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Biomedical subjects

A J Porter

Publications and source records attributed to A J Porter.

At least 37 records · Page 2Linked to original sources

Production of a functional single chain antibody attached to the surface of a plant virus.

A potato virus X (PVX) vector was used to express a single chain antibody fragment (scFv) against the herbicide diuron, as a fusion to the viral coat protein. The modified virus accumulated in inoculated Nicotiana clevelandii plants and assembled to give virus particles carrying the antibody fragment. Electron microscopy was used to show that virus particles from infected leaf sap were specifically trapped on grids coated with a diuron-BSA conjugate. The results demonstrate that the PVX vector can be used as a presentation system for functional scFv.

Cloning, Molecular↗

Binding characteristics of anti-atrazine monoclonal antibodies and their fragments synthesised in bacteria and plants.

Single-chain antibody fragments (scAb), specific for the herbicide atrazine, have been expressed in the bacterium Escherichia coli and in transgenic tobacco plants. The scAb could be purified as a monomer (monovalent) via a hexa-histidine tail or as a dimer (divalent) by antibody affinity chromatography. In competition ELISA, the bacterial scAb showed the same specificity for atrazine and related triazine herbicides as the parental mAb cell line, but both plant and bacterial monomeric scAbs showed increased sensitivity to free atrazine. Surface plasmon resonance (BIAcore 2000) analysis confirmed that purified scAb, derived from plant or bacteria, retained similar association rates as the mAb. However, the monomeric plant and bacterial scAbs showed a lower affinity for immobilised antigen, than the equivalent dimeric scAbs or mAb. This decrease in affinity was due to a 10 fold slower dissociation rate and is likely due to loss of the avidity contribution of dimeric molecules.

Antibodies, Monoclonal↗

Expression and characterisation of single-chain antibody fragments produced in transgenic plants against the organic herbicides atrazine and paraquat.

Single-chain antibody fragments (scAbs), which have a human C-kappa constant domain and a hexa-histidine tail attached to the carboxy terminus of the single-chain Fv (ScFv) fragments to facilitate purification, have been raised against the herbicides paraquat and atrazine and expressed in transgenic Nicotiana tabacum cv. Samsun NN. Prior to purification, the anti-atrazine scAb is expressed as up to 0.014% of soluble leaf protein and has a binding profile in ELISA, against an atrazine-bovine serum albumin (BSA) conjugate, similar to that of the scAb produced in Escherichia coli. Competition ELISA has shown that the plant-derived scAb also recognises free atrazine. Following antibody affinity purification to isolate dimers, the affinity for immobilised antigen approaches that of the parental monoclonal antibody. This was confirmed by surface plasmon resonance analysis. The purified scAb also recognises related triazine herbicides. When isolated from cell-suspension cultures, the anti-paraquat scAb binds to a paraquat conjugate in a concentration-dependent manner, with a profile similar to the parental monoclonal antibody. This is the first demonstration that functional scAbs against organic pollutants can be produced in transgenic plants and that the scAbs may be appropriate for the development of immunoassay-based detection systems.

Atrazine↗

Identification and Phylogenetic analysis of thermophilic sulfate-reducing bacteria in oil field samples by 16S rDNA gene cloning and sequencing.

Thermophilic sulfate-reducing bacteria (SRB) have been recognized as an important source of hydrogen sulfide (H2S) in hydrocarbon reservoirs and in production systems. Four thermophilic SRB enrichment cultures from three different oil field samples (sandstone core, drilling mud, and production water) were investigated using 16S rDNA sequence comparative analysis. In total, 15 different clones were identified. We found spore-forming, low G+C content, thermophilic, sulfate-reducing Desulfotomaculum-related sequences present in all oil field samples, and additionally a clone originating from sandstone core which was assigned to the mesophilic Desulfomicrobium group. Furthermore, three clones related to Gram-positive, non-sulfate-reducing Thermoanaerobacter species and four clones close to Clostridium thermocopriae were found in enrichment cultures from sandstone core and from production water, respectively. In addition, the deeply rooted lineage of two of the clones suggested previously undescribed, Gram-positive, low G+C content, thermophilic, obligately anaerobic bacteria present in production water. Such thermophilic, non-sulfate-reducing microorganisms may play an important ecological role alongside SRB in oil field environments.

Journal Article↗

Abnormalities of nerve fibers in the circular muscle of patients with slow transit constipation.

Abnormalities of the enteric nervous system are thought to explain the pathophysiology of motility disorders. Our aim was to determine if particular classes of enteric neurons are affected in slow transit constipation (STC). Specimens were taken from the terminal ileum and ascending, transverse and descending colon of patients undergoing subtotal colectomy for STC. Immunohistochemistry was performed using antisera to neuron-specific enolase, tachykinin, leu-enkephalin, choline acetyltransferase, vasoactive intestinal peptide, nitric oxide synthase, tyrosine hydroxylase and neuropeptide Y. The density of nerve fibres labelled with these antibodies in each layer was compared with age-matched controls. The density of nerve fibres with tachykinin and enkephalin immunoreactivity was reduced in the colonic circular muscle of the 15 patients with STC, whereas innervation of all other layers was normal. This reduction of tachykinin-immunoreactive nerve fibres also occurred in nine of the 12 specimens of terminal ileum examined. No difference was detected in the density or distribution of nerve fibres using the other antisera. Excitatory nerve fibres are present in the circular muscle in STC but they are deficient in tachykinins and enkephalin.

Adult↗

Retention of neutralising activity by recombinant anti-pneumolysin antibody fragments.

The variable domains of a neutralising (prevents erythrocyte lysis) anti-pneumolysin monoclonal antibody have been cloned and expressed as functional protein in Escherichia coli. Purification of the anti-pneumolysin single-chain antibody fragment, via antibody-affinity or metal-chelate affinity chromatography, resulted in product that was predominantly in a dimeric or monomeric form, respectively. The dimeric single-chain antibody fragment showed a higher sensitivity and affinity for immobilised antigen in both ELISA and BIAcore studies. The dimeric single-chain antibody fragment was as effective at protecting erythrocytes from lysis as the parent monoclonal. The monomeric, low affinity single-chain antibody fragment, showed reduced neutralising potency. As antibiotic resistant Streptococcus pneumoniae strains continue to show an increasing word-wide distribution, recombinant, neutralising antibody fragments, may provide an additional class of molecules useful in the treatment of toxaemia.

Animals↗

Stabilization of antibody fragments in adverse environments.

Antibody fragments have the potential to be used as sensitive and specific binding agents in a broad range of industrial applications. Genetic manipulation has been used to design a series of antibody fragment configurations with a flexible linker and/or a disulphide bond between the heavy chain and light chain of an antibody fragment against the herbicide atrazine. The thermostability and stability to a range of denaturants, polar and non-polar solvents, surfactants and proteases have been compared. It has been found that a novel antibody fragment construct (STAB: stabilized antibody) containing both a flexible linker and a disulphide bond can be effectively produced and shows greatly improved stability in these diverse environments. These STABs should be useful in environmental diagnostics and remediation, and may provide a generic approach for stabilizing antibody fragments in formulations containing detergents and penetrants for topical application in the pharmaceutical and cosmetic industries.

Atrazine↗

The neurochemical coding and projections of circular muscle motor neurons in the human colon.

BACKGROUND & AIMS: Enteric neurons can be characterized by their chemical coding, projections, and morphology. The aim of this study was to describe the different classes of human colonic circular muscle motor neurons. METHODS: Human colonic circular muscle motor neurons were identified by retrograde tracing with 1,1'-didodecyl 3,3,3',3'-indocarbocyanine perchlorate (Dil) applied to the circular muscle layer. Whole-mount preparations of the myenteric plexus were then double-labeled with antisera to choline acetyltransferase (ChAT) and/or nitric oxide synthase (NOS), or NOS and vasoactive intestinal peptide (VIP), and the position and immunoreactivity of Dil-filled neurons were recorded. RESULTS: Fifty-two percent of all Dil-filled neurons were ChAT immunoreactive, and 86% of these projected up to 11 mm orally, with 14% projecting short distances anally. Forty-eight percent of the Dil-filled neurons were NOS immunoreactive, and 77% of these projected up to 19 mm anally, with 23% projecting no more than 6 mm orally. A subpopulation of these NOS-immunoreactive motor neurons were also VIP-immunoreactive. A small population of myenteric neurons was immunoreactive for both ChAT and NOS, but none projected to the circular muscle. NOS-immunoreactive motor neurons projected for longer distances than those with ChAT immunoreactivity and were larger. CONCLUSIONS: There are two classes of human colonic motor neurons: one is excitatory (ChAT-immunoreactive) and mainly projects orally and the other is inhibitory (NOS +/- VIP immunoreactive) and projects preferentially anally.

Choline O-Acetyltransferase↗

The polarity of neurochemically defined myenteric neurons in the human colon.

BACKGROUND & AIMS: Functional classes of enteric neurons in small mammals can be determined by neurochemical coding and axonal projections. The aim of this study was to describe the projections of vasoactive intestinal peptide (VIP)-, tachykinin (TK)-, and calretinin-immunoreactive (IR) interneurons and circular muscle motor neurons in human colon. METHODS: Human colonic myenteric neurons were retrogradely filled with 1,1'-didodecyl 3,3,3',3'-indocarbocyanine perchlorate (DII) from either the myenteric plexus or the circular muscle layers in organotypic culture preparations. The preparations were then labeled with antisera, and the position and immunoreactivity of DII-filled neurons was recorded. RESULTS: Nine percent of circular muscle motor neurons were labeled with TK immunoreactivity and these projected orally; 22% were VIP-IR and projected anally. Fifty-one percent of myenteric neurons that projected anally to the myenteric ganglia were VIP-IR and none were TK-IR. In contrast, 23% of neurons projecting orally were TK-IR but only 2% were VIP-IR. Calretinin immunoreactivity was present in 23% of neurons projecting anally and 3% projecting orally within the myenteric plexus. Six percent of circular muscle motor neurons had Dogiel type II morphology, and 50% of these were TK-IR. CONCLUSIONS: Circular muscle motor neurons and myenteric interneurons with TK, VIP, and calretinin immunoreactivity in the human colon have distinct projections and polarity.

Adult↗

Choline acetyltransferase immunoreactivity in the human small and large intestine.

BACKGROUND & AIMS: Choline acetyltransferase, an enzyme involved in the synthesis of acetylcholine, is a marker of cholinergic neurons. In this study, the distribution of choline acetyltransferase immunoreactivity in human intestine is described. METHODS: Frozen-section and whole-mount preparations of human small and large bowels were made and labeled with antiserum to choline acetyltransferase. Double labeling with antiserum to neuron-specific enolase enabled the proportion of all neurons that were immunoreactive for choline acetyltransferase to be determined. RESULTS: Nerve fibers, immunoreactive for choline acetyltransferase, were frequent in the circular and longitudinal muscle layers and were widespread in the myenteric and submucous plexuses, but none was observed in the mucosa. Myenteric neurons, immunoreactive for choline acetyltransferase, showed various morphologies, the most common being unipolar and having an irregular outline with several short, lamellar processes. Sixty-four percent of all myenteric neurons were immunoreactive for choline acetyltransferase. Cholinergic submucous neurons were homogeneous in appearance with oval, smooth cell bodies and filamentous dendrites and accounted for 53% of all submucous neurons. A number of cells resembling enteroendocrine cells in the epithelium of the small and large bowels had intense choline acetyltransferase immunoreactivity. CONCLUSIONS: The majority of neurons in human small and large intestines are cholinergic.

Aged↗

Cloning, expression and characterization of a single-chain antibody fragment to the herbicide paraquat.

New cost effective methods for the detection and removal of pesticides from water samples are required to meet modern safety standards. The recent development of techniques to produce antibody fragments in bacteria has provided the opportunity to exploit antibodies as specialized chemicals for affinity detection/removal technologies. The variable heavy and light polypeptide chains of the anti-paraquat monoclonal antibody PQXB1/2 have been cloned into the single-chain antibody (ScAb) expression vector pBG1. The construct was expressed in Escherichia coli and 0.4 mg functional antibody produced from 1 dm3 of induced culture. Characterization of ScAb by antigen binding profile and competition ELISA showed it to have a sensitivity one order of magnitude below that of the parent monoclonal. ScAb was purified as a monomer or dimer and analysed by HPLC size exclusion chromatography. When immobilized on polystyrene beads the ScAb could remove 85% of a paraquat-bovine serum albumin conjugate from solution in a single step.

Amino Acid Sequence↗

Expression and purification strategies for the production of single-chain antibody and T-cell receptor fragments in E. coli.

This work describes protocols for the production of single-chain antibody and T-cell receptor fragments in E. coli. A choice of methods is given for the purification of the recombinant fragments that rely on the use of either immunoaffinity or metal chelate affinity chromatography. The TCR fragments may have to be denatured and refolded before the fragments attain their proper conformation.

Chromatography, Affinity↗

Expression of monovalent and bivalent antibody fragments in Escherichia coli.

The technology of humanization of rodent antibodies has opened the way for a broad range of therapeutic antibodies with very low immunogenicity, which are, therefore, suitable for repeated dosing. Such intact antibodies have extended serum half-lives and biodistribution profiles very similar to human antibodies. For some applications, however, the ideal therapeutic should have reduced serum half-life and altered biodistribution patterns typical of antibody fragments, such as Fab or single chain Fv. Bispecific antibody fragments offer exciting additional therapeutic possibilities, but their successful manufacture and purification on a large scale require the development of new methods. Antibody fragments often assemble in Escherichia coli as monovalent fragments with reduced affinities. We describe the spontaneous assembly of bivalent antibody fragments in E. coli and methods of purification that yield either bivalent or monovalent molecules as required.

Animals↗

Separation and concentration of bacteria with immobilized antibody fragments.

New methods to quantitatively remove bacteria from food and water samples are required to meet modern safety standards. The recent development of techniques to make Fab/Fv/scFv fragments in bacteria has provided the opportunity to exploit antibodies as specialized chemicals for affinity removal technologies. Single-chain fragments against Pseudomonas aeruginosa have been expressed in Escherichia coli, purified via a fused poly-histidine tail and immobilized upon polystyrene beads. The resulting immunoaffinity columns have been shown to effectively remove greater than 90% of an applied 10 million bacteria after a single passage through the column. Column material in the absence of single-chain retained less than 10% of the bacteria. Pseudomonas were also removed from milk, mixed bacterial cultures and when present at low cell densities.

Antibodies, Bacterial↗

Sporting 'groin strains': not always muscular!

A gluteal artery pseudoaneurysm presenting as a 'groin strain' following minor trauma is reported. Diagnosis was made by CT scan and arteriography. Treatment was with superselective catheterization and microcoil embolization.

Adolescent↗

Anal lymphoma presenting as a perianal abscess.

A case of perianal abscess secondary to anal lymphoma affecting a human immunodeficiency virus negative patient is presented. Causes of perianal abscess other than anal glandular infection are important, although the proportion of abscesses reported to be secondary to these other causes is found to vary widely in the literature. A biopsy of the wall of an anorectal abscess should be taken when suspicion of an unusual cause arises.

Abscess↗

Design of a microwave system for endoscopy: an experimental study of energy, tissue contact, and hemostatic efficacy.

BACKGROUND: A microwave generator and delivery system for endoscopic use was built. Using a 650-W, 2450-MHz magnetron, 0-160 W were generated from the tip of a 180-cm flexible coaxial cable (2.1 mm diameter). METHODS: Three methods of achieving hemostasis with microwaves were identified studying standard bleeding canine ulcers: (1) interstitial method: inserting the coaxial tip into the tissue and heating slowly until bleeding stopped; (2) contact method: tip held in contact, light pressure applied; (3) noncontact method: microwave-induced sparking (dielectric breakdown) with tip held 1 mm from tissue. RESULTS: Studies of optimal energy levels for hemostasis showed that high power (70 W) noncontact methods required significantly less energy to stop bleeding than contact or interstitial methods. The noncontact method was more effective than the contact method, stopping 20 of 20 bleeding ulcers vs. 10 of 20 (P < 0.001) and was more rapidly effective causing less tissue damage (P < 0.05) than the interstitial method. CONCLUSIONS: In a randomized comparison using a non-contact method, microwave coagulation was superior (P < 0.001) to a polidocanol 1% + adrenaline 1:10,000 injection and control treatment stopping 40 of 40 vs. 0 of 20 and 0 of 20 standard bleeding ulcers. Microwaves stopped bleeding from 10 of 10 severed mesenteric vessels, whereas injection was ineffective (0 of 10, P < 0.001). Microwaves look promising for hemostasis at flexible endoscopy.

Animals↗

Jejunal diverticulosis.

Jejunal diverticulosis is considered to be a generally asymptomatic condition in which the diverticula are pulsion mucosal herniations with walls lacking a muscle layer. These opinions in particular are examined in this review of 20 cases of jejunal diverticulosis treated during an 8 year period. The predominant complication in each case was inflammation or perforation (nine), diarrhoea or malabsorption (five), haemorrhage (three), chronic abdominal pain (two) and pseudo-obstruction (one). At least 10 patients had more than one complication, although this was often unrecognized. Ten patients complained of chronic abdominal pain, in eight of whom the cause was not diagnosed until other complications supervened. Unrecognized malabsorption may be common, as the mean serum albumin in 12 patients presenting acutely with inflammation, perforation or haemorrhage, was low (mean 32 g/L, normal 39-48 g/L). Histology sections were reviewed in the 12 cases that underwent resection, of whom 11 were suitable for classification. Four cases had narrow-mouthed pulsion diverticula with a thin or absent muscle coat, and had developed at the point of blood vessel penetration. Four cases had wide-mouthed diverticula in which the muscle layer was present, suggesting an abnormality of the intestinal wall. Two had features of both. In one case a Meckel's type diverticulum was located 10 cm from the duodenal-jejunal flexure. Seven had mucosal villous atrophy consistent with bacterial overgrowth. In conclusion, jejunal diverticulosis may be associated with complications more frequently than has been recognized and pathologically is a diverse condition.

Aged↗