CHEMICAL SIGNALS IN VERTEBRATES BRITISH WORKSHOP XIII

19-20 July, 2005

A meeting of the

at Withersdane Hall, Imperial College at Wye, Kent

 

Workshop programme

Noon – 12.55 Registration.

1.00pm LUNCH

2:20pm Welcome and Introduction

Session One

Smell & Health: chaired by Prof. Tim Jacob (School of Biosciences, Cardiff University, Cardiff) including an overview of the field today. This will include recent developments in smell and in particular the area of smell and health. Topics to include sperm "smelling" their way to the egg, smell canons, smell and sexual orientation, olfactory ensheathing cells and brain surgery. The impact of last year's Nobel prize will also be highlighted.

2:40pm Dogs as Diagnosticians I –

The Buckinghamshire Group: Bladder Cancer Proof of Principle Study.

Dr. John Church: Cancer Detecting Dogs.

Abney Court, Bourne End, Bucks SL8 5DL

Dr. Carolyn Willis: Experimental Design & Statistics and Clinical Aspects.

Amersham Hospital Whielden Street, Amersham, Bucks HP7 0JD

Dr. Claire Guest: Dog Training Aspects.

Hearing Dogs for Deaf People, The Grange, Saunderton, Princes Risborough, Bucks HP27 9NS

Discussion.

‘Can dogs smell bladder cancer?’. This was the title of the front-page illustration of the British Medical Journal of 25th September 2004, which carried the paper entitled: ‘Olfactory detection of human bladder cancer by dogs: proof of principle study’.

The historical background to this work will be briefly outlined: with the two anecdotal letters to The Lancet giving stories of pet dogs indicating on skin cancers in their owners; the ‘Biodiagnostics Workshop’ in October 2002; the visit to San Francisco in November; the establishment of the ‘Buckinghamshire’ group; and media attention to this work.

The presentations will include description of the structure to the numbers of patients to be recruited into the study, the numbers of dogs to be trained, the numbers of samples to be used in training and testing the dogs and the laboratory handling of the urine samples.

Also to be addressed will be the clinical aspects of this study; the collaboration with clinicians in the Urology Departments of the Hospitals involved, the selection of patients, both as controls and as ‘cancer’ patients, the meticulous interviewing of these patients, and the initial management of urine samples.

Lastly, Claire Guest will give an overview of dog training methods as applied to this particular study, looking at the challenges involved, the lessons learnt, and the prospects for further training studies. With special permission of the staff, Claire will also be bringing her Assistance Dog.

4:00pm Dogs as Diagnosticians II –

The Cambridge Group, represented by Dr Barbara Sommerville

Clinical Veterinary Medicine, University of Cambridge, Madingley Road, Cambs

This is an up to the minute report on the Cambridge University project to see whether dogs can detect a consistent smell in the urine of human males which indicates prostate cancer. We shall present evidence that of the 9 dogs we have attempted to train, 2 are able to distinguish established cancer urine from age matched Normal urine with a success rate greater than chance [p<0.05]. The handlers were working blind. The task seems to be difficult and requires long training and great  concentration on the part of the dogs. Six dogs were rejected as being temperamentally unsuitable and a third dog is close to success.

4:25pm TEA

4:55pm Diagnosis using a Sensor Array

Krishna Persaud

SCEAS, The University of Manchester UK

Traditional microbiological diagnostics identify bacteria through a process of ex-vivo culture in growth media followed by biochemical tests. Here we describe conducting polymer sensor arrays that have been incorporated with suitable sampling systems to enable clinical measurements to be carried out for the rapid and objective detection of urinary tract infections (UTIs) and bacterial vaginosis (BV). This technology has led to new ‘aid to diagnosis’ devices approved by the Food and Drug Administration (FDA), in the USA .

5:20pm Rapid Detection of TB using Biomimetic Sensor Arrays.

Dr Tim Gibson¹; Prof Anthony Woodman²; Dr Reinhard Fend²; Mr Rob Chandler¹ and Mr Viv Hallam¹

1) Scensive Technologies Ltd., Metic House, Ripley Drive, normalnton, West Yorkshire, WF3 4AA, UK.

2) Cranfield BioMedical Centre, Institute of BioScience and Technology, Cranfield University at Silsoe, Silsoe, Bedfordshire MK45 4DT UK

t.gibson@scensive.com

The rapid field detection of tuberculosis (TB) is a very important goal in the World diagnostics arena as the disease is increasing at an alarming rate and the level of diagnostic intervention has not been improving to match the need. Biomimicry or the use of sensors and artificial interfaces to mimic biological events is fairly well understood but as yet has not made a major impact in the diagnostic field.

This paper will present results that could change the perception of the usefulness of biomimicry in biomedical applications, as it has been possible to demonstrate the applicability of an electronic nose containing an array of 14 interfacially stimulated, conducting polymer sensors in the rapid detection of TB using clinical specimens collected directly from field sources. Laboratory trials indicate that levels of detection of 1 x 10⁴ mycobacteria per ml is possible in sputum and the technique can identify different species of respiratory micro-organism and different mycobacteria, including living and dead cells.

Single sensor biomimicry is rare and the development of 2^nd generation electronic noses that give molecular spectroscopic outputs using single conducting polymer sensor will also be presented. Such devices are a vision of the future and are being developed for use in biomedical fields and other areas where volatile chemicals need to be detected and identified.

1)     Gibson T D et al. Detection and Simultaneous Identification of Microorganisms from Headspace Samples using an Electronic Nose. Sensors and Actuators B44 (1997) 413-422.

2) Alexandros K.Pavlou, Naresh Magan, Jeff Meecham Jones, Jonathan Brown, Paul Klatser, Anthony P.F. Turner. Detection of Mycobacterium tuberculosis (TB) in vitro and in situ using an electronic nose in combination with a neural network system. Biosensors and Bioelectronics 20 (2004) 538–544

3) Clements J, Chandler R C, Gibson T D, Hulbert J N, Boden N and Ruck-Keene E A. Solid State Spectroscopy with Sub-One Second Sampling Time: A Rapid Technique for Complex Volatile Headspace Analysis. Proceedings of Eurosensors XI, 1997, Warsaw, Poland.

5:45pm Odour and the Vector of Chagas disease, Triatoma infestans

Dr Alan Cork

Natural Resources Institute, University of Greenwich, Chatham Maritime, Kent ME4 4TB, United Kingdom.

Chagas’ disease is an endemic problem in South America that affects more than 16 million people. The disease causing flagellate, Trypanosoma cruzi, is mainly transmitted through faeces deposited on potential hosts while vectors, such as Triatoma infestans (Hemiptera: Reduviidae), are blood feeding. Intradomiciliar infestations of T. infestans and other vectors in rural communities are currently controlled through coordinated fumigation programs with pyrethroid insecticides. The efficacy of such control campaigns is monitored using unbaited insect refuges and manual searching, both insensitive and time-consuming procedures when population densities are low.

There is considerable evidence in the literature to suggest that T. infestans produce a range of semiochemicals that could be used as the basis for an odour-baited trap that would improve the sensitivity and efficiency of population survey work. The results of research to identify compounds in faeces that attract and arrest conspecifics, the sex pheromone produced in copula that attract male T. infestans and compounds present in, and released from, Brindley’s gland that are thought to elicit alarm and defensive responses from conspecifics and predators respectively will be discussed. Of particular interest is the recognition that compounds used for conspecific communication may play a role in host location and have parallels with chemical cues employed by other haematophages notably, mosquitoes. However, the desire to develop odour-baited traps to monitor or control T. infestans is tempered by ethical issues associated with the use of attractant baits for disease vectors in domestic environments.

6:10 Short Break

6:20pm Smell and Taste in certain neurodegenerative disorders

Group from the Essex Neuroscience Centre, Oldchurch Hospital

Dr Christopher Hawkes , Dr Mussadiq Shah, Dr Jacqui Deeb

1. Olfactory tests distinguish Essential from Parkinsonian tremor. Evidence of enhanced detection and age resistance in familial essential tremor.

Shah M, Findley LJ, Muhammed N, Hawkes CH

Essex Neuroscience Centre, Romford, Essex. RM7 0BE

Background. It is unclear whether patients with essential tremor (ET) have intact sense of smell. If olfaction is Normal it could help distinction from tremor-dominant parkinson’s disease (PD) where smell sense is usually impaired.

Methods. 59 patients with ET were compared to 245 controls by the 40 odour University of Pennsylvania Smell Identification Test (UPSIT) and to 74 controls by olfactory event related potentials (OERP). 64 patients with tremor dominant PD were also compared to the control group by UPSIT and OERP. Mean UPSIT scores were compared between groups using multiple regression of UPSIT on group indicators with gender, age and age squared as covariates. Mean latency and amplitude was compared using multiple regression models with gender and a linear term in age as covariates.

Results. Controls and ET were indistinguishable when allowing for age, smoking and gender (p = 0.016). Mean values for ET and PD on both olfactory tests were markedly different (p =< 0.001). Familial ET patients scored higher than controls on UPSIT (p =< 0.001) and their age-related decline was slower (p = 0.035).

Conclusion. These clear differences between tremor groups could be useful clinically. Normal olfaction in suspected PD or abNormal smell in assumed ET warrants diagnostic review. The superiority of familial ET and their resistance to aging suggests a protective mechanism, possibly genetically based.

2. Taste Sense is AbNormal in Idiopathic Parkinsons’s Disease

Dr. Mussadiq Shah, Dr. Jacquie Deeb, Miss Marina Fernando and Dr. Christopher Hawkes

Essex Neuroscience Centre, Romford, Essex. RM7 0BE

Objective: To determine the prevalence of dysgeusia in idiopathic Parkinson’s disease (IPD)

Background: Patients with IPD rarely complain of difficulty with taste sensation but on pathological grounds it may be affected. We wished to measure the prevalence of dysgeusia in comparison to healthy controls using an electrogustometer.

Design/Methods: We measured taste threshold with a Rion TR-06 electrogustometer. A sterile stainless steel electrode was applied to the following areas on the tongue: a) either side of the tip, an area served by the chorda tympani (CT) nerve (VII). b) either side of the tongue base over the most lateral circumvallate papilla (VP). This is served by the glossopharyngeal nerve (IX). The stimulus current was increased using a single staircase approach until the subject reported a salt or bitter taste. This gives a threshold measurement, expressed in decibels (dB). The average of both sides was taken for each subject. The procedure was undertaken on the following groups: i) 28 healthy controls obtained from members of hospital staff or their relatives. Their mean age was 50 ±18y; 11 male, 17 female. All scored 27 or above on the mini-mental test, were not taking any drug known to interfere with taste appreciation and had good oral hygiene. ii) 52 patients with IPD all conforming to the UK Parkinsons Disease Brain Bank criteria, who were similar to controls with regard to cognition and oral hygiene. Mean age was 68 ±11.7y; 20 female, 32 male. Fourteen were taking levodopa or dopamine agonist preparations that sometimes produce a bitter sensation.

Results: Taste thresholds were plotted against age and linear regression lines constructed for Control and IPD groups for both nerves. For Controls, the mean threshold for CT was 9.2dB and for VP 12.3dB. In Patients the mean thresholds were CT: 15.8dB and VP 13.1dB. Age had minimal effect on Control threshold values. Thresholds were significantly higher in patients for CT (p=0.03) but not for VP (p=0.85). Although the abNormalities were more clear-cut for CT than VP this may have related to difficulties in accessing the posterior tongue region. Four drug naive patients had high thresholds (34dB) which equates to ageusia.

Conclusions: Taste sense is abNormal in idiopathic Parkinson’s disease.

This probably relates to the known generalised disorder of autonomic function. Taste threshold as measured at the tongue tip (chorda tympani nerve) was elevated in our patients with idiopathic parkinsons disease. Thresholds from the vallate papillae (glossopharyngeal nerve) were found to be Normal although there were 5/30 with no response. This may relate to the known autonomic dysfunction in IPD. The reason for predominant effect on just chorda tympani-mediated taste sensation is not understood but may relate to technical difficulty.

3. Smell Tests Compared to Dopamine Transporter Imaging in Diagnosis of Idiopathic Parkinson’s Disease: a Pilot Study.

Dr Jacqui Deeb

Essex Neuroscience Centre, Romford, Essex.

Background: There is no specific test for idiopathic Parkinson’s disease (IPD) and errors of diagnosis may occur in 10 - 20%. Dopamine transporter imaging (DaTScan) has high sensitivity for IPD but cannot distinguish parkinsonian syndromes. Olfactory identification is impaired in about 80% of patients with IPD but likewise has low specificity

Methods: We tested 11 patients with IPD all conforming to the UK PD Brain Bank criteria and scoring 27/30 or greater on the Minimental Test. The following procedures were used: 1) University of Pennsylvania Smell Identification Test (UPSIT; range 0-40). 2) Olfactory evoked potentials (OEP) to hydrogen sulphide using the Burghart OM2 Olfactometer. 3) [123]I FP-CIT DaTScan. For controls we used our database of 245 healthy subjects for UPSIT and 70 of these for OEP. A value exceeding two standard deviations from the mean, adjusted for age was considered abNormal.

Results. AbNormal DaTScan imaging was found in 10/11 IPD. UPSIT results were abNormal (anosmic range) also in 10/11 IPD. Two patients had discrepancy between DaTScan and UPSIT tests. OEP latency was delayed in 8/11 while OEP amplitude was low in 3/11.

Conclusion. This preliminary analysis suggests that DaTScan and UPSIT are of approximately equal diagnostic value but they may measure different aspects of IPD.

9:00 am WALL AND DESK-TOP PRESENTATIONS

Biomimetic Sensor Products for Commercial Applications.

Dr Tim Gibson

Scensive Technologies Ltd, Metic House, Ripley Drive, normalnton Business Park, normalnton, WF6 1QT,

Scensive Technologies Ltd. is a new private business established to produce biomimetic sensor products for commercial applications. The company has exclusive access to the know-how, intellectual property and resources of one of the original developers of e-nose technology, Bloodhound Sensors Ltd.

The initial products available in late 2005 will be based on the well respected e-nose, the BH114. This product has 14 conductive polymer sensors arranged into a sensor array and proprietary electronics that interrogates the surface responses of the different materials when exposed to volatile mixtures of chemicals that make up the odours being detected.

The sensors themselves are one key to the sensitivity and differentiating power of the BH114, as they are produced from ‘Analar’ quality monomers directly onto interdigitated gold electrodes as a single film in an ‘in house’ electrochemical QC procedure. This results in sensors being able to be manufactured to +/- 5% of response to a chosen volatile panel.

Also any responses coming from each sensor is directly related to the single polymer material present interacting with the volatile chemicals at the surface of the sensor. With this arrangement, it is possible to produce sensor arrays that have particular sensitivities and to ‘tune’ the sensor array used to the application area under investigation.

So far there have been at least 18 scientific publications citing the BH114 e-nose and several abstracts in conferences and proceedings. Some of the most significant results indicate the possibility of detection and identification of TB using the BH114.

Scensive is also investigating a new development in volatile detection technology, termed pulse spectroscopy. This patented technique has the potential to outperform sensor array technology, in that it can separate out the individual responses of odorants contained in a complex mixture based on their interaction with a single sensor surface. Initial results show that bacteria can be easily differentiated using such a system.

Pulse Spectroscopic Output for 3 Different Bacterial Cultures and Control Medium.

Session Two

OLFACTION: PHYSIOLOGY, PHEROMONES, AND BEHAVIOUR

chaired by Prof Krishna Persaud

SCEAS, The University of Manchester UK

10:00am GOSPEL Network Guest Lecture

Mammalian Pheromones, Audiomones, Visuomones and Snarks.

Prof. Richard L Doty, Director, University of Pennsylvania Smell and Taste Center

Since the pheromone concept, derived for insects, was first applied to mammals in the early 1960's, investigators have implicated pheromones in nearly every type of chemically-mediated mammalian social behavior and endocrine response imaginable. Pheromones have been suggested to mediate such diverse functions as maternal behavior, courtship, copulation, fighting, nesting, social communication, and endocrine function, including the acceleration of puberty, synchronization of estrus, and blockage of pregnancy. In humans, menstrual synchrony, mate choice, and even choosing a seat in a dentist's waiting room have been said to be influenced by pheromones. Recently some investigators have termed the mouse vomeronasal organ the "pheromone detector," assuming that behaviors or endocrine events mediated via the main olfactory system reflect non-pheromonal chemicals.

What, in fact, are mammalian pheromones and how can we identify them? In this presentation, I will briefly review the history of the pheromone concept, along with its myriad definitions and attempts to apply this concept to mammals. The argument is made that, despite our continued fascination with the pheromone concept and scattered reports of chemical isolation of such agents, mammalian pheromones do not, in fact, exist. It is further argued that conceptualizing chemically mediated social behaviors and endocrine responses in mammals as a hormone-like process greatly distorts and oversimplifies the nature of both the mammalian olfactory system (including the vomeronasal/accessory olfactory system) and the chemical communication process. It is my contention that chemical communication in mammals should be viewed as being more akin to communication mediated via non-chemical senses, and tempered by learning and experience, than to "releaser" or "primer" effects of hormones or ethiologically-defined releasing stimuli. It is suggested that the term pheromone is no more needed to describe chemically-mediated behavioral or endocrine responses in mammals than such hypothetical terms as "audiomones," "visuomones," and "touchomones" are needed to describe analogous phenomena mediated by sights, sounds, and tactile stimuli. Operationalism, rather than mediationism, is suggested as the appropriate means for addressing these issues.

11:00 am COFFEE

Smell and Animal Welfare I

11:30am Introduction to the Wildlife Heritage Foundation’s work.

Mark Edgerley (Director), Wildlife Heritage Foundation, Headcorn Road , Smarden, Kent

Prevention of stereotypical behaviour in caged animals

Laura Marszall, Mark Edgerley

Wildlife Heritage Foundation, Headcorn Road, Smarden, Kent

The effects of olfactory enrichment were tested on two male, 19 month old Snow Leopards (Uncia uncia) at the Wildlife Heritage Foundation in Kent. The scents used were Obsession for Men and herbivore faeces. Scents were introduced after a period of baseline observations and followed by a period of post enrichment observations.

It was found that the introduction of scent coincided with an increase in stereotypical pacing behaviour. It was later found to be coincidental and the pacing behaviour was found to be an anticipatory mechanism in relation to feeding schedule.

It was found that both scents showed a significant change in behaviour in both subjects. Activity levels increased significantly with the introduction of herbivore faeces. Activity was less affected with the introduction of Obsession for Men.

Both scents were found to have a significant effect on olfactory behaviours in the subjects. Increased incidences of scent marking, head rubbing and spraying were all noted during the enrichment period.

The herbivore faeces also showed to have longer lasting effects than Obsession for Men. Behaviour continued to show significant differences in the post enriched period.

This shows that both scents had important effects on the behaviour of the subjects and this could prove to be an effective and inexpensive method of enrichment for captive felids.

Smell and Animal Welfare II

12 noon Animal Aromatics - Caroline Ingraham

Ingraham Institute, Penwaun Farm, Clitwrch, Glasbury, Hereford HR3 5NZ

Animal Aromatics works with the principles of giving back to animals the medicinal non -food remedies that would be similar in their chemical make up to those that they would seek in the wild. This includes: a variety of essential oils, absolutes, plant extracts, macerated oils such as comfrey, tubers from the devils claw, clay, spirulina, rosehips and seaweed.

To-day many veterinary surgeons turn to pharmaceutical companies for remedies rather than to the fields or hedgerows, leaving domestic animals little choice, other than the application of synthetic chemicals to treat their ills, which most animals greatly resist, unlike the natural remedies that they select. The technical advances of the last 50 years have offered some highly effective drugs which can not be dismissed; but as always we need a balance. We need to give back to the animals what is rightfully theirs – the plants, the herbs and the fruits and we can do this by offering them the various plant extracts and oils that relate to their condition, their temperament and their environment.

Experiences and observations will be presented which demonstrate the benefit of animal aromatic practice.

Selected references:

Lyall Watson ‘Jacobson’s organ and the remarkable nature of smell’. Plum published by the Penguin group (1999, 2000)
Cindy Engel ‘Wild Health’ Weidenfeld & Nicolson (2002)
Thomas J Elpel ‘Botany in a Day’ HOPS Press(2001)

12:25pm Sniffing stategies in dogs during source location

Sara Jackson, (Cotswold Perfumery sponsored delegate) Bristol University,

The importance of sniffing

During Normal breathing little inspired air reaches the olfactory epithelium, which is situated at the posterior of the nasal cavity. When an odour is perceived sniffing can be triggered, consisting of a number of sharp inhalations followed by a single exhalation. This action changes the air flow within the nasal cavity and increases the number of odour molecules travelling over the olfactory epithelium, thereby resulting in a greater number of odour molecules coming in contact with olfactory receptors.

Sniffing is assumed to be of primary important in source location that relies on scent. The amount of sniffing used by air-trailing dogs (those dogs that primarily search air currents alone) was investigated by videotaping 25 dogs (125 trials) locating a hidden training aid, in a 30mx60m grid, while wearing a microphone.

Through observation scent detection was deemed to have occurred when the dog showed a check pace behaviour, that is a distinct, sudden, change in direction. Time spent searching prior to check was named the scent detection phase and the time spent searching afterwards, until retrieval of the training aid, was named the source location phase. The time spent sniffing within each phase was compared.

These trials showed that sniffing occurred for 21% of time in total. Within the scent detection phase they sniffed for 13% of time and with the source location phase 38% (F=23.9 (1, 22) p<0.001).

It would appear that in air trailing dogs sniffing does not play the dominant role that would be expected. As sniffing and panting can not occur simultaneously it is possible there could be a trade-off between gaining detailed olfactory information through sniffing and the need for deeper breathing while running.

This research has been funded by the MOD Corporate Research Programme.

1:00pm LUNCH

2:20pm AGM including Presentations & Announcements

3:10pm Semiochemical Update 2005, Dr. David R. Kelly,

The Tatem Laboratories, Department of Chemistry, Cardiff University, P. O. Box 912, Cardiff, CF10 3TB, Wales, UK. Tel. (44+) 029-20-874063; Home 029-20-610891; Mobile 07971-240448; FAX 029-20-874030; email KellyDR@Cardiff.ac.uk

1. Chemical Demography	Organic & inorganic substances	Sequences
Tue Jul 20 18:48:53 EDT 2004	23,635,030	44,842,870
Fri Jul 15 17:49:47 EDT 2005	26,066,252	56,595,570
Increase in period	2,431,222	11,752,700
% Increase in period	10.3	26.2

2. Bacterial quorum sensing pheromones spontaneously undergo transformation into antibacterials[1].

3. Daumone the pheromone responsible for “hibernation” of the nematode Caenorhabditis elegans has been identified. When faced with overcrowding or starvation, first stage Caenorhabditis elegans larvae switch to the dauer-form rather than take the Normal developmental pathway into adults. Dauer-larvae can survive for months without feeding, but when fed develop into Normal adults with a Normal life span (2-3 weeks)[2].

4. The sex pheromone of the German cockroach has been identified. The aggregation pheromones of the German cockroach, blattellastanoside A & B were discovered some 10 years ago[3]. Unfortunately the structures are very complex and they are non-volatile, hence there has been no practical applications. The relatively simple structure of the sex pheromone, blatellaquinone is more amenable to synthesis and is volatile[4].

5. Human pheromones and armpit (axillae) odours. There are four possible gender based permutations of pheromonal interactions: male-male, male-female, female-male and female-female. There is evidence for a male-female interaction based on androstenol and androstenone, and for a female-female interaction (menstrual synchrony[5]). But after 24 years there is still no chemical(s) associated with this phenomenon[6].

(2E)-3-Methyl-hex-2-enoic acid which was originally identified as a marker for schizophrenia (but now discredited), is a major odiferous constituent of the unpleasant odour of perspiration. It was reported to be secreted bound to a lipocalin, in common with a number of other mammalian pheromones[7]. However recent work indicates that it is released from a glutamine conjugate, by a bacterial aminoacylase[8].

A series of structurally related thio-alcohols have also been discovered in armpit secretions[9]^,[10]. One of these has the same carbon backbone as (2E)-3-methyl-hex-2-enoic acid and plausibly could have been formed by the addition of hydrogen sulfide followed by reduction of the carboxylic acid group. A similar compound involving the addition of water has also been identified.

In fact the source of the sulfur atom appears to be from cysteine, because another group has shown that the thio-alcohols can be formed from the S-cysteinyl adducts by the action of Corynebacterium striatum Ax20 lyase. The gene was expressed in E. coli and showed similar activity to that in the natural host[11]. It is appealing to link these observations into a single pathway for thioether production, but this is not warranted at this stage because these results were obtained with several different species.

Fresh apocrine sweat is odourless and many studies have shown that odour is caused by bacteria. Aerobic Corynebacteria sp. are commonly found in the armpit and their predominant action on steroids is oxidation and isomerisation to give more odiferous steroids[12]^,[13].

References