Evaluation of variable new antigen receptors (vNARs) as a novel cathepsin S (CTSS) targeting strategy.

Aberrant activity of the cysteine protease Cathepsin S (CTSS) has been implicated across a wide range of pathologies. Notably in cancer, CTSS has been shown to promote tumour progression, primarily through facilitating invasion and migration of tumour cells and augmenting angiogenesis. Whilst an attractive therapeutic target, more efficacious CTSS inhibitors are required. Here, we investigated the potential application of Variable New Antigen Receptors (vNARs) as a novel inhibitory strategy. A panel of potential vNAR binders were identified following a phage display panning process against human recombinant proCTSS. These were subsequently expressed, purified and binding affinity confirmed by ELISA and SPR based approaches. Selected lead clones were taken forward and were shown to inhibit CTSS activity in recombinant enzyme activity assays. Further assessment demonstrated that our lead clones functioned by a novel inhibitory mechanism, by preventing the activation of proCTSS to the mature enzyme. Moreover, using an intrabody approach, we exhibited the ability to express these clones intracellularly and inhibit CTSS activity whilst lead clones were also noted to impede cell invasion in a tumour cell invasion assay. Collectively, these findings illustrate a novel mechanistic approach for inhibiting CTSS activity, with anti-CTSS vNAR clones possessing therapeutic potential in combating deleterious CTSS activity. Furthermore, this study exemplifies the potential of vNARs in targeting intracellular proteins, opening a range of previously "undruggable" targets for biologic-based therapy.

Factors affecting adequacy of Pipelle and Tao Brush endometrial sampling.

OBJECTIVE: To compare factors influencing adequacy of endometrial samples obtained using two outpatient sampling devices--Pipelle and Tao Brush. DESIGN: Pragmatic unblinded trial with investigation schedule randomised separately within two groups according to endometrial cancer risk. SETTING: Gynaecology outpatient clinic of a large city hospital in Edinburgh, Scotland. POPULATION: All women referred to a gynaecology outpatient clinic during a 28-month period complaining of abnormal vaginal bleeding. METHODS: Women were assigned to two 'risk groups' for endometrial cancer ('high risk' for postmenopausal women and 'moderate risk' for premenopausal women aged over 40 years or with other risk factors). Women in each risk group had both types of biopsy and were randomised to two outpatient visualisations: hysteroscopy and/or transvaginal ultrasound scan. MAIN OUTCOME MEASURES: Completion of the investigation, adequacy of sample and acceptability of investigation to women. RESULTS: In 200 high-risk women, adequate samples were significantly more likely to be obtained by Tao Brush than Pipelle (P < 0.001). Nulliparity was strongly associated with failed insertion for both devices (P < 0.001). Inadequate samples were strongly associated with postmenopausal status only for Pipelle (P < 0.001), and among premenopausal women, for both samplers, with nulliparity (P < 0.001). A significantly greater proportion of women preferred the Tao Brush to the Pipelle endometrial sampler (P < 0.001). CONCLUSIONS: In postmenopausal women, Tao Brush sampling offers advantages over use of Pipelle, and the former should be considered as an alternative or additional sampling device in this group of women.

Difficulties in the pre-operative diagnosis of phyllodes tumours of the breast: a study of 84 cases.

Eighty-four phyllodes tumours (71 benign, eight borderline and five malignant) diagnosed over a 16-year period were studied retrospectively, to assess the diagnostic value of the pre-operative modalities used. Mammography and ultrasound appearances were non-specific. The possibility of phyllodes tumour was raised in only 23% on fine needle aspiration cytology, and in 65% on core biopsy. Accuracy was better in smaller tumours, suggesting that larger tumours need more samples. For phyllodes tumours whose growth was measured, almost all had growth rates greater than for growing fibroadenomas. The pre-operative diagnosis of phyllodes tumours is difficult, and rapid growth and/or large size of apparent fibroadenomas may be the only imaging findings to suggest phyllodes tumour. It is important to review most fibroadenomas with ultrasound, to assess the rate of growth if any. Whole breast ultrasound showed that nearly one third of women with phyllodes tumours had concurrent fibroadenomas.

A sensitive and reliable reverse transcriptase PCR-enzyme-linked immunosorbent assay for the detection of human pathogenic viruses in bivalve molluscs.

A colorimetric method, reverse transcriptase PCR with an enzyme-linked immunosorbent assay (RT-PCR-ELISA) was evaluated for ease of use, reliability, and sensitivity when detecting known human pathogenic virus present in shellfish, using a traditional polyethylene precipitation or immunocapture virus concentration method. The newly developed ELISA method could successfully detect enteroviruses and noroviruses in artificially and naturally contaminated shellfish. Overall, ELISA was shown to be a robust and sensitive method, which had a detection limit of 10 to 100 50% tissue culture infective dose enterovirus per gram of Crassostrea gigas (Pacific oyster) digestive gland and whole Mytilus edulis (common blue mussel). The technique was easily established in a new laboratory and required no specialized equipment. The method had a high sample throughput capable of screening 96 samples per run, making the technique extremely time efficient. RT-PCR-ELISA is a safe, quick, reliable technique, which has the potential for use as a standard virus detection method.

A reliable RT-PCR-ELISA method for the detection of infectious pancreatic necrosis virus (IPNV) in farmed rainbow trout.

A new method, termed RT-PCR-ELISA, was evaluated for ease of use, reliability and sensitivity when detecting infectious pancreatic necrosis virus (IPNV) present in trout kidney tissue. The method had comparable sensitivity to existing PCR assays and could successfully detect 1.5 x 10(4) pfu IPNV in artificially contaminated trout kidney samples. The technique was easily established in a new laboratory and required no specialised equipment. The method had a high sample throughput capable of screening 96 samples per run, making the technique extremely time efficient. The RT-PCR-ELISA is a safe, quick, reliable technique, which has the potential for use as a standard virus detection method.

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.

A lack of functional NK1 receptors explains most, but not all, abnormal behaviours of NK1R-/- mice(1).

Mice lacking functional neurokinin-1 receptors (NK1R-/-) display abnormal behaviours seen in Attention Deficit Hyperactivity Disorder (hyperactivity, impulsivity and inattentiveness). These abnormalities were evident when comparing the behaviour of separate (inbred: 'Hom') wildtype and NK1R-/- mouse strains. Here, we investigated whether the inbreeding protocol could influence their phenotype by comparing the behaviour of these mice with that of wildtype (NK1R+/+) and NK1R-/- progeny of heterozygous parents ('Het', derived from the same inbred strains). First, we recorded the spontaneous motor activity of the two colonies/genotypes, over 7 days. This continuous monitoring also enabled us to investigate whether the diurnal rhythm in motor activity differs in the two colonies/genotypes. NK1R-/- mice from both colonies were hyperactive compared with their wildtypes and their diurnal rhythm was also disrupted. Next, we evaluated the performance of the four groups of mice in the 5-Choice Serial Reaction-Time Task (5-CSRTT). During training, NK1R-/- mice from both colonies expressed more impulsive and perseverative behaviour than their wildtypes. During testing, only NK1R-/- mice from the Hom colony were more impulsive than their wildtypes, but NK1R-/- mice from both colonies were more perseverative. There were no colony differences in inattentiveness. Moreover, a genotype difference in this measure depended on time of day. We conclude that the hyperactivity, perseveration and, possibly, inattentiveness of NK1R-/- mice is a direct consequence of a lack of functional NK1R. However, the greater impulsivity of NK1R-/- mice depended on an interaction between a functional deficit of NK1R and other (possibly environmental and/or epigenetic) factors.

Development and application of bioluminescent Caenorhabditis elegans as multicellular eukaryotic biosensors.

We describe a novel approach to assess toxicity to the free-living nematode Caenorhabditis elegans that relies on the ability of firefly luciferase to report on endogenous ATP levels. We have constructed bioluminescent C. elegans with the luc gene under control of a constitutive promoter. Light reduction was observed in response to increasing temperature, concentrations of copper, lead and 3,5-dichlorophenol. This was due to increased mortality coupled with decreased metabolic activity in the surviving animals. The light emitted by the transgenic nematodes gave a rapid, real-time indication of metabolic status. This forms the basis of rapid and biologically relevant toxicity tests.

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.

Toxicity of the bacterial luciferase substrate, n-decyl aldehyde, to Saccharomyces cerevisiae and Caenorhabditis elegans.

This study determined that the bacterial luciferase fusion gene (luxAB) was not a suitable in vivo gene reporter in the model eukaryotic organisms Saccharomyces cerevisiae and Caenorhabditis elegans. LuxAB expressing S. cerevisiae strains displayed distinctive rapid decays in luminescence upon addition of the bacterial luciferase substrate, n-decyl aldehyde, suggesting a toxic response. Growth studies and toxicity bioassays have subsequently confirmed, that the aldehyde substrate was toxic to both organisms at concentrations well tolerated by Escherichia coli. As the addition of aldehyde is an integral part of the bacterial luciferase activity assay, our results do not support the use of lux reporter genes for in vivo analyses in these model eukaryotic organisms.

Immunocytochemical survey of putative neurotransmitters in taste buds from Necturus maculosus.

To investigate synaptic mechanisms in taste buds and collect information about synaptic transmission in these sensory organs, we have examined taste buds of the mudpuppy, Necturus maculosus for the presence of neurotransmitters and neuromodulators. Immunocytochemical staining at the light microscopic level revealed the presence of serotonin-like and cholecystokinin-like (CCK) immunoreactivity in basal cells in the taste bud. Nerve fibers innervating taste buds were immunoreactive for vasoactive intestinal peptide-like (VIP), substance P-like, and calcitonin gene-related peptide-like (CGRP) or compounds closely related to these substances. Immunoreactivity for tyrosine hydroxylase (TH) and choline acetyltransferase (ChAT) in the taste cells and nerve fibers was absent. These data suggest that serotonin, CCK, VIP, substance P, and CGRP are involved in synaptic transmission or neuromodulation in the peripheral organs of taste. No evidence was found for cholinergic or adrenergic mechanisms on the basis of the absence of immunocytochemical staining for key enzymes involved in these two transmitter systems.

Detection and quantification of microcystins (cyanobacterial hepatotoxins) with recombinant antibody fragments isolated from a naïve human phage display library.

Single-chain antibody fragments against the cyanobacterial hepatotoxin microcystin-LR were isolated from a naive human phage display library and expressed in Escherichia coli. In competition enzyme-linked immunosorbent assay (ELISA), the most sensitive antibody clone selected from the library detected free microcystin-LR with an IC(50) value of 4 microM. It was found to cross react with three other microcystin variants - microcystin-RR, microcystin-LW and microcystin-LF - and detected microcystins in extracts of the cyanobacterium Microcystis aeruginosa, found to contain the toxins by high-performance liquid chromatography (HPLC). The quantification of microcystins in these extracts by ELISA and HPLC showed good correlation. Although the antibody isolated in this study was considerably less sensitive than the polyclonal and monoclonal antibodies already available for microcystin detection, phage display technology represents a cheaper, more rapid alternative for the production of anti-microcystin antibodies than the methods currently in use.

Rapid selection of anti-hapten antibodies isolated from synthetic and semi-synthetic antibody phage display libraries expressed in Escherichia coli.

Antibody phage display libraries (Griffin and Tomlinson I) displaying antibody genes and maintained and amplified in Escherichia coli were used to isolate antibodies to the hapten target microcystin LR (1000 Da) conjugated to either bovine serum albumin or keyhole limpet haemocyanin. In competition enzyme-linked immunosorbent assay, bacterially expressed antibodies selected via the Griffin library showed at least 300 times greater sensitivity than those isolated from the Tomlinson library, for free microcystin. Bacterially expressed phage antibody libraries provide a rapid and relatively easy route for the selection of monoclonal antibodies specific for even the most difficult of antigenic targets such as free haptens.

The isolation of super-sensitive anti-hapten antibodies from combinatorial antibody libraries derived from sheep.

The complexity and expense of producing anti-hapten monoclonals via the traditional hybridoma route and the preferential selection of antibodies that recognise the conjugated form of the hapten, over antibodies that specifically recognise free hapten, are two of the more important problems that have limited the development and application of anti-hapten antibodies. The advent of phage display technology allows the rapid isolation of monoclonal antibody fragments from libraries of different antibodies (>10(8)) displayed on the surface of filamentous bacteriophages. Much of the power of this new approach lies in the flexibility with which these libraries can be screened for suitable binders. Using an optimised selection procedure, we have isolated from a sheep antibody phage display library, super-sensitive anti-hapten antibodies specific for the herbicide and environmental pollutant, atrazine. In particular, two phage clones have been isolated that can be expressed cheaply and in quantity in Escherichia coli, demonstrate excellent stability in nonphysiological conditions and are exciting prospects for immunoassay applications including ELISA, dip-stick formats, on-line monitoring and biosensor technologies. In ELISA formats they show low levels of cross reactivity with related molecules and a limit of detection of a 1-2 parts per trillion (p.p.t.), well within the 100 p.p.t. required by EC legislation.

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.

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.

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.

The diagnosis of breast cancer in women younger than 40.

This study examined how the diagnosis of breast cancer is different in young women. Records were retrieved for 239 women diagnosed with breast cancer before age 40 and compared with 2101 women aged 40 and over with breast cancer. On mammography, lesions in the younger women were more likely to be undetected or interpreted as benign, especially in women with dense breasts. However, there were 10 young women where impalpable cancers with microcalcification under 10 mm would not have been diagnosed without mammography. An abnormality was detected on ultrasound in 92.2% of cancers in young women, but was more likely to be considered benign than in older women. If ultrasound alone had been used in the young women, at least 18 cancers would have been missed. Ultrasound was useful for predicting the ultimate tumour size at pathology, and for detecting multifocality. There were 14 cases where the ultrasound appearance was indistinguishable from fibroadenoma. The importance of fine needle aspiration cytology in the diagnosis of focal lesions in young women (over 20 years) was confirmed. For symptomatic women, the proportion of breast malignancies under 10 mm was similar in the two groups. However, the younger group had significantly more poorly differentiated tumours.

Comparative sensitivity of immunoassays for haptens using monomeric and dimeric antibody fragments.

A single-chain anti-atrazine antibody fragment, scAb (single-chain Fv with a CK domain), was expressed in Escherichia coli, and monomeric and dimeric species were preferentially purified from periplasmic extracts by chromatography upon nickel chelate immunosorbent columns or by immunoaffinity purification using a constant domain (CK) tag. Recombinant monomeric and dimeric antibody fragments, Fab, and intact monoclonal antibodies were compared in assays by competition between free atrazine in solution and (a) immobilized atrazine-bovine serum albumin conjugate (indirect assay) or (b) atrazine-alkaline phosphatase (direct assay). Recombinant antibody fragments provided a lower detection limit than either Fab or intact monoclonal antibody in both assay formats. Monomeric fragments displayed a sensitivity of detection down to 0.1 ppb, compared to 1.0 ppb for dimeric fragments and the parental monoclonal.

Use of bacterial biosensors to interpret the toxicity and mixture toxicity of herbicides in freshwater.

The dose response relationship between seven commonly used herbicides and four luminescence-based bacterial biosensors was characterised. As herbicide concentration increased the light emitted by the test organism declined in a concentration dependent manner. These dose responses were used to compare the predicted vs. observed response of a biosensor in the presence of multiple contaminants. For the majority of herbicide interactions, the relationship was not additive but primarily antagonistic and sometimes synergistic. These biosensors provide a sensitive test and are able to screen a large volume and wide range of samples with relative rapidity and ease of interpretation. In this study biosensor technology has been successfully applied to interpret the interactive effects of herbicides in freshwater environments.