Biomedical subjects
J E Butler
Publications and source records attributed to J E Butler.
Differential complement activation by bovine IgG2 allotypes.
Immunoglobulin allotypes and complement (C) are known to be related to susceptibility to infection. Because bovine IgG2 is important in resistance to pyogenic infections and because its two allotypes, IgG2a and IgG2b, differ in sequence in the CH1, hinge, CH2, and CH3 regions, we tested the ability of these allotypes to initiate the bovine C cascade. Bovine IgG2a and IgG2b were standardized according to specific anti guinea pig red blood cell (GPRBC) ELISA activity using anti IgG2 reagents shown essentially unbiased for allotype. Complement activating activity of the allotypes was quantitated in a GPRBC lysis assay. With this system, IgG2b consistently had more than twice the activity in bovine C mediated lysis as compared with IgG2a. The fact that both EDTA and EGTA/Mg almost completely inhibited C mediated lysis of GPRBCs indicated that lysis was due to the classical pathway. Since antibody usually activates C by the classical pathway, this supports the supposition that activation was by the IgG2-GPRBC complexes. Flexibility analyses showed that IgG2b had a more rigid hinge than IgG2a, perhaps partially explaining the greater efficiency of IgG2b in C activation. Other mechanisms may include differences in glycosylation and in the amino acid at position 332. The difference in ability to activate C may mean that animals of the IgG2a allotype could be more susceptible to infection with extracellular pyogenic pathogens which are killed by C or by phagocytes after opsonization with IgG2 and C.
Discharge properties and recruitment of human diaphragmatic motor units during voluntary inspiratory tasks.
1. The behaviour of inspiratory motoneurones is poorly understood in humans and even for limb muscles there are few studies of motoneurone behaviour under concentric conditions. The current study assessed the discharge properties of the human phrenic motoneurones during a range of non-isometric voluntary contractions. 2. We recorded activity from 60 motor units in the costal diaphragm of four subjects using an intramuscular electrode while subjects performed a set of voluntary inspiratory contractions. These included a range of inspiratory efforts above and below the usual tidal range: breaths of different sizes (5-40 % vital capacity, VC) at a constant inspiratory flow (5 % VC s-1) and breaths of a constant size (20 % VC) at different inspiratory flows (2.5-20 % VC s-1). 3. For all the voluntary tasks, motor units were recruited throughout inspiration. For the various tasks, half-way through inspiration, 61-87 % of the sampled motor units had been recruited. 4. When the inspiratory task was deliberately altered, most single motor units began their discharge at a particular volume even when the rate of contraction had altered. 5. The initial firing frequency (median, 6.5 Hz) was consistent for tasks with a constant flow regardless of the size of the breath. However, for breaths of a constant size the initial firing frequencies increased as the inspiratory flow increased (range across tasks, 4.8-9.3 Hz). The 'final' firing frequency at the end of inspiration increased significantly above the initial frequency for each task (by 0.8-5.2 Hz) and was higher for those tasks with higher final lung volumes and higher inspiratory flows (range across tasks, 7.8-11.0 Hz). 6. There was no correlation within a task between the time of recruitment and the initial or final firing frequency for each motor unit. However, for each inspiratory task, initial and final firing frequencies were positively correlated. 7. Because the discharge of three to four units could be recorded simultaneously in a range of tasks, a quantitative 'shuffle' index was developed to describe changes in their recruitment order. Recruitment order was invariant in the task with the slowest inspiratory flow, but varied slightly, but significantly, in tasks with higher inspiratory flows. 8. The discharge rates of single motor units were compared for targeted voluntary breaths and non-targeted involuntary breaths which were matched for size. There were no significant differences in the initial or final firing frequencies, but recruitment order was not always the same in the two types of breath.
Altered responses of human elbow flexors to peripheral-nerve and cortical stimulation during a sustained maximal voluntary contraction.
The short-latency electromyographic response evoked by transcranial magnetic stimulation (MEP) increases in size during fatigue, but the mechanisms are unclear. Because large changes occur in the muscle action potential, we tested whether changes in the response to stimulation of the peripheral motor nerve could fully account for the increase in the MEP. Subjects (n=8) performed sustained maximal voluntary contractions (MVCs) of the right elbow flexors for 2 min. During the contraction, the MEP and the response to supramaximal stimulation of motor-nerve fibres in the brachial plexus were alternately recorded. During the contraction, responses to motor-nerve stimulation increased in area by 87+/-35% (mean+/-SD) in the biceps brachii and 74+/-30% in the brachioradialis, but the area of the MEPs increased by 153+/-86% and 175+/-122%, respectively. Thus, the increase in the MEP was greater than the increase in the peripheral M-wave. The onset latency of the MEP in the biceps brachii increased by 0.7+/-0.6 ms (range: -0.2 to 1.9 ms) during the sustained contraction. A smaller increase occurred in response to peripheral nerve stimulation (0.3+/-0.3 ms; from -0.3 to 0.9 ms). In the contralateral elbow flexors, neither responses to transcranial magnetic stimulation nor responses to motor-nerve stimulation changed in size or latency. During the sustained contraction, the short silent period after stimulation of the peripheral nerve (48+/-5 ms in biceps brachii and 48+/-4 ms in brachioradialis) increased in duration by about 12 ms (to 61+/-12 ms and 60+/-9 ms, respectively), whereas the silent period following transcranial magnetic stimulation increased from 238+/-39 ms in biceps brachii and 243+/-34 ms in brachioradialis to 325+/-41 ms and 343+/-42 ms, respectively. During a sustained MVC, while the motor responses to peripheral and to cortical stimulation grow concurrently, growth of the MEP cannot be entirely accounted for by changes in the muscle action potential. Hence, some of the increase in MEP size during fatigue must reflect changes in the central nervous system. Increased latency of the MEPs and lengthening of the peripherally evoked silent period are consistent with decreased excitability of the alpha motoneurone pool. Thus, an increased response from the motor cortex to the magnetic stimulus remains a likely contributor to the increase in the size of the MEP in fatigue.
Antibody repertoire development in fetal and neonatal piglets. I. Four VH genes account for 80 percent of VH usage during 84 days of fetal life.
VDJ rearrangement and VH gene usage during fetal development in 35 outbred piglets was examined by PCR amplification of VDJs; VDJs were subsequently characterized by hybridization with VH-specific gene probes and by sequencing. VDJ rearrangement was first seen in the fetal liver on day 30 of a 114-day gestation. Four VH genes (V(H)A, V(H)B, V(H)C, and V(H)E) accounted for approximately 80% of all VH gene usage regardless of gestational age, choice of piglet, or lymphoid tissue tested; D(H)A and D(H)B were used in >90% of the fetal VDJs examined. Evidence of somatic hypermutation during fetal development was not found. The proportion of the four prominent fetal VH genes did not differ significantly between cDNA and DNA, suggesting the absence of selective B cell differentiation. A comparison of recombination signal sequences, flanking sequences, and framework sequences of these fetal genes with other germline VH genes of swine offered no clue as to their selective usage. N-region additions were prominent on day 40 but not on day 30, suggesting that the onset of terminal deoxynucleotidyltransferase activity occurs after 30 days of fetal development. These collective findings indicate that the preimmune, "natural Ab" repertoire of the fetal piglet is largely restricted to the use of four nonpolymorphic and nonmutated VH genes and two nonmutated DH segments. This suggests that the preimmune repertoire of swine is either highly restricted or almost entirely determined by junctional diversity in complementarity-determining region-3.
Absence of viscerosomatic inhibition with injections of lobeline designed to activate human pulmonary C fibres.
1. Activation of pulmonary C fibres (J receptors) in animals produces inhibition of spinal motoneurones. Intravenous bolus injections of lobeline are believed to activate pulmonary C fibres (J receptors) in human subjects and to produce characteristic sensations and cardiorespiratory responses. This study quantified the respiratory sensations evoked by such injections and then used a range of suprathreshold doses of lobeline and tested for the presence of reflex or descending inhibition of motoneuronal output. 2. Injections of lobeline produced dose-dependent sensations of respiratory discomfort referred to the throat and upper chest beginning within about 10 s and often associated with coughing. As the dose increased the latency for the sensations decreased while their duration and intensity increased. Reflex changes in blood pressure, heart rate and ventilation also occurred. 3. Injections of lobeline at doses sufficient to evoke respiratory discomfort lasting 25-32 s (37-73 microgram kg-1) increased the size of the H reflex in soleus with an onset latency of about 10 s and lasting about 20 s. 4. The size of EMG responses evoked in upper limb muscles by transcranial magnetic stimulation of the motor cortex increased shortly after injections and remained elevated for about 30-35 s. 5. Injections of lobeline during sustained voluntary contractions of the elbow flexors at submaximal or maximal levels did not impair the ability to produce force. 6. Walking was not disrupted by repeated suprathreshold doses of lobeline. 7. It is concluded that injections of lobeline sufficient to evoke cardiorespiratory reflexes and sensations of severe respiratory discomfort are not associated with functionally important inhibition of motor performance. The results cast doubt on the ability of the J reflex to limit exercise in humans.
Determination of gene usage by differential polymerase chain reaction product hybridization.
All swine VH genes belong to a highly homologous family and have identical leader sequences, and the swine VH locus contains a single JH. The small number of VH genes used by the fetus and neonate in the first 6 weeks have unique CDR1 and CDR2 sequences, permitting each to be identified using specific oligonucleotide probes. We have used this system as a model for the development of a rapid method for determining the proportional usage of closely related genes based on differential polymerase chain reaction (PCR) product hybridization (DPPH). The validity of the method is demonstrated using mixtures of PCR product containing known amounts of VH gene DNAs and by comparing data obtained by this method with those obtained by enumeration of individual hybridizing clones from lymphoid tissue and peripheral blood B cells. Since DPPH is at least 100-fold more efficient than the enumeration of individual hybridizing clones, it is especially useful for analyzing large numbers of samples in population studies. The possible extension of this method to the usage of other genes is discussed.
Identification, characterization and expression of Toxocara canis nematode polyprotein allergen TBA-1.
We have cloned the cDNA of TBA-1, the Nematode polyprotein allergen (NPA) of Toxocara canis and found it to be most similar to ABA-1, the Ascaris NPA, on the basis of amino acid sequence. We could study the antigenic properties of an E-coli synthesized fusion protein prepared with the cloned gene since no glycosylation site was expected from the deduced amino acid sequence. Although no IgE responses to TBA-1 were detected, recombinant TBA-1 was differently recognized by serum IgG antibodies when the recombinant TBA-1 was directly adsorbed vs when immobilized via a streptavidin linkage on polystyrene microtitre wells. One group of sera recognized TBA-1 directly immobilized while the second only recognized TBA-1 immobilized via streptavidin linkage. The former were from rodents immunized with a Toxocara sp. adult worm extract while the latter were obtained from rodents infected with T. canis larva or immunized with a Anisakis simplex L3 larval extract. These observations suggest that the two in vivo forms of TBA-1 are expressed, but during different stages of the parasite's life cycle.
The DPE, a conserved downstream core promoter element that is functionally analogous to the TATA box.
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Human respiratory muscles: sensations, reflexes and fatiguability.
1. Given the importance of the ventilatory 'pump' muscles, it would not be surprising if they were endowed with both sensory and motor specializations. The present review focuses on some unexpected properties of the respiratory muscle system in human subjects. 2. Although changes in blood gas tension were long held not to influence sensation directly, studies in subjects who are completely paralysed show that increases in arterial CO2 levels elicit strong sensations of respiratory discomfort. 3. Stretch reflexes in human limb muscles contain a monosynaptic spinal excitation and a long-latency excitation. However, inspiratory muscles show an initial inhibition when tested with brief airway occlusions during inspiration. This inhibition does not depend critically on input from pulmonary or upper airway receptors. 4. Human inspiratory muscles (including the diaphragm) have been considered to fatigue during inspiratory resistive loading. However, recent studies using phrenic nerve stimulation to test the force produced by the diaphragm show that carbon dioxide retention (hypoventilation) and voluntary cessation of loading occur before the muscles become overtly fatigued.
Immunoglobulin diversity, B-cell and antibody repertoire development in large farm animals.
The B-cell lineage, the antibodies produced by these cells and the diversification of the antibody repertoire are essential for the health and survival of all mammals. Cattle, sheep, swine and horses, unlike rodents and primates, develop their antibody repertoire from a relatively small number of VH (variable heavy) genes of one or several families and cattle, sheep and horses use almost exclusively lambda-light chains. These large farm animals appear to use templated mutation (gene conversion) in addition to untemplated (point) mutation in repertoire development; this may occur predominantly in the ileal Peyer's patches. Whether B-cell lymphogenesis is continuous throughout life--as in rodents and primates--or whether B cells are largely of the B-1 lineage and develop only in foetal and neonatal life, is uncertain. The fact that immunoglobulin D (IgD) is totally absent from swine and ruminants may be significant, as IgD is expressed weakly on rodent B-1 cells. Information on IgG subclass diversity in large farm animals is incomplete, except for sheep and cattle, and no information is available for any large farm animal to show whether T helper 1 (Th1) and Th2 responses correlate with the expression of any subclass antibody response, as is the case in rodents. All large farm animals exclusively use the mammary gland to transfer immunity to offspring, although the receptor involved in the transport of IgG into colostrum and milk has not been characterised. Efforts to standardise the nomenclature and measurements of antibodies and immunoglobulins in large farm animals are discussed, and a proposal currently under review concerning the standardisation of the nomenclature for bovine immunoglobulins is presented as a model.
Contraction of the human diaphragm during rapid postural adjustments.
1. The response of the diaphragm to the postural perturbation produced by rapid flexion of the shoulder to a visual stimulus was evaluated in standing subjects. Gastric, oesophageal and transdiaphragmatic pressures were measured together with intramuscular and oesophageal recordings of electromyographic activity (EMG) in the diaphragm. To assess the mechanics of contraction of the diaphragm, dynamic changes in the length of the diaphragm were measured with ultrasonography. 2. With rapid flexion of the shoulder in response to a visual stimulus, EMG activity in the costal and crural diaphragm occurred about 20 ms prior to the onset of deltoid EMG. This anticipatory contraction occurred irrespective of the phase of respiration in which arm movement began. The onset of diaphragm EMG coincided with that of transversus abdominis. 3. Gastric and transdiaphragmatic pressures increased in association with the rapid arm flexion by 13.8 +/- 1.9 (mean +/- S.E.M.) and 13.5 +/- 1.8 cmH2O, respectively. The increases occurred 49 +/- 4 ms after the onset of diaphragm EMG, but preceded the onset of movement of the limb by 63 +/- 7 ms. 4. Ultrasonographic measurements revealed that the costal diaphragm shortened and then lengthened progressively during the increase in transdiaphragmatic pressure. 5. This study provides definitive evidence that the human diaphragm is involved in the control of postural stability during sudden voluntary movement of the limbs.
Effect of contraction strength on responses in biceps brachii and adductor pollicis to transcranial magnetic stimulation.
The sizes of the motor-evoked potentials (MEPs) and the durations of the silent periods after transcranial magnetic stimulation were examined in biceps brachii, brachioradialis and adductor pollicis in human subjects. Stimuli of a wide range of intensities were given during voluntary contractions producing 0-75% of maximal force (maximal voluntary contraction, MVC). In adductor pollicis, MEPs increased in size with stimulus intensity and with weak voluntary contractions (5% MVC), but did not grow larger with stronger contractions. In the elbow flexors, MEPs grew little with stimulus intensity, but increased in size with contractions of up to 50% of maximal. In contrast, the duration of the silent period showed similar changes in the three muscles. In each muscle it increased with stimulus intensity but was unaffected by changes in contraction strength. Comparison of the responses evoked in biceps brachii by focal stimulation over the contralateral motor cortex with those evoked by stimulation with a round magnetic coil over the vertex suggests an excitatory contribution from the ipsilateral cortex during strong voluntary contractions.
Polymorphism of the IGHG3 gene in cattle.
We cloned and analyzed the gene (IGHG3) encoding cattle IgG3 using a spleen cDNA library and a genomic phage clone. IGHG3 differs most remarkably from IGHG1 and IGHG2 in the structure of its hinge. The IGHG3 hinge is encoded by two exons, one of which encodes a 22 amino acid 3' extension of the CH1 exon. The IGHG3 gene is highly homologous to the C-gamma pseudogene previously reported. Seventy-one DNA samples from different cattle were tested by pulsed-field gel electrophoresis and Southern blot to detect genetic polymorphisms. This analysis revealed two different alleles which we designated IGHG3(a) and IGHG3(b). The two putative alleles differ from each other by a 6 amino acid substitution in the coding region and an 84 base pair insertion in the intron between the CH2 and CH3 exons. Our data also indicate that the cattle heavy chain locus spans approximately 400 kilobases from IGHM to IGHA and encodes only three IgG subclasses, all of which are transcriptionally active.
Sequence analysis of pig switch mu, C mu, and C mu m.
The complete sequence of pig switch mu (S mu) and the various domains of C mu, a total of ca 8 kilobases (kb) of sequence, are presented and comparatively analyzed. S mu starts about 2.5 kb downstream of the single pig JH gene and ends 0.4 kb upstream of C mu; the length of S mu is 3.2 kb. Three major pentameric repeats, gagct, gggct, and gggtt, comprise more than half of the entire S mu sequence. The two most frequent decamers are gagctgagct and gagctgggct. The longest single repeated sequence is 93 base pairs. The frequency of the various pentamers indicates that pig S mu is more similar to human S mu than to mouse S mu. The sequence of genomic pig C mu and its deduced amino acid sequence are similar to the C mu sequences published for other vertebrates and are most similar to those of sheep, another artiodactyl. The membrane and secreted tailpiece exons are highly conserved (ca > 90% similarity) and are actually identical to those of the sheep. The degree of similarity to other mammalian IgMs generally decreases from the carboxyl to the amino terminal domains, with the exception of C mu 2, which shows the lowest similarity among species (61% DNA, 44% aa). As in human C mu, we identified five potential N-linked glycosylation sites, two cysteines involved in forming interchain disulfide bonds, and the two additional cysteines involved in the linkage of IgM pentamers. This is only the third species for which a S mu sequence has been reported but the first from a species lacking the second virgin B-cell receptor, i.e., IgD. IgD in mice and humans lacks a switch region and its expression depends on the splicing of C mu-bearing mRNA with C delta. However, we found no evidence that the lack of C delta influenced the genomic structure of S mu, C mu, or C mu m.
Characterization of the bovine C alpha gene.
The complete genomic sequence of a bovine C alpha gene is reported here. The genomic sequence was obtained from a C alpha phage clone that had been cloned from a genomic EMBL4 phage vector library. The C alpha sequence had previously been expressed as a chimeric antibody and identified as IgA using IgA-specific antibodies. Intron/exon boundaries were determined by comparison of the genomic sequence with an expressed bovine C alpha sequence obtained from spleen by reverse transcription-polymerase chain reaction (RT-PCR). Analysis of 50 Swedish bovine genomic DNA samples using genomic blots and five different restriction enzymes failed to detect evidence of polymorphism. However, PstI digests of Brown Swiss DNA showed a restriction fragment length polymorphism (RFLP), suggesting that at least two allelic variants of bovine IgA exist. Comparison of the deduced amino acid sequence of bovine IgA with sequences available for other species indicated that the highest homology was with that of swine, another artiodactyl. This was the highest homology observed for all mammalian IgA compared except for that between IgA1 and IgA2 in humans. Bovine IgA shares with rabbit IgA3 and IgA4, an additional N-linked glycosylation site at position 282. However, the collective data indicate that cattle are like swine and rodents and unlike rabbits in having a single locus of the gene encoding IgA of this species.
Immunoglobulin gene organization and the mechanism of repertoire development.
Recombinant DNA technology has made it possible to comparatively examine the immunoglobulin (Ig) gene constitution of numerous species. These studies reveal that many species diverge from the pattern seen in rodents and primates while revealing a number of interesting correlations involving Ig gene organization, B-cell lymphogenesis and the mechanism of repertoire development; many of these fail to correlate with traditional phylogenetic relationships. These correlates pose new questions regarding repertoire developments, which suggest that considerable diversity exists in the process. These findings also allow new questions to emerge regarding the role of IgD, the size of the B-cell repertoire, and the role that antigen may or may not play in repertoire development. These issues are discussed with the intention of stimulating experimentation to test a number of speculative hypotheses regarding B-cell and repertoire development.
Task failure with lack of diaphragm fatigue during inspiratory resistive loading in human subjects.
Task failure during inspiratory resistive loading is thought to be accompanied by substantial peripheral fatigue of the inspiratory muscles. Six healthy subjects performed eight resistive breathing trials with loads of 35, 50, 75 and 90% of maximal inspiratory pressure (MIP) with and without supplemental oxygen. MIP measured before, after, and at every minute during the trial increased slightly during the trials, even when corrected for lung volume (e.g., for 24 trials breathing air, 12.5% increase, P < 0.05). In some trials, task failure occurred before 20 min (end point of trial), and in these trials there was an increase in end-tidal PCO2 (P < 0.01), despite the absence of peripheral muscle fatigue. In four subjects (6 trials with task failure), there was no decline in twitch amplitude with bilateral phrenic stimulation or in voluntary activation of the diaphragm, even though end-tidal PCO2 rose by 1.6 +/- 0.9%. These results suggest that hypoventilation, CO2 retention, and ultimate task failure during resistive breathing are not simply dependent on impaired force-generating capacity of the diaphragm or impaired voluntary activation of the diaphragm.