IL-12 cells and IL-15 activated not only a local response against tumour, but also induced systemic antitumour immunity that led to a delay or inhibition of tumour development at a distant site. In vitro studies demonstrated that when used together, B78/IL-12 cells and IL-15 induced a shift from a type Th2 to a type Th1 response. Activation of the antitumour immune response in double-treated mice resulted, in part, from stimulation of IFN- production and was accompanied by the development of cytotoxic effectors in the spleen. As shown in a macrophage tumouricidal assay, macrophages could also play a role in the antitumour effects. The results confirmed that vaccination with IL-12 gene-modified tumour cells is superior to the treatment with unmodified tumour cell vaccine and, additionally, showed that IL-15 is an excellent candidate for adjuvant therapy, inducing synergistically not only a delay of tumour growth but also its complete eradication.     

New imidazolidinedione derivatives as antimalarial agents

A series of new N-alky- and N-alkoxy-imidazolidinediones was prepared and assessed for prophylactic and radical curative activities in mouse and Rhesus monkey models. New compounds are generally metabolically stable, weakly active in vitro against Plasmodium falciparum clones (D6 and W2) and in mice infected with Plasmodium berghei sporozoites. Representative compounds 8e and 9c showed good causal prophylactic activity in Rhesus monkeys dosed 30 mg/kg/day for 3 consecutive days by IM, delayed patency for 19-21 days and 54-86 days, respectively, as compared to the untreated control. By oral, 9c showed only marginal activity in causal prophylactic and radical curative tests at 50 mg/kg/day×3 and 30 mg/kg/day×7 plus chloroquine 10 mg/kg for 7 days, respectively.     

Membrane Damage by an α-Helical Pore-forming Protein,Equinatoxin II,Proceeds through a Succession of Ordered Steps

Actinoporin equinatoxin II (EqtII) is an archetypal example of α-helical pore-forming toxins that porate cellular membranes by the use of α-helices. Previous studies proposed several steps in the pore formation: binding of monomeric protein onto the membrane, followed by oligomerization and insertion of the N-terminal α-helix into the lipid bilayer. We studied these separate steps with an EqtII triple cysteine mutant. The mutant was engineered to monitor the insertion of the N terminus into the lipid bilayer by labeling Cys-18 with a fluorescence probe and at the same time to control the flexibility of the N-terminal region by the disulfide bond formed between cysteines introduced at positions 8 and 69. The insertion of the N terminus into the membrane proceeded shortly after the toxin binding and was followed by oligomerization. The oxidized, non-lytic, form of the mutant was still able to bind to membranes and oligomerize at the same level as the wild-type or the reduced form. However, the kinetics of the N-terminal helix insertion, the release of calcein from erythrocyte ghosts, and hemolysis of erythrocytes was much slower when membrane-bound oxidized mutant was reduced by the addition of the reductant. Results show that the N-terminal region needs to be inserted in the lipid membrane before the oligomerization into the final pore and imply that there is no need for a stable prepore formation. This is different from β-pore-forming toxins that often form β-barrel pores via a stable prepore complex.     

Effect of 14 days of bed rest in older adults on parameters of the body sway and on the local ankle function

This study explored the effects of a 14-day horizontal bed rest (BR) without countermeasures on postural sway, maximal voluntary torque and precision of voluntary torque matching. Sixteen subjects were tested before, immediately after and two weeks after BR. The increase in frequency and amplitude after BR was comparable for both sway subcomponents (rambling and trembling) in medial-lateral direction. But in anterior–posterior direction, rambling increased more in frequency (?7% vs. +31%, p < 0.05) while trembling increased more in amplitude (+35% vs. +84%, p < 0.05). The drop in maximal voluntary torque after BR was present for plantar flexion (p < 0.05) but not for dorsal flexion. After the BR, the subjects were less precise in the dorsal flexion torque matching task (p < 0.05). All the observed parameters, except the dorsal flexion torque matching error, returned back to the pre-BR values after the two weeks of re-conditioning. Results of this study indicate that body sway subcomponents responded differently to BR. Based on these findings, it was not possible to draw clear assumptions on the effects of neural and structural changes on body sway.     

Characterization of the Lipid-Binding Site of Equinatoxin II by NMR and Molecular Dynamics Simulation

Equinatoxin II (EqtII) is a soluble, 20 kDa pore-forming protein toxin isolated from the sea anemone Actinia equina. Although pore formation has long been known to occur in distinct stages, including monomeric attachment to phospholipid membranes followed by detachment of the N-terminal helical domain and oligomerization into the final pore assembly, atomistic-level detail of the protein-lipid interactions underlying these events remains elusive. Using high-resolution solution state NMR of uniformly-¹⁵N-labeled EqtII at the critical micelle concentration of dodecylphosphocholine, we have mapped the lipid-binding site through chemical shift perturbations. Subsequent docking of an EqtII monomer onto a dodecylphosphocholine micelle, followed by 400 ns of all-atom molecular dynamics simulation, saw several high-occupancy lipid-binding pockets stabilized by cation-π, hydrogen bonding, and hydrophobic interactions; and stabilization of the loop housing the conserved arginine-glycine-aspartate motif. Additional simulation of EqtII with an N-acetyl sphingomyelin micelle, for which high-resolution NMR data cannot be obtained due to aggregate formation, revealed that sphingomyelin specificity might occur via hydrogen bonding to the 3-OH and 2-NH groups unique to the ceramide backbone by side chains of D109 and Y113; and main chains of P81 and W112. Furthermore, a binding pocket formed by K30, K77, and P81, proximate to the hinge region of the N-terminal helix, was identified and may be implicated in triggering pore formation.     

Role of the carotid chemoreceptors in regulation of inspiratory onset

We studied the effect of intermittent tidal breaths of CO2-enriched air (3-9% CO2) on the duration of expiratory time (TE) in five trained dogs, before and after (3 dogs) bilateral surgical denervation of the carotid bodies (CBD). During studies the dogs lay quietly, either awake or in nonrapid-eye-movement sleep, and breathed through a cuffed endotracheal tube inserted via a chronic tracheostomy. Studies were conducted during bilateral blockade of the cervical vagus nerves (VB), achieved by circulating cold alcohol through radiators placed around exteriorized vagal skin loops. Prior to CBD, single breaths of CO2 significantly shortened TE and thus advanced the onset of the subsequent inspiration. Further, the decrease in TE induced by the CO2 stimulus was in direct proportion to the inspired CO2 concentration. Thus 3% CO2 shortened TE by 1.82 +/- 0.93 (SD) s, and 9% CO2 by 3.44 +/- 1.53 s. Changes in TE occurred in the absence of associated changes in either tidal volume or inspiratory time. After CBD, test breaths of CO2 failed to shorten TE during VB. We conclude that the carotid bodies have the ability to mediate changes in the timing of inspiratory onset in response to a transient CO2 stimulus.