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Biomedical subjects

A J Jones

Publications and source records attributed to A J Jones.

At least 37 records · Page 2Linked to original sources
PubMed1996

OX-40 antibody enhances for autoantigen specific V beta 8.2+ T cells within the spinal cord of Lewis rats with autoimmune encephalomyelitis.

The V beta 8.2 T cell receptor (TCR) component is the predominant V beta gene product associated with antigen specific CD4+ T cell response to the major encephalitogenic epitope of myelin basic protein (MBP) in Lewis rats. Lewis rats were actively immunized with MBP in complete Freund's adjuvant and the V beta 8.2 positive and negative cells were analyzed for IFN-gamma mRNA production and OX-40 cell surface expression during the onset of EAE. The V beta 8.2+ T cells isolated from the spinal cord produced the majority of mRNA for IFN-gamma and also showed a marked enhancement for OX-40 expression compared to V beta 8.2+ T cells isolated from the lymph nodes. Only a fraction of IL-2 receptor positive T cells examined ex vivo from the inflammatory compartments co-expressed the OX-40 antigen. These results suggested that OX-40 cell surface expression could be used to identify and isolate the most recently activated T cells ex vivo. OX-40+ T cells isolated from the spinal cord were highly enriched for the V beta 8.2 T cell receptor component compared to OX-40- or unsorted spinal cord lymphocytes. OX-40+ T cells isolated from the spinal cord had an enhanced response to MBP, whereas OX-40+ cells isolated from the lymph nodes responded to both MBP and purified protein derivative. These data suggest that activated T cells can be isolated and characterized with the OX-40 antibody which only respond to the antigens present at the local site. The data also imply that isolation of OX-40+ T cells will be useful in identifying V beta biases and autoantigen specific cells within inflamed tissues even when the antigen specificity is unknown.

Animals↗
PubMed1996

Stable formulations of recombinant human growth hormone and interferon-gamma for microencapsulation in biodegradable microspheres.

PURPOSE: The successful development of controlled release formulations for proteins requires that the protein not be denatured during the manufacturing process. The major objective was to develop formulations that stabilize two recombinant human proteins, human growth hormone (rhGH) and interferon-gamma (rhIFN-gamma), at high protein concentrations (> 100 mg/mL) in organic solvents commonly used for microencapsulation, methylene chloride and ethyl acetate. METHODS: Several excipients were screened to obtain the maximum solubility of each protein. These formulations (aqueous, lyophilized, milled, spray dried, or isoelectric precipitate) were then rapidly screened by emulsification in the organic solvent followed by recovery into excess buffer. Additional screening was performed with solid protein that was suspended in the organic solvent and then recovered with excess buffer. The recovery of native protein was determined by native size exclusion chromatography (SEC-HPLC) and circular dichroism (CD). The selected formulations were encapsulated in polylactic-coglycolic acid (PLGA) microspheres by either water-in-oil-in-water (W/O/W) or solid-in-oil-in-water (S/O/W) methods. The initial protein released from the microspheres incubated at physiological conditions was analyzed by SEC-HPLC, CD, and biological assays. RESULTS: The stability of a given formulation in the rapid screening method correlated well with stability during encapsulation in PLGA microspheres. Formulations of rhGH containing Tween 20 or 80 resulted in lower recovery of native protein, while trehalose and mannitol formulations (phosphate buffer, pH 8.0) yielded complete recovery of native rhGH. Other additives such as carboxymethyl cellulose, gelatin, and dextran 70 were not effective stabilizers, and polyethylene glycol provided some stabilization of rhGH. Trehalose/rhGH (1:4 mass ratio) and mannitol/rhGH (1:2 mass ratio) formulations (potassium phosphate buffer, pH 8.0) were lyophilized, reconstituted to 200 and 400 mg/mL rhGH, respectively, and then encapsulated in PLGA microspheres. The protein was released from these microspheres in its native state. Lyophilized formulations of rhGH yielded analogous results indicating the ability of trehalose and mannitol to stabilize the protein. Small solid particles of rhGH generated by spray drying (both air and freeze-drying) formulations containing Tween 20 or PEG were stable in ethyl acetate, but not methylene chloride. Similar results were also obtained with rhIFN-gamma (137 mg/mL in succinate buffer, pH 5.0), where both mannitol and trehalose were observed to stabilize the protein during exposure to the organic solvents resulting in the release of native rhIFN-gamma from PLGA microspheres. CONCLUSIONS: The rapid screening method allowed the development of stable concentrated protein solutions or solid protein formulations that could be successfully encapsulated in PLGA microspheres. The excipients observed to stabilize these proteins function by preferential hydration of the protein, and in the dry state (e.g., trehalose) may stabilize the protein via water substitution yielding a protective coating around the protein surface. Studies of other proteins should provide further insight into this mechanism of protein stabilization during encapsulation.

Chemical Precipitation↗
PubMed1996

A month-long effect from a single injection of microencapsulated human growth hormone.

An injectable sustained-release form of human growth hormone (hGH) was developed by stabilizing and encapsulating the protein, without altering its integrity, into biodegradable microspheres using a novel cryogenic process. A single injection of microspheres in monkeys resulted in elevated serum levels of recombinant hGH (rhGH) for more than one month. Insulin-like growth factor-I (IGF-I) and its binding protein IGFBP-3, both of which are induced by hGH, were also elevated for four weeks by the rhGH containing microspheres to a level greater than that induced by the same amount of rhGH administered by daily injections. These results show that, by using appropriate methods of stabilization and encapsulation, the advantages of sustained-release formulations previously demonstrated for low-molecular-weight drugs can now be extended to protein therapeutics.

Animals↗
PubMed1995

Complications of day-case angiography using 5F catheters.

Questionnaires were sent retrospectively to patients who had undergone day-case angiography at a British teaching hospital to evaluate the complications suffered after leaving hospital. In 1992, 5F catheters were used in 61 day-case patients. On analysis of the responses and detailed follow-up in areas of concern, no unexpected complication was found. It is concluded that day-case angiography using 5F catheters is associated with low morbidity after discharge home.

Ambulatory Care↗
PubMed1995

Non-invasive aortoiliac assessment.

OBJECTIVES: To assess the accuracy of Duplex ultrasound in the assessment of aortoiliac disease. DESIGN: Prospective, semi-blind study. SETTING: Vascular laboratory and radiology departments, University Hospital. MATERIALS AND METHODS: Ninety-two patients underwent assessment of the aortoiliac segment by femoral pulse palpation, Duplex ultrasound and biplanar arteriography. Of these 184 aortoiliac segments, 68 were also assessed by intraarterial pressure measurements and 80 by magnetic resonance angiography (MRA). MAIN RESULTS: Femoral pulses were abnormal in all 32 occluded aortoiliac segments. Of 152 patent segments, femoral pulse palpation was misleading in 50 (33%). MRA detected all occlusions and had a sensitivity of 71% and specificity of 68% for stenoses, compared to arteriography. Colour flow Duplex misdiagnosed four occlusions as stenoses. Duplex had a sensitivity of 91% and specificity of 93% for stenoses when compared to arteriography. Two stenoses, detected by Duplex and confirmed by pressure gradients, were missed by arteriography. CONCLUSIONS: Pressure measurements remain the gold standard for aortoiliac examination, arteriography providing only morphological information. The limitations of femoral pulse palpation should be appreciated. Although MRA was faster, Duplex examination proved slightly more sensitive to stenoses. At present, colour Duplex provides the best non-invasive assessment of aortoiliac disease and could prevent unnecessary arteriograms.

Aorta, Abdominal↗
PubMed1994

Magnetic resonance angiography or IADSA for diagnosis of carotid pseudo occlusion?

Accurate diagnosis of internal carotid artery (ICA) occlusion is essential in the investigation of carotid disease yet may be difficult using Duplex. Traditionally contrast arteriography has been used to confirm the diagnosis despite its cost and potential dangers. Twenty-one patients with 23 ICA occlusions were evaluated by a 3-dimensional time of flight magnetic resonance angiography (MRA) technique. The cervical carotids and circle of Willis were imaged during the MRA examination which lasted 30 minutes. Confirmatory conventional angiography was performed in all patients. Using angiography as the gold standard, all occlusions were correctly diagnosed by MRA and 22 of 23 occlusions correctly diagnosed by Duplex. There was good agreement between MRA and angiography for all 42 ICAs imaged (Kappa statistic 0.83). Diagnosis of internal carotid artery occlusion is critical as it determines the need for operation. In this situation MRA provides a useful non-invasive complement to Duplex. A combination of non-invasive studies may enable arteriography to be rejected with greater confidence in this high risk group.

Angiography, Digital Subtraction↗
PubMed1991

High resolution analysis of functional determinants on human tissue-type plasminogen activator.

Sixty-four variants of human tissue-type plasminogen activator (tPA) were produced using recombinant DNA techniques. Charged residues were converted to alanine in clusters of from one to four changes per variant; these clusters spanned all the domains of the molecule. The variants were expressed by mammalian cells and were analyzed for a variety of properties. Variants of tPA were found that had reduced activity with respect to each tested property; in a few cases increased activity was observed. Analysis of these effects prompted the following conclusions: 1) charged residues in the nonprotease domains are less involved in fibrin stimulation of tPA activity than those in the protease domain, and it is possible to increase the fibrin specificity (i.e. the stimulation of tPA activity by fibrin compared to fibrinogen) by mutations at several sites in the protease domain; 2) the difference in enzymatic activity between the one- and two-chain forms of tPA can be increased by mutations at several sites on the protease domain; 3) binding of tPA to lysine-Sepharose was affected only by mutations to kringle-2, whereas binding to fibrin was affected most by mutations in the other domains; 4) clot lysis was influenced by mutations in all domains except kringle-2; 5) sensitivity to plasminogen activator inhibitor-1 seems to reside exclusively in the region surrounding residue 300. A model of the tPA protease domain has been used to map some of the critical residues and regions.

Amino Acid Sequence↗
PubMed1991

Morphine, but not sodium cromoglycate, modulates the release of substance P from capsaicin-sensitive neurones in the rat trachea in vitro.

1. Opioids have been shown to inhibit substance P (SP) release from primary afferent neurones (PAN). In addition, opioid receptors have been identified on PAN of the vagus nerves. Sodium cromoglycate (SCG) decreases the excitability of C-fibres in the lung of the dog in vivo. We have utilised a multi-superfusion system to investigate the effect of opioids and SCG on the release of SP from the rat trachea in vitro. 2. Pretreatment of newborn rats with capsaicin (50 mg kg-1 s.c. at day 1 and 2 of life) resulted in a 93.2 +/- 6.3% reduction in tracheal substance P-like immunoreactivity (SP-LI) content when determined by radioimmunoassay in the adult. 3. Exposure to isotonically elevated potassium concentrations (37-90 mM), capsaicin (100 nM-10 microM), and bradykinin (BK; 10nm-1 microM) but not des-Arg9-BK (1 microM) stimulated SP-LI release by a calcium-dependent mechanism. 4. SCG (1 microM and 100 microM) did not affect spontaneous, potassium (60 mM)- or BK (1 microM)-stimulated SP-LI release. 5. Morphine (0.1-100 microM) caused dose-related inhibition of potassium (60 mM)-stimulated SP-LI release with the greatest inhibition of 60.4 +/- 13.7% at 100 microM. The effect of morphine was not mimicked by the kappa-opioid receptor agonist, U50,488H (10 microM) or the delta-opioid receptor agonist, Tyr-(D-Pen)-Gly-Phe-(D-Pen) (DPDPE). 6. The effect of morphine was totally abolished by prior and concomitant exposure to naloxone (100 nM) which had no effect on control release values. 7. We conclude that opioid receptors, predominantly of the MM-opioid receptor subtype, inhibit SP-LI release from PAN in the rat trachea and suggest that centrally inactive MM-opioid receptor agonists may have therapeutic potential in the treatment of asthma.

3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cycloh↗
PubMed1991

GABAB receptor modulation of the release of substance P from capsaicin-sensitive neurones in the rat trachea in vitro.

1. The role of gamma-aminobutyric acid (GABA) as an inhibitory transmitter in the central nervous system is well documented. Recently, GABAA and GABAB receptors have been identified in the peripheral nervous system, notably on primary afferent neurones (PAN). We have utilised a multi-superfusion system to investigate the effect of selective GABA receptor agonists and antagonists on the release of substance P (SP) from the rat trachea in vitro. 2. GABA (1-100 microM) did not affect spontaneous release of SP-like immunoreactivity (LI) but caused dose-related inhibition of calcium-dependent potassium (60 mM)-stimulated SP-LI release. The greatest inhibition of 77.7 +/- 18.8% was observed at 100 microM. 3. The inhibitory effect of GABA was mimicked by the GABAB receptor agonist, (+/-)-baclofen (1-100 microM), but not the GABAA receptor agonist, 3-amino-1-propane-sulphonic acid (3-APS, 1-100 microM). Baclofen (100 microM) had no effect on SP-LI release stimulated by capsaicin (1 microM). 4. The inhibitory effect of baclofen (30 microM) was significantly reduced by prior and concomitant exposure to the GABAB receptor antagonist, phacolofen (100 microM) but not the GABAA receptor antagonist, bicuculline (10 microM). Neither antagonist, alone, affected spontaneous or potassium-stimulated SP-LI release. 5. We conclude that activation of pre-synaptic GABAB receptors on the peripheral termini of PANs in the rat trachea inhibits SP-LI release and suggest that GABAB receptor agonists may be of value in the therapeutic treatment of asthma.

Animals↗
PubMed1990

A precise and rapid microtitre plate clot lysis assay: methodology, kinetic modeling and measurement of catalytic constants for plasminogen activation during fibrinolysis.

A rapid and precise turbidimetric clot lysis assay employing a microtitre plate reader and personal computer is described in detail. The use of such widely available instrumentation, the convenience and rapid throughput suggest the assay could be developed as a reference method with which to measure the potency of tissue plasminogen activator (t-PA) in conjunction with the WHO reference preparation. The method has been used to investigate molecular parameters involved in fibrinolysis. Aggregation status of the fibrin does not appear to influence the mechanism of plasminogen activation and clot lysis by plasmin. High ratios of plasminogen to fibrin resulted in a change in clot turbidity and in a change in the lysis profile of turbidity versus time. This is probably the result of plasminogen binding to fibrin and consequent restriction of the access of plasmin to its sites of cleavage in the fibrin. A simple model is proposed, and equations have been derived, for the kinetics of lysis which adequately describe the mechanism and which are confirmed by experimental data. This model results in estimates of the Km and kcat for the activation of plasminogen by t-PA during clot lysis of approximately 150 nM and 0.1 s-1, respectively, in excellent agreement with published values. The assay should therefore prove useful in quantitative evaluations of the molecular phenomena occurring during fibrinolysis. The more rapid activation of lys-plasminogen than glu-plasminogen by t-PA was confirmed. However, evidence was obtained that the lys-form binds more tightly to fibrin by the same factor. This observation suggested that the appropriate substrate in the kinetic model is fibrin-bound plasminogen.(ABSTRACT TRUNCATED AT 250 WORDS)

Buffers↗
PubMed1990

Comparison of fine needle aspiration cytology, radioisotopic and ultrasound scanning in the management of thyroid nodules.

In 175 patients presenting with thyroid nodules, the diagnostic value in management of fine needle aspiration and cytology (FNAC), pertechnetate (99mTc) scanning and ultrasound imaging was examined. In 82 patients, the diagnosis was confirmed at operation; in the remaining 93, there was a follow-up period of at least 2 years. Thyroid cancer was found in 13 patients. For FNAC the sensitivity, specificity and positive predictive value for thyroid cancer were 92%, 85% and 41% respectively compared with 82%, 34% and 11% for 99mTc pertechnetate scanning, 75%, 61%, 19% for ultrasound and 73%, 58% and 19% for combined pertechnetate and ultrasound scanning. In 14% of patients, the aspirates were inadequate for cytology at the first examination. FNAC is therefore the preferable initial investigation and usually gives results adequate for a decision on surgical or medical management. With medical management and follow-up, ultrasound is of value in defining the nodule and the appearance of the rest of the gland.

Biopsy, Needle↗
PubMed1990

Age- and position-related heterogeneity of equine tendon extracellular matrix composition.

The digital flexor tendons of the neonate and adult horse have been compared with respect to variation in extracellular matrix composition along their length. Two pepsin-sensitive, acetic acid soluble proteins, molecular weight (Mr) 52 kD (np 52) and Mr 54 kD (np 54), were prominent throughout the length of neonatal tendons. In adult tendon, np 52 and np 54 were less abundant and restricted to the cannon (metacarpal) region. In contrast, a single pepsin- and collagenase-resistant protein of Mr 55 kD (fp 55) was exclusive to the fetlock (metacarpophalangeal joint) region regardless of age, although more distinct in the adult. Pepsin extracted fp 55 precipitated at 2.0 M sodium chloride: 0.5 M acetic acid and was further purified to homogeneity by bacterial collagenase digestion. Analysis of fp 55 amino acid composition revealed the presence of a large proportion of glycine residues (379 of 1001), suggesting a possible homology with the collagen family. These data demonstrate that the composition of equine digital flexor tendons varies with age, is heterogeneous along its length, and suggests that variation in tendon extracellular matrix composition is influenced by functional requirements.

Acetates↗
PubMed1989

Study of the primary structure of recombinant tissue plasminogen activator by reversed-phase high-performance liquid chromatographic tryptic mapping.

Two high-resolution tryptic maps have been developed for recombinant tissue plasminogen activator (rt-PA) that separate the expected 51 tryptic peptides. The trypsin digestion was performed after reduction and S-carboxymethylation of the protein. The high-performance liquid chromatographic separation of the tryptic peptides used a Nova-Pak C18 (5 microns) column with a mobile phase that contained 0.1% aqueous trifluoroacetic acid (TFA) or 50 mM sodium phosphate (pH 2.85) and a linear gradient of acetonitrile. A TFA solvent system was also used for re-purification and for characterization of the peptides isolated from the phosphate-based separation. All of the isolated peptides had compositions consistent with the sequence proposed for rt-PA. The identities of the glycopeptides were confirmed by lectin chromatography on concanavalin A-Sepharose. The mixture of tryptic peptides was also treated with endo-beta-N-acetylglucosaminidase H and peptide:N-glycosidase F to locate the position of either high mannose or complex oligosaccharides. These studies demonstrated that a high mannose oligosaccharide is attached to Asn-117 while complex carbohydrate side-chains are attached to Asn-184 and Asn-448. The residue Asn-184 is the site of optional glycosylation that results in the formation of two rt-PA variants that contain either two or three oligosaccharides.

Amino Acid Sequence↗
PubMed1989

Immunization procedures for E. coli proteins.

Three immunization procedures were compared for the production of antibodies to the minor components of a complex E. coli protein (ECP) mixture: a conventional protocol and two methods that allow for the selective in vitro (cascade) or in vivo (passive) depletion of highly immunogenic proteins. An indirect ELISA showed that a maximum ELISA antibody titer was obtained with all the procedures 60 d after immunization. Analysis of these antisera by two-dimensional SDS-PAGE immunoblots, however, demonstrated that antibody reactivity to minor components in the mixture was not achieved until 112 d. This analysis also showed that a marked improvement in antibody response to minor components was obtained with the cascade immunization procedure. The mean titer and spectrum of antibody reactivity was similar for each group, and suggested that, although some individual variation was noted, the improvements observed were the result of the protocol used. Thus, for these ECPs, and multiple antigen mixtures in general, the preferred immunization protocol should employ at least three hosts and utilize the cascade immunization of Thalhamer and Freund. Characterization of the resulting antisera is best performed by use of silver stained two-dimensional SDS-PAGE and immunoblotting.

Animals↗