Biomedical subjects

D C Jackson

Publications and source records attributed to D C Jackson.

At least 73 records · Page 4Linked to original sources

PubMed1994

Antigenic competition in a multivalent foot rot vaccine.

The antigenic competition that occurs when pilus antigens of different serogroups are combined in multivalent vaccines for foot rot has been investigated using recombinant pilus antigens. Our prototype vaccine contains pili from nine serogroups of Dichelobacter nodosus which are expressed in Pseudomonas aeruginosa. Sheep inoculated with this multivalent vaccine were not as well protected against foot rot as those given the monovalent vaccine. Levels of agglutinating and total antibody specific for any particular pili serogroup were found to be significantly reduced in sheep vaccinated with six or more closely related pili. This effect was more pronounced for agglutinating antibody, which is thought to mediate protection, but was also observed with total antibody levels measured by ELISA. The antigenic competition was not associated with the total antigen load as a tenfold higher dose of monovalent pili induced high titres of antibody. Furthermore, distributing the vaccine to four sites, each draining to a different lymph node, failed to overcome the competition. Experiments with mixtures of monospecific sera indicate that the phenomenon is unlikely to be due to blocking of serogroup-specific protective antibodies by an excess of cross-reactive non-protective antibody elicited by heterologous pili.

Agglutinins↗

PubMed1994

Lactic acidosis transiently increases metabolic rate of turtle myocytes.

We measured O2 consumption as an estimate of metabolic rate in isolated calcium-tolerant ventricular myocytes of turtles (Chrysemys picta belli) at control pH 7.8 and in the same solution brought to pH 7.4 and 7.0 with additions of lactic acid. Our aim was to test the hypothesis that lactic acidosis caused metabolic depression by initiating downregulation of Na+ channels, and thus Na(+)-K(+)-ATPase (Na+ pump) activity, which we would measure as a decrease in O2 consumption. Myocyte O2 consumption was measured in reptilian N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid-buffered Ringer solution and in nomially Na(+)- and Ca(2+)-free solution, thus estimating the Na+ pump component of metabolic rate. Lowering extracellular pH from 7.8 to 7.0 resulted in a significant increase in metabolic rate of cells in Ringer solution but not those in Na(+)- and Ca(2+)-free solution. This result was unchanged by the addition of 2 mM Ca2+ to Na(+)-free cell suspensions, indicating that the difference was due to the presence of Na+. Addition of 100 microM amiloride to cells in Ringer solution at pH 7.0 abolished the increase in O2 consumption, suggesting that the apparent increase in Na(+)-K(+)-ATPase activity was secondary to Na(+)-H+ exchange. Intracellular pH was measured using 5,5-dimethyl[14C]oxazolidine-2,4-dione. Cells treated with amiloride and those in Na(+)- and Ca(2+)-free solution did not regulate intracellular pH following acidosis and maintained basal metabolic rate. These data suggest that the Na(+)-H+ exchanger is an important contributor to intracellular pH regulation in the myocyte but increases Na+ pump activity and metabolic rate immediately following acidosis.

Acidosis, Lactic↗

PubMed1993

Maintenance of high extracellular pH does not influence cell pH or metabolism in submerged anoxic bullfrogs.

We compared extracellular and intracellular acid-base states in paralyzed bullfrogs subjected to 4 h of anoxic submergence at 15 degrees C with or without maintenance of extracellular pH at preanoxic levels by bicarbonate infusion. We also assessed anaerobic metabolism under these conditions by measuring tissue lactate and glycogen concentrations in liver, heart, and skeletal muscle. Although bicarbonate infusion resulted in a significantly higher arterial blood pH (pHe) than saline infusion, intracellular pH (pHi) of heart and skeletal muscle, as determined by the DMO equilibration technique, were not significantly different after 4 h of anoxia. We were also unable to demonstrate any differences in anaerobic metabolic rate, since both tissue lactate accumulation and glycogen depletion were identical in bicarbonate- and saline-infused frogs in the tissues studied. We conclude that (1) alterations in the extracellular acid-base state by bicarbonate infusion are not necessarily reflected in the intracellular compartment, perhaps due to powerful intracellular buffering processes, and (2) maintenance of an alkaline extracellular pH during anoxia in bullfrogs does not influence the anaerobic metabolic rate. We could not, however, rule out a possible role for intracellular pH in regulating anaerobic metabolism during anoxia in frogs.

Anaerobiosis↗

PubMed1993

A study of the advantages and limitations of immunoblotting procedures for the detection of antibodies against influenza virus.

An immunoblotting procedure was used to determine the specificity and examine some of the properties of antibodies produced following infection of mice with influenza virus or inoculation with noninfectious material with Alhydrogel or complete Freund's adjuvant. The noninfectious material used was beta-propiolactone-inactivated influenza virus and a preparation (HANA) enriched for the surface glycoproteins, hemagglutinin (HA) and neuraminidase (NA). When influenza viral proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under nonreducing conditions, each of the anti-viral antisera tested exhibited strong binding. Under reducing conditions, however, much weaker binding was observed especially towards the HA1 subunit of HA. This was particularly apparent with antisera raised to virus or HANA in the absence of adjuvant. A panel of monoclonal antibodies directed to HA also bound well to viral HA separated by SDS-PAGE under nonreducing conditions but failed to recognize epitopes on HA1 separated under reducing conditions. These results suggest that when HA is reduced and immobilized on a solid support, it does not display the conformational features essential for the integrity of all epitopes. The immunoblotting procedure was also used to determine the isotype of anti-viral antibody directed against individual viral proteins and to detect matrix protein 2 (M2) in purified influenza virions and influenza-infected cells using antisera raised to a synthetic peptide representing a sequence within the M2 protein.

Amino Acid Sequence↗

PubMed1993

Modulation of CD4+ T-cell recognition of influenza hemagglutinin by carbohydrate side chains located outside a T-cell determinant.

Two distinct T-cell clones raised to the H3 subtype influenza virus A/Memphis/71 fail to proliferate in response to other H3 viruses that contain the amino acid substitution Asp63-->Asn within the heavy (HA1) chain of the hemagglutinin (HA). These nonstimulatory viruses nevertheless have an intact sequence corresponding to the determinants recognized by the clones. The substitution at residue 63 results in the creation of a potential glycosylation site which occurs outside the boundaries of the T-cell determinants. A synthetic peptide encompassing the two determinants and extended to include residue Asn63 stimulated levels of proliferation of the clones similar to those obtained with a peptide containing Asp63. These results indicate that the presence of Asn rather than Asp does not in itself affect T-cell recognition. Partial enzymatic deglycosylation of isolated HA possessing the glycosylation site restored the ability of the T-cell clones to proliferate. More extensive deglycosylation of HA1 had a differential effect on the clones, allowing one to proliferate but not the other. These results illustrate that even when attached to residues outside a determinant, the carbohydrate of a glycoprotein antigen can influence T-cell recognition. In this way, changes in glycosylation patterns of HA from different isolates of the virus may influence the efficacy of preexisting T-cell immunity.

Amino Acid Sequence↗

PubMed1993

Binding affinity of influenza virus N9 neuraminidase with Fab fragments of monoclonal antibodies NC10 and NC41.

Sedimentation equilibrium centrifugation has been applied to determine the affinity and stoichiometry of the interaction between Fab fragments, derived from monoclonal antibodies NC10 and NC41, with influenza virus neuraminidase N9 isolated from either tern or whale. Although the two neuraminidase epitopes recognized by NC10 and NC41 Fab overlap, crystallographic studies have shown that the modes of binding of each Fab are different. The sedimentation equilibrium experiments described here reveal that the binding affinities are also different, with NC10 Fab binding more strongly to each neuraminidase. Furthermore, comparison of the affinity of binding of each antibody fragment reveals a stronger interaction with tern neuraminidase than with whale neuraminidase. Although the respective epitopes recognized by each antibody on the two antigens are similar, this technique shows that they do nevertheless possess sufficient differences to affect significantly the binding of antibody.

Animals↗

PubMed1993

Quantitative analysis of the interaction between lysozyme and monoclonal antibody D1.3.

The method of sedimentation equilibrium has been used to determine the stoichiometry and binding constant for the interaction between hen egg white lysozyme and monoclonal antibody D1.3. The procedures described allow the relative binding affinities of 125I-labelled lysozyme and unlabelled lysozyme to be compared. The data indicate that labelled and unlabelled lysozyme bind to monoclonal D1.3 with similar affinity (binding constant, K = 1.6 x 10(9)/M). Using solid-phase methods estimates obtained for the binding constant were lower and dependent both on the amount of antigen coated on the plate and the dilution of primary antibody (D1.3). These data were not consistent with a simple equilibrium binding model, suggesting kinetic or orientation effects. In contrast sedimentation equilibrium experiments provide a rapid and accurate method for determining both the stoichiometry and binding constants for the interaction between antigens and antibodies.

Animals↗

PubMed1993

Studies on the antibody response of mice and humans after immunization with potential influenza virus A (H1N1) vaccines.

The antibody response of mice and adult humans to immunization with subunit vaccines derived from a pair of antigenically distinct influenza A H1N1 viruses isolated in eggs was investigated. Although the haemagglutinin molecule of each virus differed by only three amino acid residues, highly specific antibody responses were elicited in mice as determined by haemagglutination inhibition and radioimmunoprecipitation assays. Results from competitive radioimmunoassays using monoclonal antibodies of known specificity and a study of the reactivity of mouse antisera with H1N1 field strains indicated that the marked differences in the antibody responses to the two vaccines was due to an amino acid substitution in the distal tip of the haemagglutinin molecule. In contrast, crossreactive antibody responses were elicited in humans presumably due to exposure to viruses related to the candidate vaccine prior to vaccination. Although immunogenic differences are apparent in this pair of antigenically distinct viruses in naive laboratory animals, these differences are not apparent following vaccination of humans that had prior exposure to related viruses.

Amino Acid Sequence↗

PubMed1993

Prevention of hypoxemia during lumbar puncture in infancy with preoxygenation.

Hypoxemia has previously been reported during lumbar puncture (LP) in infancy. The purpose of this study was to determine whether preoxygenation before the LP would reduce hypoxemia during the procedure in infants. Twenty-one infants (one to 15 weeks of age) undergoing LP for evaluation of possible sepsis were randomly assigned to the control group (12) or treatment group (9). The treatment group was preoxygenated breathing oxygen (FiO2 = 1.0) spontaneously via snug face mask for three minutes prior to being positioned for the LP. The control group spontaneously breathed room air during this interval. Oxyhemoglobin saturation was measured prior to, and continuously during, the LP with pulse oximetry. The groups were comparable in age, resting respiratory rate, baseline saturation, and duration of the procedure. The treatment group developed significantly less desaturation during the procedure than the control group (P < 0.05). We conclude that preoxygenation prior to LP prevents most of the hypoxemia resulting from the procedure in infants.

Female↗

PubMed1993

Conservation of determinants for class II-restricted T cells within site E of influenza virus hemagglutinin and factors influencing their expression.

The determinants recognized by helper T cells specific for the site E region of H3 subtype influenza virus hemagglutinin (HA) have been defined by examining the reactivity of T-cell clones with sets of overlapping peptides of various lengths covering the site. Two overlapping sequences, TLIDALLG and LIDALLGDP, were identified as the minimal determinants for four of five representative clones. These sequences are located within a loop of the molecule closed by a disulfide bond and presumably require cleavage of this bond for interaction with the class II major histocompatibility molecule. In contrast, the determinant recognized by the fifth clone was dependent on the presence of an intact disulfide bond for its expression and could not be represented by a synthetic peptide homolog of the linear sequence. Both TLIDALLG and LIDALLGDP are conserved within all field strains of the H3 subtype. Nevertheless, recognition of these sequences by the T-cell clones is affected by the glycosylation pattern of the hemagglutinin and by residues lying outside the minimal determinant. Three distinct clones directed towards the sequence LIDALLGDP were remarkably similar in their pattern of response to a set of synthetic analogs of the determinant, suggesting that residues of the T-cell receptor other than those contacting the minimal determinant may be responsible for the different specificities observed for these clones with different field strains of virus.

Amino Acid Sequence↗

PubMed1992

Western blot analysis of antibody responses to influenza virion proteins.

An immunoblotting procedure was developed to detect antibody responses in mice and humans to influenza virion proteins. The technique was capable of detecting 1.5 micrograms of haemagglutinin (HA) on nitrocellulose strips at a 1:5000 dilution of a mouse serum with an initial haemagglutination inhibition titre of 20. The effects of the use of the blocking agent Tween-20 on virion proteins were also studied. The commonly used concentration of 0.05% (v/v) Tween-20, when included in blocking and incubation buffers, greatly reduced the amount of detectable matrix protein but caused no detectable loss of HA and neuraminidase/nucleoprotein proteins. If virion proteins were separated by polyacrylamide gel electrophoresis under reducing conditions, antibody bound to HA2 more strongly than to HA1. Under non-reducing conditions, more antibody bound to the uncleaved HA protein than to other proteins. IgG1 and IgG2a antibody responses in mice to each protein were stronger than IgG2b and IgG3 responses.

Animals↗

PubMed1992

Two distinct serum mannose-binding lectins function as beta inhibitors of influenza virus: identification of bovine serum beta inhibitor as conglutinin.

Normal bovine and mouse sera contain a component, termed beta inhibitor, that inhibits the infectivity and hemagglutinating activity of influenza A viruses of the H1 and H3 subtypes. We have previously shown these beta inhibitors to be mannose-binding lectins that apparently act by binding to carbohydrate on the viral hemagglutinin, blocking access of the receptor-binding site to receptors on host cells (E. M. Anders, C. A. Hartley, and D. C. Jackson, Proc. Natl. Acad. Sci. USA 87:4485-4489, 1990). For the H3-subtype virus A/Memphis/1/71 x A/Bel/42 (H3N1), sensitivity to beta inhibitors is determined by the oligosaccharide at residue 165 of the hemagglutinin, this glycosylation site being lost in a resistant mutant selected by growth in the presence of bovine serum. In the present study, we sequenced the hemagglutinin genes of additional bovine serum-resistant mutants derived from influenza viruses A/Philippines/2/82 (H3N2) and A/Brazil/11/78 (H1N1). The results confirm the importance of carbohydrate at residue 165 for inhibitor sensitivity of H3 viruses and implicate carbohydrate at residue 87 (94a in the H3 numbering system) as an important determinant in the sensitivity of H1-subtype viruses to the bovine inhibitor. Unlike the two H3 mutants, which had also gained resistance to hemagglutination inhibition by mouse serum, the H1 bovine serum-resistant mutant remained sensitive to the mouse beta inhibitor, suggesting that inhibition by the two types of sera is mediated by distinct mannose-binding lectins. In support of this hypothesis, the beta inhibitors in bovine and mouse sera were shown to differ in their pattern of inhibition by monosaccharides and in their sensitivity to 2-mercaptoethanol. In these and other properties, the bovine inhibitor closely resembled conglutinin, a Ca(2+)-dependent N-acetylglucosamine- and mannose-binding lectin present in bovine serum but absent from the serum of other species. Furthermore, polyclonal and monoclonal anticonglutinin antibodies abrogated the hemagglutination-inhibiting activity of bovine serum. Direct binding of conglutinin to the parent viruses and reduced binding to their respective mutants were confirmed by radioimmunoassay.

Animals↗

PubMed1992

Metabolic and cardiodynamic responses of isolated turtle hearts to ischemia and reperfusion.

We used 31P and 1H nuclear magnetic resonance spectroscopy to measure intracellular pH, high energy phosphates, and lactate levels in hearts of turtles (Chrysemys picta bellii) subjected to 1.5 h of global ischemia followed by reperfusion. We simultaneously monitored maximum ventricular developed pressure (Pmax), maximal rate of pressure development (dP/dtmax), rate-pressure product (RPP), cardiac output, and heart rate and also measured lactate efflux from the hearts during reperfusion. Our goal was to test the hypothesis that turtle hearts would prove tolerant of prolonged global ischemia at 20 degrees C and would recover completely on reperfusion without any indication of ischemia-or reperfusion-related injury. The 1.5 h of ischemia resulted in decreases in phosphocreatine and ATP to 31.4 +/- 2.8 and 87.3 +/- 6.3% of control, respectively, while Pi rose to 236.6 +/- 26.3%. Intracellular pH decreased during this period from 7.38 +/- 0.02 to 6.87 +/- 0.04. Most of these changes occurred during the first 30 min. Tissue lactate rose during 1.5 h of ischemia from approximately 1.5 to 22.3 mumol/g wet tissue wt. However, the rate of lactate production was much higher during the first 21 min of ischemia (0.41 mumol.g-1.min-1) than during the remaining 70 min (0.10 mumol.g-1.min-1). With the onset of ischemia, Pmax, dP/dtmax, RPP, and heart rate all decreased dramatically with roughly the same time course as the changes in high-energy phosphates and intracellular pH. On reperfusion, turtle hearts rapidly restored high-energy phosphates, intracellular pH, lactate, and cardiodynamics to control levels, usually within 15-30 min, with no evidence of reperfusion injury.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1992

Apneic oxygen uptake in the torpid bat, Eptesicus fuscus.

Like many mammalian heterotherms, the big brown bat, Eptesicus fuscus, breathes intermittently during torpor. By exploiting this bat's preference to roost in crevices, we could separately measure O2 uptake during ventilatory bouts and apneic periods using a flow-through metabolic chamber with a small dead space volume and short time constant. Oxygen uptake was measured during apneas ranging from 10 to 150 min duration at body temperatures of 20, 10 and 5 degrees C. The fraction of total O2 uptake acquired during apnea was 0.26 +/- 0.03 (9), 0.54 +/- 0.10 (5) and 0.35 +/- 0.04 (3) for body temperatures of 20, 10 and 5 degrees C, respectively. Cardiogenic pulsations during apnea visible on plethysmographic pressure traces and theoretical calculations of airway and cutaneous diffusion potentials support the notion that apneic O2 uptake occurs down an open airway by both diffusion and bulk convection.

Animals↗

PubMed1991

Electron microscopic evidence for the association of M2 protein with the influenza virion.

Immunogold electron microscopy revealed that site-specific antibodies elicited by a synthetic peptide representing the N-terminal sequence (residues 2-10) of influenza virus M2 protein were capable of binding to the surface of virions. Antibody binding was observed with two human influenza virus strains but not with an avian virus strain which has amino acid substitutions in the appropriate sequence of M2. These results provide direct evidence for the presence of M2 in the influenza virion.

Amino Acid Sequence↗

PubMed1991

Three antibody molecules can bind simultaneously to each monomer of the tetramer of influenza virus neuraminidase and the trimer of influenza virus hemagglutinin.

Trimeric hemagglutinin and tetrameric neuraminidase molecules isolated from influenza virus bind an average of 9 and 13 molecules respectively of monovalent antibody fragments prepared from IgG isolated from polyclonal sera. In each case this represents an average of approximately three molecules of antibody binding to each protomer. Although there is compelling evidence for the presence of multiple adjacent and overlapping epitopes covering the surface of these two viral antigens, steric hindrance ensures that even under saturating conditions only three molecules of monovalent antibody fragments can be simultaneously accommodated on each monomer.

Animals↗

PubMed1991

Extracellular and intracellular acid-base effects of submergence anoxia and nitrogen breathing in turtles.

We compared extracellular and intracellular acid-base state in turtles (Chrysemys picta bellii) subjected to anoxic submergence to turtles made anoxic by N2-breathing. Measurements made on control animals and on animals after 1, 2, 4, or 6 h of anoxia included blood pH, PO2, PCO2, and lactate as well as liver, heart, skeletal muscle, and brain pHi (using DMO equilibration), lactate, and glycogen concentrations. We hypothesized that the anaerobic metabolic rate of submerged turtles would be depressed by the more severe extra- and intracellular acidosis, and that this would be indicated by reduced lactate accumulation and glycogen depletion. Submerged turtles became extremely acidemic due to a combined metabolic and respiratory acidosis and had significantly lower arterial pH than N2-breathing animals (6.98 and 7.34, respectively, after 6 h). In spite of this disparity in pHa, 6 h pHi values for liver, heart, and brain were similar. Likewise, our data on glycogen depletion and lactate accumulation at h 6 in these tissues suggest no dramatic differences in anaerobic metabolic rate. While skeletal muscle pHi was somewhat lower at h 6 in the submerged group (6.73 vs 6.91 for N2-breathers), we observed no differences in either glycogen depletion or lactate accumulation in this tissue between our two treatments. Thus, at h 6, in spite of a 0.37 pH unit difference in pHa and a nearly 70 mm Hg difference in arterial and presumably cytosolic PCO2, pHi and tissue lactate and glycogen concentrations were similar. These results can be explained if the in vivo intracellular buffer values (beta) of turtle tissues are very high. We conclude that extracellular acid-base state is not necessarily reflected intracellularly in vivo in turtles and care must be taken in extrapolating from one compartment to another when attempting to make inferences about metabolic depression or acid-base regulation in this species.

Acid-Base Equilibrium↗

PubMed1991

Effects of anoxia and graded acidosis on the levels of circulating catecholamines in turtles.

We measured circulating levels of catecholamines in painted turtles subjected to anoxia with different degrees of concomitant acidosis at 20 degrees C and in turtles subjected to long-term submergence at 3 and 10 degrees C. Blood levels of both epinephrine (E) and norepinephrine (NE) increased during N2-breathing, N2/CO2 breathing and submergence, with NE generally being present in higher concentrations than E. During submergence at 20 degrees C, anoxic turtles experienced an extreme acidosis and NE levels exceeded 18,000 pg/ml. The greater the degree of acidosis in anoxic turtles the higher were the levels of plasma NE (log [NE; pg/ml] = 1.640 x pHa + 15.776, r = -0.826). Elevation of plasma E under anoxic conditions was more modest and the correlation between plasma E and pHa was less pronounced (log [E; pg/ml] = -0.329 x pHa + 6.069, r = -0.285). Submergence at lower temperatures also resulted in increases in plasma levels of NE, but while plasma E generally increased during anoxia, this elevation was less dramatic than that observed for NE. Exposure of turtles to either mild (6.5% CO2) or severe (14.5% CO2) normoxic hypercapnia resulted in no increase in E and only modest increases in NE. Upon resumption of air-breathing in all of the 20 degrees C protocols, turtles rapidly restored E and NE to control levels. The function of elevated plasma catecholamines during anoxia and acidemia in turtles is unknown but may be important in stimulating respiratory and cardiovascular recovery once air-breathing is resumed. Catecholamines may also play a role in mediating the rise in blood glucose we observed in this study, which may be an important factor in maintaining tissue viability during anoxic stress.

Acidosis↗