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.

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.

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.

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.

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.

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.

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.

Studies on the physico-chemical characteristics of polymorph migration stimulator.

Polymorph migration stimulator is a supernatant factor produced by the interaction between glucocorticosteroids and human blood monocytes in culture. Studies on the physical characteristics of this factor show that it is soluble and stable at high and low temperatures. Its activity is reduced by acid and alkali treatment and destroyed by the proteolytic enzyme protease. Experiments involving dialysis, ultrafiltration and Sephadex G-100 gell filtration indicate that the molecular weight of the polymorph migration stimulator is between 12,000 and 15,000. It is suggested that this factor may mediate the anti-inflammatory effects of glucocorticosteroids on phagocytic cells.

Evaluation of rosette inhibition test in renal transplantation.

Serial rosette inhibition tests were performed on 11 renal transplant patients in an attempt to predict graft rejection. The rosette inhibition titre was higher in immunosuppressed patients than in normal subjects. The test was of predictive value in only two out of 12 rejection episodes, where a fall in titre to normal levels occurred 48 hours and 24 hours, respectively, before biochemical evidence of rejection. In two further rejection episodes the titre fell at the time of rejection. The titre changes in all tests were small and there were frequent inconsistencies in the results of individual tests. A study of the variables was undertaken, with standardization of the technique, and improvements were made in reading the test. Despite these changes the test was still not sufficiently accurate or reliable to be used as the basis of treatment of rejection episodes.