Structural features of aminoquinolines necessary for antagonist activity against botulinum neurotoxin

Certain aminoquinoline antimalarial compounds, such as chloroquine, antagonize the paralytic actions of botulinum neurotoxins (BoNT). These studies have been extended to determine the critical structural groups necessary for synthetic aminoquinolines to have antagonist activity against BoNT. Isolated mouse hemidiaphragms were maintained at 36 degrees C and indirectly stimulated; the resulting isometric twitch tensions were recorded as a measure of synaptic function. The muscles were exposed to the test compounds before being treated with a challenge concentration of BoNT (typically 0.2 nM of serotype A). The time to onset of 50% muscle paralysis due to BoNT was used to assess quantitatively the efficacy of the test compounds, which were then ranked on the basis of the concentrations necessary to delay paralysis by a specified time increment. Of the compounds tested, those having a 7-chloro-4-aminoquinoline configuration, similar to chloroquine (or the structurally similar 6-chloro-9-amino acridine group in quinacrine), were most effective. Truncation of the alkyl-amino-alkyl group from chloroquine and conversion of the 4-amino nitrogen to a primary amine did not significantly alter its effectiveness as a BoNT antagonist. However, the 6-chloro- or 8-chloro- isomers of chloroquine were essentially ineffective. These results suggest that aminoquinolines antagonize the paralytic actions of BoNT through interaction with a selective, stereospecific site that is not well correlated with antimalarial activity.     

Effect of 3,4-diaminopyridine on the time course of decay of compound muscle action potential augmentation in the Lambert-Eaton myasthenic syndrome

3,4-Diaminopyridine (3,4-DAP) is known to be beneficial in the symptomatic treatment of the Lambert-Eaton myasthenic syndrome (LEMS). The effects of 3,4-DAP on the decay of postexercise augmentation were observed in 6 patients with LEMS. After 10 s maximal voluntary contraction, the amplitude of the compound muscle action potential (CMAP) recorded from abductor digiti minimi decayed exponentially after an initial rise. The rate of decay in CMAP amplitude was increased after treatment with 3,4-DAP, suggesting that this drug has an effect on the efflux of calcium ions from the presynaptic nerve terminal.     

Cytotoxicity of murine B lymphocytes induced by human VH4-34 (VH4.21) gene-encoded monoclonal antibodies

We previously found that a ventricular isovolumic pressure-time curve could be well fitted by the difference between two S-shaped logistic curves for the pressure rising and falling components, and called it "hybrid logistic" function: P(t)=A/[1+exp[-(4B/A)(t-C)]]-D/[1+exp[-(4E/D)(t-F)]]+G. We reported that the parameters of this hybrid logistic function are useful to characterize left ventricular contraction and relaxation comprehensively. In this study, we investigated how well this hybrid logistic function could fit the isometric twitch force-time curves of cross-circulated right ventricular papillary muscles of 7 dogs. This function precisely fitted the isometric force curves with correlation coefficients above 0.9996, much better than another fitting function (F(t)=C(t/A)(B)exp[1-(t/A)(B)]) proposed by Nwasokwa. The present results indicate that our hybrid logistic function can also reasonably express the canine right ventricular papillary muscle isometric twitch force-time curve. We suggest the possibility that the parameters of this hybrid logistic function are also useful to comprehensively characterize right ventricular papillary muscle twitch contraction and relaxation.     

Depletion of neutralising antibodies resensitises a secondary non-responder to botulinum A neurotoxin

The objective was to evaluate whether removal of neutralising antibodies potentially resensitises a secondary non-responder to botulinum neurotoxin A (BoNT/A). Neutralising antibodies directed against BoNT/A are produced during long term treatment with BoNT/A-hemagglutinin complex in up to 10% of patients with cervical dystonia. These patients become secondary non-responders. Other serotypes of BoNT are not yet generally available and may also bear the risk of inducing antibody formation. Plasma exchange (PE) (one treatment cycle) and immunoadsorption on a protein A column (IA-PA; three treatment cycles) was employed over 15 months to remove neutralising antibodies from a severely disabled secondary non-responder with cervical dystonia. After plasma exchange or IA-PA, BoNT/A was reinjected. Antibodies were measured with a sensitive functional toxin neutralising test. Repeated use of plasma exchange and IA-PA depleted neutralising antibodies to below the detection limit and subsequently allowed successful BoNT/A injection into dystonic muscles. No serious side effects were found related to the depletion of IgG. In conclusion PE or IA-PA performed before BoNT/A readministration may provide an alternative strategy in treating selected secondary non-responders who are severely disabled.     

Presynaptic protein interactions in vivo: evidence from botulinum A, C, D and E action at frog neuromuscular junction

The present study examines the paralytic action of botulinum neurotoxins at their natural target, the neuromuscular junction. We asked whether syntaxin, synaptosome-associated protein of 25 kDa (SNAP-25) and vesicle-associated membrane protein (VAMP/synaptobrevin), the proteins proteolysed by botulinum, are susceptible to cleavage in frog nerve terminals, and whether they form complexes in vivo. In control terminals, the three SNAREs were distributed in broad bands at 1 micrometer intervals, at sites consistent with presynaptic Ca2+ channels. Within 3 h, botulinum A, C, D and E (BoNT/A/C/D/E) blocked nerve-evoked muscle contractions but their effects on substrate immunoreactivity varied. The effect of BoNT/A on either C-terminus or N-terminus immunoreactivity of SNAP-25 was undetectable after 3-h incubation, although C-terminus immunoreactivity was reduced after 24 h; N-terminus immunoreactivity was not affected even after 36 h. BoNT/E reduced C-terminus immunoreactivity of SNAP-25 1.5 h after toxin application when transmitter release was blocked, but required 24 h to reduce N-terminus immunoreactivity. BoNT/C reduced syntaxin immunoreactivity after 24-h incubation but did not affect SNAP-25. BoNT/D reduced VAMP immunoreactivity at 3 h while it increased SNAP-25 C-terminal staining fourfold. BoNT/A and BoNT/C applied together for 24 h reduced syntaxin immunoreactivity and that of both C- and N-terminus of SNAP-25, indicating that retention of SNAP-25 N-terminus after cleavage by BoNT/A depended on intact syntaxin. Therefore, we infer that SNAP-25 interacts with VAMP and with syntaxin in vivo. Neurotoxin action abolished only 40-60% of SNAP-25, VAMP or syntaxin immunoreactivity suggesting that distinct pools of these proteins, not immediately involved in triggered exocytosis, are resistant to proteolysis.     

Sodium withdrawal contractures in developing and regenerating rat extensor digitorum longus muscles

During postnatal development of extensor digitorum longus (EDL) muscle, sodium withdrawal contractures were observed during the first 6 days after birth, and not after this time. In regenerating EDL muscles, zero-Na contractures were demonstrated: (1) 7 days after bupivacaine injection, but not 14 or 90 days after this injection; (2) 7, 14, and 90 days after autotransplantation; and (3) 7, 14, and 90 days after the intervention in sliced muscles. The present findings emphasize the role of the denervation in the development of zero-Na contractures in the regenerating muscles and suggest that a calcium-sodium exchange across the sarcolemma may appear in these muscles.     

Physostigmine, but not 3,4-diaminopyridine, improves radial maze performance in memory-impaired rats

The results of some studies suggest that 3,4-diaminopyridine (3,4-DAP), a drug that enhances the release of acetylcholine, may improve memory. The present study examined the ability of 3,4-DAP to reverse the memory impairment produced by scopolamine and the ability of 3,4-DAP and physostigmine to reverse the memory impairment produced by quinolinic acid lesions of the nucleus basalis magnocellularis (nbm) in rats. Mnemonic functioning was assessed with the use of a partially baited eight-arm radial maze. Entries into arms that were never baited were defined as reference memory errors; entries into baited arms from which the food already had been eaten were defined as working memory errors. In Experiment 1, 0.1 mg/kg scopolamine produced a significant increase in working and reference memory errors. Various doses of 3,4-DAP had no significant ameliorative effect on the mnemonic deficit. In Experiment 2, cholinergic function was impaired using a unilateral intra-nbm injection of quinolinic acid (120 nmol in 1.0 microliter). These lesions reduced the levels of the cholinergic marker, choline acetyltransferase, in the cortex by more than 40%. Results showed that the nbm lesion animals were significantly more impaired on the working than reference memory component of the task. Physostigmine (0.01, 0.05, 0.10, 0.20, 0.50 mg/kg) dose-dependently decreased the number of working but not reference memory errors. 3,4-DAP (10(-8), 10(-6), 10(-4), 10(-2), 10(0) mg/kg) had no reliable effect. It was concluded that physostigmine, but not 3,4-DAP, ameliorates memory impairments following decreases in cholinergic function.     

Antibody response to pneumococcal polysaccharide vaccine in young, adult and old mice

The anti-pneumococcal antibody response was studied in young (5-week-old) and adult (10-week-old) BALB/c and CBA/J mice and in adult (9-10-week-old) and old (12-, 18- and 24-month-old) AB6F1 and B6D2F1 mice after s.c. immunization with a 23-valent pneumococcal polysaccharide vaccine. Both young and adult mice showed a significant IgM antibody response to the vaccine 6 days after immunization with 1-11 micrograms antigen. There were significant immune responses to serotypes 1, 2, 4 and 7F in contrast to small responses to serotypes 14, 19F and 23F after immunization with the vaccine. One month after immunization, there were only marginal differences in IgM anti-pneumococcal antibody levels to the vaccine (anti-PPS) between immunized and unimmunized BALB/c mice, whereas in CBA/J mice the anti-PPS remained higher in immunized than in unimmunized mice. Immunization of old mice induced a significant IgM antibody response 6 days after immunization, but the anti-PPS thereafter decreased rapidly towards preimmunization values in AB6F1 mice. A significant IgG anti-PPS was not detected in any of the mice studied. The IgA anti-PPS tended to vary over time with no consistent pattern. It is important to carefully consider age and strain of the mice used when studying the immune response to pneumococcal polysaccharide antigens.     

Effects of feeding conditions on sensitivity to tubocurarine of nerve-muscle preparations from the mouse

1. The effects of feeding conditions on the sensitivity to the effect of tubocurarine (dTc) in vitro were compared among various nerve-muscle preparations from mice. The mice were fed under conditions that restricted or compelled their movement for 64 days and controls were fed conventionally. 2. The sensitivity to the effect of dTc differed considerably among preparations. It was much higher in the sciatic nerve-extensor digitorum longus muscle (EDL), moderately higher in the sciatic nerve-soleus muscle (SOL) and lower in the phrenic nerve-diaphragm (DPH) in control mice. 3. The order of the sensitivity was not altered by either type of conditioning. Constant restriction of movement or compelled movement did not modify the sensitivity of DPH to the effect of dTc in vitro. 4. Compulsion facilitated the sensitivity in both SOL and EDL. Restriction selectively increased the sensitivity of EDL. Both types of conditioning selectively and significantly reduced twitch development in EDL. 5. These results indicate that the sensitivity to dTc of neuromuscular transmission reflects constant states of motor activity.     

A comparison of the electrophysiological effects of two organophosphates, mipafox and ecothiopate, on mouse limb muscles

Adult male albino mice were given single subcutaneous injections of either mipafox (110 mumol/kg) or ecothiopate (0.5 mumol/kg), two organophosphorus compounds (OPs). Acetylcholinesterase activity was measured in the soleus (slow-twitch) and extensor digitorum longus (EDL; fast-twitch) muscles. At 7 and 28 days after dosing, in vitro electrophysiological measurements were carried out in the soleus and EDL. Action potentials and end-plate potentials were evoked at 30 Hz and recorded intracellularly from single muscle fibers. The amplitudes, time course, and latencies of these potentials were measured and the variability (jitter) of latencies was calculated. Recordings after mipafox were also made with 3-Hz stimulation. Acetylcholinesterase activity was inhibited by mipafox (65% in the soleus; 76% in the EDL) and ecothiopate (59% in the soleus; 42% in the EDL). Mipafox and ecothiopate both increased postjunctional (muscle action potential) jitter in the soleus and EDL at 7 days after dosing. Organophosphates caused an increase in end-plate potential amplitudes in the soleus. Mipafox caused an increase in prejunctional (end-plate potential) jitter at 28 days after dosing in both muscles. A single dose of ecothiopate also caused an increase in prejunctional jitter at 28 days in the soleus. The OP-induced increase in jitter was different at different frequencies of stimulation. The results show that there are electrophysiological changes in both muscles after administration of organophosphorus compounds. The slow-twitch soleus appears more sensitive to prejunctional changes caused by OPs than the fast-twitch EDL.     

Distinct exocytotic responses of intact and permeabilised chromaffin cells after cleavage of the 25-kDa synaptosomal-associated protein (SNAP-25) or synaptobrevin by botulinum toxin A or B

Botulinum neurotoxin (BoNT) types A and B are Zn2+-requiring endoproteases which potently block neurotransmitter release by cleavage of a 25-kDa synaptosomal-associated protein (SNAP-25) and synaptobrevin, respectively. Synaptobrevin is important for the exocystosis of catecholamines from dense-core granules and evidence is presented here for the involvement of SNAP-25 in this process in neuroendocrine cells. The effects of BoNT/A and BoNT/B on regulated secretion were compared in intact bovine chromaffin cells to investigate the consequences of cleavage of the different targets. Catecholamine secretion elicited by Ba2+, by elevated K+ concentrations or by nicotine was prevented by each toxin. A very good correlation was observed between the extents of SNAP-25 cleavage or synaptobrevin cleavage and inhibition of secretion by BoNT/A or BoNT/B, respectively, which indicates the importance of SNAP-25 and synaptobrevin in regulated exocytosis. Despite truncation of almost the entire SNAP-25 pool by exposure of the cells to BoNT/A, a residual fraction of secretion persisted that was induced by 20microM Ca2+ (and to a lesser extent by 1 mM Ba2+) following permeabilisation. Addition of more BoNT/A failed to reduce this level of secretion. Inclusion of Mg.ATP, which greatly enhanced secretion from permeabilised cells, was required for Ca2+-stimulated or Ba2+-stimulated BoNT/A-resistant secretion. Furthermore, synaptobrevin is essential for this response because the response was not observed in BoNT/B treated cells. In view of the ability of BoNT/E to abolish secretion from permeabilised cells and to delete 26 amino acids from the C-terminus of SNAP-25, it can be deduced that cleavage of only nine residues by BoNT/A does not prevent the resultant truncated form exhibiting attenuated activity under the conditions created by permeabilisation. This identification of a novel component of secretion from permeabilised cells should facilitate investigation of the functional interaction of SNAP-25 with other proteins involved in regulated exocytosis.     

Proteolysis of SNAP-25 isoforms by botulinum neurotoxin types A, C, and E: domains and amino acid residues controlling the formation of enzyme-substrate complexes and cleavage

Tetanus toxin and the seven serologically distinct botulinal neurotoxins (BoNT/A to BoNT/G) abrogate synaptic transmission at nerve endings through the action of their light chains (L chains), which proteolytically cleave VAMP (vesicle-associated membrane protein)/synaptobrevin, SNAP-25 (synaptosome-associated protein of 25 kDa), or syntaxin. BoNT/C was reported to proteolyze both syntaxin and SNAP-25. Here, we demonstrate that cleavage of SNAP-25 occurs between Arg198 and Ala199, depends on the presence of regions Asn93 to Glu145 and Ile156 to Met202, and requires about 1,000-fold higher L chain concentrations in comparison with BoNT/A and BoNT/E. Analyses of the BoNT/A and BoNT/E cleavage sites revealed that changes in the carboxyl-terminal residues, in contrast with changes in the amino-terminal residues, drastically impair proteolysis. A proteolytically inactive BoNT/A L chain mutant failed to bind to VAMP/synaptobrevin and syntaxin, but formed a stable complex (KD = 1.9 x 10(-7) M) with SNAP-25. The minimal essential domain of SNAP-25 required for cleavage by BoNT/A involves the segment Met146-Gln197, and binding was optimal only with full-length SNAP-25. Proteolysis by BoNT/E required the presence of the domain Ile156-Asp186. Murine SNAP-23 was cleaved by BoNT/E and, to a reduced extent, by BoNT/A, whereas human SNAP-23 was resistant to all clostridial L chains. Lys185Asp or Pro182Arg mutations of human SNAP-23 induced susceptibility toward BoNT/E or toward both BoNT/A and BoNT/E, respectively.     

Na(+)-K+ ATPase concentration in different adult rat skeletal muscles is related to oxidative potential

To investigate the relationship among fibre type, oxidative potential, and Na(+)-K+ ATPase concentration in skeletal muscle, adult male Wistar rats weighing 259 +/- 8 g (mean +/- SE) were sacrificed and the soleus (SOL), extensor digitorum longus (EDL), red vastus lateralis (RV), and white vastus lateralis (WV) removed. These muscles were chosen as being representative of the two major fibre type populations: slow twitch (SOL) and fast twitch (EDL, RV, WV) and exhibiting either a high (SOL, EDL, RV) or low (WV) oxidative potential. Na(+)-K+ ATPase concentration (pmol.g-1 wet weight), measured by the [3H]ouabain binding technique, differed (p < 0.01) only between the WV (238 +/- 7.9) and the SOL (359 +/- 9.6), EDL (365 +/- 10), and RV (403 +/- 12). Similarly, muscle oxidative potential as measured by the maximal activity of citrate synthase was different (p < 0.01) only between the WV and the other three muscles. Citrate synthase activity (mumol.min-1.g-1 wet weight) was 4.0 +/- 0.7, 12.3 +/- 0.9, 9.1 +/- 0.7, and 11.3 +/- 1.0 in the WV, SOL, EDL, and RV, respectively. These results indicate that Na(+)-K+ ATPase concentration is not related to the speed of contraction but to the oxidative potential of the muscle. Since chronic activity is a primary determinant of oxidative potential, it would be expected that increases in Na(+)-K+ ATPase would accompany increases in muscle utilization.     

Gating and permeability of ion channels produced by botulinum toxin types A and E in PC12 cell membranes

Botulinum neurotoxin (BoNT) is known to produce cationic channels in artificial bilayers. This study examined ion channels formed by BoNT in native membranes from cultured PC12 cells under conditions approximating those thought to occur during toxin internalization. Membrane patches were excised from PC12 cells using patch electrodes and exposed to symmetrical solutions containing either 200 mM CsCl, RbCl or KCl. The patch pipettes also contained 1-5 microg/ml BoNT buffered to pH 5.3 while the bath solutions were buffered to pH 7.0. In the presence of toxin, bursts of ion channel openings were observed. These toxin-induced channels were most active with a negative voltage applied to the same side as the toxin (cis). The increased activity at negative voltages was due to an increase in mean open time of e-fold per 120 mV and a decrease in mean closed time between bursts of e-fold per 110 mV. The shorter mean closed time within a burst was independent of membrane voltage. While BoNT-induced ion channels started as a single conductance level of 27 pS (KCl), 34 pS (RbCl) or 46 pS (CsCl) they typically increased in roughly equal steps to five or more times the original channel conductance. These higher conductance BoNT 'channels' opened and closed synchronously and could be distinguished from superposition of multiple independent channels. Despite differences in putative transmembrane sequences between BoNT/A and BoNT/E, both serotypes evidenced the same channel conductance and mean open time.     

Effects of Mg2+ on Ca2+ handling by the sarcoplasmic reticulum in skinned skeletal and cardiac muscle fibres

The influence of myoplasmic Mg2+ (0.05-10 mM) on Ca2+ accumulation (net Ca2+ flux) and Ca2+ uptake (pump-driven Ca2+ influx) by the intact sarcoplasmic reticulum (SR) was studied in skinned fibres from the toad iliofibularis muscle (twitch portion), rat extensor digitorum longus (EDL) muscle (fast twitch), rat soleus muscle (slow twitch) and rat cardiac trabeculae. Ca2+ accumulation was optimal between 1 and 3 mM Mg2+ in toad fibres and reached a plateau between 1 and 10 mM Mg2+ in the rat EDL fibres and between 3 and 10 mM Mg2+ in the rat cardiac fibres. In soleus fibres, optimal Ca2+ accumulation occurred at 10 mM Mg2+. The same trend was obtained with all preparations at 0.3 and 1 microM Ca2+. Experiments with 2,5-di-(tert-butyl)-1,4-benzohydroquinone, a specific inhibitor of the Ca2+ pump, revealed a marked Ca2+ efflux from the SR of toad iliofibularis fibres in the presence of 0.2 microM Ca2+ and 1 mM Mg2+. Further experiments indicated that the SR Ca2+ leak could be blocked by 10 microM ruthenium red without affecting the SR Ca2+ pump and this allowed separation between SR Ca2+ uptake and SR Ca2+ accumulation. At 0.3 microM Ca2+, Ca2+ uptake was optimal with 1 mM Mg2+ in the toad iliofibularis and rat EDL fibres and between 1 and 10 mM Mg2+ in the rat soleus and trabeculae preparations. At higher [Ca2+] (1 microM), Ca2+ uptake was optimal with 1 mM Mg2+ in the iliofibularis fibres and between 1 and 3 mM Mg2+ in the EDL fibres.(ABSTRACT TRUNCATED AT 250 WORDS)     

Proteolysis of SNAP-25 by types E and A botulinal neurotoxins

Clostridial neurotoxins, tetanus toxin (TeTx) and the seven related but serologically distinct botulinal neurotoxins (BoNT/A to BoNT/G), are potent inhibitors of synaptic vesicle exocytosis in nerve endings. Recently it was reported that the light chains of clostridial neurotoxins act as zinc-dependent metalloproteases which specifically cleave synaptic target proteins such as synaptobrevin/VAMPs, HPC-1/syntaxin (BoNT/C1), and SNAP-25 (BoNT/A). We show here that BoNT/E, like BoNT/A, cleaves SNAP-25, as generated by in vitro translation or by expression in Escherichia coli. BoNT/E cleaves the Arg180-Ile181 bond. This site is different from that of BoNT/A, which cleaves SNAP-25 between the amino acid residues Gln197 and Arg198. These findings further support the view that clostridial neurotoxins have evolved from an ancestral protease recognizing the exocytotic fusion machinery of synaptic vesicles whereby individual toxins target different members of the membrane fusion complex.     

Effects of ovariectomy and hindlimb unloading on skeletal muscle

Female rats (7-8 mo old, n = 40) were randomly placed into the intact control (Int) and ovariectomized control (Ovx) groups. Two weeks after ovariectomy, animals were further divided into intact 2-wk hindlimb unloaded (Int-HU) and ovariectomized hindlimb unloaded (Ovx-HU). We hypothesized that there would be greater hindlimb unloading-related atrophy in Ovx than in Int rats. In situ contractile tests were performed on soleus (Sol), plantaris (Plan), peroneus longus (Per), and extensor digitorum longus (EDL) muscles. Body weight and Sol mass were approximately 22% larger in Ovx than in Int group and approximately 18% smaller in both HU groups than in Int rats (Ovx x HU interaction, P < 0.05), and there was a similar trend in Plan muscle (P < 0.07). There were main effects (P < 0.05) for both ovariectomy (growth) and hindlimb unloading (atrophy) on gastrocnemius mass. Mass of the Per and EDL muscles was unaffected by either ovariectomy or hindlimb unloading. Time to peak twitch tension for EDL and one-half relaxation times for Sol, Plan, Per, and EDL muscles were faster (P < 0.05) in Ovx than in Int animals. The results suggest that 1) ovariectomy led to similar increases of approximately 20% in body weight and plantar flexor mass; 2) hindlimb unloading may have prevented ovariectomy-related muscle growth; 3) greater atrophy may have occurred in Sol and Plan of Ovx animals compared with controls; and 4) removal of ovarian hormonal influence decreased skeletal muscle contraction times.     

Summation of extraocular motor unit tensions in the lateral rectus muscle of the cat

Contractile measures on 67 single muscle units in the cat lateral rectus muscle were made in response to motoneuron stimulation. Simultaneous activation of four to five additional units, using muscle nerve stimulation, allowed an examination of unit force summation. Linear force addition was found in 73% of the units, while 25% added only about half of their twitch force to the twitch force of the nerve-activated units. "Nonadditive" units had significantly weaker twitch tensions than the units which added linearly. Lengthening or shortening the whole muscle, from maximal isometric settings, reduced whole muscle twitch tension as well as muscle unit tension. Injury to the lateral rectus muscle did not significantly alter whole muscle tension. These findings suggest that the known serial and branching arrangement of these muscle fibers, as well as the complex interfiber matrix, may help explain the force reduction in some muscle units and the whole muscle's resistance to insult.     

Oligoclonality of serum immunoglobulin G antibody responses to Streptococcus pneumoniae capsular polysaccharide serotypes 6B, 14, and 23F

Serum antibodies (Abs) specific for the capsular polysaccharides of Streptococcus pneumoniae provide protection against invasive pneumococcal disease. Previous studies indicate that Abs to pneumococcal polysaccharide (PPS) serotypes 1 and 6B have limited clonal diversity. To determine if restricted diversity was a feature common to other PPS specificities, we examined the light (L)-chain expression and isoelectric heterogeneity of type 6B, 14, and 23F Abs elicited in 15 adults following PPS vaccination. At the population level, both PPS-6B and PPS-14 Abs expressed kappa and lambda chains, although 6B Abs more frequently expressed lambda chains lambda and 14 Abs more frequently expressed kappa chains. In individual sera, Abs were generally skewed towards either kappa or lambda expression. 23F-specific Abs had predominantly kappa chains. Isoelectric focusing analyses showed that sera contained one or at most a few immunoglobulin G Ab spectrotypes to all three respective capsular serotypes, a result indicative of oligoclonality. A sequence analysis of a purified PPS-14-specific Ab having a single spectrotype gave uniform amino-terminal sequences for both the heavy chain (V(H)III subgroup) and the L chain (kappaIII-A27 V region). From these results we conclude that within individual adults, serum Ab responses to PPS serotypes 6B, 14, and 23F derive from a small number of dominant B-cell clones, and consequently variable-region expression is probably individually limited as well. Oligoclonality appears to be a general characteristic of human PPS-specific Ab repertoires, and we suggest that this property could lead to individual differences in Ab fine specificity and/or functional activity against encapsulated pneumococci.     

Potentiation of mivacurium by rocuronium is age- and time-dependent: a study in children, adolescents, and young and elderly adults

Potentiation occurs when the steroidal muscle relaxant, rocuronium, is coadministered with the benzylisoquinolinium relaxant, mivacurium. The effect of time and age on this interaction was evaluated in four predetermined groups: children, adolescents, young adults, and elderly adults (15 per group) by monitoring the ulnar nerve-evoked force of contraction of the adductor pollicis (twitch response). During recovery from paralysis induced by 800 micrograms/kg of rocuronium, an infusion of mivacurium was started and maintained for at least 90 minutes to retain the twitch response at 1% to 9% of baseline tension (95 +/- 4% paralysis). Rocuronium at 600 micrograms/kg induced greater than 95% paralysis in 57 of the 60 patients within 2.2 +/- 0.4 (mean +/- SE) minutes. The period of recovery from rocuronium-induced paralysis to 5% of baseline twitch height was longest in the elderly (30.1 +/- 2.9 minutes) and shortest in the adolescents (16.5 +/- 2.4 minutes). The mivacurium infusion requirements to maintain 95 +/- 4% paralysis was highest in children and progressively increased with time. In young and elderly adults, the infusion rates remained lower than that of children and did not change with time. The incidence of satisfactory spontaneous recovery within 20 minutes (train-of-four ratio > 75%) was the highest in children, followed by adolescents and young adults, and was least in the elderly. The residual neuromuscular effect of rocuronium on the subsequent mivacurium infusion was most pronounced in the elderly, followed by young adults, then adolescents, and was least in children.