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Biomedical subjects

R J Fulton

Publications and source records attributed to R J Fulton.

At least 19 recordsLinked to original sources
PubMed1997

Advanced multiplexed analysis with the FlowMetrix system.

The FlowMetrix System is a multiplexed data acquisition and analysis platform for flow cytometric analysis of microsphere-based assays that performs simultaneous measurement of up to 64 different analytes. The system consists of 64 distinct sets of fluorescent microspheres and a standard benchtop flow cytometer interfaced with a personal computer containing a digital signal processing board and Windows95-based software. Individual sets of microspheres can be modified with reactive components such as antigens, antibodies, or oligonucleotides, and then mixed to form a multiplexed assay set. The digital signal-processing hardware and Windows95-based software provide complete control of the flow cytometer and perform real-time data processing, allowing multiple independent reactions to be analyzed simultaneously. The system has been used to perform qualitative and quantitative immunoassays for multiple serum proteins in both capture and competitive inhibition assay formats. The system has also been used to perform DNA sequence analysis by multiplexed competitive hybridization with 16 different sequence-specific oligonucleotide probes.

Alleles↗
PubMed1996

Selective depletion of myelin-reactive T cells with the anti-OX-40 antibody ameliorates autoimmune encephalomyelitis.

The OX-40 protein was selectively upregulated on encephalitogenic myelin basic protein (MBP)-specific T cells at the site of inflammation during the onset of experimental autoimmune encephalomyelitis (EAE). An OX-40 immunotoxin was used to target and eliminate MBP-specific T cells within the central nervous system without affecting peripheral T cells. When injected in vivo, the OX-40 immunotoxin bound exclusively to myelin-reactive T cells isolated from the CNS, which resulted in amelioration of EAE. Expression of the human OX-40 antigen was also found in peripheral blood of patients with acute graft-versus-host disease and the synovia of patients with rheumatoid arthritis during active disease. The unique expression of the OX-40 molecule may provide a novel therapeutic strategy for eliminating autoreactive CD4+T cells that does not require prior knowledge of the pathogenic autoantigen.

Animals↗
PubMed1995

Opposing neuroendocrine actions of the natriuretic peptides: C-type and A-type natriuretic peptides do not interact with the same hypothalamic cells controlling prolactin secretion.

The two major members of the family of natriuretic peptides (NPs) in brain, A-type natriuretic peptide (ANP) and C-type natriuretic peptide (CNP) exert opposing actions on the neuroendocrine regulation of prolactin (PRL) secretion. We have targeted for compromise and destruction cells within the diencephalon which bear receptors for ANP (NRP-A receptors), CNP (NRP-B receptors), or both peptides (NPR-C receptors) using novel cytotoxin cell targeting methodology in order to determine if the neuroendocrine effects of these two peptides are exerted on similar cell systems. In animals pretreated with ANP conjugated to the cytotoxic A chain of ricin, central administration of a dose of ANP which is known to inhibit PRL secretion did not alter PRL levels in plasma; however, subsequent administration of CNP elicited the stimulation of PRL secretion. In rats pretreated with CNP-ricin A chain conjugate, a treatment we hypothesize targets for destruction CNP responsive cells, ANP injection did inhibit PRL secretion, while the stimulatory effect of CNP was absent. These results suggest that the neuroendocrine effects of these two natriuretic peptides on PRL secretion are expressed on different cellular elements of the hypothalamo-pituitary axis. Furthermore, they reveal that neither peptide acts directly on the tuberoinfundibular dopamine system since pretreatment with either cytotoxin conjugate failed to alter basal PRL levels. Thus ANP and CNP do not appear to express opposing actions on the same cell systems, suggesting the recruitment of each peptide individually by differing, unique stimuli for PRL release.

Animals↗
PubMed1995

Central oxytocin and ANP receptors mediate osmotic inhibition of salt appetite in rats.

These studies evaluated the involvement of central oxytocin (OT) and atrial natriuretic peptide (ANP) receptors in the osmotic inhibition of hypovolemia-induced salt appetite. Rats were pretreated centrally with the A chain of the cytotoxin ricin conjugated to OT (rAOT) or ANP (rAANP) to selectively inactivate cells bearing these respective receptors, or rats were pretreated with the unconjugated A chain (rA) as a control. Hypovolemia was induced with subcutaneous colloid injections, and rats then were given either 2 M mannitol, which raises plasma osmolality but lowers plasma sodium, or 1 M NaCl, which raises both. Hypertonic mannitol inhibited saline ingestion in rA-treated control rats but stimulated ingestion in rAOT- and rAANP-treated rats, whereas hypertonic NaCl blunted saline ingestion in rA- and rAOT-treated rats but stimulated ingestion in rAANP-treated rats. Angiotensin II-induced saline intake was similarly potentiated in rAOT- and rAANP-treated rats, indicating that this treatment also activates central inhibitory OT and ANP pathways. These data suggest that central ANP receptors mediate both Na(+)- and osmolality-induced inhibition of NaCl ingestion, whereas central OT receptors primarily mediate osmolality-induced inhibition of NaCl ingestion in rats.

Animals↗
PubMed1994

Pyodermatitis-pyostomatitis vegetans: a specific marker for inflammatory bowel disease.

In pyodermatitis-pyostomatitis vegetans annular pustular cutaneous lesions may precede, accompany, or follow the usually extensive vegetating oral disease. Sometimes only cutaneous or only oral lesions occur and previously have been described as separate entities. Clinical, histopathologic, and immunopathologic evidence clearly indicates this is one disease and suggests that it is distinct from pemphigus vegetans. The association between pyodermatitis-pyostomatitis vegetans and inflammatory bowel disease, most commonly ulcerative colitis, has been amply confirmed. Pyodermatitis-pyostomatitis vegetans should be considered a marker for inflammatory bowel disease.

Adolescent↗
PubMed1993

Central oxytocin inhibition of salt appetite in rats: evidence for differential sensing of plasma sodium and osmolality.

Sodium chloride ingestion is stimulated during conditions of sodium deficiency to maintain body fluid and electrolyte balance. Recent studies have indicated that salt appetite in rats is often inversely related to peripheral and central secretion of the hormone oxytocin (OT). We studied the potential role of central OT on salt and water ingestion by treating rats intracerebroventricularly with OT conjugated to the A chain of the plant cytotoxin ricin (rAOT) to produce a chronic selective inactivation of brain cells containing OT-receptive elements. The rats treated with rAOT and control rats treated with the ricin A chain alone were given 5-hr two-bottle (water and 0.5 M NaCl) drinking tests 30 min after they were made hyperosmolar by injections of hypertonic (2M) mannitol solution, which elevated plasma osmolality but reduced plasma Na+ concentration. In the control rats only water intake was stimulated in response to the induced hyperosmolality, but in the rAOT-treated rats hypertonic mannitol caused a robust salt appetite as well as thirst. Analogous results were obtained in rats treated with two different OT-receptor antagonists prior to induction of hyperosmolality with mannitol. In contrast to these results, when hyperosmolality was induced by administration of equivalently hypertonic (1M) NaCl, which elevated both plasma osmolality and plasma Na+ concentration, only water intake but not salt intake was stimulated in both control and OT-receptor antagonist-treated rats. When salt appetite was stimulated by the physiological stimulus of polyethylene glycol-induced hypovolemia, hypertonic mannitol similarly inhibited salt ingestion in control animals but not in rAOT-treated rats, whereas hypertonic NaCl inhibited subsequent salt ingestion in both groups. These results suggest that salt appetite is regulated by both Na(+)- and osmolality-sensing mechanisms in rats. In addition, they indicate that central OT likely mediates a significant component of osmolality-related inhibition of salt appetite but does not appear to be essential for Na(+)-related inhibition of this important homeostatic behavior.

Animals↗
PubMed1993

A variant of exotoxin A that forms potent and specific chemically conjugated immunotoxins.

To introduce a free sulfhydryl into Pseudomonas aeruginosa exotoxin A (ETA), methionine 161 in domain I of the toxin was changed to cysteine by site-directed mutagenesis. The free sulfhydryl provides a convenient site for covalent attachment of ETA to other proteins in the production of chimeric toxins. The mutation was then introduced into a variant of ETA that is impaired in receptor binding, termed ETA-60EF61, that has the dipeptide Glu-Phe inserted between residues 60 and 61. The resulting double mutant, ETA-60EF61 Cys161, was conjugated to three different monoclonal antibodies via a thioether linkage, and the immunotoxins were tested for cytotoxicity with cells in culture. Each immunotoxin was extremely potent against cells that expressed surface determinants for the monoclonal antibodies but had little cytotoxicity for cells that did not bind the antibodies. For comparison, we also conjugated ricin A chain to each of the three monoclonal antibodies and found that the resulting immunotoxins were at least two-orders of magnitude less potent than the corresponding immunotoxins made with ETA-60EF61Cys161. This study demonstrates that ETA-60EF61Cys161 makes potent and specific immunotoxins and may potentially be useful in selectively eliminating subpopulations of cells in vitro and in vivo.

ADP Ribose Transferases↗
PubMed1993

C-type natriuretic peptide mediates the hypothalamic actions of the natriuretic peptides to inhibit luteinizing hormone secretion.

Both A- and C-type natriuretic peptides (ANP and CNP, respectively) significantly reduce LH secretion when injected into the third cerebral ventricle of conscious rats. To establish which natriuretic peptide receptor subtype transduces these inhibitory messages, we have employed novel cytotoxin cell targeting techniques to selectively destroy cells in the hypothalamus that respond to ANP or CNP. Rats pretreated with ANP conjugated to the toxic A-chain of the plant cytotoxin ricin failed 1 week later to respond to central injection of ANP with the normal inhibition of LH secretion. These rats did, however, respond with significant inhibition of LH secretion to central injection of CNP. In fact, the LH inhibition observed after CNP injection was significantly greater than that expressed after similar injection of CNP in rats pretreated with unconjugated ricin A-chain (toxin control). Those control rats displayed significant reduction of LH levels in response to ANP injection as well. Plasma LH levels were not significantly affected by central administration of either ANP or CNP in rats pretreated with ricin A-chain conjugated to CNP. These results further demonstrate the power of this novel technology and provide positive evidence supporting our hypothesis that ANP exerts its LH-inhibiting effect by displacing endogenous CNP from clearance receptors within the brain. This endogenous CNP, then, like exogenously applied CNP, activates the guanyl cyclase-B receptors on cells, which are part of the network controlling the release of LHRH.

Animals↗
PubMed1992

Central peptidergic mechanisms controlling reproductive hormone secretion: novel methodology reveals a role for the natriuretic peptides.

A variety of neural factors can influence reproductive hormone secretion by neuromodulatory actions within the hypothalamus or neuroendocrine actions within the anterior pituitary gland. Passive immunoneutralization and antagonist administration protocols have suggested physiological roles for a number of these factors; however, both experimental approaches have severe technical limitations. We have developed novel methodology utilizing cytotoxin cell targeting with neuropeptides linked to the toxic A chain of the plant cytotoxin ricin. With this methodology we can target and destroy in vivo or in vitro cells bearing receptors for that peptide. Ricin A chain conjugated to atrial natriuretic peptide (ANP), a neuropeptide known to pharmacologically inhibit luteinizing hormone-releasing hormone (LHRH) release, was injected into the cerebroventricular system of intact, cycling rats and ovariectomized rats. Cytotoxin conjugate treatment significantly lengthened the estrous cycle. In ovariectomized rats the luteinizing hormone surge induced by steroid priming was completely inhibited. LHRH content of the median eminences of these rats was not significantly altered. These data suggest that ANP binding to clearance receptors in the hypothalamus displaces the C-type natriuretic peptide (CNP) from the shared clearance receptor, making more CNP available to inhibit LHRH release. In the absence of cells bearing the clearance receptor all available CNP binds to the ANPR-B receptor and exerts its effect via an inhibitory interneuron, since LHRH fibers are spared by this treatment.

Animals↗
PubMed1990

A nonoxytocinergic prolactin releasing factor and a nondopaminergic prolactin inhibiting factor in bovine neurointermediate lobe extracts: in vitro and in vivo studies.

Several peptidergic PRL-releasing factors (PRFs) have been described; however, none have been proven to be of primary physiological importance in the control of hormone release. Similarly dopamine withdrawal alone cannot completely explain the profiles of PRL secretion observed under a variety of conditions. We describe here the isolation in semipurified form of both a PRF and a PRL-inhibiting factor (PIF) from bovine neurointermediate lobe (NIL) extracts. Acid extracts of bovine NILs stimulated, in a dose-related manner, PRL release from cultured anterior pituitary cells, even after immunoabsorption of endogenous oxytocin from the extract. PIF and PRF activities were semipurified from NIL extracts by Sephadex chromatography and detected by in vitro and in vitro bioassays. The PRF material could be separated from oxytocin by gel sieving and was active in the presence of dopamine in vitro unlike synthetic oxytocin and in cell preparations in which the oxytocin-responsive lactotrophs had been removed by selective cytotoxin cell targeting using an oxytocin-ricin A chain cytotoxic conjugate. The PRF material stimulated PRL secretion in a dose-dependent fashion in conscious male rats after iv injection. The PIF material comigrated on sizing gel chromatography with immunoreactive oxytocin and was active in vitro during dopamine blockade with domperidone and in vivo in the presence of endogenous dopaminergic tone. These data suggest that novel factors present in the NIL might exert physiologically relevant control over lactotroph function and add to the growing literature on the presence of a PRF in the NIL.

Animals↗
PubMed1988

Pharmacokinetics of tumor-reactive immunotoxins in tumor-bearing mice: effect of antibody valency and deglycosylation of the ricin A chain on clearance and tumor localization.

The blood clearance and tissue distribution of immunotoxins composed of either intact or Fab' fragments of tumor-reactive, monoclonal rat antimurine immunoglobulin D (anti-delta) or nonreactive, normal rat immunoglobulin G and ricin A chain (native or chemically deglycosylated) were determined in BCL1 tumor-bearing mice. In the presence of accessible target cells, neither the valency of the antibody nor deglycosylation of the A chain affect the initial rate of clearance (alpha phase) of immunotoxins from the blood; however, both factors affect the levels of tumor-specific and nonspecific localization of the immunotoxins. Thus, the use of immunotoxins with deglycosylated A chain greatly reduced the levels of nonspecific uptake by the liver and concomitantly increased tumor-specific localization. Immunotoxins prepared with Fab' fragments of anti-delta antibody and deglycosylated A chain were twice as effective at localizing to splenic tumor than immunotoxins prepared with intact immunoglobulin G and deglycosylated A chain. Approximately 90% of tumor-specific localization of anti-delta immunotoxins occurred within 1 h after injection and less than 5% of the immunotoxin remained in the circulation 4 h after injection. In addition, the antigen-binding capacity of the remaining circulating immunotoxins decreased in a linear manner over the first 10 h to approximately 20% of the initial binding activity. Thus, by 10 h after injection, only 2-3% of injected immunotoxin remained in the circulation and this material expressed little antigen reactivity. Analysis of the in vivo stability of circulating 125I-labeled immunotoxins in tumor-bearing mice demonstrated that Fab' immunotoxins are more stable than IgG immunotoxins prepared with N-succinimidyl-3-(2-pyridylthio)propionate. In BCL1-bearing mice, significant splitting of the anti-delta immunotoxins did not occur until tumor localization was virtually complete.

Animals↗
PubMed1988

In vivo therapy of the BCL1 tumor: effect of immunotoxin valency and deglycosylation of the ricin A chain.

The in vivo therapeutic effects of Fab' and IgG anti-delta (anti-IgD)-ricin A chain immunotoxins were compared in mice bearing the surface IgD-positive BCL1 leukemia. The immunotoxins were prepared with either native or deglycosylated ricin A chain. Immunotoxin therapy was assessed both by the number of cells bearing the BCL1 immunoglobulin idiotype which remained in the spleen 24-48 h after injection with immunotoxin and by adoptive transfer of these spleen cells into normal mice. Immunotoxins prepared with either Fab'-anti-delta or IgG-anti-delta and native A chain induced a dose-dependent reduction in the number of idiotype-positive BCL1 cells present in the spleens of the tumor-bearing mice. The maximal therapeutic response was achieved with 250 micrograms of immunotoxin (containing 80-100 micrograms A chain) per mouse for both immunotoxins, resulting in tumor reduction of approximately 90%. This represents an elimination of 3-4 X 10(8) tumor cells. Reduction in the number of tumor cells was not observed with control reagents including antibody alone, antibody mixed with A chain, or an immunotoxin of irrelevant specificity. A Fab' immunotoxin prepared with deglycosylated ricin A chain was approximately 5-fold more effective as an antitumor reagent than the same immunotoxin prepared with native A chain; thus, optimal therapy was achieved after injection of 50 micrograms of immunotoxin (containing 15-20 micrograms A chain). Since the immunotoxins prepared with deglycosylated A chain were only 2-3-fold more toxic to the mice than those prepared with native A chain, the former resulted in a 2-3-fold increase in the therapeutic index.

Animals↗
PubMed1987

Redesigning nature's poisons to create anti-tumor reagents.

Immunotoxins are conjugates of cell-reactive antibodies and toxins or their subunits. In this report, the chemistry, biology, pharmacokinetics, and anti-tumor effects of first generation immunotoxins; the preparation of improved second generation immunotoxins that display greater anti-tumor efficacy; and the role of genetic engineering in creating third-generation immunotoxins are discussed.

Animals↗
PubMed1987

Inhibition of the helper function of murine T cells with Fab'-anti-L3T4 ricin A chain immunotoxin.

The ability of Fab'-anti-L3T4 A chain-containing immunotoxins to inhibit the helper function of keyhole limpet hemocyanin-specific T helper lymphocytes was evaluated. Keyhole limpet hemocyanin-specific T helper cells were prepared from the lymph nodes of primed mice and were enriched for T cells. Enriched populations of trinitrophenyl-specific B cells were prepared from spleens of normal mice. In the presence of antigen, the keyhole limpet hemocyanin-specific T helper cells were able to induce proliferation and differentiation of the trinitrophenyl-specific B cells. However, when the T helper cells were first treated with an immunotoxin composed of Fab' fragments of anti-L3T4 antibody coupled to ricin A chain (Fab'-anti-L3T4-A), they failed to induce proliferation and differentiation of the antigen-specific B cells. The concentrations of Fab'-anti-L3T4-A required to inhibit T cell help for the proliferation and differentiation of trinitrophenyl-specific B cells by 50% were 1 X 10(-9)M and 4 X 10(-10) M, respectively. Fab'-anti-L3T4 antibody alone did not inhibit T cell-induced B cell proliferation and differentiation by 50% at greater than 100-fold higher concentrations.

Animals↗
PubMed1987

Molecular dissection of the murine antibody response to streptococcal group A carbohydrate.

Monoclonal antibodies (mAb) to streptococcal group A carbohydrate (GAC) are encoded by a minimum of two VH, four JH, four V kappa, three J kappa, one V lambda, and one J lambda gene segments. The IdX, IdI-1, and Id5 idiotypic determinants are expressed by anti-GAC mAb and are found on free kappa chains. Each pattern of these determinants is encoded by a distinct V kappa gene segment, apparently without the requirement for a particular J kappa, VH, or JH gene segment, or somatic mutation. In contrast, the binding site-associated idiotypic determinant IdI-3a does not correlate with any single V or J gene segment.

Animals↗
PubMed1986

Analysis of anti-streptococcal group A carbohydrate idiotope levels in sera: correlation of magnitude of expression with idiotope position and VK haplotype.

We have employed monoclonal anti-idiotopes to map the corresponding idiotopes on the variable domain of a prototype antibody specific for streptococcal group A carbohydrate (GAC). Idiotope variability, as assessed by direct and competitive binding assays, previously was shown to correlate with idiotope position; determinants farther from the binding site were less variable. We now describe a relationship between idiotope position and idiotope concentration in normal and GAC-immune sera from mice of several inbred and recombinant inbred strains. Employing sera as inhibitors in a competitive radioimmunoassay, we demonstrate that idiotopes farther from the binding site (more proximal) tend to be present at higher levels in GAC-hyperimmune sera. Only the most proximal idiotope was detected in normal serum, and this idiotope was present in normal sera from all 12 strains tested. Finally, significant interstrain differences in patterns of idiotope expression were observed, and some of these differences appear to be correlated with allelic differences at immunoglobulin structural gene loci.

Alleles↗