Characterization of an IL-2 mimetic with therapeutic potential.

Human interleukin-2 (IL-2) interacts with two types of functional receptors (IL-2R alpha betagamma and IL-2R betagamma) and acts on a broad range of target cells involved in inflammatory reactions and immune responses. IL-2 is also used in different clinical trials aimed at improving the treatment of some cancers and the recovery of CD4 lymphocytes by HIV patients. The therapeutic index of IL-2 is limited by various side effects dominated by the vascular leak syndrome. We have shown that a chemically synthesised fragment of the IL-2 sequence can fold into a helical tetramer likely mimicking the quatemary structure of an hemopoietin. Indeed, peptide p1-30 (containing amino acids 1 to 30, including the sequence corresponding to the entire alpha helix A of IL-2) spontaneously folds into an alpha-helical homotetramer and stimulates the growth of T-cell lines expressing human IL-2R beta, whereas shorter versions of the peptide lack helical structure and are inactive. At the cellular level, p1-30 induces lymphokine-activated killer (LAK) cells and preferentially activates CD8 low lymphocytes and natural killer cells, which constitutively express IL-2R beta. A significant IFN-gamma production is also detected following p1-30 stimulation. A mutant form of p1-30 (Asp20-->Lys) which is likely unable to induce vascular leak syndrome remains capable to generate LAK cells like the original p1-30 peptide. Altogether our data suggest that p1-30 has therapeutic potential.

The first alpha helix of interleukin (IL)-2 folds as a homotetramer, acts as an agonist of the IL-2 receptor beta chain, and induces lymphokine-activated killer cells.

Interleukin (IL)-2 interacts with two types of functional receptors (IL-2Ralphabetagamma and IL-2Rbetagamma) and acts on a broad range of target cells involved in inflammatory reactions and immune responses. For the first time, we show that a chemically synthesized fragment of the IL-2 sequence can fold into a molecule mimicking the quaternary structure of a hemopoietin. Indeed, peptide p1-30 (containing amino acids 1-30, covering the entire alpha helix A of IL-2) spontaneously folds into an alpha-helical homotetramer and stimulates the growth of T cell lines expressing human IL-2Rbeta, whereas shorter versions of the peptide lack helical structure and are inactive. We also demonstrate that this neocytokine interacts with a previously undescribed dimeric form of IL-2Rbeta. In agreement with its binding to IL-2Rbeta, p1-30 activates Shc and p56(lck) but unlike IL-2, fails to activate Janus kinase (Jak)1, Jak3, and signal transducer and activator of transcription 5 (STAT5). Unexpectedly, we also show that p1-30 activates Tyk2, thus suggesting that IL-2Rbeta may bind to different Jaks depending on its oligomerization. At the cellular level, p1-30 induces lymphokine-activated killer (LAK) cells and preferentially activates CD8(low) lymphocytes and natural killer cells, which constitutively express IL-2Rbeta. A significant interferon gamma production is also detected after p1-30 stimulation. A mutant form of p1-30 (Asp20-->Lys), which is likely unable to induce vascular leak syndrome, remains capable of generating LAK cells, like the original p1-30 peptide. Altogether, our data suggest that p1-30 has therapeutic potential.

Types, numbers and distribution of synapses on the dendritic tree of an identified visual interneuron in the brain of the locust.

The descending contralateral movement detector (DCMD), an identified descending interneuron in the brain of the locust Schistocerca gregaria has been investigated by using light and electron microscopy. We describe the fine structure, distribution and numbers of synapses that it receives from another identified brain neuron, the lobular giant movement detector (LGMD), and from unidentified neurons. The DCMD dendrites emerging from the integrative segment vary in form and number between individuals and sexes but always form a flattened dendritic domain. The arborizations and the integrative segment appear to be exclusively postsynaptic. Two types of synaptic contacts (Type 1 and 2) onto the DCMD can be discerned as having either round (Type 1) or pleiomorphic synaptic vesicles (Type 2) and by large (Type 1) or small (Type 2) subsynaptic appositions. Contact zones of Type 1 synapses are smaller than those of Type 2. LGMD-synapses are of Type 1 and occur intermingled with presynaptic sites of unidentified units. Some branches of the DCMD receiving input from unidentified units are devoid of contacting LGMD processes. Synapses of both types are randomly distributed over the DCMD integrative segment and at fibres with similar sizes. Type 1 synapses are much more frequent than Type 2 synapses and their number is negatively correlated with fibre diameter. For a whole DCMD dendritic arborization, a total of 8500 active zones of chemical synapses has been calculated, including a minimum of 2250 LGMD-synapses and about 1000 Type 2 synapses. The DCMD may thus receive a considerable amount of input from as yet unidentified neurons.

Plasmodium falciparum sporozoite invasion is inhibited by naturally acquired or experimentally induced polyclonal antibodies to the STARP antigen.

Antibody(Ab)-mediated inhibition of sporozoite invasion of hepatocytes is a mechanism that has been clearly demonstrated to act upon Plasmodium falciparum pre-erythrocytic stages in humans. Consequently we have analyzed the Ab response to a recently identified P. falciparum sporozoite surface protein, STARP, in malaria-exposed individuals and tested the inhibitory effect of these Ab upon hepatocyte invasion in vitro. STARP-specific IgG were detected in 90 and 61% of sera from regions where individuals were exposed to 100 and 1-5 infectious bites per year, respectively. These IgG were predominantly of the cytophilic IgG1 or IgG3 type. STARP and the major sporozoite surface protein, CS, elicited equivalent IgG levels in adults. When affinity purified from either African immune sera or the serum of an individual experimentally protected by irradiated sporozoite immunization, STARP-specific Ab prevented up to 90% of sporozoites from invading human hepatocytes. The dose-dependent and reproducible inhibition was more pronounced than that observed with human CS-specific Ab affinity purified under identical conditions. Substantial reduction of sporozoite invasion was also observed with Ab induced by artificial immunization with recombinant STARP protein and reactive with the native protein. Taken together with recent findings of human cytotoxic T lymphocytes specific for this antigen, these results promote the interest of studying the efficacy of STARP as a target for immune effector mechanisms operating upon pre-erythrocytic stages.

A novel Plasmodium falciparum sporozoite and liver stage antigen (SALSA) defines major B, T helper, and CTL epitopes.

In the search for subunit vaccines that are able to induce the type of sterile, protective immunity achieved by irradiated sporozoites, there is increasing evidence that defense mechanisms directed at the intrahepatic stage and Ags expressed at this stage are critical. We have initiated a systematic search for such molecules and report here the identification and partial characterization of a novel Plasmodium falciparum gene encoding a 70-kDa protein, expressed in both sporozoite and liver stages (SALSA), with a vaccine potential that stems from its antigenic features. Antigenicity and immunogenicity studies were conducted in individuals exposed to malaria, in immunized mice, and in chimpanzees, using a recombinant protein and two synthetic peptides. Results show that the SALSA nonrepetitive sequence defines 1) major B cell epitopes, as shown by a high prevalence of Abs to each peptide in three African areas differing in their level of endemicity; 2) Th epitopes, as demonstrated by lymphoproliferation and IFN-gamma secretion in cells from the individuals from one of the low transmission areas, as well as helper effect upon Ab secretion in mice; and 3) epitopes for cytolytic lymphocytes, demonstrated in immunized and sporozoite-challenged chimpanzees, and associated with MHC class I leukocyte Ags. The latter are of particular importance, because this is the only part of the malaria life cycle in which the parasite is located in a cell expressing class I Ags and because CD8+ lymphocytes were found to be responsible for protection in experimental models.

The Mycobacterium bovis 32-kilodalton protein antigen induces human cytotoxic T-cell responses.

The 30-kDa protein (P32) is a mycobacterial secreted antigen which is homologous in Mycobacterium bovis and M. tuberculosis. In vitro, P32 induced T-cell proliferation. M. tuberculosis- or P32-stimulated T-cell lines lysed macrophages pulsed with P32 or M. tuberculosis, respectively. We conclude that P32 stimulates cytotoxic T cells specifically.

ACTIVITY OF GIANT INTERNEURONES AND OTHER WIND-SENSITIVE ELEMENTS OF THE TERMINAL GANGLION IN THE WALKING CRICKET

Using intracellular recording techniques in stationary walking crickets (Gryllus bimaculatus), we have investigated the relationship between locomotion and the activity of interneurones ascending from the terminal ganglion. Nine different types of giant interneurones (GI) were characterized during walking and standing. One third of them reduced their activity, while the others enhanced their spike rate, during walking. These physiological properties were strictly correlated with morphological characteristics such as axon position in the longitudinal tracts of the terminal ganglion. In general, ventral GIs reduced and dorsal GIs increased their spike frequency during walking. In some of them, there was a weak but significant correlation between the spike rate and translational speed, but no correlation with rotational speed. In all GIs except 10-3a, the changes in activity occurred at the start of walking. In GI 10-3a, an increase in membrane potential and spike rate was observed before the start of locomotion. Therefore, an intrinsic mechanism within the central nervous system operating on GI 10-3a is suggested. Additionally, the activities of filiform hair receptors and of previously undescribed small ascending interneurones (SAI) have been studied during walking. About 80 % of the receptors slightly increased their spike rate during walking, while one SAI became more active during walking and another one was hardly affected. The physiological properties of ascending interneurones are discussed with respect to their modulation and particular function during walking.

Fast immunopurification of small amounts of specific antibodies on peptides bound to ELISA plates.

ELISA is widely used as a means to detect antibodies, but the potential of ELISA plates as an immunosorbent for the purification of specific antibodies does not seem to have been evaluated. In this study, ELISA plates coated with peptides representing short sequences of various antigens from Plasmodium falciparum, the etiologic agent of human malaria, have been successfully used as a means to purify small amounts of the corresponding antibodies. ELISA plates, identical to those used for antibody detection, also permitted the evaluation of various elution conditions for each pairing of peptide and serum; we tested four eluting buffers (0.2 M glycine, pH 2.5; 0.2 M lysine, pH 11.5; 3.0 M MgCl2, 0.075 M Hepes, 25% ethylene glycol, pH 7.1-7.2 and 4 M NH4SCN in 0.1 M NaH2PO4, pH 6.0) with four pairs of peptides and sera. The ELISA plates could also be used to estimate the affinity of the eluted antibodies by the technique of Pullen et al. (1986). The eluted antibodies were compared to those obtained by immunopurification on recombinant proteins adsorbed on nitrocellulose filters. In contrast to the latter, they were not contaminated by antibodies directed against the carrier moiety of the recombinant protein. When used in immunofluorescence assays with various stages of the parasite the antibodies immunopurified on peptides bound to ELISA plates were able to react with the native antigens in the parasite.

RTIME: a program for time-series measurements and evaluation in electrophysiology with the AT-PC.

This work describes a program which uses a standard RS232-serial interface of an IBM-AT-compatible microcomputer to receive via four data lines input from any laboratory equipment (spike discriminators, stimulus equipment, etc.) generating pulses between 0 to -15 V (low) and +5 to +15 V (high). Therefore, program operation is independent from any hardware extensions of the computer. The time of occurrence of input pulses is recorded with a resolution of 10 microseconds. Depending on processor speed and optional on-line display of interval histograms, a maximum sampling rate of 1.3-6 kHz is attained. Designed primarily for electrophysiological applications, the program comprises an extensive set of functions for off-line analysis of data either in the time- or in the phase-domain. The program is controlled by menu-selectable commands with detailed on-line explanation and is therefore suited for use even by operators without computer experience, e.g., students on courses in experimental physiology. Elaborate schemes of evaluation can be defined as macro commands to speed up and simplify complex data analysis in actual research.

[Electrophysiological study of pro-arrhythmogenic effects of erythromycin]. / Etude électrophysiologique des effets pro-arythmogènes de l'érythromycine.

Three cases of torsades de pointe induced by erythromycin have recently been reported. After observing a new case, the authors tried to demonstrate the possible mechanism of the arrhythmogenic action of this molecule. Twenty-two patients undergoing electrophysiological studies in the catheter laboratory to determine the cause of syncope were given an intravenous injection of 10 mg/Kg of erythromycin lactobionate. The drug was injected in 1 minute (bolus) in 11 patients (Group A). The other 11 patients (Group B) received the drug by slow intravenous infusion (20 minutes). Electrophysiological parameters were measured before and after erythromycin. A significant prolongation of the atrial refractory periods (+39 ms), ventricular refractory periods (+20 ms), QT (+20 ms) and QTC intervals (+42 ms) was observed in Group A. These electrophysiological effects could explain an arrhythmogenic action similar to that of antiarrhythmic drugs in Group I of Vaughan-Williams' classification. The slow intravenous infusion of erythromycin in Group B considerably reduced these undesirable secondary effects. This difference was directly related to serum concentrations of the molecule.

The neurotrophic factor neuroleukin is 90% homologous with phosphohexose isomerase.

Neuroleukin (NLK) is a protein of relative molecular mass (Mr) 56,000 (56K) secreted by denervated rat muscle and found in large amounts in muscle, brain, heart and kidneys. The protein is a neurotrophic factor for spinal and sensory neurons and a lymphokine product of lectin-stimulated T-cells. It also induces immunoglobulin secretion by human mononuclear cells. Molecular clones of NLK have been expressed in monkey COS cells and the product was shown to have the same biological and biochemical properties as the extracted protein. NLK is abundant in muscle, brain and kidney, but is active at concentrations of 10(-9) to 10(-11) M, similar to those for other polypeptide factors. We have cloned the gene for pig muscle phosphohexose isomerase (PHI) (EC 5.3.1.9) which catalyses the conversion of glucose-6-phosphate to fructose-6-phosphate, an obligatory step in glycolysis, and determined its amino-acid sequence. Surprisingly, it is 90% homologous to the sequence of mouse neuroleukin.

[Study of electrophysiological effects of betaxolol]. / Etude des effets électrophysiologiques du bétaxolol.

Betaxolol is a new cardioselective beta-blocker without any intrinsic sympathomimetic activity. A study of the cardiac electrophysiological effects of this molecule used intravenously was carried out in 20 patients. Betaxolol prolongs the sinus cycle (+ 25%, p 0.001), prolongs the sino-atrial conduction time (+ 20.1%) and atrio-hissian conduction time (+ 10.8%, p 0.001), and prolongs the refractory periods at all levels: atrial (+ 8%), nodal (+ 20.8%), right ventricular (+ 4.3%). These findings confirm the electrophysiological characteristic effects of the beta-blockers series; however, the moderate but significant increase of the refractory ventricular period, seems to provide this molecule with a certain degree of originality in this therapeutic class.

[Acute myocarditis in Lyme's syndrome. Value of myocardial scintigraphy with gallium 67]. / Myocardite aiguë au cours d'un syndrome de Lyme. Intérêt de la scintigraphie myocardique au gallium 67.

The authors report the case of a 35 year old man with no known previous cardiac disease. One month after a tic bite causing diffuse abdominal erythema, he was admitted to hospital with fever, breathlessness and bradycardia. The electrocardiogramme showed first degree atrioventricular block with a sinoatrial block (SA = 200 ms, AH = 240 ms). Echocardiography eliminated the diagnosis of pericardial effusion. Thallium 201 myocardial scintigraphy was pathological and showed irregular global uptake suggesting cardiomyopathy. Gallium 67 scintigraphy showed increased uptake in the left ventricle. The evolution was uncomplicated with normalisation of clinical, ECG and radiological changes. Cardiac catheterisation and angiography eliminated ischaemic and primary cardiomyopathy. Control radionuclide investigations were normal at one month: there was no persistent abnormal Gallium uptake. The diagnosis of Lyme's syndrome was confirmed by positive serology with successive titres of 1/1024 and 1/2048 (significant at titres over 1/256). This unusual case illustrates: the risk of myocardial disease in Lyme's syndrome; the diagnostic value of Gallium 67 scintigraphy in acute myocarditis: Gallium seems to fix specifically on inflamed tissues, so distinguishing myocarditis from primary cardiomyopathies.

The tail flattening reflex in Lumbricus: reconstitution after tail amputation and modifications in segmental nerve roots.

On stimulation of the head, the caudal 20-30 segments of intact earthworms exhibit a marked dorso-ventral flattening reflex which is an element of the escape response. When the tail is removed this reflex is reconstituted gradually in segments anterior to the amputation level without regeneration of new tail segments. It takes longer to reestablish the flattening if larger parts of the body are cut off, but after 60 days p.o. all animals regained the full response independent of the number of segments amputated. Extirpation of three consecutive ganglia from the ventral nerve cord also suffices to develop flattening anterior to the operated segments, but the reaction is less pronounced and appears later than after tail amputation. Moreover, it vanishes at the same time the normal tail flattening is observed again, presumably after regeneration across the gap in the cord. In different regions of the body, area and perimeter distributions of transversely sectioned axons in the three pairs of segmental nerves (SN I-III) of the cord were measured. Characteristic changes along the length of the animal were found for SN I and SN III. In tail segments the axonal size distributions are more variable than "typical" segments. The flattening reflex is mediated by axons in SN III (Pallas and Drewes, 1981). In body segments having reconstituted behavioral characteristics of the lost tail segments, the area of the largest axons and the general size distribution in SN III approximate those found in normal tail segments.

[Early diagnosis of postinfarction ventricular aneurysm. Value of contrast scintigraphy]. / Le diagnostic précoce de l'anévrysme ventriculaire post-infarctus. Intérêt de la scintigraphie de contraste.

Early diagnosis of a ventricular aneurysm after myocardial infarction may provide a valuable means of preventing certain complications. We studied 55 patients with acute myocardial infarction on the 8th day by Technetium 99 angioscintigraphy with study of wall motion and ejection fraction at equilibrium. The results were compared with left ventriculography performed three months after infarction. Angioscintigraphy demonstrated 17 cases of dyskinesia, 16 cases of hypokinesia, and 22 cases of akinesia, divided into two subgroups depending on whether the ejection fraction was lower (n = 11) or higher than 40% (n = 11). Contrast angiography at 3 months confirmed the presence of 27 aneurysms, 17 akinetic plaques and 11 cases of hypokinesia. Comparison between the two series of investigations showed excellent correlations: in the group of dyskinesia (17 cases) an aneurysm was confirmed in 15 cases; in the group of hypokinesia (16 cases) only one aneurysm was demonstrated by the reference angiography. The results were less uniform in the group with akinesia. In the subgroup with a low ejection fraction, 9 out of 11 cases progressed to aneurysm and, in retrospect, should be considered as likely aneurysms. There were only 2 aneurysms (2/11) in the subgroup with localised akinesia and a high ejection fraction. The overall results suggest that angioscintigraphy at the 8th day of myocardial infarction is a reliable means of detecting left ventricular aneurysm; in cases of dyskinesia or widespread akinesia with an ejection fraction of less than 40% the presence of an aneurysm was confirmed in 24 out of 28 patients (86%).(ABSTRACT TRUNCATED AT 250 WORDS)

A 'hidden line' algorithm for 3D-reconstruction from serial sections--an extension of the NEUREC program package for a microcomputer.

An algorithm is described to generate pictures in arbitrary perspective from serially sectioned biological material. Only those parts of the object which lie on a direct line of sight to the observer are included into the computed paper-plot, while hidden lines are omitted. The program was written for an inexpensive Apple II+ microcomputer as an extension of an earlier program library (NEUREC) for three-dimensional reconstruction. Application examples of neuronal and cardiac tissues are presented.

NEUREC - a program package for 3D-reconstruction from serial sections using a microcomputer.

A software package is described to reconstruct three-dimensional pictures in true perspective from a series of parallel sections using a low-cost computer system (Apple II plus). Data sampling via a graphic tablet and graphical output on the monitor screen or a digital plotter are assigned to different programs under control of a menu program. The number of data representing the object under study is unlimited. Originally written in BASIC, the programs were translated to machine language. As an application of the package, reconstructions of an identified large interneuron of the locust brain are presented.

[The electrophysiologic effects of intravenously administered bepridil]. / Etude des effets électrophysiologiques du bépridil utilisé par voie veineuse.

The electrophysiological effects of intravenous bepridil were studied at doses of 2 mg/kg and 3 mg/kg. The drug was tested on 25 patients of both sexes who were undergoing electrophysiological investigation for other reasons (11 normal ECGs, 11 pathological ECGs, 3 WPW syndromes). Electrophysiological parameters before and after bepridil were compared. The following changes were noted: - the refractory period was significantly prolonged at all conducting levels; - the sinus cycle was significantly lengthened in a practically constant fashion (+9,2 p. 100); - conduction in the sinoatrial and atrioventricular nodes was significantly prolonged (+15 p. 100 and + 11 p. 100); - the Luciani-Wenckebach point was significantly decreased in the anterograde, and even more so in the retrograde, directions (-20 and -23 p. 100 respectively); - however, infrahisian conduction was not affected either in patients with normal or pathological ECGs. The drug was effective in blocking the accessory pathway in two of the three patients with the WPW syndrome. A study of drug plasma levels did not show a dose-effect relationship. The plasma levels were extremely variable from one subject to another, suggesting tissue fixation of the active part of the molecule. These results demonstrate the valuable electrophysiological effects of bepridil which, by its mode of action, is very similar to amiodarone.