Self-Interaction Chromatography of mAbs: Accurate Measurement of Dead Volumes.

PURPOSE: Measurement of the second virial coefficient B22 for proteins using self-interaction chromatography (SIC) is becoming an increasingly important technique for studying their solution behaviour. In common with all physicochemical chromatographic methods, measuring the dead volume of the SIC packed column is crucial for accurate retention data; this paper examines best practise for dead volume determination. METHOD: SIC type experiments using catalase, BSA, lysozyme and a mAb as model systems are reported, as well as a number of dead column measurements. RESULTS: It was observed that lysozyme and mAb interacted specifically with Toyopearl AF-Formyl dead columns depending upon pH and [NaCl], invalidating their dead volume usage. Toyopearl AF-Amino packed dead columns showed no such problems and acted as suitable dead columns without any solution condition dependency. Dead volume determinations using dextran MW standards with protein immobilised SIC columns provided dead volume estimates close to those obtained using Toyopearl AF-Amino dead columns. CONCLUSION: It is concluded that specific interactions between proteins, including mAbs, and select SIC support phases can compromise the use of some standard approaches for estimating the dead volume of SIC columns. Two other methods were shown to provide good estimates for the dead volume.

Decreased spinal cord motor neuron numbers in mice depleted of central nervous system copper.

Disrupted copper availability in the central nervous system (CNS) is implicated as a significant feature of the neurodegenerative disease amyotrophic lateral sclerosis (ALS). Solute carrier family 31 member 1 (Slc31a1; Ctr1) governs copper uptake in mammalian cells and mutations affecting Slc31a1 are associated with severe neurological abnormalities. Here, we examined the impact of decreased CNS copper caused by ubiquitous heterozygosity for functional Slc31a1 on spinal cord motor neurons in Slc31a1+/- mice. Congruent with the CNS being relatively susceptible to disrupted copper availability, brain and spinal cord tissue from Slc31a1+/- mice contained significantly less copper than wild-type littermates, even though copper levels in other tissues were unaffected. Slc31a1+/- mice had less spinal cord α-motor neurons compared to wild-type littermates, but they did not develop any overt physical signs of motor impairment. By contrast, ALS model SOD1G37R mice had fewer α-motor neurons than control mice and exhibited clear signs of motor function impairment. With the expression of Slc31a1 notwithstanding, spinal cord expression of genes related to copper handling revealed only minor differences between Slc31a1+/- and wild-type mice. This contrasted with SOD1G37R mice where changes in the expression of copper handling genes were pronounced. Similarly, the expression of genes related to toxic glial activation was unchanged in spinal cords from Slc31a1+/- mice but highly upregulated in SOD1G37R mice. Together, results from the Slc31a1+/- mice and SOD1G37R mice indicate that although depleted CNS copper has a significant impact on spinal cord motor neuron numbers, the manifestation of overt ALS-like motor impairment requires additional factors.

Use and abuse of faecal occult blood tests in an acute hospital inpatient setting.

BACKGROUND/AIMS: The role of the faecal occult blood test (FOBT) is untested. The aims of this study were to define the use of FOBT in a general hospital setting and to determine its influence on patient management. METHODS: Case notes and laboratory reports were retrospectively reviewed in all FOBTs performed in 2006 across three acute hospitals, with specific reference to clinical setting, indication, influence over clinical decision-making and management. Both guaiac and immunological tests were performed on all specimens. RESULTS: A total of 330 patients aged 2-104 (mean 74) years, 47% men, had 461 tests performed. A positive result was recorded in one or both tests in 64% of patients. Evidence of dietary restriction was found in only eight (2%) of patients and 218 (66%) patients took one or more medications that could have caused a false positive result. Indications were mostly for overt or suspected gastrointestinal blood loss with or without anaemia and/or iron deficiency, but 5% were for non-bloody diarrhoea and 3% screening for colorectal cancer. Patient care was adversely affected or delayed in 54 patients (16%), mostly because of the result being the stimulus for the decision to refer or not for endoscopy. Only one was considered appropriate as a screening test for colorectal cancer. CONCLUSIONS: The FOBT was applied in clinically inappropriate settings without consideration to confounding issues, and often led to inappropriate clinical decisions with considerable cost to hospital and patient. There is no place for FOBT in an acute hospital setting.

Principal component score modeling for the rapid description of chromatographic separations.

This paper describes the use of Principal Component Analysis (PCA) as a tool for modeling chromatographic separations. PCA is an analytical technique developed to extract key information out of large data sets and to develop relationships and correlations. The basis of the proposed model is the use of PCA to correlate experimental chromatographic data across different process variables or scales. The generated correlations are then used to provide for the simulation of additional chromatographic runs not included in the initial dataset. The approach is demonstrated by application to the cation exchange separation of a four protein component feed comprising ovalbumin, ovatransferrin, lysozyme, and myoglobin. A good fit between modeled and experimental data was found, and the ability of the method to model additional chromatographic separations not within the original dataset is demonstrated. The technique has the potential to accommodate changing system variables such as column dimensions as well as process variables including sample volume and salt gradient. It provides a potentially powerful tool for the rapid investigation of scale-up effects and for the minimization of the material inventories needed for such studies.

Patient and community attitudes toward perioperative biobanking and genomic research.

We surveyed hospital patients and clinicians to ascertain their attitudes to the establishment of a perioperative biobank for future genomics research, and whether the requirements for an opt-out approach to consent can be met. We enrolled hospital patients (n=187), patient spouse/family members (n=64), ethics committee members (n=14), and clinical staff (doctors and nurses [n=67]), and unspecified community members (n=10). They were asked to rate and describe their views on medical research and biobanking, the need for individual consent, and the importance of confidentiality. Of 406 survey forms distributed, 342 (84%) were returned. Nearly all participants (98%) indicated that a perioperative biobank is important, 93% were comfortable with de-identified genetic research, and 90% indicated that the hospital should be able to use leftover blood for medical research, provided the research has been approved by an ethics committee and personally identifying information has been removed. Participants were more likely to support biobanking if it used de-identified samples, and if, for this reason, their consent was not sought. Participants with chronic medical and surgical conditions were significantly more supportive and comfortable with genetic research, as were most in the hospital community. Most hospital patients, community members and clinicians are supportive of the development of a perioperative biobank used for genomic research. This supports the adoption of an opt-out approach to consent model.

An antibody to the beta-secretase cleavage site on amyloid-beta-protein precursor inhibits amyloid-beta production.

Proteolytic cleavage of amyloid-beta-protein precursor (AbetaPP) by beta- and gamma-secretases results in production of the amyloid-beta peptide (Abeta) that accumulates in the brains of sufferers of Alzheimer's disease (AD). We have developed a monoclonal antibody, 2B12, which binds in the vicinity of the beta-secretase cleavage site on AbetaPP but does not bind within the Abeta region. We hypothesised that this antibody, directed against the substrate rather than the enzyme, could inhibit cleavage of AbetaPP by beta-secretase via steric hindrance and thus reduce downstream production of Abeta. The antibody would enter cells by binding to AbetaPP when it is at the cell surface and then be internalised with the protein. We subsequently demonstrated that, after addition of 2B12 to standard growth media, this antibody was indeed capable of inhibiting Abeta40 production in neuroblastoma and astrocytoma cells expressing native AbetaPP, as measured by an ELISA. This inhibition was both concentration- and time-dependent and was specific to 2B12. We were only able to inhibit approximately 50% of Abeta40 production suggesting that not all AbetaPP is trafficked to the cell surface. We propose that this antibody could be used as a novel, putative therapy for the treatment of AD.

Micro scale self-interaction chromatography of proteins: A mAb case-study.

Self-interaction chromatography is known to be a fast, automated and promising experimental technique for determination of B22, but with the primary disadvantage of needing a significant amount of protein (>50 mg). This requirement compromises its usage as a technique for the early screening of new biotherapeutic candidates. A new scaled down SIC method has been evaluated here using a number of micro LC columns of different diameters and lengths, using typically 10 times less stationary phase than traditional SIC. Scale-down was successfully accomplished using these micro-columns, where the SIC results for a range of differing columns sizes were in agreement, as reflected by k', B22 and column volumes data. The results reported here demonstrate that a scaled down version of SIC can be easily implemented using conventional liquid chromatography system where the final amount of mAbs used was 10 times less than required by conventional SIC methodologies.

Crystallization of a type III chloramphenicol acetyl transferase.

A type III variant of chloramphenicol acetyl transferase was purified from Escherichia coli and crystallized in the presence of cobaltic hexamine chloride and 2-methyl-2, 4-pentandiol. Two crystal forms were obtained, one of which proved to be suitable for high-resolution X-ray diffraction studies. The space group of this form is R32 with aR = 74.5 A, alpha R = 92.5 degrees, with a monomer (Mr 25,000) in the asymmetric unit. The crystals diffract to 1.7 A resolution. The crystal symmetry has led to a re-evaluation of the oligomeric symmetry of the enzyme and the proposal that it is a trimer rather than a tetramer, the quaternary structure predicted previously from studies of the association of hybrid s subunits.

Electroacoustic production of murine hybridomas.

The optimal conditions for the production of murine hybridomas by electroacoustic fusion of cells in sugar solutions and of cells in ionic strength media (up to 115 mM NaCl) have been investigated. In the electroacoustic fusion technique cells were brought into contact in a 3 MHz ultrasonic standing wave field and were fused by application of an electric pulse. Hybridomas of murine splenocytes and X63-Ag8.653 mouse myelomas were successfully produced in low ionic strength mannitol solutions and in a range of salt concentrations up to 115 mM. The yield of hybridomas by electroacoustic fusion of cells in mannitol was at least as good as that obtained when cells were fused following conventional dielectrophoresis induced contact. The electroacoustic fusion yield was also comparable to conventional yields when cells were exposed to a pulse in higher ionic strength media where dielectrophoresis induces heating effects. Hybridomas were produced at similar electric field strengths when cells were suspended in high ionic strength media (up to 115 mM NaCl) or in mannitol. The effective electric field strength for hybridoma production was close to that at which trypan blue tests indicated membrane damage.

Amplified detection, using a monoclonal antibody, of an aphid-specific epitope exposed during digestion in the gut of a predator.

Monoclonal antibodies are invaluable tools for identifying and quantifying prey remains in the fore-guts of predators. However, they must be target-specific, detect an epitope that is well replicated within the prey (to enhance assay sensitivity) and, critically, recognise a site that can resist digestion. A monoclonal antibody is reported that proved to be aphid-specific and capable of detecting, and accurately identifying, as little as 16.5 ng of aphid protein within a heterologous mixture of invertebrate material. The antibody was selected by screening hybridoma lines for antibodies that bound with semi-digested aphid proteins. The antibody detected an epitope that was found, against expectation, to significantly increase in concentration with time (by approx. 50% over 6 h) in the gut of the carabid predator Pterostichus melanarius. The resultant extended antigen detection period and half-life, and the high specificity of this antibody, showed it to be an enhanced tool for studying interactions between aphids and their predators in the field. It was concluded that the antibody was initially generated to a surface epitope on a high molecular weight native protein (> 200 kD). This epitope, however, was then either replicated on internal sites progressively revealed by digestion, or new epitopes became available as the conformation of the protein changed during digestion.

Electroacoustic fusion of millilitre volumes of cells in physiological medium.

A technique is described in which erythrocytes suspended in 1.1 ml of 145 mM NaCl, have been fused by electrofusion. The cells in suspension were brought into close contact by setting up a 3 MHz ultrasonic standing wave in a cylindrical cell container. The aluminium foil base of the container served both to transmit ultrasound and as an electrode for electrofusion. The electric pulse was generated by a capacitor discharge system. The electric field strength required to fuse cells increased as the ionic strength of the cell suspending phase increased. Cells in physiological saline fused at an electric field strength of 7.3 kV/cm with a 50 microseconds pulse.

Fixed dilated pupils in association with a thiopentone infusion in severe head injury.

We report a case of thiopentone induced mydriasis in a 17-year-old, head injured patient. Two initial 50 mg intravenous boluses and 12 h of a 125 mg/h infusion (total of 1500 mg) of thiopentone were used to reduce intracranial pressure in this patient. The patient developed fixed dilated pupils (> 8 mm) and hypothermia (27 degrees C). Thiopentone plasma concentrations were performed giving a level of 309 mumol/1; withdrawing the thiopentone infusion resulted in the pupil diameter returning to 5 mm within 24 h. A new drug plasma concentration was performed revealing a level of 198 mumol/1. We discuss the clinical implications of such a drug response and review the relevant literature.

Adult polycystic kidney disease and arachnoid cyst formation: Case report.

A 34 year old man is reported with adult polycystic kidney disease with an associated extensive arachnoid cyst occupyingmost of his left hemicranium. The aetiology of arachnoid cyst formation is discussed.

Non-neoplastic demyelinating process mimicking a disseminated malignant brain tumour.

Non-neoplastic demyelinating processes of the brain with ring enhancing lesions and mass effect on MRI imaging, mimicking malignant brain tumours, are rare phenomena. We document the case of a 32 year old male with clinical, radiological and initial histological findings, suggestive of a malignant brain tumour. Additional investigations confirmed the diagnosis of multiple sclerosis. This case is significant as the lesion could not be easily distinguished from a malignant brain tumour on imaging alone. Cases such as this illustrate the importance of considering a demyelinating process in the differential diagnosis of tumour-like brain lesions.

Infectious bovine keratoconjunctivitis: isolation of Moraxella bovis from two groups of young beef cattle in fly control field trials during 1981.

The rate of infection with Moraxella bovis was determined for two herds of calves during the course of a year. High infection rates with the haemolytic variant were found in the herds while they were housed during their first winter but this infection rate fell steadily after turnout. A rise in infections with the non-haemolytic variant was observed during this time. The animals remained free from clinical infectious bovine keratoconjunctivitis during the summer, unlike similar groups of animals in previous years. Immunity, weather and flies are considered for their possible relevance to this observation.

Mitochondrial metals as a potential therapeutic target in neurodegeneration.

Transition metals are critical for enzyme function and protein folding, but in excess can mediate neurotoxic oxidative processes. As mitochondria are particularly vulnerable to oxidative damage due to radicals generated during ATP production, mitochondrial biometal homeostasis must therefore be tightly controlled to safely harness the redox potential of metal enzyme cofactors. Dysregulation of metal functions is evident in numerous neurological disorders including Alzheimer's disease, stroke, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis and Friedrich's ataxia. This review describes the mitochondrial metal defects in these disorders and highlights novel metal-based therapeutic approaches that target mitochondrial metal homeostasis in neurological disorders.

X-ray fluorescence imaging reveals subcellular biometal disturbances in a childhood neurodegenerative disorder.

Biometals such as zinc, iron, copper and calcium play key roles in diverse physiological processes in the brain, but can be toxic in excess. A hallmark of neurodegeneration is a failure of homeostatic mechanisms controlling the concentration and distribution of these elements, resulting in overload, deficiency or mislocalization. A major roadblock to understanding the impact of altered biometal homeostasis in neurodegenerative disease is the lack of rapid, specific and sensitive techniques capable of providing quantitative subcellular information on biometal homeostasis in situ. Recent advances in X-ray fluorescence detectors have provided an opportunity to rapidly measure biometal content at subcellular resolution in cell populations using X-ray Fluorescence Microscopy (XFM). We applied this approach to investigate subcellular biometal homeostasis in a cerebellar cell line isolated from a natural mouse model of a childhood neurodegenerative disorder, the CLN6 form of neuronal ceroid lipofuscinosis, commonly known as Batten disease. Despite no global changes to whole cell concentrations of zinc or calcium, XFM revealed significant subcellular mislocalization of these important biological second messengers in cerebellar Cln6nclf (CbCln6nclf ) cells. XFM revealed that nuclear-to-cytoplasmic trafficking of zinc was severely perturbed in diseased cells and the subcellular distribution of calcium was drastically altered in CbCln6nclf cells. Subtle differences in the zinc K-edge X-ray Absorption Near Edge Structure (XANES) spectra of control and CbCln6nclf cells suggested that impaired zinc homeostasis may be associated with an altered ligand set in CbCln6nclf cells. Importantly, a zinc-complex, ZnII(atsm), restored the nuclear-to-cytoplasmic zinc ratios in CbCln6nclf cells via nuclear zinc delivery, and restored the relationship between subcellular zinc and calcium levels to that observed in healthy control cells. ZnII(atsm) treatment also resulted in a reduction in the number of calcium-rich puncta observed in CbCln6nclf cells. This study highlights the complementarities of bulk and single cell analysis of metal content for understanding disease states. We demonstrate the utility and broad applicability of XFM for subcellular analysis of perturbed biometal metabolism and mechanism of action studies for novel therapeutics to target neurodegeneration.

The accurate measurement of second virial coefficients using self-interaction chromatography: experimental considerations.

Measurement of B22, the second virial coefficient, is an important technique for describing the solution behaviour of proteins, especially as it relates to precipitation, aggregation and crystallisation phenomena. This paper describes the best practise for calculating B22 values from self-interaction chromatograms (SIC) for aqueous protein solutions. Detailed analysis of SIC peak shapes for lysozyme shows that non-Gaussian peaks are commonly encountered for SIC, with typical peak asymmetries of 10%. This asymmetry reflects a non-linear chromatographic retention process, in this case heterogeneity of the protein-protein interactions. Therefore, it is important to use the centre of mass calculations for determining accurate retention volumes and thus B22 values. Empirical peak maximum chromatogram analysis, often reported in the literature, can result in errors of up to 50% in B22 values. A methodology is reported here for determining both the mean and the variance in B22 from SIC experiments, includes a correction for normal longitudinal peak broadening. The variance in B22 due to chemical effects is quantified statistically and is a measure of the heterogeneity of protein-protein interactions in solution. In the case of lysozyme, a wide range of B22 values are measured which can vary significantly from the average B22 values.

Anti-amyloid precursor protein immunoglobulins inhibit amyloid-ß production by steric hindrance.

The cleavage of amyloid precursor protein (APP) by ß- and γ-secretases results in the production of amyloid-ß (Aß) in Alzheimer's disease. We raised two monoclonal antibodies, 2B3 and 2B12, that recognize the ß-secretase cleavage site on APP but not Aß. We hypothesized that these antibodies would reduce Aß levels via steric hindrance of ß-secretase. Both antibodies decreased extracellular Aß levels from astrocytoma cells, but 2B3 was more potent than 2B12. Levels of soluble sAPPα from the nonamyloidogenic α-secretase pathway and intracellular APP were not affected by either antibody nor were there any effects on cell viability. 2B3 exhibited a higher affinity for APP than 2B12 and its epitope appeared to span the cleavage site, whereas 2B12 bound slightly upstream. Both of these factors probably contribute to its greater effect on Aß levels. After 60 min incubation at pH 4.0, most 2B3 and 2B12 remained bound to their antigen, suggesting that the antibodies will remain bound to APP in the acidic endosomes where ß-secretase cleavage probably occurs. Only 2B3 and 2B12, but not control antibodies, inhibited the cleavage of sAPPα by ß-secretase in a cell-free assay where the effects of antibody internalization and intracellular degradation were excluded. 2B3 virtually abolished this cleavage. In addition, levels of C-terminal APP fragments, generated following ß-secretase cleavage (ßCTF), were significantly reduced in cells after incubation with 2B3. These results strongly suggest that anti-cleavage site IgGs can generically reduce Aß levels via inhibition of ß-secretase by steric hindrance and may provide a novel alternative therapy for Alzheimer's disease.