WO1995015494A1

WO1995015494A1 - Neutrophil determination

Info

Publication number: WO1995015494A1
Application number: PCT/GB1994/002595
Authority: WO
Inventors: Ian Stoakes; Thomas Gairdner Price; Patrick John Kingsley
Original assignee: Nutron Limited
Priority date: 1993-11-30
Filing date: 1994-11-28
Publication date: 1995-06-08
Also published as: AU1194395A

Abstract

The present invention relates to a method of determining the environmental intolerogens to mammalian blood which method comprises sampling blood, preparing a sample extract of each intolerogen to be tested, mixing a portion of the intolerogen extract with a proportion of sample blood, incubating the mixture and probing the mixture to determine changes in the neutrophil population when compared with an incubated control blood portion devoid of intolerogens, whereby significant changes in the neutrophil population indicate an intolerance to said intolerogens.

Description

NEUTROPHIL DETERMINATION The present invention relates to a method of determining neutrophil degranulation in the blood of mammals. The invention has particular reference to the treatment of inflammatory conditions in mammals by removing from the mammalian diet those foods to which an individual sufferer is determined to be intolerant.

Inflammatory illness may manifest itself in many forms with a result that its prevalence can be obscured. Pharmacological interventions are not free from unwanted side effects, require long term usage and do little to stem the growing tide of sufferers requiring treatment at the expense of health care providers.

In the 1980's a number of papers were published in The Lancet on the subject of rheumatoid arthritis and irritable bowel syndrome. In particular, see:-

Darlington, L. G et al, Placebo controlled blind study of dietary manipulation therapy in the treatment of rheumatoid arthritis. The Lancet 1986, Feb 1, 126.

Little, C. H. et al Platelet serotonin release in rheumatoid arthritis; a study in food intolerant patients. The Lancet 1983, August 6, Alun-Jones, V. et al Food intolerance; a major factor in the pathogenesis of Irritable Bowel Syndrome, The Lancet 1982 ii, 115-17.

Bentley S. et al, Food hypersensitivity in Irritable Bowel Syndrome, The Lancet 1983 ii, 295.

In each of these papers it was shown that the removal of foods to which the individual had proved to be intolerant resulted in a cessation of symptoms, in approximately 30% of the subjects in the case of rheumatoid arthritis and 70% of subjects with irritable bowel syndrome. Many similar papers have been published demonstrating improvements in inflammatory conditions and in others in which an inflammatory pathogenesis is not immediately apparent. Despite this amount of published information, there is little evidence of any change in the management or treatment of inflammatory illnesses at general practice or consultant level. This has been due mainly to the fact that individual intolerances could only be determined by a lengthy elimination diet procedure. The constraints of general practice and consultant practice within the health care system is such as not to permit either the time or the expenditure required for a procedure requiring such painstaking and time-consuming efforts by both carer and patient. There is, therefore, a long-felt need for a test to determine such intolerances in a simple, quick and straightforward manner.

Mammalian blood comprises a fluid plasma containing red cells, (erythrocytes) platelets and white cells, (leucocytes). The white cell, leucocytes, can be sub¬ divided into lymphocytes, monocytes and granulocyteε and the granulocytes themselves comprise three further separate groups, namely eosinophils, basophils and neutrophils. It is well known that the white blood cells are part of the body's immune system and in particular the neutrophil has a vital role in protecting the body against invading organisms. An awareness of the neutrophil's potential to mediate acute inflammatory tissue damage has increased with a demonstration that certain environmental factors can prime the neutrophil. See for example, the paper by Haslett, C. et al, "The neutrophil, current opinion in immunology" 2; 10-18, 1989. The injurious potential of the neutrophil does not, however, appear to become apparent unless the cell is activated and when this happens a process occurs which is similar to the reaction following the ingestion of bacteria, except that it appears to be directed extra-cellularly into surrounding tissues rather than internally into phagocytic vacuoles (i.e. the area of the plasma membrane that encircles a target small enough to be ingested). With the rupture or degranulation of a neutrophil at least 50 toxins are released from the plasma membrane and another 20 from the granules themselves, including oxygen radicals, arachidonic acid products and granular substances .

It is the synergistic interaction of these toxic elements which constitutes a major part of the body's immune defence system. At the same time, if wrongly triggered it is believed that these toxic elements greatly increase their damaging effects . When the neutrophil is specifically triggered by any one of a number of pro-inflammatory signals, enzymes of the mixed function oxidase system begin to generate and release oxygen metabolites (namely superoxide anion, hydrogen peroxide and hypochlorite) . The release of superoxide anions from the primed neutrophils can be enhanced by chemotactic factors including N-formyl- methionyl-leucyl-phenylalanine. However, recent research has determined that there is no detectable N-formyl- methionyl-leucyl-phenylalanine stimulated superoxide release unless the neutrophils have been previously primed. It would appear, therefore, that it is the state of the neutrophil rather than its mere presence in the tissues that determines whether it injures the tissues or not. At the same time as oxygen metabolites are released, the granules fuse with the plasma membrane discharging their contents both into the extracellular area and into the phagocytic vacuole. In pathological states, tissues rather than microbes may bear the brunt of an attack heralded by the release of a complex mix of oxidase-derived oxygen metabolites and granulocyte-based toxins. Inflammation itself then leads to the release of chemotactic factors which attract further neutrophils to the site of tissue injury. The knowledge that chemicals are released from the activated, primed neutrophil has led to the desire to develop therapeutic interventions which could interrupt the cell's unwanted destructive activities. So far, this has been unsuccessful because research has concentrated on developing drugs that reverse, or inactivate, these various chemicals.

Rather than look at the therapeutic interventions to prevent the cells' unwanted destructive activities, the present Applicants have concentrated on investigating the causes of neutrophil priming rather than a search for drugs which would antagonise any chemicals released from the activated neutrophil.

The present Applicants have realised that lung mast cells have been shown to degranulate leading to the symptoms of asthma. Prior to that earlier research had concentrated on the development of bronchodilators and steroids to correct the damage done by the chemicals released by the degranulation. The development of sodium cromoglycate allowed mast cells to be stabilised, thus preventing them from degranulating on challenge with specific antigens .

A mammalian mast cell contains granules which contain histamine and other vaso-active substances which are released from the granules when an antigen combines with an antibody bound to the plasma membrane of the mast cell . These chemicals are vaso-active in that they cause dilatation and an increase in the permeability of blood capillaries, and are anaphylactic agents in that they cause contraction of the smooth muscle in viscera. The release of these chemicals has been implicated in patients who suffer from extrinsic asthma; it is thought that airborne antigens combine with mast cell-bound IgE in the mucous membrane of the lungs to cause degranulation of the mast cells . In intrinsic asthma histamine and other chemical release has also been implicated often without any specific antigen which causes degranulation of the mast cells being identifiable.

Furthermore, with the role of IgE mechanisms being less obvious, bronchial hypereactivity is of great importance, and inflammation in the lungs can only be controlled, to a certain extent, by corticosteroids .

Inhaled sodium cromoglycate can effectively control the symptoms of extrinsic, IgE-mediated asthma. In intrinsic asthma where bronchial hypereactivity plays such an important role, sodium cromoglycate controls the symptoms far less effectively.

Furthermore, the release of histamine, etc, from mast cells is thought to give rise to diseases other than asthma such, for example, as hay fever, urticaria, perennial and seasonal rhinitis, and conjunctivitis.

The disodium salt of 1,3-bis (2-carboxychromon-5-yloxy) propan-2-ol (sodium cromoglycate) is thought to inhibit the release of chemicals from mast cell granules (see e.g. Biochemical Pha acology, volume 21, pages 171-179 (1972)).

British Patent No. 1144905 - Fisons Pharmaceuticals Limited discloses the use of substituted bis-(2-carboxy- chromonyl-oxy) derivatives, including sodium cromoglycate, as inhibitors of the effects of certain types of antigen- antibody reaction e.g. the combination of reaginic antibody with specific antigen.

WO-A-90/13284 discloses a pharmaceutical solution containing 0.1-6% w/w sodium cromoglycate, 0.1-2.0% w/w carbomer and an inorganic base which is particularly suitable for ophthalmic use for the treatment of vernal kerato-conjunctivitis and the ocular effects of hayfever. WO-A-87/03197 discloses a pharmaceutical composition comprising microspheres incorporating sodium cromoglycate; the microspheres comprise a material having ion-exchange properties and can be used for the treatment of diseases of the eye, nose and lungs in which mast cell granulation is implicated.

EP-A-0183457 discloses an aqueous formulation of sodium cromoglycate which, in some embodiments, contains hydroxypropylmethycellulose as a viscosity increasing agent. EP-A-0084898 discloses a pharmaceutical composition comprising liposomes and sodium cromoglycate for the treatment of allergic conditions. EP-A-0212927 discloses a pharmaceutical composition comprising sodium cromoglycate and a mucolytic agent such, for example, as N-acetyl-1- cysteine. EP-A-00551207 discloses a mixture of sodium cromoglycate of mass median diameter from 2 to 30 microns with a pharmaceutically acceptable water soluble carrier of sieve size from 200 to 1000 microns which is disclosed as being useful in the treatment of the gastrointestinal tract. EP-A-0153003 discloses a pharmaceutical formulation comprising a suspension of fine divided sodium cromoglycate in an oily non-aqueous vehicle which may also contain an antibiotic such, for example, as chloramphenicol; the formulation of EP-A-0153003 is disclosed as being useful in the treatment of conditions of the eye or nose in which allergy or immune reactions play a contributory part. Sodium cromoglycate is available commercially from Messrs. Fisons under the trade name "INTAL" which, in addition to sodium cromoglycate, also contains an amount of isoprenaline, a bronchodilator, when called INTAL Compound for the management of allergic asthma (see Unlisted Drugs, volume 19, No. 11, page 150 (November 1967)).

A disadvantage associated with sodium cromoglycate, however, is that it is considered to be only suitable for the management of extrinsic asthma and relatively minor degrees of intrinsic asthma. In spite of the considerable commercial effort to improve its effectiveness, sodium cromoglycate has been found not to be suitable for use in the treatment of severe attacks for which powerful bronochodilators such, for example, as salbutamol or anti- flammatory steroids are typically prescribed. It will be appreciated by a person skilled in the art, however, that the use of bronchodilators and anti-inflammatory steroids is not wholly satisfactory in view of the well-known disadvantages associated with these drugs and there is a requirement, therefore, to improve the effectiveness of sodium cromoglycate as a treatment for e.g. asthma, in which mast cell degranulation is implicated.

Whereas specific future research might understandably concentrate on developing a drug to stabilise the neutrophil, if the environmental factors that cause it to become activated are identified and controlled, priming may not occur in the first place, thus providing an immediate opportunity for prevention and treatment.

The present Applicants have appreciated in one aspect of their invention, that the neutrophil is the key to the prevention and treatment of inflammatory diseases.

According to one aspect of the present invention, therefore, there is provided a method of determining the environmental intolerogens to mammalian blood which method comprises sampling the mammalian blood, preparing a sample extract of each intolerogen to be tested, mixing a portion of the intolerogen extract with a portion of sample blood, incubating the mixture, and probing the mixture to determine changes in the neutrophil population when compared with an incubated control blood portion devoid of intolerogens whereby changes in the neutrophil population indicate intolerance to the said intolerogen.

The present Applicants consider that the inflammatory cycle begins with the intake of intolerogens such as a food and ends with ill-health. This involves a local IgE related reaction with gut walls and mast cells. The subsequent release of acidic chemicals from the mast cells causes increased local vascular and intestinal permeability allowing comparatively or partially undigested food particles, especially proteins, to gain access directly to the blood stream.

Upon detection of the protein material within the blood stream, it is thought that the neutrophil releases a permeability increasing protein which exacerbates the problem. Once in the blood stream these food particles engage the appropriate receptors on the plasma membrane causing the neutrophil to degranulate. The neutrophil binds to the endothelial lining of the blood vessel as if attempting to engulf it while discharging its toxins into the endothelial membrane. The host tissue through which these particular blood vessels are passing are consequently damaged and inflammation results. In effect, host tissues are directly attacked by powerful, neutrophil derived, toxins. If the tissue is a joint, rheumatoid arthritis will result; if the lungs, the result may be asthma. The pancreas may also be a target organ of the blood borne neutrophil reaction leading inevitably to a reduced output of digestive enzymes and sodium bicarbonate necessary to create and maintain the correct alkalinity within the duodenum. As a result, foods, most importantly proteins, will escape complete breakdown and will be presented to the absorptive area of the jejunum and the ileum in a partially digested form.

Where food intolerogens have produced gut permeability these undigested particles will be absorbed into the blood stream where they will then prime neutrophils . The resultant neutrophil-derived toxic attack is further exacerbated by the release of chemotactic factors from the site of the inflammation. These chemotactic factors attract further neutrophils. If this involves infective agents it is a natural effect and entirely beneficial. If, however, the stimulus cannot be eliminated, as in the case of a food intolerance, further damage to the host tissue will result and a continuous cycle would appear to be initiated. Neutrophils so attracted, which have already been primed by food particles, will degranulate, thus increasing the seriousness of the situation.

In another aspect of the present invention, therefore, the present Applicants have determined that the neutrophil is important to the disease producing inflammatory cascade and that if intolerogenic foodstuffs are avoided, the neutrophil will not be primed and will confine itself to a purely defensive role and the inflammatory cascade will be prevented. As a result of this, the present Applicants have found that many clinical conditions in which the neutrophil is currently acting as an inflammatory mediator will resolve ,

The present Applicants have found that the removal of intolerogenic food can be of considerable assistance in treating a large number of inflammatory conditions, typically those set out in Table 1 below and also can have a direct effect on other inflammatory conditions in which an inflammatory pathogenesis is not immediately apparent, see for example, those conditions set out in Table 2 below.

Table 1 Table 2

Adult respiratory Peptic Irritable bowel distress syndrome ulcers syndrome

Myocardial Stomatitis Anxiety infarction

Rheumatoid Gingivitis Depression arthritis

Osteo-arthritis Nephritis Cystic fibrosis

Asthma Cystitis Fibro-myalgia

Bronchitis Rhinitis Migraine

Dermatitis Sinusitis Emphysema

Otitis media Gout Psoriasis

Diverticulitis Pre-menstrual syndrome

Crohn's disease Idiopathic oedema

Ulcerative Hyperkinesis colitis

Eczema

Hay fever

The step of identifying environmental intolerogens to which a patient exhibits a response, requires the drawing of a blood sample and the testing of that sample against a number of potential intolerogens. In general, these may be of foodstuffs, although they may also be of other materials likely to be introduced into the blood stream. The present Applicants are aware that the investigation of neutrophil degranulation may be of considerable value in assessing, for example, a patient's potential intolerance to drugs, anaesthetics and the like.

The means of analysing and determining the degranulation of the neutrophil will be well known to those skilled in the art. In one aspect, the process of the invention comprises drawing a blood sample from a patient, preparing a plurality of test samples, at least one control sample, preparing an extract of the intolerogen, such as a food extract to be tested by dispersing it in a suitable carrier, mixing an intolerogen extract with a corresponding blood sample, mixing the carrier devoid of intolerogens with a control blood sample, incubating both intolerogens blood mixture and the control and thereafter analysing and comparing both the intolerogen mixture and the control to determine any significant changes in the neutrophil populations in terms of blood cell size, density, and/or apparent degranulation of the intolerogen mixture relative to the incubated control sample.

In some aspects of the invention, the amount of change may then be further compared with the statistical mean derived from testing a plurality of other blood samples of the same individual or of the same mammal group thereby to determine whether the amount of the change is significant as compared with the mean change.

The amount of change or degranulation of the neutrophil population may be determined in some embodiments by direct measurement. For example, specific toxins known to be released by neutrophils may be determined specifically for the increase or change in presence in blood plasma in the test sample compared with the control. The presence or amount of one or more such toxins may be determined directly and then compared with a control sample. Alternatively, the amount of degranulation of the neutrophil population may be determined indirectly by measuring either the loss of neutrophils in the granulocyte population in general, or by a relative shift in the populations of granulocytes, neutrophils, basophils, and eosinophils compared with other populations within the blood sample.

In a particular aspect of the present invention, the degranulation of the neutrophils may be assessed using a haematology analyser. Haematology analysers are normally used to determine the total white blood cell populations of blood samples using a combination of radio frequencies (alternating current) and direct current. A typical haematology analyser is commercially available from Messrs. Toa of Japan under the trade name "SYSMEX NE-8000". The principle and method of operation of this haematology analyser is comprehensively described in the accompanying manual available from Messrs. Toa under the reference NE- 8000/11.88 (revised to 8.89,12.89), see particularly section 4 "Principles of Operation".

The use of a haematology analyser permits the examination and testing of samples substantially automatically and over a timescale measured in minutes compared with many hours for visual observations under a microscope by examining total white blood cell morphology as hitherto employed. Furthermore, such automated techniques are more reproducible and are not susceptible to human error.

In a particular aspect of the present invention, there is provided a method of testing the effect of intolerogens on a sample of mammalian blood which method comprises the steps of preparing an intolerogen extract, adding the intolerogen extract to a blood sample, incubating the blood sample mixture, sub-dividing the sample into aliquots, treating a first aliquot with a lysing agent to lyse all the cells in the sample except the granulocytes, treating a second aliquot with a lysing agent to lyse all the cells except the eosinophils, treating a third aliquot with a lysing agent to lyse all the cells except the basophils, lysing an aliquot of each control sample in a corresponding manner, probing each lysed aliquot to determine the degranulation of the granulocyte population relative to the incubated and correspondingly lysed intolerogen free control, thereby to determine the degranulation of the neutrophil population.

Each aliquot may be suspended in an electrolytic diluent and disposed in a transducer chamber having a partition dividing the transducer chamber into two regions. The partition may have a small aperture extending therethrough between the two regions and each region may be provided with an electrode. The size of the aperture may be of the same order as the size of the granulocyte cells in the blood sample and an electrolytic diluent may be selected to have a significantly different electrical conductance from that of said granulocyte cells.

The granulocyte cells in each aliquot may be aspirated through the aperture from one region of the transducer chamber to the other region. A substantially constant electrical current may be applied to the electrodes . As a cell passes through the aperture, the voltage between the electrodes may change transiently by a detectable amount and any changes may be monitored continuously during aspiration of the cells to count the number of cells passing through the aperture. In the case of the second and third aliquots, the applied constant current may be a constant direct current; the total volume of each cell passing through the aperture may be proportional to the transient change in voltage. The numbers of basophils and eosinophils in the second and third aliquots respectfully may thus be counted. On aspirating the cells in the first aliquot, namely the granulocytes, a constant direct current and a constant radio frequency alternating current may be applied across the electrodes; as before, the transient change in d.c. current may be proportional to the total volume of granulocyte cells as it passes through the aperture while the total nuclear size and density of each cell may be proportional to the transient change in the r.f. signal as the cell passes through the aperture.

The neutrophil population in the blood sample may, therefore be calculated by subtracting the counted numbers of basophils and eosinophils in the second and third aliquots from the calculated granulocyte population in the first aliquot.

The present applicants have found that the use of the SYSMEX-NE8000 machine can be simplified. The machine as supplied by Messrs.Toa has 5 test channels. Each channel provides a different function for use in a full haematology analysis. In practice only one channel of this machine is used in the practice of the invention; this is the channel for the RF/DC transducer, the other channels being provided with de-ionized water.

Furthermore, in the determination of neutrophils, because of a lack at the present time of a lysing agent for eosinophils and basophils, together, or for neutrophils directly, means that the determination of the neutrophil is effected by determining the total granulocyte population and then subtracting from it the eosinophil and basophil populations to arrive at the neutrophil population. The eosinophil content is very small, typically about 0.5% of the granulocyte population. In practice, the present applicants have found that by lysing the basophils and determining the remaining granulocytes, they can obtain satisfactory results at less cost.

In a further aspect of the invention, therefore, there is provided a method which comprises adding the intolerogen extract to a blood sample, incubating the blood sample mixture, subdividing the sample into aliquots, treating a first aliquot with a lysing agent to lyse all the cells except granulocytes and treating a second aliquot with a lysing agent to lyse all the cells except the basophils, probing each lysed aliquot to determine the degranulation of the granulocyte population relative to the incubated and correspondingly lysed intolerogen free control; whereby the apparent change in the total granulocyte less the basophil population is an indication of intolerance to the intolerogen added to the sample.

In a further aspect of the invention, the present applicants have found that the lysing of samples for both eosinophil and basophil population counts can be dispensed with, and the degranulation of the granulocyte population at large can be measured. This provides an operational improvement in the test in that the test is significantly cheaper to perform.

According to one further aspect of the invention, there is provided a method of determining the environmental intolerogens to mammalian blood which method comprises sampling the mammalian blood, preparing a sample extract of each intolerogens to be tested, mixing a portion of the intolerogen extract with a portion of sample blood, incubating the mixture, and probing the mixture to determine changes in the neutrophil population when compared with an incubated control blood portion devoid of intolerogens whereby changes in the granuloctye population indicate intolerance to the said intolerogen. It will be appreciated by the man skilled in the art that as the SYSMEX NE8000 machine is primarily a haematology analyser, a degranulated granulocyte may appear under the d.c. and r.f. analysis as a monocyte or a lymphocyte. The determination in this invention is not concerned with monocytes and lymphocytes, it is concerned solely with the degranulation of one or more of the granulocyte population and specifically the neutrophil.

The control sample does not contain any intolerogen extract, but should contain and be treated in exactly the same way as each of the intolerogens/blood mixtures, that is to say, the lysing steps should be carried out on a corresponding aliquot of the control.

In a particular aspect of the present invention, a plurality of blood samples are prepared and each sample is challenged with a different intolerogen or food extract. The plurality of food intolerogens employed may be selected to include sufficient foods to constitute a complete and balanced diet. In these circumstances, the intolerogens noted as having a significant effect on the neutrophil population of the blood tested should be removed from that diet and a patient is then placed on a diet constituting food or other environmental factors which have been shown to produce little or no priming or degranulation of the neutrophils. The intolerogen or food extracts may be presented to the blood samples in the form of the suspension or a solution extract. The suspension may comprise a carrier medium, such as phenol benzyl solvent or saline buffered saline.

In a particular embodiment the intolerogens may comprise a freeze-dried food sample obtained from Messrs. Dome Hollister-Stier. The freeze-dried food sample may be diluted with saline buffered saline mixed well, centrifuged and filtered to give a particle free extract of a food substance. The liquid food extract may include a small amount of phenol to prevent bacterial contamination. Where food extracts from Messrs. Dome/Hollister-Stier are employed, the liquid extract may comprise one gram of food extract, 20 mis of liquid diluent as a carrier. This may be further diluted and it will be understood that the present invention comprehends dilutions of the initial solution in the range of 1:5 to about 1:50. The diluted food suspension may be added to an undiluted blood sample and the correct ratio of blood to food antigen is of importance, in relation to the analysis technique employed, since if too little blood is used, the resultant analysis may be non-specific where a haematology analyser is being employed.

In some embodiments, the food extracts obtained from Messrs . Holister-Stier may be diluted and the required concentration with saline/buffered saline (which may contain phenol) and may then be mixed with undiluted blood in the ratio 50:50. When using this diluted suspension of food extract, a ratio of extracted blood in the range of 25:75 and 75:25 may be satisfactory.

After introduction of the food extract into the food sample, the mixture may be incubated. The period of incubation will, to some extent, depend on the dilution ratios and the relative concentration of food extracts to the blood sample. If, however, the mixture is incubated too long, the reaction of the neutrophil to the granulocytes in general to the food antigen may be non¬ specific. Typical periods of incubation lie within the range of 30 minutes to 90 minutes with a typical period of incubation of 60 minutes.

It will be apparent to the man skilled in the art that each intolerogen challenged blood sample will show some decrease or change in the neutrophil population with a decrease in the measured non-degranulated neutrophil population which may serve to indicate an intolerance to that particular intolerogen.

In a specific embodiment the Applicants have found, for example, that by eliminating the food to which an asthma patient exhibits a reaction from the patients diet, any remaining sensitivity in the patient to mast cell degranulation responds more readily to treatment with a therapeutic substance which is indicated for inhibiting mast cell degranulation as compared with the case where the patient's diet includes food to which the patient has a reaction. Thus, pharmaceutical substances such as disodium 1,3-bis(2-carboxychromon-5-yloxy) propan-2-ol (sodium cromoglycate) in a pharmaceutically accepted carrier has been found to be particularly beneficial in supporting or inhibiting mast cell degranulation. The present applicants have also found that where a patient is placed on a diet in accordance with this invention, and infringements of that diet are likely to occur, a substantial dose of sodium cromoglycate will assist in reducing (although not necessarily eliminating totally) the effects of that infringement.

In a particular embodiment of the present invention, the therapeutic substance may contain 0.1 to 7.5 weight for weight of sodium cromoglycate. The pharmaceutically acceptable carrier may be an aqueous solution as defined in British Patent Specification No. 1144905 or alternatively the pharmaceutically acceptable carrier may be a liquid propellant such as microspheres of the type disclosed in WO-A-87/03197 or an oily, non-aqueous vehicle of the type disclosed in EP-A-0153003. Typically, the therapeutic substance may be an aqueous solution containing about 2% w/w sodium cromoglycate. In some embodiments, the therapeutic substance may contain a thickening agent e.g. hydroxypropyl methylcellulose as disclosed in EP-A-0183457 or a carbomer and inorganic base as disclosed in WO-A-90/13284. For some applications, the therapeutic substance may comprise an aqueous suspension comprising sodium cromoglycate partitioned between a free aqueous phase and a liposome phase as disclosed by EP-A- 0084898, or a mixture of sodium cromoglycate and a pharmaceutically acceptable water soluble carrier as disclosed by EP-A-0051207.

In a different aspect of the present invention, the therapeutic substance may also contain a bronchodilator, a mucolytic agent and/or an antibiotic. Said bronchodilator may be selected from salbutamol, isoprenaline, adrenaline, orciprenaline, isoetharine and pharmaceutically acceptable salts thereof. Said mucolytic agent may be N-acetyl-1-cysteine. The antibiotic may be chloramphenicol.

Typically, the pharmaceutical substance may be the anti- asthma drug which is commercially available from Messrs . Fisons under the trade name "INTAL".

Said pharmaceutical substance may be administered to the patient by inhalation in the form of a fine suspension e.g. by delivery from a nebuliser to the mucous membrane in the bronchi and bronchioles of the lungs or in the post-nasal cavity. Alternatively, the pharmaceutical substance may be administered parentally e.g. by injection or through the gastrointestinal tract. Alternatively, the pharmaceutical substance may be applied topically in the form of a cream, gel, ointment or drops e.g. in the eye for the treatment of conjunctivitis.

Although the above discussion is set forth primarily with respect to asthma, the method of the present invention may be used for the treatment of any disease in which the degranulation of mast cells is implicated and also has particular application in the treatment of hayfever and the like where the degranulation of mast cells causes membrane inflammation and anaphylaxis .

The analysis of the results in any test in accordance with the present invention can be performed in an number of ways depending on the object of the test. In general, the test may be employed to detect "injurious" intolerances, i.e. those foodstuffs or intolerogens to which a patient is particularly intolerant and to which the patient is likely to exhibit an adverse reaction.

Another possible use of the test is to identify the broader range of intolerances, including those to which the patient has only a slight intolerance, but which could be significant if that food features to excess in that patients diet.

The accompanying drawings is a histogram of a typical test, showing the distribution of granulocyte counts for each food sample. The low counts are indicative of intolerance. Where a granulocyte (neutraphil) count is clearly below the main distribution curve for the bulk of foods then that food sample is indicative of probable "injurious" intolerance. The interpretation of granulocyte counts near or at the bottom of the distribution curve is more difficult since the boundary between tolerance and intolerance may be at a different position on the distribution curve for different individuals.

In a preferred aspect of the invention the test should be conducted several times and the intolerances compared in order to determine the common intolerances in each test. This has the disadvantage of increasing the cost. However, for the majority of patients the results, of multiple tests will be substantially identical.

Where this latter practice is desirable is where the patient has a low granulocyte count, since in this case the difference in the cell counts between a normal reaction and an intolerogenic reaction will be much less marked for a given intolerance level . This arises since the total number of granulocytes in any sample of given volume and capable of being counted is less .

Following is a description by way of example only of methods of carrying the invention into effect.

In the accompanying examples, percentages are percent by weight unless otherwise stated.

Specific Examples The method of the invention was applied to a number of food extracts. 92 food extracts were selected, being extracts of food which were reasonably commonplace and which could be used as a basis for a healthy diet. The extracts used were supplied by Messrs . Dome/Hollister-Stier and were supplied in a glycerol suspension.

Each food extract was diluted with a phenolised saline in a proportion of 1 part of the extract as obtained from the suppliers in this case Hollister-Stier to 20 parts of saline. The phenolised saline, comprised an aqueous solution of 0.9% sodium chloride with 0.2% of phenol, the percentages being by weight 0.5 g of extract was mixed with 10 ml of the saline and was allowed to mix with some shaking for a period of 24 to 36 hours. At the end of that period, the mixture was centrifuged and the supernatant liquor decanted for use in the test. Any residue is discarded. The resultant extract has an effective shelf life of about 7 days. The foods to be tested are as follows:

Beef Barley Cheese Beetroot

Chicken Corn Egg White Celery

Lamb Oats Egg Yolk Cucumber

Pork Rice Whole Milk B e l l pepper

Rabbit Rye Whey Lettuce

Turkey Wheat Lactose Onion

Cod Gluten Apple Radish

Crab Malt Banana Tomato

Haddock Aubergine Grape Almonds

Halibut Baked bean Grapefruit Brazil nut

Herring Broccoli Lemon Coconut

Mackerel Brussels sprout Mango Peanut

Salmon Cabbage Melon Sesame

Shrimp Carrot Orange seed

Sole Cauliflower Olive Sunflower

Trout Courgette Peach seed

Tuna Garlic Pear Walnut

Baker's Lentil Pineapple Chocolate yeast Mushroom Plum Coffee

Brewer's Parsnip Raspberry Black yeast Pea Rhubarb pepper

Tea Potato Strawberry White

Chilli Runner bean Sugar beet pepper pepper Soya bean Sugar cane Candida/

Curry Spinach Turnip albicans powder

About 20 ml of blood was taken from the patient from which 96 portions each of 100 μl were prepared using a Tecan Multidispenser, the portions being measured in to a microtitre dish. Four portions were isolated for use as controls; each of the remainder was mixed with a different food intolerogen. A 100 μl proportion of extract, diluted as described above, is then added to each of the test portions. The control portions were treated with a 100 μl portion of the phenolised saline. The samples and controls were then incubated at room temperature on a shaker for a period of about one hour.

The samples and controls were then subject to analysis using a Sysmex NE 8000 haematology analyser. The Sysmex NE 8000 analyser has five analysis channels, each 100 μl sample portion is divided into five and is treated with reagents within the machine to determine certain parameters within the blood sample. The five channels are as follows:- 1. 4 μl to the Red Blood Counting channel

2. 6 μl to the Haemoglobin transducer

3. 24 μl to the basophil transducer

4. 12 μl to the Eosinophil transducer 5. 12 μl to the White Blood Cell Counting

RF/DC transducer.

Channels one and two are of no interest in the test of the present invention and the reagent supplies to each of these channels is de-ionised water. The reagent supplies to each of channels 3 to 5 are as follows:-

Channel 3. A lysing reagent for basophils available under the Trade Name Stromatolyser-BA (SBA-200A). This is a lyse reagent to permit the absolute count of basophils by the elimination of the red blood cell stroma and white blood cells other than basophils. Stromatolyser-BA is an acid surfactant comprising:-

Hydrochloric acid 0.18%

Polyoxethylene alkyl ether 2.00%

Channel 4. A lysing reagent for Eosinophils available under the Trade Name Stromatolyser-EO for enabling the absolute count of Eosinophols by eliminating the red blood cell stro a and the white blood cells other than the eosinophils. Stromatolyser-EO is an alkaline surfactant comprising:-

Sodium hydroxide 0.057%

Polyoxyethylene alkyl ether 1.0%

Channel 5. A reagent under the Trade Name Cellpack-GL which is used to dilute whole blood specimens and to size the white blood cells into three populations namely lymphocyte, granulocyte and monocyte. Cellpack-GL comprises an aqueous solution of 3.1% of formaldehyde and 0.14% sodium chloride.

In passage through the Sysmex Analyser, each sample is subdivided as indicated and then treated with the reagents for the appropriate channel. The aspiration of the cells through the Sysmex Analyser, results in the passage of the cells through a small aperture formed in a partition in a , chamber, the test fluid being passed from a first side of the partition to the second side. Electrodes are disposed on each side of the partition. The aperture in the partition has a diameter of the same order as that of the cells in the portion. A constant DC current is passed between the electrodes. As a cell passes through the aperture, the resistance of the electrical path between the electrodes increases significantly and as a result the voltage drop across the electrodes increases by a detectable amount. At the same time a radio frequency alternating current (r.f.) is applied across the electrode. As a cell passes through the aperture, a transient change occurs in the measured r.f. voltage, the measured voltage change being proportion to the total nuclear and cytoplasmic granular volume and density of the cell. The analyser, used in this novel way can both count cells and determine their nature, i.e. whether granulocyte or agranulocyte (non-granular) .

The results can be plotted as a scattergram or can be recorded as a set of comparative figures. The analyser is coupled to a computer, programmed to record the results and then to analyze them to provide a print out of the significant changes between the control and the samples. The computer further compares the results with the list of food extracts tested and then prints out a list of foods which showed an intolerant reaction sorted in the order of the severity of the reaction.

The test described above was carried out on a number of patients. Each patient was asked to set out their various symptoms, they were then given their test results and were asked to alter their diets:-

a. to avoid the particular foods to which an intolerance had been shown, and

b. to maintain a diet on only those foods which had been indicated not to show intolerance.

Each patient was asked to continue with the dietary restriction for a period of 6 to 8 weeks; they were then asked to complete a questionnaire setting out the changes in their health notice over the period of the diet. The results are set out in Table 3 below.

Example 2 Example 1 was repeated but in this case the Stromatolyser-EO in Channel 4 was substituted by de- ionised water, and the results from the Channel were ignored. Example 1 was repeated on the same blood sample, the intolerances revealed by the test were identical. The reason for this is that the proportion of eosinophils in blood is very small, of the order of 0.5%, and in consequence, the results are not significantly affected. This does allow, however, a considerable saving both in time and in cost.

Example 3 Example 2 was repeated using de-ionised water for the reagent in each of Channels 3 and 4. The test in this case in on the total granulocyte population as measured by the White Blood Cell count in Channel 5. The results were identical to those of Example 1 in terms of the intolerances measured. This further modified procedure provided a further reduction in time and cost of operation of the test.

The above examples show that using the Sysmex haematology analyser, the benefits of the invention can be obtained using various modes of operating the machine. The essence of the invention is however the automated measurement of the neutrophil degranulation in reaction to an intolerogen. The above examples show that the test for neutrophil degranulation is not masked by the presence of eosinophil and basophil populations of the blood. This allows for a simplified test procedure in the determination of intolerogens to a patient's blood.

The results set out in Table 3 accompanying this specification also demonstrate the advantages of having knowledge of the intolerogenic response of patients blood to intolerogens used to challenge blood samples. The effect of avoiding known intolerogens in diet, produces a remarkable reduction in many symptoms in many patients and provides clinical practitioners with an efficient tool for reducing the effect of dietary introduced aggravation in common ailments such as rheumatism, arthritis, asthma, and irritable bowel syndrome.

TABLE 3

TEST NO. START INITIAL FINAL TIME IMPROVEMENT WEIGHT SYMPTOMS WEIGHT

B002489 08st 121b IBS & fluid retent 08st.l01b 2mnts General feeling of well being/no longer bloated

B000907 14st 081b IBS & fluid retent/ 13st 081b 9wks No bloating/ lethargy clearer skin/ more energy

B002742 lOst 001b IBS (undiagnosed) 09st 071b 14wks No bloating or U) stomach ache

B001061 lOst 121b IBS lOst 091b lOwks Symptoms cleared - some weight loss

B002231 12st 061b IBS & overweight 12st 071b IBS & overweight

B002991 93kg IBS/breathlessness/ 87.5kg 2mnts IBS & overweight/general breathlessness cleared

B001126 list 061b Overweight lOst 061b 8wks Sinusitis greatly improved

B000740 13st 121b Overweight/chronic 12st 051b 9wks Weight loss & sinusitis sinusitis gone

B002741 16st 101b Overweight/hay fever 15st 071b 2mnts Weight loss/hay fever cleared

B002765 13st 171b Overweight/runny nose 12st 071b 2mnts Weight & headaches loss/healthier and more active

B001516 16st 031b Overweight/IBS and 15st 021b Weight loss/IBS ecezma gone/ sleeping better

B000723 16st 041b Overweight 15st 071b 3mnts Weight loss and improvement in asthma CO

B000829 15st 021b Overweight 12st 101b Weight loss. 'Diabetes' appears to have disappeared

B003204 lOst 081b Overweight/bloated 09st 051b 9wks Weight loss with PMT/fatigue no 'dieting' other than this

B001477 15st 081b Overweight/arthritis 14st 091b 8wks Weight loss. General feeling of well being, good attitude

B001320 22st 041b Overweight 20st 061b 9wks Weight loss/generally healthier

B000795 13st 021b Overweight list 061b 8wks Weight loss/generally healthier

B000735 16st 081b Overweight 14st 021b 9wks Weight loss

B000478 list 101b Overweight/water lOst 061b 7wks Weight loss/no retention water retention

B002251 lOst 081b Overweight/water 09st 001b 2mnts Weight loss/no retention water retention

B002250 lOst 041b Hiatus Hernia/ lOst 011b 3mnts Cough heartburn/water disappeared/digest retention ion improved

B001840 12st 081b Eczyma/IBS/overweight 12st 001b 3.5 Eczema mnts improved/small weight loss

B000578 Eczyma/asthma 7wks Eczema and asthma improved/weight loss

B002436 list 091b Water retention/ lOst 121b 9wks Feel improved, general lethargy, more headaches/overweight energy/headaches gone some weight loss

B000644 13st 101b Arthritis/tiredness 13st 101b 6wks Great improvement in arthritis/more

energy

B001114 17st 041b Arthritis/overweight 15st 091b 6wks Arthritis improved/weight loss

B001083 09st 001b Arthritis 08st 081b 4/5 Arthritis wks improved/no more migraines

B002145 12st 001b Arthritis/overweight list 001b 3mnts Arthritis improved

o

Claims

1. A method of determining the environmental intolerogens to mammalian blood which method comprises sampling blood, preparing a sample extract of each intolerogen to be tested, mixing a portion of the intolerogen extract with a proportion of sample blood, incubating the mixture and probing the mixture to determine changes in the neutrophil population when compared with an incubated control blood portion devoid of intolerogens, whereby significant changes in the neutrophil population indicate an intolerance to said intolerogens.

2. A determination method as claimed in claim 1 which method comprises drawing a blood sample from a patient, preparing a plurality of test samples and at least one control sample, preparing an extract of the intolerogen by dispersing it in a suitable carrier, mixing the intolerogen extract with the corresponding blood sample, mixing the carrier for the intolerogen with the control blood sample, incubating both the mixture and the control and thereafter analysing and comparing both the intolerogen mixture and the control to determine any significant changes in the neutrophil population in terms of blood cell size, density and/or apparent degranulation of the intolerogen mixture relative to the incubated control sample.

3. A method as claimed in claim 1 or claim 2 which comprises adding the intolerogen extract to a blood sample, incubating the blood sample mixture, sub-dividing the sample into aliquots, treating the first aliquot with a lysing agent to lyse all the cells except granulocytes, treating a second aliquot with a lysing agent to lyse all the cells except basophils, probing each lysed aliquot to determine the degranulation of the granulocyte population relative to the incubated, corresponding lysed intolerogen free control, whereby the apparent change in the total granulocyte less the basophil population is an indication of the intolerance to the intolerogen added to the sample.

4. A method as claimed in any preceding claim characterised in that the changes in the neutrophil/granulocyte populations in terms of blood cell size, density and/or apparently degranulation is compared with the statistical mean derived from testing a plurality of other blood samples of the mammal group thereby to determine whether the amount of the change is significant as compared with the mean change.

5. A method as claimed in any preceding claim characterised in that the degranulation of the neutrophils is determined by a relative shift in the degranulation of the population of granular size being neutrophils, basophils and eosinophiles compared with other populations within the blood sample.

6. A method as claimed in any preceding claim characterised in that the degranulation of the neutrophils/granulocytes is determined using a haematology analyzer.

7. A method as claimed in any preceding claim characterised in that the intolerogen is a food extract which is added to a blood sample, incubating the blood/food sample, sub-dividing the sample into aliquots, treating a first aliquot with a lysing agent to lyse all the cells in the sample except for granulocytes, treating a second aliquot with a lysing agent to lyse all the cells except the eosinophils, treating a third aliquot with a lysing agent to lyse all the cells except the basophils and lysing an aliquot of each control sample in a corresponding manner, probing each lysed aliquot to determine the degranulation of the granulocyte population relative to the incubated and correspondingly lysed intolerogen free control thereby to determine the degranulation of the neutrophil population.

8. A method as claimed in any preceding claim characterised in that each sample aliquot is probed by being suspended in electrolytic diluent and disposed in the transducer chamber for an analyzer having a partition dividing the transducer into two regions .

9. A method as claimed in claim 8 characterised in that the partition has a small aperture extending therethrough between two regions and each region is provided with an electrode further characterised in that the size of the aperture is of the same order of the size of the granulocyte cells in the blood sample and an electrolytic diluent is selected to have a significantly different electro conductance from said granulocyte cells .

10. A method as claimed in claim 9 characterised in that the granulocyte cells in each aliquot are aspirated through the aperture from one region of the transducer chamber to the other region while a substantially constant electrical current is applied across the electrodes whereby as a cell passes through the aperture the voltage between the electrodes changes transiently by a detectable amount and any changes in the voltage is monitored continuously during aspiration of the cells to count the number of cells passing the aperture.

11. A method as claimed in claim 10 characterised in that in the case of the second and third aliquots the applied constant current is a constant direct current and the total volume of each cell passing through the aperture is proportional to the transient change in the voltage.

12. A method as claimed in claim 10 or 11 characterised in that on aspiration the cells in the first aliquot a constant direct current and a constant radio frequency alternating current is applied across the electrodes; the transient change in DC current being proportional to the total volume of granulocytes cells as they pass through the aperture, and the transient change in the ratio frequency signal as the cells pass the aperture is proportional to the total nuclear size and density of each cell.

13. A method as claimed in any preceding claim whereby the method of the invention is conducted with only two aliquots, namely by lysing to determine the basophils and determining the remaining granulocytes .

14. A method as claimed in any preceding claim characterised in that the lysing of the eosinophil and basophil populations is dispensed with and the measurement of the degranulation of the granulocyte population is taken as a significant measure of neutrophil degranulation.

15. A method as claimed in any preceding claim characterised in that the intolerogens are food intolerogens and the food intolerogens to test are those which are selected to provide sufficient foods to constitute a complete and balanced diet.

16. A method as claimed in claim 15 characterised in that the intolerogen or food extract is presented to the blood samples in the form of a solution.

17. A method as claimed in claim 16 characterised in that the suspension may comprise a carrier medium selected from phenolbenzyl solvent or buffered saline.

18. A method as claimed in any one of claims 15 to 17 characterised in that the food sample is a freeze dried food sample.

19. A method as claimed in claim 18 characterised in that the food dried food sample is diluted with buffered saline, mixed well, centrifuged and filtered to give a particle free extract of food substance.

20. A method as claimed in claim 19 characterised in that the liquid food extract includes a proportion of phenol to prevent bacterial contamination.

21. A method as claimed in any preceding claim characterised in that a period of incubation is allowed after challenging the blood sample with the intolerogen.

22. A method as claimed in claim 21 characterised in that the typical period of incubation is within the range of 30 minutes to 90 minutes.

23. A method as claimed any preceding claim characterised in that the diet is applied in combination with a therapeutic treatment to improve the effect of the therapeutic substance.

24. A method as claimed in claim 23 characterised in that the therapeutic substance is sodium cromoglycate.