WO2014037858A1

WO2014037858A1 - Method and kit for the diagnosis and monitoring of sensitivity and intolerance to foods

Info

Publication number: WO2014037858A1
Application number: PCT/IB2013/058148
Authority: WO
Inventors: Enzo SPISNI; Massimo CAMPIERI
Original assignee: Alma Mater Studiorum - Universita' Di Bologna
Priority date: 2012-09-04
Filing date: 2013-08-30
Publication date: 2014-03-13
Also published as: ITVR20120176A1

Abstract

The present invention relates to a method of extracorporeal diagnosis of food sensitivity or intolerance to gluten in a patient, comprising the following steps in sequence: - providing a sample of peripheral blood from the patient, or from mononuclear cells (PBMC) peripheral blood derived from the patient's blood; - adding at least one food containing gluten to a portion of the peripheral blood sample; - adding at least one gluten-free protein extract to at least another portion of the peripheral blood sample; - letting the thus added portions of the sample to rest for a predetermined time; - measuring the amount of at least one chemokine produced in the samples after the resting interval, the at least one chemokine being selected from the group comprising CXCL10 (IP10), CXCL9 (MIG) and CXCL11 (l-TAC); - determining a reference value (PG) related to the chemokine production caused by gluten, based on the amount of chemokine produced in the portions of the sample, and - checking whether the reference value (PG) is greater than a maximum threshold value or lower than a minimum threshold value, or is an intermediate value.

Description

METHOD AND KIT FOR THE DIAGNOSIS AND MONITORING OF SENSITIVITY AND INTOLERANCE TO FOODS

METHOD AND KIT FOR THE DIAGNOSIS AND MONITORING

SENSITIVITY AND INTOLERANCE TO FOODS

The present invention relates to a method and a kit for the diagnosis and monitoring of sensitivity and intolerances to food, and in particular to food containing gluten.

State of the Art

Most gastroenterologists agree on the possibility that there may be personal sensitivities to certain foods, probably linked to phenomena of innate immunity. These sensitivities differ from allergies and adverse reactions to foods, which can be demonstrated by tests accepted by conventional science (immunological, biochemical and histological). To date, their existence cannot be demonstrated with certainty, nor is it possible to diagnose it with conventional methods.

For such sensitivities only unconventional methods exist, such as the EAV test of Voll or the Cytotest™, that are not accepted by the scientific community since they are characterized by very poor reproducibility.

Of all these supposed sensitivities, the only one that has been ascertained is the gluten sensitivity, which has been recently defined as non-celiac gluten sensitivity (see "Spectrum of gluten-related disorders: consensus on new nomenclature and classification "Sapone A. et al. BMC Med 2012; 7: 10-13).

Patients with this disease are defined as sensitive to gluten or "gluten sensitive"

(GS) or non-celiac gluten sensitive (NCGS).

Gluten sensitivity has been defined as a condition of intolerance to gliadin and glutenin present in grains. For a long time it has been discussed about the existence of this disease, in which there is a loss of tolerance to these proteins (or their digested fragments) with symptoms only partly similar to celiac disease, which, however, can also involve extra intestinal symptoms.

Sensitivity to gluten, that for a long time has been considered to be a psychosomatic reaction, has become an ascertainable disease only recently, as a result of studies in "double-blind" that have demonstrated its existence (see "Gluten causes gastrointestinal symptoms in subjects without celiac disease: a double-blind randomized placebo-controlled trial "of Biesiekierski JR et al., Am J Gastroenterol. 201 1 ; 106:508-14).

For carrying out studies or analyses in "double-blind" as they are termed, investigations are carried out by administering substances to patients, of which neither the administering doctors nor the patients know the composition, although, of course, the substances are identified by a code or abbreviation, so that, at the end of the treatment, it is possible to ascertain the substances administered, whereas studies or analyses in "blind", may be defined as the investigations carried out by administering substances to patients, whose composition is known only to the administering doctors.

Recently, the scientific community has accepted the existence of non-celiac gluten sensitivity as a medical condition separate from celiac disease on the basis of histopathological and serological tests (see "Spectrum of gluten- relateddisorders: new consensus on nomenclature and classification" Sapone A. et al. BMC Med 2012; 7:10-13).

The pathogenesis of gluten sensitivity is still unknown (see "New understanding of gluten sensitivity" of Volta U, D, and De Giorgio R. - Nat Rev Gastroenterol Hepatol. 2012; 9:295-9). In a study of not celiac GS patients and healthy controls, it was observed that the intestinal permeability of celiac patients is significantly increased compared with the GS. Within GS patients instead it appears to be normal (see "Divergence of gut permeability and mucosal immune gene expression in two gluten-associated conditions: celiac disease and gluten sensitivity " A. Sapone, A. Fasano et al. BMC Med 2011 ; 9:23). In the same study, it was shown that the GS subjects show an increased expression of some adhesion molecules expressed by the intestinal cells, such as claudin-1 , ZO-1 and claudin-4, and that the expression of markers of innate immunity such as Toll like -2 receptor is altered in the intestinal mucosa of GS patients.

Finally, the expression of the marker termed FOXP3 on regulatory T cells was found to be decreased in GS patients. No other biomarker for the diagnosis of gluten sensitivity has been proposed up to now.

The symptoms of gluten sensitivity are rarely comparable to those of the celiac disease and allergy to gluten, and resolve after discontinuation of gluten taking. The main symptoms reported by patients suffering from gluten sensitivity are: abdominal pain (68%), eczema and/or rash (40%), headache (35%), clouded mind (34%), fatigue (33%), diarrhoea (33%), depression (22%), anaemia (20%), numbness in the legs, arms or fingers (20%) and joint pain (1 1 %) (see "Spectrum of gluten-related disorders: consensus on new nomenclature and classification " A. Sapone et al. BMC Med 2012; 7: 10-13).

The current diagnosis of gluten sensitivity is exclusively done by using criteria of exclusion of other diseases such as:

- exclusion of allergic mechanisms typical of gluten allergies (allergic to wheat immunoassay negative); - exclusion of autoimmune mechanisms typical of celiac disease (anti-EMA and / or anti tTG negative), and

- exclusion of duodenal mucosal damage typical of celiac disease (duodenal histopathology in the norm).

In addition, patients defined GS must show a regression of symptoms after a gluten-free diet is initiated, whereas symptoms reappear when the patients are fed (blinded) with a load of gluten.

As for the other pathologies linked to gluten assumption, a gluten-free diet which involves the elimination of cereals containing gluten and any derivatives thereof appears to be actually the best therapeutic solution. However, for the gluten sensitivity the gluten-free diet is effective although less severe than the diets prescribed for celiacs who must also avoid cross-contamination by foods containing gluten. It is believed that, after a period of gluten-free diet of 90-120 days, it is possible to try gradual reintroduction of gluten, thereby reactivating a sort of tolerance that makes it possible to avoid gluten-free diets that last a lifetime (see "Spectrum of gluten-related disorders: consensus on new nomenclature and classification " A. Sapone et al. BMC Med 2012; 7: 10-13).

The international application WO2011 163258 describes a method of diagnosing and monitoring diseases and food sensitivity, which is based on the use of synthetic peptides having a known sequence and on the response of lymphocytes, separated from the blood of the patient, to such synthetic molecules. The response is evaluated as lymphocyte proliferation or lymphocyte migration, and also includes the measurement of cytokine secretion in the group including: IL-1 b, IL-4, IL-6, IL-8, IL-10, IL-13, MCP-1 , G-CSF, IFN-g and TNFa. FFrroomm tthhee aarrttiiccllee ""AAnnaallyyssiiss ooff tthhee ppaatthhoopphhyyssiioollooggiiccaall mmeecchhaanniissmmss uunnddeerrllyyiinngg tthhee sseennssiittiivviittyy ttoo gglluutteenn"" bbyy EEnnzzoo SSppiissnnii,, aavvaaiillaabbllee oonnlliinnee aatt tthhee wweebbssiittee::

hh††ttppss..////wwww^yyii??..aannccwweebb uunniibboojjtt//AAsssseeggnniiRRiicceetteeaa RRiicchh;;eessttee cciioocc//22001122//IIDD%%55BB99

a method of diagnosis of gluten intolerance is known which is based on adding protein extracts, obtained from flour containing gluten (e.g. Manitoba), to mononuclear cells separated from peripheral blood (or PBMC) and on the dosage of the amount of 42 cytokines and chemokines.

However, it has not been suggested so far a diagnostic tool more objective than the symptoms, and indicating whether the reintroduction of gluten is well tolerated or not by the patients.

The main object of the present invention is to provide a method of carrying out an objective diagnosis and monitoring food sensitivity and food intolerance, and particularly the sensitivity to gluten-containing foods.

Another object of the present invention is to provide a method, as indicated above, that is both highly reliable and easy to be carried out. In addition, the method makes it possible to compare the responses of individual patients to foods containing different quantities and quality of gluten (i.e.: flour of different cereals).

Another object of the present invention is to provide a kit for carrying out the method according to the present invention of diagnosing and monitoring sensitivity and intolerances, particularly gluten sensitivity.

According to a first aspect thereof, the present invention provides a method of extracorporeal diagnosing food intolerance or sensitivity of a patient, comprising the following steps in sequence: - providing a sample of peripheral blood derived from the patient or a sample of peripheral blood mononuclear cells (PBMC) from the venous blood of the patient;

- adding at least one food including gluten to a portion of such peripheral blood sample:

- adding at least one gluten-free protein extract to at least another portion of such peripheral blood sample;

- letting such sample portions thus prepared to rest for a predetermined time interval;

- measuring the amount of at least one chemokine formed in such samples after the resting time interval, such at least one chemokine being selected from the group comprising CXCL10 (IP10), CXCL9 (MIG) and CXCL11 (l-TAC);

- determining a reference value (PG) correlated to the production of chemokine caused by the gluten in the sample portions; and

checking whether such a reference value (PG) is greater than a maximum threshold value, or lower than a minimum threshold value, or intermediate such values.

Advantageously, the method makes it possible to diagnose gluten sensitivity induced by gluten-containing foods.

Even more advantageously, at least one chemokine is selected from the group consisting of CXCL10 (IP10), CXCL9 (MIG), and CXCL1 1 (l-TAC).

Preferably, the food is a total protein extract from at least one gluten- containing food. According to another aspect of the present invention, there is provided a kit for carrying out a method according to the present invention, comprising the following steps:

- at least one multiwell plate with plastic or PVDF bottom, some of such wells having a total of proteins extracted from flour with gluten pre-adsorbed on the bottom, whereas other wells have total proteins extracted from gluten-free flours pre-adsorbed on the bottom;

- at least a first package of synthetic branched copolymer having a high molecular weight for the separation of the PBMCs;

- at least one second package of RPMI 1640 medium containing 10% FBS and at least one antibiotic; and

- a plurality of test tubes.

Further aspects and advantages of the present invention will become apparent from the following detailed description of specific examples of a method of diagnosing, the description being made with reference to the accompanying drawings, in which:

- Figure 1 is a diagram illustrating the results of tests conducted on samples of peripheral blood of healthy patients and GS patients, evaluating CXCL10 as chemokine;

- Figure 2 shows the results of tests conducted on a not-GS patient treated with flour bags containing flour with gluten, or placebo;

- Figure 3 show the results obtained on a borderline patient treated with flour bags containing flour with gluten, or placebo;

- Figure 4 shows the results obtained in a GS patient treated with flour bags containing flour with gluten, or placebo; - Figure 5 schematically shows the method or operating sequence by means of a kit according to the present invention; and

- Figure 6 shows the loading scheme of a multiwell plate provided in a kit according to the present invention.

A method according to the present invention is an extracorporeal method that is based on the evaluation of immunological responses of peripheral blood mononuclear cells (PBMC) in vitro, when exposed to contact with protein extracts, preferably total protein extracts, of foods to which sensitivity or intolerance is expected. The same extracts are put in direct contact with the PBMC, by solubilizing the total protein extracted in the culture medium in which PBMC are cultured (proteins in suspension), or by causing the total protein to adhere to a substrate, e. g. a substrate of PVDF (polyvinylidene fluoride), or nitrocellulose, or plastic materials suitable for holding the proteins that are available on the bottom of the wells in which the PBMCs are successively kept.

PBMCs are then incubated for 4-24 hours, in the presence of the protein extracts. The incubation time depends on the sensitivity of the adopted method of dosing the available chemokines. As a matter of fact, the PBMCs of GS patients, when in contact with the protein extracts, produce chemokines that accumulate in the PBMC culture medium. After incubation with these extracts, the immunological responses of PBMC are evaluated through the dosage of the chemokines secreted by the PBMCs exposed to the total protein extracts of foods subjected to analysis.

Example 1 given below refers to an incubation time of 16 hours, that is an intermediate value between the two limit values, time after which the amount of chemokines accumulated in the culture medium can be dosed with Elisa or Luminex systems having a sensitivity of about 10 pg / ml. Longer times can be used for assays with less sensitive methods, shorter times can be used combined with more sensitive assay methods.

This method has been tested and tuned for gluten sensitivity by using protein extracts from flours containing gluten, although it can be easily adopted for any food (grains and their derivatives), from which total protein extracts containing gluten can obtained.

With a high degree of probability, since no contraindication is foreseen, the above described method is also applicable to other types of sensitivity (e. g. sensitivity to tomato, soya, etc.). or food intolerances

Currently, it is not been possible so far to test the above method for other types of sensitivity, since the diagnostic criteria for sensitivities that differ from gluten sensitivity have not been defined yet.

The method according to the present invention is intended to identify some markers of sensitivity or intolerance to the protein component of foods (such as gluten), as well as to develop a test kit for the diagnosis, therapy and follow-up of these feelings.

The method according to the present invention is based on the separation of mononuclear cells (PBMC) obtained from a peripheral blood sample of the patient. The PBMCs thus obtained are put in direct contact, in vitro, with total protein extracts of foods, such as flour containing gluten (e. g. Manitoba wheat flour, Claudio wheat flour or a flour of other types of cereals containing gluten), and total extracts of gluten-free foods (such as rice flour and corn flour). Then the changes in differential secretion of chemokines are measured, which are produced by PBMC cells as a result of their direct contact with the food proteins containing gluten. Chemokines, whose secretion could be altered upon contact of the PBMC cells with food proteins, such as gluten, are the following:

- CXCL10 (IP10);

- CXCL9 (MIG), and

- CXCL1 1 (l-TAC).

Among these, in so far as the sensitivity to gluten is concerned, the chemokine CXCL10 has proved to be very sensitive, whereas CXCL9 and CXCL1 1 have a high homology with the CXCL10 and bind the same cellular receptor. It is very likely then that also their secretion could be altered in PBMCs of patients sensitive to a specific food (such as wheat) exposed to proteins extracted from that food (such as gluten).

The peripheral blood sample, preferably venous, for carrying out the extracorporeal method according to the present invention was obtained by collecting, for example, 5 ml of peripheral venous blood from each patient undergoing the test, in tubes containing an anticoagulant, such as for example EDTA (ethylenediaminetetraacetic acid).

For implementing the method according to the present invention, arterial blood or other blood type, if not contaminated (eg faecal blood) could also be used.

Total proteins from commercial flours have been extracted according to the method termed "Osborne fractionation", a method well known to a skilled person in the art, modified according to van den Broeck et al. for the extraction of gliadin and glutenin (see "A modified extraction protocol enables detection and quantification of celiac disease-related gluten proteins from wheat" Di van den Broeck et al. - Journal of Chromatography B, 2009; 877:975-982). The used flours having a high gluten content are Manitoba flour and Claudio flour. The used flour containing lower levels of gluten are the so-called ancient grains flours (e.g. Senatore Cappelli), or flour of other cereals (e.g. Farro). The used gluten- free flours are rice flour and corn flours, certified as food for celiacs. Protein extracts thus obtained are stored in the form of pellet dried in a freezer at -20 ° C.

The peripheral blood mononuclear cells (PBMC) are obtained from a normal separation on density gradient of collected blood, for example Ficoll gradient. To separate the PBMCs from peripheral blood commercial kits such as the SepMate™ of Voden Spa, or of Ficoll-Paque™ gradients can also be used, although other suitable procedures are not excluded. As it is known, Ficoll is a synthetic water-soluble branched copolymer having a quite high molecular weight, synthesized from sucrose and epichlorohydrin, and used for preparing density gradients for cell separation. Example 1

PBMC cells from each single patient were cultured using commercial land RPMI 1640 (Sigma, USA) with the addition of 10% heat-inactivated foetal bovine serum (a reagent well known to those skilled in the art and commonly used for cell cultures), and antibiotics (such as penicillin 100 U/ml and streptomycin 100 mg / ml).

PBMCs obtained from each patient were distributed in 5 identical wells in a 24-well plate, each well containing 300,000 cells and 1.5 ml of complete medium (RPMI 1640 + heat inactivated serum + antibiotics). The first well was added nothing (control), whereas 40 micrograms of total protein, , Rice, Corn, Manitoba and Claudio, respectively, dissolved in 500 microliters of complete medium, were added to each of the remaining 4 wells.

The 24-well plates were placed for 16 hours in an C0₂ incubator (Heraeus- b-5060-ek) in controlled atmosphere (37 ° C, 6% C02) with slight agitation of an orbital shaking plate.

After incubation, the contents of the wells were transferred into 2 ml eppendorf tubes. The eppendorf tubes were then centrifuged at 500 g/min for 10 minutes at 4 ° C. After centrifugation, the supernatants or conditioned medium (1 ml) were collected and transferred into new eppendorf tubes. The pellet, i. e. the semisolid gathering of cells on the bottom of the tubes after centrifugation, containing the PBMC, was discarded.

The above described embodiment was developed using 24-well plates and protein extracts were added in suspension. It is also possible to adopt 96-well micro-plate with plastic or PVDF bottom, in which proteins have been previously adsorbed and immobilized at the bottom of the well. The test performed in this manner has shown to provide identical results to those described above.

The quantification of inflammatory cytokines in conditioned media can be performed by using well-known technologies, such as the ELISA technology, the Luminex™ technology the radioimmunoassay (RIA) methods, or other suitable quantitative methods with analogue sensitivity. The inventors of the present invention have used the Luminex™ technology with the reagents provided by the company BioRad. The control wells, containing PBMC in the absence of protein extracts, provide the baseline value of chemokine secretion by PBMCs, in the absence of any protein extracts.

The wells in which protein extracts of rice and corn are added constituted the negative controls (stimuli gluten-free). The wells with protein extracts of Manitoba and Claudio formed the production of chemokines in the presence of gluten. The reference value used is given by the following formula:

PG = P (Manitoba)-P (rice)

PG = chemokine production caused by gluten

P (Manitoba) = production of the chemokine in the presence of proteins extracted from Manitoba (pg/ml)

P (rice) = production of the chemokine in the presence of protein extracted from rice (pg/ml).

For the chemokine CXCL10 with reference to gluten sensitivity, based on the values obtained from the analysed samples, it was established that:

PG <50 pg / ml = negative patient to gluten sensitive test (healthy)

PG> = 100 positive patient to gluten sensitive test (affected by GS)

PG≤ 50≤ 100 = borderline patient (not determined)

The values obtained for Corn and Claudio were used as internal controls for the experiment and must be similar: the first to the value of the rice, and the second to the value of the Manitoba.

For a sample of 19 healthy individuals and 22 patients already diagnosed as GS, based on the exclusion criteria, the method according to the present invention was carried out. By dosing the chemokine CXCL10 (see Figure 1), the results did show that, while the PBMC obtained from the blood of healthy controls, and exposed to protein extracts containing gluten, have secretion values lower than the threshold value of 100pg/ml and in most cases lower than the minimum threshold value of 50 pg/ml, the PBMC obtained from GS patients and exposed to contact with gluten-containing protein extracts showed much higher values of secretion, with peaks that often exceed 250 pg/ml. Moreover, the data obtained from assays of CXCL10, by applying a statistic T-test between the two groups, a well known test to skilled persons in the art, have shown to be statistically quite significant with P values (probability of error) lower than 0,001 , as shown in the Table 1 below.

With reference to Figure 1 , the indications "healthy patient" and "GS patient" have been established on the basis of conventional exclusion diagnostic criteria. More particularly, if the patients suffered from disorders due to intake of food containing gluten, but were considered to be healthy in so far as all other pathologies were concerned, with the sole exclusion of gluten sensitivity, then these were referred to as GS patients. On these GS subjects a diagnostic test according to the present invention was conducted, after which values of chemokine CXCL10 indicated in Figure 1 were detected, and then it was assessed to be:

- healthy, i.e. not sensitive to gluten, the patients 1 , 3, 4, and 6 to 19, who were also previously considered to be healthy, as well as patients from 4 to 7, 16, 18, 19, and 22 previously deemed to be sensitive to gluten,

- borderline, the patient 2 previously considered to be healthy, as well as the patient 14 previously considered to be GS, and - gluten sensitive, the patient 5 previously considered to be healthy, as well as patients 1 to 3, 8 to 13, 15, 17, 20, and 21 previously considered to be GS.

A testing based on the taking of gluten or placebo in double-blind manner was then performed on the same patients. The testing confirmed the results obtained with the method according to the present invention and belied most of the conclusions reached on the basis of simple diagnosis of gluten sensitivity by exclusion of other illnesses/ailment.

Table 1

Statistical analysis (Unpaired T-test) applied to the obtained values of CXCL10 in the "healthy patients" compared with the group of patients confirmed as GS

The obtained P value (two-tailed P value) is lower than 0.001.

Group Healthy controls GS Patients

Mean 18.30 pg/ml 269,64pg/ml

SD 28.12 140, 15

SEM 6.45 37.46

N 19 14

In order to further validate the method according to the present invention, a blind study was carried out, where patients, classified as GS or healthy were treated with bags containing flour with gluten, or placebo (gluten-free flours) to be added to foods at meals. The study was carried out in cooperation with the Unit of Gastroenterology (SVD), Prof. Alberto Lanzini and Dr. Chiara RICCI - AO Civil Hospital of Brescia, University of Brescia. The results of the analysed samples, once the blind was opened, have shown that the test based on CXCL10 is suitable for accurately discriminating between healthy and GS patients, and shows highly positive values upon taking gluten and the appearance of symptoms as declared by the patients. They also showed that the responses to the different used flours are affected by the content of gluten present therein.

Figure 2 illustrates the test results obtained on a patient not suffering from GS, who had declared moderate symptoms when taking placebo and absent symptoms during the intake of gluten. More particularly, the same Figure shows the differential analysis of the secretion of CXCL10 (PG, in pg/ml) by the PBMCs of a patient considered to be GS, who later was classified as not GS. This patient was stimulated with protein extracts from flour with a high gluten content (Manitoba and Claudio), low gluten content (Kamut and Senatore Cappelli) or gluten-free (mais). Rice flour (gluten free) was used as a control value. At time T1 the patient underwent a gluten-free diet for at least 90 days. At time T2 he took placebo (declaring moderate symptoms), whereas at T1 a he underwent a gluten- free diet, and at T1 b he took daily a gluten load of 2 plates each containing 100 g of pasta (declaring the disappearance of symptoms).

Figure 3 shows the results obtained for a border-line patient, who did state moderate symptoms when taking gluten. More particularly, this Figure shows the differential analysis of the secretion of CXCL10 (PG, in pg/ml) by the PBMCs of a border-line GS patient, stimulated with protein extracts having a high gluten content (Manitoba and Claudio), low gluten content (Kamut and Senatore Cappelli) or gluten-free (mais). Also in this case, the rice flour (gluten free) was used as a control value. At time T1 the subject underwent a gluten-free diet for at least 90 days. At time T2 he took placebo (no symptoms). At T1 he underwent again a gluten-free diet and at T1 b he took daily a gluten load corresponding to 2 plates each containing 100 g of pasta (mild symptoms).

Figure 4 shows the results obtained with a patient suffering from GS, who declared strong symptoms when taking gluten. Also for this patient it the differential secretion of CXCL10 (PG, in pg/ml) by the PBMCs stimulated with protein extracts of flour with a high gluten content (Manitoba and Claudio), low gluten content (Kamut and Senatore Cappelli) or gluten-free (mais) was analysed, whereas the rice flour (gluten-free) was used as a control value. As can be seen, at time T1 the patient followed a gluten-free diet for at least 90 days. At time T2 he took placebo (no symptoms). At T1 a he was again kept subjected to a gluten-free diet, and at T1 b he took a gluten load corresponding to 2 plates each containing 100 g of pasta (strong symptoms). With this patient, it was observed that grains with a high gluten content (Manitoba) give more positive answers, whereas grains with a lower gluten content (e.g. Senatore Cappelli) give less positive responses.

Mais gives no response, thereby confirming the fact that the responses are gluten-dependent.

Compared to the method disclosed in WO201 1163258, the method according to the present invention differs both for the starting population and the used stimulus, and above all for what is measured as a response. Chemokines dosed according to the present invention are in fact essentially produced by monocytes and granulocytes (see: Molecular mechanisms underlying the synergistic induction of CXCL10 by LPS and IFN-gamma in human neutrophils. N. Tamassia, F. Calzetti, T. Ear, A. Cloutier, S. Gasperini, F. Bazzoni, PP. McDonald , MA. Cassatella, Eur J Immunol. 2007 Sep, 37; 9 :2627-34), whereas according to WO2011 163258 dosed cytokines are obtained from lymphocytes. Furthermore, in the present invention total protein extracts from food are used, whereas according to WO2011 163258 synthetic peptides with known sequences are used.

A kit according to the present invention preferably comprises:

- a 96 wells multiwell plate with plastic or PVDF bottom and total proteins extracted from flour with and without gluten pre-adsorbed on the bottom of each well (as in the diagram of Figure 5);

- a first pack of the polymer solution for the separation of PMBCs by using a density gradient, example, g. a 100 ml bottle of Ficoll ® ;

- a second package, e. g. a bottle of RPMI 1640 medium containing 10% FBS and antibiotics, and

- 200 eppendorf tubes (0.5 ml).

For carrying out a diagnostic method according to the present invention with the kit set out above, one proceeds as follows:

Step 1 : Separation of PBMCs

5 ml of whole blood are diluted with 5 ml of PBS, as shown in the first image on the left of Figure 5.

The blood diluted in PBS on 5 ml of Ficoll ® (second image from the left in Figure 5) is stratified. And then centrifuged for 30 minutes at 900 r / min, the centrifuge being set at "with no brake" mode. The ring of PBMC as shown in the third image from the left in Figure 5 is collected, and transferred to a new tube containing 10 ml of PBS to wash the PBMCs, and centrifuged at 500g/min for 15 minutes, with no brake.

The previous step is repeated, while discarding the supernatant and washing again the pellet in 10 ml of PBS. Then one centrifuges for 15 minutes at 500g/min "with no brake".

The pellet is re-suspended in 1 ml of complete medium RPMI 1640 (supplied in the kit). Step 2: Seeds of PBMC

The cells are counted with a counting chamber. RPMI 1640 is added to achieve the concentration of 300,000 cells per ml for each patient. While maintaining the cell suspension obtained in the previous phase under agitation, seed 200μΙ of PBMCs are seeded in each of the six wells relating to the patient, according to the scheme shown in Figure 6.

In Figure 6 the loading scheme of a 96-well multiwell plate included in the kit is shown. The wells marked with M have proteins extracted from flour with gluten (Manitoba) adsorbed on the bottom, those marked with R have proteins extracted from gluten-free rice flour were adsorbed on the bottom. Whereas those marked with B have no protein adsorbed on the bottom of the well. The kit allows PBMCs from 16 patients (indicated by the numbers 1-16) to be loaded.

Step 3: Incubation and dosage of CXCL10

Once seeding is complete, incubation of the multiwell plate is carried out for 16 hours in a humidified incubator with 5% C02 with moderate agitation. The content of the wells is collected and transferred to 0.5 ml eppendorf tubes. Centrifugation is carried out for 10 minutes at 100 r/min to eliminate the PBMCs. The supernatant is collected and stored at -80 °C until dosage is carried out (120 days maximum), or dosage is carried out immediately.

Chemokines (CXCL10) are dosed with a high-sensitivity method (Elisa, Luminex, RIA) in each collected supernatant.

Step 4: Analysis of results

Stimulation of CXCL10 from protein extracts containing gluten:

Patient 1 = (average values M1) - (average values R1)

The values are deemed to be:

- positive, if the result exceeds the maximum threshold value of 100 pg / ml;

- borderline for values from 50 to 100 pg / ml,

- negative, if the result is lower than the minimum threshold value of 50 pg / ml.

The values of B act as controls and must be lower than the minimum threshold value of 50 pg / ml.

Higher values of B indicate that the production of CXCL10 in that patient is high even in the absence of protein extracts, and thus they make the result unreliable for that patient.

The above described invention is susceptible to numerous modifications and variations within the protection scope as defined by the claims.

Claims

1. A method of extracorporeal diagnosis of food sensitivity or intolerance to gluten in a patient, comprising the following steps in sequence:

- providing a sample of peripheral blood from the patient, or from mononuclear cells (PBMC) peripheral blood derived from the patient's blood;

- adding at least one food containing gluten to a portion of said peripheral blood sample;

- adding at least one gluten-free protein extract to at least another portion of said peripheral blood sample;

- letting said thus added portions of said sample to rest for a predetermined time;

- measuring the amount of at least one chemokine produced in said samples after said resting interval, said at least one chemokine being selected from the group comprising CXCL10 (IP10), CXCL9 (MIG) and CXCL1 1 (l-TAC);

- determining a reference value (PG) related to the chemokine production caused by gluten, based on said amount of chemokine produced in said portions of said sample, and

- checking whether said reference value (PG) is greater than a maximum threshold value or lower than a minimum threshold value, or is an intermediate value.

2. A method according to claim"! , is characterized in that said food is a total protein extract from at least one food.

3. A method according to claim 2, characterized in that at least one food comprises flour.

4. A method according to claim 3, characterized in that said flour comprises cereal flour containing gluten.

5. A method according to any preceding claim, characterized in that said peripheral blood sample is an extract of venous blood containing monocytes and granulocytes.

6. A method according to any preceding claim, characterized in that said peripheral blood mononuclear cells are obtained by means of a density gradient separation?. A method according to any preceding claim, characterized in that said sample during the resting interval is incubated and the amount of at least one chemokine produced in the incubated sample is measured.

8. A method according to claim 7, characterized in that it comprises:

providing said mononuclear cells extracted from peripheral blood (PBMC) in a culture medium;

adding heat-inactivated foetal bovine serum and at least one antibiotic and protein extract from foods to the culture medium;

incubating the said culture medium in a temperature-controlled incubator; centrifuging the incubation product, and

quantifying the inflammatory chemokines been separated following the centrifugation in the supernatant.

9. A method according to claim 8, characterized in that said at least one antibiotic is selected from the group comprising penicillin and streptomycin.

10. A method according to claim 8 or 9, characterized in that approximately 300,000 PBMC cells are used in approximately 1.5 ml of culture medium.

11. A method according to any claim, characterized in that at least one gluten-free protein extract is obtained from rice flour or mais flour.

12. A kit for carrying out the method according to any claim 1 to 11 , characterized in that it comprises: - at least one multiwell plate with plastic or PVDF bottom, some of such wells having total proteins extracted from flour with gluten pre-adsorbed on the bottom, whereas other wells have total proteins extracted from gluten-free flours pre- adsorbed on the bottom;

- at least a first pack of synthetic branched high-molecular weight polymer solution for the purification of PMBCs;

- at least one second pack of RPMI 1640 medium containing 10% FBS and at least one antibiotic, and

- a plurality of tubes.

13. A kit according to claim 12, characterized in that at least one first pack is a 100 ml bottle.

14. A kit according to claim 12 or 13, characterized in that at least one first pack is a synthetic branched copolymer having a relatively high molecular weight, being water-soluble and synthesized from sucrose and epichlorohydrin.

15. A kit according to any claim 12, 13 or 14, characterized in that each tube is of eppendorf-type.