DIAGNOSTIC METHOD FOR ALGIC PATHOLOGIES

Abstract: The invention in question concerns a diagnostic method which allows diagnosis of some algic diseases by measurement of the hypofunctionality of or decrease in G-inhibitory proteins.

Description: DIAGNOSTIC METHOD FOR ALGIC PATHOLOGIES Field of the invention The invention in question refers to a diagnostic method for some algic pathologies by measurement of the hypofunctionality or decrease in G-inhibitory protein (hereinafter "Gi/o") levels. State of the art A serious, debilitating headache occurs in the lifetime of approx. 40% of the world population. In Italy 10 million people are periodically subject to headache attacks. Two essential types of headache may be distinguished: primary headache, which is generally a symptom of a debilitating but benign disease; and secondary headache, which is instead a symptom of very serious illnesses such as brain tumour, giant cell arthritis, intracranial aneurysm, subarachnoid haemorrhage, meningitis etc. To these two types of headache must be added the common headache which almost everybody experiences at least once in a lifetime. The state-of-the-art diagnosis of the various forms of headache is carried out exclusively on a clinical basis, i.e. examining only the features with which the attack manifests itself (number and duration of the attacks, description of the associated symptomatology, familial anamnesis, exclusion of mass-occupying endocranial pathologies, of various metabolic disorders, of both brain and systemic infections etc.). Fibromyalgia is characterised by diffuse pain and hyperalgesia of prevalently muscular location. The epidemiology is approximately 5% of the population. To date numerous neurophysioiogical, haematological, bioptic and psychological studies aimed at identifying the pathogenesis of the fibromyalgic syndrome have yielded no objective, reliable results which can differentiate the fibromyalgic patient from the healthy subject. It would thus seem to be of great importance to have a simple, reliable diagnostic method which is able to discriminate between primary headaches, headaches which are secondary to other pathologies and the common headache, and also to give a sure diagnosis of fibromyalgia. 2 The G-inhibitory proteins are ubiquitous proteins located on the inner wall of the cytoplasmic membrane. Their inhibitory effect on various cell functions is activated consequent to the stimulation of suitable membrane receptors to which the proteins are bound. This inhibitory effect is exerted through reduction of cAMP levels, the opening of potassium channels and the closure of calcium channels. In other words the cells (particularly nerve cells) become hyperpolarised and hence less excitable. State-of-the-art knowledge has not linked any human pathology to a reduction in Gi/o protein functionality or cell levels. Brief description of the figures Fig. 1 shows on the ordinate the levels of basal cAMP in lymphocytes of headache and fibromyalgic patients as compared to healthy subjects; Fig. 2 shows the effect of pretreatment with pertussis toxin (PTX) on the capacity for inhibiting stimulated cAMP production in intact human lymphocytes. As can be seen, the incubation of lymphocytes with forskolin strongly increases basal cAMP levels. This increase is prevented (see blank columns) by Gpp(NH)p (Gi activator) added in suitable concentrations (shown on the abscissa); in the case of lymphocyte pretreatment with PTX (which selectively inactivates the Gi) this inhibition is absent (black columns). Fig. 3 shows the findings on the inhibition of stimulated cAMP production in the lymphocytes of headache subjects; Fig. 4 shows the findings on the inhibition of stimulated cAMP production in the lymphocytes of fibromyalgic subjects. Detailed description of the invention The invention in question allows the above problems to be overcome, in that it presents a simple laboratory diagnostic method which can rapidly and accurately identify the presence of the above-mentioned pathologies. It has in fact surprisingly been found, and this is the object of the present invention, that the different forms of primary headache and fibromyalgia can be related to a deficit of and/or reduction in Gi/o protein functionality. The deficit of Gi/o proteins can be determined with the appropriate analytical 3 methods which are well known in the literature (e.g. Western Blot and immunofluorescence). Gi/o protein functionality can be evaluated by measuring their capacity to inhibit cAMP production (in other words, by measuring basal cAMP levels, which in ill patients are higher due to lack of inhibition), or by measuring the inhibition of stimulated cAMP production, or by measuring the levels of mRNA for Gi/o proteins. These measurements too are performed with methods which are already known in the literature (e.g. Northern Blot, in situ hybridisation, RT-PCR). One of the important advantages of the present diagnostic method is that the ubiquitous nature of G-inhibitory proteins allows measurements to be performed on various types of figured elements of the blood and on various types of cells: muscular, nervous etc. It is only for reasons of convenience that the present description particularly refers to analyses performed on blood cells (leukocytes), since these are most easily removed from the patient. Analyses on other types of cells of figured blood elements can be applied using the well-known appropriate technologies. The study was particularly carried out on lymphocytes obtained from peripheral venous blood, since these are seen from the examination of the literature to be the most suitable for studies on G-inhibitory proteins. Some methods are herewith described as examples for the evaluation of the functionality and cell levels of Gi/o proteins, but obviously other methods for the determination of G-inhibitory proteins which are known in the literature can be used in the diagnostic method according to the invention. Evaluation of Gi/o protein functionality The heparinised (500 I.U. heparin:10 ml blood) blood samples (15-20 ml) are diluted 1 :1 with physiological solution. The lymphocytes are then isolated by centrifugation in density gradient as described by Bόyum (Scand. J. Clin. Lab. Invest., 21 (Suppl. 97): 77, 1968) and permeabilised with digitonin (10 μg/ml) under such conditions as maintain the biological functions intact (Corey et al., J. Biol. Chem., 264: 14165, 1989). The lymphocytes from healthy subjects are then preincubated with or without 4 pertussis toxin (PTX) at the concentration of 100 ng/ml at 37o for 90 min. After preincubation, the lymphocytes are divided into samples containing 1-2x10 6 intact cells and the various reagents are added in amounts giving a final volume of 300 μl. For the determination of cAMP content the lymphocytes are resuspended in an incubation mixture containing 100 aeM theophylline (or 3-isobutyl-1- methylxanthine) to prevent degradation of cAMP by the phosphodiesterases. To measure the basal cAMP level the vehicle (DMSO) is added to the sample. The formation of cAMP is started by adding 10-4M forskolin, which directly stimulates the adenylcyclase. To obtain the inhibition curve of cAMP production the non-hydrolysable analogue of GTP Gpp(NH)p (guanilyl 5'-imidophosphate) must be added to the forskolin- containing samples in the range of concentrations from 10 nM to 100 μM. After a 15-minute incubation at 37°, the cells are lysed and the sampled centrifuged. The measurement of cAMP content is then carried our on the supranatant using an ELISA method. Quantification of Gi/o proteins The quantification of Gi/o proteins is effected on the isolated lymphocytes according to the Western Blot analysis or using immunofluorescence. a) Western blot analysis The cells are broken up by sonication in 25 mM Hepes buffer and 10% sucrose. The cells are then centrifuged at 105,000 rpm for 60 min. The membranes thus obtained are resuspended in 1mM Tris-HCI buffer (pH 7.4) containing 1 mM EDTA, 5 aeg/ml leupeptin, 0.3 mg/ml phenylmethylsulphonylfluoride (PMSF) and I mM dithiothreitol (DTT). The membrane proteins are first separated over acrylamide gel (10 or 12%), after having been diluted 1 :3 in the sample buffer according to Laemli (Nature 227: 680-685; 1971). Gels 1.5 mm thick and 8 x 10 cm in size are prepared and submitted to a current of 30 or 50 mA (double-course) for 45-60 min. After electrophoretic separation the proteins are electrically transferred (1 h at 100 V, 400 mA) onto nitrocellulose according to the technique described by Towbin et 5 al. (Proc. Natl. Acad. Sci. USA 76: 4350-4354; 1979), using T s-glycine buffer - containing 10% of methanol. Part of the filters containing the reference standards or the marker molecular weights are stained with starch black for verify the transfer. The filters containing the lymphocyte proteins are washed twice with TBS (50 mM Tris, 138 mM NaCI, 2mM MgCI2, p.H 7.4) then incubated in the same buffer containing 3% of serum albumin or gelatine for 2h at room temperature. Then they are washed once with TBS (5 min) and once with the same buffer (5 min) containing 0.05% Tween 20 (TTBS). After this washing the filters are incubated overnight with the primary antibody (anti Giα according to Laemli, rabbit serum) diluted 1 :500 in TTBS. The antibody is then removed and the filters washed twice with TTBS (5 min). The second antibody, anti-rabbit goat igG conjugated with alkaline phosphatase, diluted 1 :3000, is then added and is kept in contact with the filters for 1 h at room temperature. After this contact the filters are abundantly washed with TTBS and then 4 times with TBS (5 min per washing). Parallel to the reaction with anti-Giα serum some filters are incubated with rabbit serum (preimmune) to verify the specificity of the reaction of immunorecognition with specific anti-Giα serum. The reaction is performed through addition to the filters of a chromogenous substrate for alkaline phosphatase and the bands obtained are visualised and identified by comparison with the standard Giα protein band. All the washing and incubation operations with specific serum and conjugated antibody are carried out by placing the filters on a rotary shaker, b) Immunofluorescence The density of the Gi/o membranes is calculated qualitatively also on whole lymphocytes from control samples and from patients. The cells are fixed in paraformaldehyde and treated for antibody recognition (anti-Giα IgG, dilution 1 :100) , then conjugation with IgG FITC (1 :500). The reaction is visualised through a fluorescence microscope after a series of washings. Quantification of levels of the mRNA codifying G-inhibitory proteins 6 The determination of the message levels for the G proteins is performed with 2 fundamental methods: the Northern test and in situ hybridisation. a) Northern analysis Total RNA is prepared according to the techniques described by the various manufacturers of preparation kits. RNA nucleic acid is then separated over agarose gel in denaturing conditions, in parallel to RNA standard. The gel is then transferred onto nitrocellulose and prepared for hybridisation according to classic methods. Hybridisation is performed though recognition with a DNA probe, consisting of oligonucleotides from the terminal sequence LKDCGLF, LKECGLY or from cDNA for the Gi2α and Gi3α clone. b) in situ hybridisation Preparations of approx. 10 μm are prepared from the blood cells or tissues. The preparations are treated with stringent hybridisation buffer conditions so as to lower the response background. The hybridisation is performed using oligonucleotide probes or cDNA. Experimental data The results obtained using the above methods have shown that patients affected with the various forms of primary headache and fibromyalgia have a reduced functionality of the Gi/o proteins. This hypofunctionality appears to be correlated to a reduction in Gi/o protein levels in the lymphocytes of ill subjects. It has moreover been observed that, due to this reduced Gi/o protein functionality, the lymphocytes of ill subjects have higher basal levels of cyclic AMP than in healthy controls. The experimental data obtained are summarised in Figures 1-4 which were already illustrated hereabove. On the basis of the results shown in the figures it may thus be said that the present method allows diagnosis of primary headache and fibromyalgia by measurement of one or more of the above-mentioned parameters. The diagnostic method according to the invention thus allows differential diagnosis on a biochemical basis between primary headaches due to a deficit of inhibitory G proteins and the numerous forms of secondary headache. These methods may also be extremely useful for the diagnosis of fibromyalgia. 7 On the basis of results obtained in rats rendered diabetic with alloxan and streptazocine, in which a decrease in Gi/o protein functionality was demonstrated (Gawler et al., Nature 327: 229-232, 1987; Bushfield et al., Biochem. J. 271 : 365- 372, 1990; Lacombe et al., Diabetes Metab. 22: 432-438, 1996; Gando et al., J. Pharmacol. Exp. Ther. 282: 475-484, 1997), it is predictable that the pain from diabetic neuropathy can also be detected by using the diagnostic method (or methods) according to the invention.

Claims: 8 CLAIMS 1. Diagnostic method for the determination of algic diseases in which the deficit and/or hypofunctionality of G-inhibitory proteins are measured in figured blood elements and/or human cells. 2. Diagnostic method according to claim 1 , in which the increase in basal cAMP levels is measured. 3. Diagnostic method according to claim 1 , in which the deficit of G-inhibitory proteins is measured with the Western-blot analysis. 4. Diagnostic method according to claim 1 in which the deficit of G-inhibitory proteins is measured by immunofluorescence. 5. Diagnostic method according to claim 1 in which the deficit is measured by determining the levels of the mRNA codifying the Gi proteins. 6. Diagnostic method according to claim 1 in which the hypofunctionality is measured by evaluating the inhibition of cAMP production. 7. Diagnostic method according to claims 1-6 in which blood cells are used. 8. Diagnostic method according to claims 1-7 in which the algic diseases identified are: primary headaches and fibromyalgic syndrome.

Inventors: BARTOLINI ALESSANDRO, BENEFORTI ELISABETTA, GALEOTTI NICOLETTA, GHELARDINI CARLA, ZOPPI MASSIMO, BARTOLINI, ALESSANDRO, BENEFORTI, ELISABETTA, GALEOTTI, NICOLETTA, GHELARDINI, CARLA, ZOPPI, MASSIMO
Applicants: BARTOLINI ALESSANDRO, BENEFORTI ELISABETTA, GALEOTTI NICOLETTA, GHELARDINI CARLA, UNIV FIRENZE, ZOPPI MASSIMO, BARTOLINI, ALESSANDRO, BENEFORTI, ELISABETTA, GALEOTTI, NICOLETTA, GHELARDINI, CARLA, UNIVERSITA' DEGLI STUDI DI FIRENZE, ZOPPI, MASSIMO
Application year: 1999
Main classification: G01N33
IPC codes: G01N33