Animal-Friendly Affinity Reagents: Replacing the Needless in the Haystack.

The multibillion-dollar global antibody industry produces an indispensable resource but that is generated using millions of animals. Despite the irrefutable maturation and availability of animal-friendly affinity reagents (AFAs) employing naïve B lymphocyte or synthetic recombinant technologies expressed by phage display, animal immunisation is still authorised for antibody production. Remarkably, replacement opportunities have been overlooked, despite the enormous potential reduction in animal use. Directive 2010/63/EU requires that animals are not used where alternatives exist. To ensure its implementation, we have engaged in discussions with the EU Reference Laboratory for alternatives to animal testing (EURL ECVAM) and the Directorate General for Environment to carve out an EU-led replacement strategy. Measures must be imposed to avoid outsourcing, regulate commercial production, and ensure that antibody producers are fully supported.

The administration of intermittent parathyroid hormone affects functional recovery from trochanteric fractured neck of femur: a randomised prospective mixed method pilot study.

AIMS: We wished to assess the feasibility of a future randomised controlled trial of parathyroid hormone (PTH) supplements to aid healing of trochanteric fractures of the hip, by an open label prospective feasibility and pilot study with a nested qualitative sub study. This aimed to inform the design of a future powered study comparing the functional recovery after trochanteric hip fracture in patients undergoing standard care, versus those who undergo administration of subcutaneous injection of PTH for six weeks. PATIENTS AND METHODS: We undertook a pilot study comparing the functional recovery after trochanteric hip fracture in patients 60 years or older, admitted with a trochanteric hip fracture, and potentially eligible to be randomised to either standard care or the administration of subcutaneous PTH for six weeks. Our desired outcomes were functional testing and measures to assess the feasibility and acceptability of the study. RESULTS: A total of 724 patients were screened, of whom 143 (20%) were eligible for recruitment. Of these, 123 were approached and 29 (4%) elected to take part. However, seven patients did not complete the study. Compliance with the injections was 11 out of 15 (73%) showing the intervention to be acceptable and feasible in this patient population. TAKE HOME MESSAGE: Only 4% of patients who met the inclusion criteria were both eligible and willing to consent to a study involving injections of PTH, so delivering this study on a large scale would carry challenges in recruitment and retention. Methodological and sample size planning would have to take this into account. PTH administration to patients to enhance fracture healing should still be considered experimental. Cite this article: Bone Joint J 2016;98-B:840-5.

The stress response to bilateral femoral fractures: a comparison of primary intramedullary nailing and external fixation.

OBJECTIVES: Concern has recently been expressed with regard to the physiologic effects of primary intramedullary femoral nailing in seriously injured patients. "Damage control orthopaedics" techniques have been proposed, which comprise principally the use of primary external fixation. The aim of this study was to compare the physiologic effects of external femoral fixation with those of intramedullary stabilization over the first 24 hours after femoral fracture using an established large animal (ovine) trauma model. METHODS: Under terminal anesthesia, bilateral high-energy femoral fractures and hypovolemic shock were produced using a pneumatic actuator. Twenty-four sheep were randomized into 4 groups and monitored for 24 hours. Group 1--control, group 2--trauma only, group 3--trauma and external fixation, and group 4--trauma and reamed intramedullary nailing. Outcome measures included the following: pulmonary embolic load (transesophageal echocardiography), metabolic base excess, plasma coagulation markers, and polymorphonuclear cell counts obtained from bronchoalveolar lavage samples. RESULTS: The total embolic load was significantly higher (P < 0.001) in the intramedullary nailing group. All trauma groups had a significant increase (P < 0.05) in prothrombin times with a fall in antithrombin III and fibrinogen levels. However, the type of fracture stabilization used did not significantly affect any of the other outcome measurements. CONCLUSIONS: A higher pulmonary embolic load can be expected during early intramedullary femoral fracture stabilization compared with primary external fixation. However, the degree of stimulation to systemic coagulation and pulmonary inflammation by each type of surgery was comparable.

Cognitive function and cerebral emboli after primary hip arthroplasty.

Transcranial Doppler ultrasound has been used to detect cerebral emboli after hip arthroplasty. The cognitive effects of these embolic events are unclear. The aim of this study was to assess cognitive change after primary cemented hip arthroplasty using a range of neuropsychological tests and to measure intraoperative cerebral embolic load. Twenty primary cemented total hip arthroplasties underwent a series of cognitive tests before and at four days after surgery. A range of validated tests assessed: global cognitive function; verbal fluency and speed; immediate and delayed memory recall; attention and mental processing speeds. Intra-operative transcranial Doppler ultrasound monitoring of the middle cerebral artery for embolic signals was also performed. A one-sample Wilcoxon signed-rank test compared median cognitive results post-pre surgery and a Mann-Whitney U test established if there was a cognitive difference between those patients who had detectable cerebral emboli and those who did not. Scatter plot graphs were also used to establish any correlation between the embolic load and clinical cognitive dysfunction. A significant (p<0.01) difference was noted in specific tests assessing mental processing speed, visual searching and sustained and divided attention following surgery. Intra-operative cerebral embolic signals were detected in 11 out of 20 patients and the majority occurred with femoral component cementation and hip reduction. There was no difference in cognitive dysfunction between those patients who had detectable cerebral embolic signals and those who did not and there appeared to be no direct correlation between the size of the embolic load and the level of cognitive dysfunction.

Comparison of opioid receptor distributions in the rat ileum.

The cellular expression patterns of mu-, delta- and kappa-opioid receptors in the rat ileum were examined using fluorescence immunohistochemistry. Double-labelling was used to examine cellular receptor co-localisation as a pre-requisite for intracellular molecular interactions, such as heterodimerisation. Tissues were stained as whole-mount preparations. Strong, broadly distributed immunoreactivity (ir) was observed for each receptor in the myenteric and submucous plexuses. Although intracellular mu- and delta-ir patterns differed in ganglion neurons, mu/delta co-expression was extensive in these cells. mu/delta co-expression was also observed in interstitial cells, which were diffusely distributed in submucous plexus preparations but generally located adjacent to myenteric plexus structures. Punctate kappa-ir was seen broadly in nerve fibres in both plexuses, suggesting localisation in varicosities. Neuronal mu/kappa co-localisation was not apparent, although kappa-ir fibres were often apposed against mu-ir cells. mu/kappa co-localisation was detected in interstitial cells in submucous plexus preparations. Similarities in mu and delta expression patterns might reflect similar functional properties previously detected for these receptors. This study indicates that the rat gastrointestinal tract might provide a useful tool for the future study of molecular interactions between opioid receptor types.

The use of an in vitro submerged keratinocyte model to predict induction of squamous metaplasia.

An in vitro submerged keratinocyte model of squamous metaplasia (SQ) in epithelia is being developed to assess the risk associated with exposure to certain environmental agents. Tracheobronchial epithelium (TBE) in vivo can respond to airborne environmental insult by becoming squamous. Epidemiological evidence suggests that cigarette smoke is capable of inducing this change. Retinoic acid has been shown to maintain cells in the mucociliary state. SQ is considered protective and adaptive but potentially preneoplastic if unrelenting and is used histologically in the diagnosis of squamous cell carcinoma. SQ is characterised by upregulation of the expression of transglutaminase I (TGI), TGI activity leading to the formation of isopeptide cross-linked envelopes and replacement of the mucociliary cell type with non-polar squamous cells out of contact with the basal lamina. The ability of the in vitro keratinocyte submerged model to predict the squamous metaplastic response in vivo has been investigated in vitro using TG catalysed fluorescein cadaverine incorporation as a measure of cross-linked envelope formation, Alamar blue conversion to measure viability and Coomassie blue incorporation to measure total cellular protein. The modulation of the squamous condition by retinoic acid (RA), cigarette smoke condensate (CSC) and nicotine has been assessed in keratinocytes cultured in Green's medium. RA inhibited FC incorporation by 95% at 1 x 10(-5) M and simultaneously increased cell viability providing evidence to support its role in the regulation of the non-differentiated state. Nicotine (0-1 mg/ml) induced a dose-dependent increase in viability at 6 days, a response that was accompanied by an increase in FC incorporation at 12 days. CSC (0-5 microg/ml) increased FC incorporation after 12 days. Hence, nicotine modulated the squamous condition by up-regulating TGI activity following a period of hyperactivity. CSC induced a gradual change to the differentiated state and RA served to maintain the cells in an undifferentiated state.

Segregation of somatosensory activation in the human rolandic cortex using fMRI.

The segregation of sensory information into distinct cortical areas is an important organizational feature of mammalian sensory systems. Here, we provide functional magnetic resonance imaging (fMRI) evidence for the functional delineation of somatosensory representations in the human central sulcus region. Data were collected with a 3-Tesla scanner during two stimulation protocols, a punctate tactile condition without a kinesthetic/motor component, and a kinesthetic/motor condition without a punctate tactile component. With three-dimensional (3-D) anatomical reconstruction techniques, we analyzed data in individual subjects, using the pattern of activation and the anatomical position of specific cortical areas to guide the analysis. As a complimentary analysis, we used a brain averaging technique that emphasized the similarity of cortical features in the morphing of individual subjects and thereby minimized the distortion of the location of cortical activation sites across individuals. A primary finding of this study was differential activation of the cortex on the fundus of the central sulcus, the position of area 3a, during the two tasks. Punctate tactile stimulation of the palm, administered at 3 Hz with a 5.88(log10.mg) von Frey filament, activated discrete regions within the precentral (PreCG) and postcentral (PoCG) gyri, corresponding to areas 6, 3b, 1, and 2, but did not activate area 3a. Conversely, kinesthetic/motor stimulation, 3-Hz flexion and extension of the digits, activated area 3a, the PreCG (areas 6 and 4), and the PoCG (areas 3b, 1, and 2). These activation patterns were observed in individual subjects and in the averaged data, providing strong evidence for the existence of a distinct representation within area 3a in humans. The percentage signal changes in the PreCG and PoCG regions activated by tactile stimulation, and in the intervening gap region, support this functional dissociation. In addition to this distinction within the fundus of the central sulcus, the combination of high-resolution imaging and 3-D analysis techniques permitted localization of activation within areas 6, 4, 3a, 3b, 1, and 2 in the human. With the exception of area 4, which showed inconsistent activation during punctate tactile stimulation, activation in these areas in the human consistently paralleled the pattern of activity observed in previous studies of monkey cortex.

Effects of resistive breathing on exercise capacity and diaphragm function in patients with ischaemic heart disease.

BACKGROUND: Muscle weakness has been suggested to result from the deconditioning that accompanies decreased activity levels in chronic cardiopulmonary diseases. The benefits of standard exercise programmes on exercise capacity and muscular strength in disease and health are well documented and exercise capacity is a significant predictor of survival in patients with chronic heart failure (CHF). Selective respiratory muscle training has been shown to improve exercise tolerance in CHF and such observations have been cited to support the suggestion that respiratory muscle weakness contributes to a reduced exercise capacity (despite biopsies showing the metabolic profile of a well trained muscle). AIMS: This study aimed to determine the effects of selective inspiratory muscle training on patients with chronic coronary artery disease to establish if an improved exercise capacity can be obtained in patients that are not limited in their daily activities. METHODS: Nine male patients performed three exercise tests (with respiratory and diaphragm function assessed before the third test) then undertook a 4-week programme of inspiratory muscle training. Exercise tolerance, respiratory and diaphragmatic function were re-assessed after training. RESULTS: Exercise capacity improved from 812+/-42 to 864+/-49 s, P<0.05, and velocity of diaphragm shortening increased (during quiet breathing from 12.8+/-1.6 to 19.4+/-1.1 mm s(-1), P<0.005, and sniffing from 71.9+/-9.4 to 110.0+/-12.3 mm s(-1), P<0.005). In addition, five from nine patients were stopped by breathlessness before training; whereas only one patient was stopped by breathlessness after training. CONCLUSION: The major findings in this study were that a non-intensive 4-week training programme of resistive breathing in patients with chronic coronary artery disease led to an increase in exercise capacity and a decrease in dyspnoea when assessed by symptom limited exercise testing. These changes were associated with significant increases in the velocity of diaphragmatic excursions during quiet breathing and sniffing. Patients that exhibited small diaphragmatic excursions during quiet breathing were most likely to improve their exercise capacity after the training programme. However, the inspiratory muscle-training programme was not associated with any significant changes in respiratory mechanics when peak flow rate, forced expiratory volume and forced vital capacity were measured. The resistive breathing programme used here resulted in a significant increase in the velocity of diaphragm movement during quiet breathing and sniffing. In other skeletal muscles, speed of contraction can be determined by the relative proportion of fibre types and muscle length (Jones, Round, Skeletal Muscle in Health and Disease. Manchester: University Press, 1990). The intensity of the training programme used here, however, is unlikely to significantly alter muscle morphology or biochemistry. Short-term training studies have shown that there can be increases in strength and velocity of shortening that do not relate to changes in muscle biochemistry or morphology. These changes are attributed to the neural adaptations that occur early in training (Northridge et al., Br. Heart J. 1990; 64: 313-316). Independent of the mechanisms involved, this small, uncontrolled study suggests that inspiratory muscle training may improve exercise capacity, diaphragm function and symptoms of breathlessness in patients with chronic coronary artery disease even in the absence of heart failure.

Fluorescein cadaverine incorporation as a novel technique for the characterization of terminal differentiation in keratinocytes.

A novel technique for detecting transglutaminase activity and the production of cornified envelopes in keratinocytes has been devised. This was based on the enzymatic incorporation of fluorescein-labelled cadaverine (FC) into cornified envelopes. The addition of FC (20mum) to the incubation medium served as an amine donor for transglutaminase reactions in place of protein lysine residues. Cells incorporating the label became visible with fluorescence microscopy and were quantified by fluorimetry. There was a significant difference in the level of FC incorporation into cornified envelopes under the various media conditions and time points employed. The greatest incorporation was observed by keratinocytes cultured in Green's medium and fluorescent intensity decreased in the order: Green's> KGM with calcium>KGM. Confocal imaging of keratinocytes dual stained with FC and propidium iodide revealed the presence of distinct layers and demonstrated how FC was incorporated into differentiating cells and not the basal layer. FC incorporation has the potential to serve as a rapid assessment of terminal differentiation in keratinocytes. It is simple and less time-consuming than currently available alternative techniques. This approach also has the advantage of combining microscopic and quantitative data.

Germ cell tumors of the testis overexpress wild-type p53.

Several recent studies have suggested that testicular germ cell tumors express high levels of wild-type p53 protein. To clarify and confirm this unexpected result, we have investigated seminomatous and nonseminomatous germ cell tumors at the genomic, mRNA, and protein levels. Thirty-five tumors were examined for p53 overexpression using antibodies directed against the p53 (PAb1801, PAb240, and CM1), mdm2 (IF2), and p21Waf1/Clp1 (EA10) proteins. Thirty-two tumors were screened for p53 mutations by single-strand conformation polymorphism analysis. Eighteen tumors were screened with a functional assay that tests the transcriptional competence of human p53 protein expressed in yeast. On frozen sections, 100, 65, 35, 73, and 0% of tumors reacted with the CM1, PAb240, PAb1801, IF2, and EA10 antibodies, respectively. No p53 mutations were detected by single-strand conformation polymorphism or by functional assay. The fact that many tumors overexpress wild-type p53 but not mdm2 rules out mdm2 overexpression as a general explanation for the presence of wild-type p53 in these tumors. The absence of p21 overexpression suggests that p53 may be unable to activate transcription of critical target genes, which may explain why the presence of wild-type p53 is tolerated in this tumor type, although the mechanism for this transcriptional inactivity remains to be established.

Phantom evaluation of heating of superficial tissues located above interstitial microwave antennas.

PURPOSE: A technique that improves heating of superficial tissues above an implant of microwave interstitial antennas is presented. METHODS AND MATERIALS: Adequate heating of tumor margins is achieved by extending an implant of microwave antennas beyond the tumor boundary by 1-2 cm. When the tumor infiltrates the superficial tissues including the skin, the implant cannot even reach the superficial margin of the tumor since it requires tissue to support the catheters. This may yield cold spots in the tissues above the implant. Measurements in a phantom with varying thickness of the superficial layer above the implant demonstrated inadequate Specific Absorption Rates of energy distribution in this layer. A method that improves these distributions in the superficial layers was developed and tested in this work. This method requires placing a deionized water bolus on the phantom (patient) surface. Additional microwave antennas are placed on top of the bolus above and parallel to the implanted antennas. The Specific Absorption Rates distributions were evaluated for the thicknesses of superficial layer ranging from 1.5 mm to 16 mm and two bolus thicknesses (5 and 10 mm). RESULTS: The adequate Specific Absorption Rates distributions were achieved for all tested thicknesses of the superficial layer (1.5, 4, 8, 12, and 16 mm). The use of the 5 mm bolus versus 10 mm bolus is discussed. The use of additional antennas did not significantly increase stray radiation. CONCLUSION: This method has the potential to optimize heating of superficial tissues located above a microwave antenna implant.

Bifunctional protein in carrot contains both aspartokinase and homoserine dehydrogenase activities.

We have purified homoserine dehydrogenase to homogeneity and subjected polypeptide fragments derived from digests of the protein to amino acid sequencing. The amino acid sequence of homoserine dehydrogenase from carrot (Daucus carota) indicates that in carrot both aspartokinase and homoserine dehydrogenase activities reside on the same protein. Additional evidence that aspartokinase and homoserine dehydrogenase reside on a bifunctional protein is provided by coelution of activities during purification steps and by enzyme-specific gel staining techniques. Highly purified fractions containing aspartokinase activity were stained for aspartokinase activity, homoserine dehydrogenase activity, and protein. These gels confirmed that aspartokinase activity and homoserine dehydrogenase activity were present on the same protein. This arrangement of aspartokinase and homoserine dehydrogenase activities residing on the same protein is also found in Escherichia coli, which has two bifunctional enzymes, aspartokinase I-homoserine dehydrogenase I and aspartokinase II-homoserine dehydrogenase II. The amino acid sequence of the major form of homoserine dehydrogenase from carrot cell suspension cultures most closely resembles that of the E. coli ThrA gene product aspartokinase I-homoserine dehydrogenase I.

Purification and Interconversion of Homoserine Dehydrogenase from Daucus carota Cell Suspension Cultures.

Homoserine dehydrogenase from cell suspension cultures of carrot (Daucus carota L.) has been purified to apparent homogeneity by a combination of selective heat denaturation, ion exchange and gel filtration chromatographies, and preparative gel electrophoresis. Carrot homoserine dehydrogenase is composed of subunits of equal molecular weight (85,000 +/- 5,000). During purification, the enzyme exists predominantly in two molecular weight forms, 180,000 and 240,000. The enzyme can be reversibly converted from one form to the other, and each has different regulatory properties. When the enzyme is dialyzed in the presence of 5 millimolar threonine, the purified enzyme is converted into its trimeric form (240,000), which is completely inhibited by 5 millimolar threonine and is stimulated 2.6-fold by K(+). When the enzyme is dialyzed in the presence of K(+) and absence of threonine, the purified enzyme is converted into a dimer (180,000), which is not inhibited by threonine and is only stimulated 1.5-fold by K(+). The enzyme also can polymerize under certain conditions to form higher molecular weight aggregates ranging in size up to 720,000, which also are catalytically active. This interconversion of homoserine dehydrogenase conformations may reflect the daily stream of events occurring in vivo. When light stimulates protein synthesis, the threonine pool decreases in the chloroplast, while K(+) concentrations increase. The change in threonine and K(+) concentrations shift the homoserine dehydrogenase from the threonine-sensitive to the threonine-insensitive conformation resulting in increased production of threonine, which would meet the demands of protein synthesis. The reverse process would occur in the dark.

Effects of theophylline on capillary tube leucocyte migration.

Theophylline stimulates the capillary tube migration of human peripheral blood mixed leucocytes. Minor stimulation of polymorph migration is produced directly by theophylline and dibutyryl cyclic AMP, but polymorph migration is markedly stimulated by mononuclear leucocyte culture supernatants to which theophylline has been added. These results suggest that polymorph migration is stimulated when intracellular cyclic AMP increases, and that mononuclear leucocytes produce a potential migration stimulator whose activity is enhanced by theophylline.

Effect of prednisolone on the growth of human bone marrow cells in vitro.

The addition of prednisolone to autostimulatory cultures of human bone marrow in agar results in the formation of an increased number of granulocytic aggregates. The effect is dependent on the concentration of cultured cells and does not occur at low cell concentration. The increase in aggregate numbers is maximal early in the culture and occurs at steroid concentrations which are comparable with pharmacological levels. Prednisolone directly inhibits the responsiveness of granulocytic precursors to colony-stimulating activity (CSA) and it is suggested that the stimulatory effect is indirect and may be caused by a steroid action on mediator production. These findings may be relevant to the polymorphonuclear leucocytosis induced by glucocorticoids.