Production of experimental chimeras in livestock by blastocyst injection.
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Animals↗
Biomedical subjects
J E Butler
Publications and source records attributed to J E Butler.
The metabolism and transport of bovine serum SIgA.
Experiments were undertaken in six Holstein-Friesian cattle to determine whether secretory IgA (SIgA) could be transported from serum into exocrine body fluids. Preliminary data indicated that when IgG1, IgG2, IgM and SIgA were administered i.v., only SIgA and IgG1 appeared in bile 90 min later at concentrations equal to or exceeding those in serum at the same time. Two hours post-injection, 70% of the SIgA recovered in bile was intact however only 30% co-precipitable with anti-secretory component (SC) while greater than 90% of the administered IgA was precipitated by this method. All recovered IgG1 was of low molecular weight. More detailed studies indicated that the IgA recovered in bile 7 h post-injection or in milk 3 h post-injection, was predominantly lower molecular weight than intact SIgA. Most of this low molecular weight radioactivity was TCA precipitable and ca 50% was dialyzable; these data indicate that TCA-precipitability is an inadequate criterion for determining whether intact SIgA is transported. The radioactivity recovered in parotid saliva was almost entirely non-TCA precipitable and dialyzable. Almost all SIgA recovered in bovine serum remained intact and had a t1/2 of 15.7 h. When transport into milk and bile was calculated from total, recovered radioactivity (i.e. 29% and 2.7, respectively), data compared favorably with those conducted in sheep in which dimeric IgA (without SC) was administered i.v. When we calculated transport on the basis of recovered intact IgA, only 1.47 and 0.54% of the injected dose had been transported into milk and bile, respectively, 24 h later. Most IgA in ruminant bile may be of serum origin although the same appears to be unlikely for the IgA in milk.
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Heterogeneity of bovine IgG2--I. The A1 allotypic determinant is the major antigenic determinant recognized on bovine IgG2a by polyclonal rabbit anti-IgG2a.
Rabbit antisera specific for the IgG2a subclass of bovine immunoglobulins also contain antibodies which recognize the A1 allotype of this immunoglobulin subclass. In many sera anti-Al constitutes the major specificity of these rabbit antisera and all of the 10 rabbits immunized with Al(+) IgG2a produced some antibodies which recognized the allotype. The determinant is the same as recognized by a bovine alloantiserum to Al. The Al allotype is shared by the intact and the Fc portion of IgG2a, but rabbits immunized with the Fc fragment also recognize an Al-related determinant which is not exposed on intact Al(+) IgG2a. None of the four rabbits immunized with Al(-) IgG2a produced precipitating antibodies which recognized the gene product of the Al(-), i.e. A2 allele, suggesting that only Al is an immunodominant antigenic determinant for rabbits. Data reported here help to explain the antigenic heterogeneity seen among the IgG2 populations from different cattle when they are tested by immunoprecipitation using rabbit anti-IgG2 reagents.
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Dimeric M315 is transported into mouse and rat milk in a degraded form.
The controversial issue of serum to milk transport of IgA in rodents was addressed in experiments that evaluated the molecular integrity and antigen-binding ability of the dimeric IgA (dIgA) recovered in the stomachs of rat and mouse pups suckling dams which had been administered homogeneous, dimeric M315 intravenously (i.v.). Four rat and three mice dams were i.v. administered affinity-purified, 125I-dIgA anti-DNP (M315) of which greater than 83% could bind DNP after iodination, and of which greater than 82% could be captured by solid phase anti-M315. Dams were given the labelled dIgA either 1-day or 8-days postpartum. Twenty-three hours after i.v. administration, the radioactivity of each pup, its stomach contents and an aliquot of serum were analyzed for total radioactivity, anti-DNP activity and for the molecular integrity of the recovered I-125. Data showed that 11-43% of 125I-activity given to the dam could be recovered from the stomach contents and sera of the pups after this time. Immunoassay revealed that less than 2% of the recovered radioactivity could bind DNP, i.e. a loss of 98% of functional antibody. It was calculated from ultracentrifugational analyses that less than 0.7% of the 125I-dIgA had been transported intact to the suckling neonates. Analyses of stomach milk and neonatal sera by sucrose density gradient ultracentrifugation revealed that almost all recovered radioactivity was in the form of low mol. wt fragments. Data fail to support the concept of an active mechanism for the transport of intact IgA from serum to milk in rodents during early or mid-lactation.
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The immunochemistry of sandwich ELISAs--I. The binding characteristics of immunoglobulins to monoclonal and polyclonal capture antibodies adsorbed on plastic and their detection by symmetrical and asymmetrical antibody-enzyme conjugates.
Radiolabelled bovine IgG1, IgG2, SIgA and IgM and heavy-chain specific polyclonal and monoclonal antibodies to these isotypes were employed as models to investigate immunochemical aspects of sandwich enzyme immunoassays (ELISAs). The titration plots obtained by measuring enzyme activity paralleled those obtained when the binding of radiolabelled immunoglobulins to solid-phase capture antibodies was quantitated. As predicted from the Mass Law, the percentage of labelled immunoglobulin which was bound remained constant over the range in which the sandwich ELISA titration was linear on a log-log plot. Also as predicted from the Mass Law, increasing the solid-phase concn of polyclonal antibodies by affinity purification increased the linear region of the log-log ELISA plot and the corresponding region over which a constant percentage of immunoglobulin binding was observed. When used as capture antibodies adsorbed on plastic at equal concns, the best monoclonal antibodies were 1/8- less than 1/16 as effective as their polyclonal counterparts in binding iodinated bovine immunoglobulins; these differences can be directly interpreted to result from an 8 and greater than 16-fold higher functional, relative affinity of the polyclonal reagents. Steric hindrance was shown to occur when symmetrical sandwich ELISAs, i.e. capture and detection antibody are both heavy-chain specific, are used to measure monomeric but not IgM immunoglobulins. The use of an asymmetrical configuration, i.e. anti-Fab antibody-enzyme conjugates, avoids this problem. Symmetrical conjugates based on the avidin-biotin system, horseradish peroxidase or alkaline phosphatase, were less effective than their asymmetrical (anti-Fab) counterparts. Evidence that the lower activity of symmetrical conjugates was due to steric hindrance was illustrated using horseradish peroxidase-antibody conjugates of different sizes. Sandwich assays using affinity-purified, polyclonal solid-phase antibodies and an asymmetrical conjugate were judged to be immunochemically and economically optimal. Using an asymmetrical configuration, the non-linear nature of sandwich ELISA titration plots is the predictable result of changing antibody to antigen ratios in an antibody-limiting system, and not the result of steric hindrance of the detection system.
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Altered recognition of surface-adsorbed compared to antigen-bound antibodies in the ELISA.
We found in preliminary studies using 125I-labelled antibodies that an antibody bound to a solid-phase antigen was recognized more efficiently than an antibody adsorbed directly to the solid phase. The present study was designed therefore to quantitate the differential recognition of an antibody adsorbed directly to the solid phase and an antibody bound to antigen on the solid phase using the amplified enzyme-linked immunosorbent assay (a-ELISA), and to compare results with the amounts of specific antibody determined by quantitative immunoprecipitation. The degree of differential recognition was quantitated for rabbit IgG and SIgA anti-ovalbumin (anti-OA) and anti-fluorescein, and was found to be dependent upon the isotype of the antibody and not its specificity. The ratio describing the differential recognition of SIgA antibodies (1.8) was much less than for IgG antibodies (greater than 30) and remained constant over the titration range analyzed while the ratios obtained for IgG varied substantially (25-60) over the same range. These ratios of differential recognition were used to estimate rabbit IgG antibody levels to OA, bovine serum albumin, ferritin and alpha-lactalbumin. The estimates obtained were consistently much less than total antibody levels measured by quantitative precipitation. The use of glutaraldehyde-aggregated OA in the ELISA, however, increased the amount of IgG anti-OA and SIgA anti-OA capable of recognizing OA adsorbed on plastic from 12 to 50 and from 30 to 80%, respectively.
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The proteolytic activity of milk fat, whey and casein for iodinated, extrinsic bovine IgG1, IgG2, SIgA and IgM.
Purified, iodinated bovine immunoglobulins (Igs) were incubated with fresh Guernsey milk or with the casein, fat and whey fraction of such milk for up to 12 hr at 37 degrees C. Igs incubated in whole milk, showed little evidence of proteolysis in either the whey, fat or casein fractions although the amount of radioactivity which became associated with the latter two fractions prevented adequate analysis. When the individual milk fractions were first prepared and then incubated with iodinated Igs, we found no evidence for proteolysis of any Ig in whey or casein but ca. 25% breakdown or dissociation of the IgM and SIgA which had been incubated with milk fat. Breakdown of these Igs in fat was not inhibited with benzamidine-HCl, sodium azide or EDTA. These data show that: only those Igs which associate with milk fat are degraded or dissociated by it and the Ig fragments described from cows milk or recovered during studies on Ig transport cannot be ascribed to the proteolytic activity of fresh milk.
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The association of extrinsic bovine IgG1, IgG2, SIgA and IgM with the major fractions and cells of milk.
The association of purified, iodinated bovine immunoglobulins with the various fractions of whole milk and with cell-free skim milk and with two different mammary cell populations was studied. Associations in whole milk were concentration independent over a 6-fold range and revealed that SIgA and IgM were 5-fold more prevalent in milk fat than IgG1 and IgG2; the concentration of IgM and SIgA was 3-fold and 2-fold higher in fat than whey, respectively. A significant proportion of the IgM, ca 20% and to a lesser extent IgG2, ca 10%, were found in association with the casein pellet. Greater than 85% of the IgG2, greater than 90% of the IgG1, approximately equal to 80% of the SIgA and 70% of the IgM were found in milk whey. Isoelectric precipitation of casein significantly reduced the amount of IgM which associated with fat. When labelled Igs were incubated with milk leukocytes alone, only SIgA and IgM became significantly associated with them. However, when 10(6) cells were added, the amount of SIgA and IgM in the casein-cell pellet was not additive, although the increase for IgM was significant. These Igs also associated with the casein pellet of cell-free skim milk. When whole milk was used, the milk fat competed with cells and casein for association with SIgA and IgM. Homogenization of the fat layer from normal milk with or without added cells, caused significant release of the Igs which sedimented in the pellet.
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Maternal-neonatal immunoregulation in swine. II. Influence of multiparity on de novo immunoglobulin synthesis by piglets.
In the first-litter sows lower serum levels were found for all three Ig classes as compared to multiparous sows. The same was true for IgA in lacteal secretions and in piglet serum during the first days of life, while no differences were found for IgG levels. In contrast to these findings, IgM levels were found to be higher in lacteal secretions of first-litter sows and in piglet serum during the first days of life as compared to their counterparts. From three weeks after birth Igs found in piglet serum mainly originate from de novo synthesis. In this period piglets of first litter sows showed a higher IgA level up to the 6th week of life and higher IgG and IgM levels up to the end of the investigation period. Results are discussed in terms of maternal-neonatal immune regulation, focussing on the apparent suppressive role of maternally-derived IgG on total de novo Ig synthesis by suckling piglets.
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Detection of pregnancy by radioimmunoassay of a novel pregnancy-specific protein in serum of cows and a profile of serum concentrations during gestation.
The development of a double antibody radioimmunoassay for a bovine pregnancy-specific protein (pregnancy-specific protein B; PSPB) is presented. By means of this assay, PSPB could be measured in serum of pregnant cows. Five dairy cows were bled throughout gestation to measure serum levels of PSPB. Serum concentrations (means +/- SE) exceeded 1 ng/ml by 30 days postbreeding and increased gradually through three months (9 +/- 0.6 ng/ml), six months (35 +/- 6 ng/ml), and nine months (150 +/- 75 ng/ml) of gestation. Maximum levels of PSPB (542 +/- 144 ng/ml) were reached two days before parturition and then steadily declined to less than 78 ng/ml by 21 days postpartum. In 21 cows bled daily from 15 through 30 days postbreeding, PSPB could be measured in a few cows before and in most cows by 24 days after breeding. In a commercial herd of 102 beef cows, the assay could detect pregnancy earlier and more accurately than the routine method of rectal palpation. This radioimmunoassay measures a unique antigen that, for the first time, provides a serological method for detecting pregnancy in cows.
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Dimeric mouse IgA is transported into rat bile five times more rapidly than into mouse bile.
The kinetics of the biliary dimeric IgA transport in rats is well documented from studies on animals with cannulated bile ducts, while in mice such information rests on the analysis of samples periodically removed from the gall bladder and on serum disappearance rates. We have quantitatively compared the kinetics of IgA transport in these two rodents by using affinity-purified radiolabelled monomeric and dimeric M315 IgA which specifically binds dinitrophenyl (DNP). Bile collection procedures, sample analysis, and administration of the radiolabelled M315 were the same for both species, although in mice the cystic duct was ligated to prevent reflux of any bile or IgA from the gall bladder into the common bile duct. A pronounced selection for transport of dimeric M315 was seen in both species. When dimeric M315 was administered, it was recovered in the dimeric form from the bile of both species, and 60-80% could specifically bind to DNP-gelatin. When monomeric M315 was administered, very little was transported, and the IgA recovered in bile had a much lower capacity to bind DNP-gelatin and appeared to be of low molecular weight. Approximately 60% of the intravenously administered dimeric IgA was transported into bile of both species but biliary transport of the heterologous dimeric IgA into rat bile was five times more rapid than the transport of the dimeric M315 into its homologous species, the mouse. Hence, IgA transport in these species is similar in specificity, integrity of the transported IgA and in the percentage cleared, while differing significantly in the rate of transport. This cannot be ascribed to the species source of the IgA.
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Injuries to the metatarsophalangeal joints in athletes.
Injury to the metatarsophalangeal joints in sports has become an increasing problem with the advent of more flexible shoewear and artificial playing surfaces. These injuries vary from mild sprains to severe tearing of the capsuloligamentous complex including avulsion fractures. This may include a compression fracture of the metatarsal head in the more severe cases. At the extreme, dislocation or fracture-dislocation of the metatarsophalangeal joint occurs. The findings in 62 cases of these injuries in Rice University athletes from 1971 to 1985 are presented. Treatment follows a standard regimen of ice, taping and anti-inflammatory medication with gradual return to sports activity as symptoms allow. Recommendations for preventive therapy include stiffening the forefoot in athletic shoes or the use of an orthotic device. This should reduce the incidence of long-term sequelae.
Adult↗
Biochemistry and biology of ruminant immunoglobulins.
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Animals↗
Preliminary evidence for a diagnostic immunoglobulin G1 antibody response among culture-positive cows vaccinated with Brucella abortus strain 19 and challenge exposed with strain 2308.
The sera of cows inoculated with Brucella abortus have a characteristically high titer of immunoglobulin (Ig) G1 antibodies to a soluble brucella antigen compared with sera of noninoculated vaccinated cattle. Concentrations of antigen-specific IgG1 were greater than 10-fold higher than those for IgG2, even though total IgG2 concentrations were higher than total IgG1 concentrations. Increases in IgG1 antibodies to Brucella abortus soluble antigen were detected shortly after vaccination in those cows from which strain 19 was isolated and by 28 weeks in cows from which strain 2308 was isolated. Increases in specific antibodies were not paralleled by increases in either total IgG1 or total IgG2 concentrations. Rather, there was a 15-fold to greater than 200-fold increase in specific activity, with up to 16% of the IgG1 specific for the brucella antigen used in the assay. Thus, measurement of changes in total IgG1 concentrations is not a reliable method to identify brucellosis-associated anti-Brucella abortus soluble antigen activity. Only one cow in a panel of 10 selected for detailed study showed a false-positive IgG1 titer, whereas some serologic assays showed as many as 4 or 5 false-positives. Results of the complement-fixation test, among the battery of serologic tests used for detection of brucellosis, best agreed with the occurrence of increased IgG1 antibody levels.
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The relationship between the binding of primary antibody to solid-phase antigen in microtiter plates and its detection by the ELISA.
Radioiodinated monomeric and dimeric M315 (mM315 and dM315) prepared from BALB/c ascites fluid by gel filtration and affinity chromatography were used to study the relationship between primary antibody binding to solid-phase dinitrophenylated gelatin in microtiter ELISAs and its indirect detection by enzyme-antibody conjugates and complexes. The relationship between the amount of mM315 or dM315 which binds to dinitrophenylated gelatin to the amount added is linear over a nearly 3-log range with a slope of 1; the amount of M315 which binds in this linear range after 24 h represents all of the active antibody in the system. On a binding site basis, mM315 was inhibited by a significantly lower amount of dinitrophenyl-(DNP)glycine than was dM315. The indirect detection of bound M315 over the same 3-log range using ELISA yielded a sigmoidal titration curve which encompassed a short linear region that had a slope of 0.9 or less. Plateauing of the titration plots at high input of both mM315 and dM315 was shown to be progressively exaggerated in direct relationship to the size of the enzyme-antibody conjugate used for their detection. The data show that the upper region of the sigmoidal ELISA titration plot is the result of steric hindrance of the detection system. Through the combined use of an 131I-enzyme-antibody immune complex (EIC) detection system and 125I-M315, it was shown that the deviation from the linear binding of 125I-M315 observed during its indirect detection in the so-called linear region of the ELISA titration curve was the result of changing ratios of bound EIC: bound primary antibody, not altered enzymic activity.
Alkaline Phosphatase↗
Identification and characterization of a hapten-modifiable TEPC 15 cross-reactive idiotype in swine.
Rabbits and swine immunized with TEPC 15 IgA, goats immunized with T15-positive IgM and swine immunized with affinity-pure swine anti-phosphorylcholine (PC) all produce antibodies which recognize a hapten-inhibitable idiotypic determinant on swine anti-PC. The similarity in reactivity and order of inhibitability with various PC analogs of the heterologous (swine anti-TEPC 15) and isologous (swine anti-swine anti-PC) reagents indicates that they recognize a related idiotype and suggest it may be the predominant idiotype expressed on swine anti-PC antibodies. The heterologous and isologous anti-idiotypic reagents generated in this study recognize swine and mouse anti-PC but not normal swine IgM, IgG or MOPC 460. Only reactions with swine anti-PC and mouse T15-positive anti-PC proteins are hapten-inhibitable. The greater inhibitory capacity of trimethylammonium and acetylcholine than PC suggests that the idiotope(s) recognized on swine anti-PC by the anti-idiotypic reagents is integral rather than peripheral to the anti-PC binding site. The nearly exclusive IgM anti-PC response of swine to Streptococcus pneumoniae R36A and PC-Brucella have so far hindered attempts to study the isotypic distribution of the idiotype.
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Quantitation of bovine beta 2-microglobulin: occurrence in body fluids, on milk fat globules and origin in milk.
The concns of bovine beta 2-microglobulin (beta 2M) and selected control proteins were measured using a competitive immunoassay to determine the origin of beta 2M in cows' milk. Using milk samples collected at various times, separated into different fractions and treated with protease inhibitors, it was established that beta 2M appears in cows' milk by protease-dependent degradation of the cellular fraction of milk, probably mononuclear cells, but is not derived from milk fat globules (MFG) or from polymorphonuclear leukocytes despite positive immunofluorescence of the former. The latter source could be eliminated by the induction of neutrophilia which produced no changes in beta 2M levels. Our data also indicate a minimal contribution by MFG to levels of secretory component despite its detection by indirect immunofluorescence on MFG but are consistent with the view that the milk fat globule membrane protein, bovine-associated microprotein, is derived from MFG by proteolysis. Protease-dependent degradation of milk components is an in vivo storage effect which occurs as early as the first 3 hr of in vivo storage. Compared to three other secretions tested, beta 2M was most concentrated in lacteal secretions and codistributed with bovine lymphocyte antigen in milk.
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In vivo proteolytic activity of the mammary gland. Contribution to the origin of secretory component, beta 2-microglobulin and bovine-associated mucoprotein (BAMP) in cows milk.
Milk samples were collected from Holstein-Friesian cows at various times after milking (10-30 min; 30 min-10 hr) and treated with a protease inhibitor or control solution. Samples were then fractionated into whole, skimmed and cell-free skimmed milk aliquots. Some animals were treated with E. coli endotoxin prior to sample collection. The concentrations of three membrane-associated proteins (MAP), beta 2-microglobulin (beta 2M), secretory component (SC) and bovine-associated mucoprotein (BAMP) as well as albumin were measured in each aliquot to determine if in vivo proteolysis of milk elements could explain the origin of these MAP in milk. All three MAP could be localized on milk fat globules (MFG) and alveolar epithelial cells of the gland. Data revealed that all BAMP in milk can be accounted for by in vivo proteolytic degradation of MFG while most beta 2M is derived by similar degradation, from cellular elements in milk, presumably monocytes. Experiments with endotoxin which elevate PMN levels, failed to influence the release of any MAP while elevating albumin levels by greater than 10-fold. Based on these studies, SC release into milk cannot be ascribed to a protease-dependent mechanism.
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