ITVR20120176A1 - METHOD AND KIT FOR DIAGNOSIS AND SENSITIVITY MONITORING AND INTOLERANCES IN FOOD - Google Patents
METHOD AND KIT FOR DIAGNOSIS AND SENSITIVITY MONITORING AND INTOLERANCES IN FOOD Download PDFInfo
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Classifications
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
- G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
- G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
- G01N33/5047—Cells of the immune system
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6863—Cytokines, i.e. immune system proteins modifying a biological response such as cell growth proliferation or differentiation, e.g. TNF, CNF, GM-CSF, lymphotoxin, MIF or their receptors
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/52—Assays involving cytokines
- G01N2333/521—Chemokines
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/02—Nutritional disorders
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- Biomedical Technology (AREA)
- Urology & Nephrology (AREA)
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- Medicinal Chemistry (AREA)
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- Tropical Medicine & Parasitology (AREA)
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Description
METODO E KIT PER LA DIAGNOSI ED IL MONITORAGGIO DI SENSIBILITÀ ED INTOLLERANZE AGLI ALIMENTI. METHOD AND KIT FOR DIAGNOSIS AND MONITORING OF SENSITIVITY AND INTOLERANCES TO FOOD.
La presente invenzione riguarda un metodo e un kit per la diagnosi ed il monitoraggio di sensibilità ed intolleranze agli alimenti, più in particolare al glutine. The present invention relates to a method and a kit for the diagnosis and monitoring of sensitivities and intolerances to foods, more particularly to gluten.
Stato della tecnica State of the art
La maggior parte dei gastroenterologi concorda sulla possibilità che possano esistere sensibilità personali ad alcuni alimenti, probabilmente collegate a fenomeni di immunità innata. Queste sensibilità sono diverse dalle intolleranze e dalle reazioni avverse agli alimenti, che sono evidenziabili mediante test accettati dalla scienza ufficiale (immunologici, biochimici ed istologici). Ad oggi, la loro esistenza non à ̈ appurabile con certezza, né à ̈ possibile diagnosticarle con metodiche convenzionali. Most gastroenterologists agree on the possibility that there may be personal sensitivities to certain foods, probably related to innate immunity phenomena. These sensitivities are different from intolerances and adverse reactions to foods, which are evidenced by tests accepted by official science (immunological, biochemical and histological). To date, their existence cannot be ascertained with certainty, nor is it possible to diagnose them with conventional methods.
Per queste sensibilità esistono quindi soltanto metodiche non convenzionali, non accettate dalla comunità scientifica, che hanno riproducibilità molto scadenti, quali ad esempio: il test EAV di Voll od il Cytotest<TM>. For these sensitivities there are therefore only unconventional methods, not accepted by the scientific community, which have very poor reproducibility, such as: the Voll EAV test or the Cytotest <TM>.
Di tutte queste sensibilità presunte, l’unica ad essere stata accertata à ̈ quella al glutine, che à ̈ stata definita recentemente come sensibilità al glutine o “gluten sensitivity†(si veda “Spectrum of gluten-related disorders: consensus on new nomenclature and classification†di Sapone A. et al. BMC Med. 2012;7:10-13). I pazienti affetti da questa patologia sono definiti come sensibili al glutine o “gluten sensitive†o GS. Of all these presumed sensitivities, the only one to have been ascertained is that of gluten, which has recently been defined as gluten sensitivity or â € œgluten sensitivityâ € (see â € œSpectrum of gluten-related disorders: consensus on new nomenclature and classificationâ € by Sapone A. et al. BMC Med. 2012; 7: 10-13). Patients with this pathology are defined as gluten sensitive or â € œgluten sensitiveâ € or GS.
La sensibilità al glutine à ̈ stata definita come una condizione di intolleranza alla gliadina e alla glutenina presenti nei cereali. Per lungo tempo si à ̈ discusso sull’esistenza o meno di questa patologia, nella quale si riscontra una perdita di tolleranza verso tali proteine (o loro frammenti digeriti) con una sintomatologia soltanto in parte simile alla malattia celiaca, che tuttavia si può esplicitare anche a livello extraintestinale. Sensitivity to gluten has been defined as a condition of intolerance to gliadin and glutenin present in cereals. For a long time there has been discussion about the existence or otherwise of this pathology, in which there is a loss of tolerance towards these proteins (or their digested fragments) with symptoms only partially similar to celiac disease, which however can be explained also at the extraintestinal level.
La sensibilità al glutine, per molto tempo considerata una reazione psicosomatica, à ̈ divenuta una patologia accertabile solo recentemente, in seguito a studi in “doppio cieco†che ne hanno dimostrato l’esistenza (si veda “Gluten causes gastrointestinal symptoms in subjects without celiac disease: a double-blind randomized placebo-controlled trial†di Biesiekierski JR et al., Am J Gastroenterol.2011;106:508-14). Sensitivity to gluten, for a long time considered a psychosomatic reaction, has only recently become a detectable pathology, following â € œdouble blindâ € studies that have shown its existence (see â € œGluten causes gastrointestinal symptoms in subjects without celiac disease: a double-blind randomized placebo-controlled trialâ € by Biesiekierski JR et al., Am J Gastroenterol. 2011; 106: 508-14).
Per studi o analisi in “doppio cieco†, come à ̈ noto, si intendono indagini effettuate somministrando a pazienti sostanze, delle quale né i medici curanti né i pazienti conoscono la composizione, sebbene, naturalmente, le sostanze siano identificate da un codice o sigla, che consenta, alla fine del trattamento, di appurare le sostanze somministrate, mentre per studi o analisi in “cieco†, si intendono invece indagini effettuate somministrando a pazienti sostanze, di cui solo i medici curanti conoscono la composizione. By `` double-blind '' studies or analyzes, as is known, we mean investigations carried out by administering to patients substances, of which neither the treating physicians nor the patients know the composition, although, of course, the substances are identified by a code or abbreviation, which allows, at the end of the treatment, to ascertain the substances administered, while for studies or analyzes in `` blind '', we mean investigations carried out by administering substances to patients, of which only the treating doctors know the composition.
Recentemente, la comunità scientifica ha accettato la gluten sensitivity come condizione patologica distinta dalla malattia celiaca in base ad indagini istopatologiche e sierologiche (si veda “Spectrum of gluten-related disorders: consensus on new nomenclature and classification†di Sapone A. et al. BMC Med.2012;7:10-13). Recently, the scientific community has accepted gluten sensitivity as a pathological condition distinct from celiac disease on the basis of histopathological and serological investigations (see â € œSpectrum of gluten-related disorders: consensus on new nomenclature and classificationâ € by Sapone A. et al. BMC Med. 2012; 7: 10-13).
La patogenesi della sensibilità al glutine risulta ad oggi ancora sconosciuta (si veda “New understanding of gluten sensitivity†di Volta U, D e Giorgio R. - Nat Rev Gastroenterol Hepatol. 2012;9:295-9). In uno studio condotto su celiaci, pazienti GS e controlli sani, à ̈ stato osservato che la permeabilità intestinale dei pazienti celiaci appare notevolmente aumentata rispetto ai GS, nei quali invece risulta normale (si veda “Divergence of gut permeability and mucosal immune gene expression in two gluten-associated conditions: celiac disease and gluten sensitivity†di Sapone A, Fasano A. et al. BMC Med. 2011;9:23). Nello stesso studio à ̈ stato evidenziato che i soggetti GS mostrano un aumento dell’espressione di alcune molecole di adesione espresse dalle cellule intestinali, come claudina-1, ZO-1 e claudina-4, e che l’espressione del marker di immunità innata denominato Toll-like receptor 2 risulta alterata nella mucosa intestinale di pazienti GS. The pathogenesis of gluten sensitivity is still unknown today (see â € œNew understanding of gluten sensitivityâ € by Volta U, D and Giorgio R. - Nat Rev Gastroenterol Hepatol. 2012; 9: 295-9). In a study conducted on celiacs, GS patients and healthy controls, it was observed that the intestinal permeability of celiac patients appears significantly increased compared to GS, in which instead it is normal (see â € œDivergence of gut permeability and mucosal immune gene expression in two gluten-associated conditions: celiac disease and gluten sensitivityâ € by Sapone A, Fasano A. et al. BMC Med. 2011; 9: 23). In the same study it was shown that GS subjects show an increase in the expression of some adhesion molecules expressed by intestinal cells, such as claudin-1, ZO-1 and claudin-4, and that the expression of the innate immunity referred to as Toll-like receptor 2 is altered in the intestinal mucosa of GS patients.
Infine, l’espressione del marker denominato FOXP3 sui linfociti T regolatori, à ̈ stato trovato diminuito nei pazienti GS. Nessun altro marker identificativo risulta essere stato fino ad ora proposto per la diagnosi della gluten sensitivity. Finally, the expression of the marker named FOXP3 on regulatory T lymphocytes was found to be decreased in GS patients. Until now, no other identifying marker has been proposed for the diagnosis of gluten sensitivity.
La sintomatologia della sensibilità al glutine risulta alle volte sovrapponibile a quella della malattia celiaca e dell’allergia al glutine e si risolve con l’interruzione dell’assunzione del glutine. I sintomi principalmente riportati da pazienti affetti dalla sensibilità al glutine sono: dolore addominale (68%), eczema e/o eruzione cutanea (40%), cefalea (35%), mente annebbiata (34%), affaticamento (33%), diarrea (33%), depressione (22 %), anemia (20%), sensazione di intorpidimento alle gambe, alle braccia o alle dita (20%); e dolori articolari (11%) (si veda “Spectrum of gluten-related disorders: consensus on new nomenclature and classification†di Sapone A. et al. BMC Med.2012;7:10-13). The symptomatology of gluten sensitivity is sometimes similar to that of celiac disease and gluten allergy and is resolved with the interruption of gluten intake. Symptoms mainly reported by patients with gluten sensitivity are: abdominal pain (68%), eczema and / or rash (40%), headache (35%), clouded mind (34%), fatigue (33%), diarrhea (33%), depression (22%), anemia (20%), feeling of numbness in the legs, arms or fingers (20%); and joint pain (11%) (see â € œSpectrum of gluten-related disorders: consensus on new nomenclature and classificationâ € by Sapone A. et al. BMC Med. 2012; 7: 10-13).
L’attuale diagnosi della gluten sensitivity viene effettuata esclusivamente utilizzando criteri di esclusione di altre patologie ovvero: The current diagnosis of gluten sensitivity is carried out exclusively using criteria for the exclusion of other pathologies, namely:
- l’esclusione di meccanismi allergici tipici delle allergie al glutine (test immuno-allergici al grano negativi); - the exclusion of allergic mechanisms typical of gluten allergies (negative immuno-allergic tests to wheat);
- l’esclusione dei meccanismi autoimmuni tipici della celiachia (anti –EMA e/o anti tTG negativi); e - the exclusion of the autoimmune mechanisms typical of celiac disease (anti - EMA and / or anti tTG negative); And
- l’esclusione di danni alla mucosa duodenale tipici della celiachia (istopatologia duodenale nella norma). - the exclusion of damage to the duodenal mucosa typical of celiac disease (normal duodenal histopathology).
Inoltre, i pazienti definiti GS devono mostrare una regressione dei sintomi dal momento in cui à ̈ stata loro introdotta una dieta senza glutine o gluten free e la loro ricomparsa quando viene loro fornito (in cieco) un carico di glutine. Furthermore, patients defined as GS must show a regression of symptoms from the moment they were introduced to a gluten-free or gluten-free diet and their reappearance when they are provided (blinded) with a load of gluten.
Come per le altre patologie collegate all’assunzione di glutine, una dieta priva di queste proteine, che prevede cioà ̈ l’eliminazione dall’alimentazione dei cereali contenenti glutine e di tutti i loro derivati, risulta essere ad oggi la miglior soluzione terapeutica. Tuttavia, per la sensibilità al glutine la dieta gluten free risulta efficace anche se meno severa rispetto a quella prescritta ai celiaci, i quali devono evitare anche eventuali contaminazioni incrociate o cross-contaminations da alimenti contenenti glutine. Si ritiene che, dopo un periodo di dieta priva di glutine di 90-120 giorni, si possa tentare una reintroduzione graduale di glutine, riattivando così una sorta di tolleranza che permette di evitare diete aglutinate che durino tutta la vita (si veda “Spectrum of gluten-related disorders: consensus on new nomenclature and classification†di Sapone A. et al. BMC Med. As for the other pathologies connected to the intake of gluten, a diet free of these proteins, which provides for the elimination from the diet of cereals containing gluten and all their derivatives, is currently the best solution therapeutic. However, for gluten sensitivity, the gluten free diet is effective even if less severe than that prescribed for celiacs, who must also avoid any cross-contamination or cross-contamination from foods containing gluten. It is believed that, after a period of 90-120 days of a gluten-free diet, a gradual reintroduction of gluten can be attempted, thus reactivating a kind of tolerance that allows avoiding gluten-free diets that last a lifetime (see â € œSpectrum of gluten-related disorders: consensus on new nomenclature and classificationâ € by Sapone A. et al. BMC Med.
2012;7:10-13). 2012; 7: 10-13).
La domanda internazionale WO2011163258 insegna un metodo per diagnosticare e monitorare malattie e sensibilità alimentari, il quale à ̈ basato sull’utilizzo di peptidi sintetici a sequenza nota e la risposta dei linfociti estratti dal paziente a questi antigeni sintetici. La risposta viene valutata come proliferazione linfocitaria o migrazione linfocitaria e comprende anche la misurazione della secrezione di citochine comprese nel gruppo: IL-1b, IL-4, IL-6, IL-8, IL-10,IL-13, MCP-1, G-CSF, IFN-g eTNFa. The international application WO2011163258 teaches a method for diagnosing and monitoring food diseases and sensitivities, which is based on the use of synthetic peptides of known sequence and the response of the lymphocytes extracted from the patient to these synthetic antigens. The response is evaluated as lymphocyte proliferation or lymphocyte migration and also includes the measurement of the secretion of cytokines included in the group: IL-1b, IL-4, IL-6, IL-8, IL-10, IL-13, MCP-1 , G-CSF, IFN-g and TNFa.
Non à ̈ stato, peraltro, finora suggerito uno strumento diagnostico più oggettivo della sintomatologia, che indichi se la reintroduzione di glutine sia tollerata bene o no dal singolo paziente. However, a more objective diagnostic tool than symptoms has not been suggested so far, which indicates whether the reintroduction of gluten is tolerated well or not by the individual patient.
Scopo principale della presente invenzione à ̈ quello di fornire un metodo per una diagnosi obiettiva ed il monitoraggio di sensibilità ed intolleranze alimentari, in particolare la sensibilità al glutine. The main purpose of the present invention is to provide a method for an objective diagnosis and monitoring of food sensitivities and intolerances, in particular sensitivity to gluten.
Un altro scopo della presente invenzione à ̈ quello di fornire un metodo come sopra indicato che sia al contempo altamente affidabile e di facile realizzazione. Another object of the present invention is to provide a method as indicated above which is both highly reliable and easy to implement.
Un altro scopo della presente invenzione à ̈ quello di fornire un kit per la realizzazione del metodo per diagnosticare e monitorare sensibilità ed intolleranze alimentari, in particolare la sensibilità al glutine, secondo la presente invenzione. Another object of the present invention is to provide a kit for carrying out the method for diagnosing and monitoring food sensitivities and intolerances, in particular sensitivity to gluten, according to the present invention.
Secondo un primo aspetto della presente invenzione si fornisce un metodo di diagnosi extracorporeo della sensibilità od intolleranza alimentare di un paziente, comprendente le seguenti fasi in sequenza: According to a first aspect of the present invention, an extracorporeal diagnosis method of the food sensitivity or intolerance of a patient is provided, comprising the following sequential steps:
- additivare almeno un alimento ad un campione di sangue periferico derivato dal paziente o di cellule mononucleate da sangue periferico (PBMC) derivanti dal paziente; - add at least one food to a sample of patient-derived peripheral blood or patient-derived peripheral blood mononuclear cells (PBMC);
- lasciare a riposo il campione così additivato per un predeterminato intervallo di tempo; e - leave the sample thus added to stand for a predetermined time interval; And
- misurare la quantità di almeno una chemochina prodotta nel campione dopo l’intervallo di riposo. - measure the quantity of at least one chemokine produced in the sample after the rest interval.
Vantaggiosamente, il metodo à ̈ per la diagnosi della sensibilità al glutine e l’alimento contiene glutine. Advantageously, the method is for the diagnosis of gluten sensitivity and the food contains gluten.
Ancora più vantaggiosamente, l’almeno una chemochina à ̈ scelta dal gruppo costituito da CXCL10 (IP10), CXCL9 (MIG) e CXCL11 (I-TAC). Even more advantageously, at least one chemokine is chosen from the group consisting of CXCL10 (IP10), CXCL9 (MIG) and CXCL11 (I-TAC).
Preferibilmente, l’alimento à ̈ un estratto proteico totale da almeno un alimento. Preferably, the food is a total protein extract from at least one food.
Secondo un altro aspetto della presente invenzione si fornisce un kit per la realizzazione di un metodo secondo la presente invenzione, comprendente le seguenti fasi: According to another aspect of the present invention, a kit is provided for carrying out a method according to the present invention, comprising the following steps:
- almeno una piastra a più pozzetti (multiwell) con fondo in PVDF, alcuni di tali pozzetti presentando proteine totali estratte da farine con glutine preadsorbite sul fondo, mentre altri pozzetti presentano proteine totali estratte da farine senza glutine pre-adsorbite sul fondo; - at least one multi-well plate (multiwell) with a PVDF bottom, some of these wells having total proteins extracted from gluten-free flours pre-absorbed on the bottom, while other wells have total proteins extracted from gluten-free flours pre-adsorbed on the bottom;
- almeno una prima confezione; - at least one first pack;
- almeno una seconda confezione di terremo RPMI 1640 contenente FBS al 10% ed almeno un antibiotico; e - at least a second pack of RPMI 1640 terremo containing 10% FBS and at least one antibiotic; And
- una pluralità di provette. - a plurality of test tubes.
Ulteriori aspetti e vantaggi della presente invenzione appariranno maggiormente dalla seguente descrizione dettagliata di specifici esempi di realizzazione di un metodo di diagnosi, la descrizione essendo fatta con riferimento agli uniti disegni, nei quali: Further aspects and advantages of the present invention will become more apparent from the following detailed description of specific examples of embodiment of a diagnostic method, the description being made with reference to the accompanying drawings, in which:
- la Figura 1 Ã ̈ un diagramma che illustra risultati di prove condotte su campioni di sangue periferico di pazienti sani e di pazienti GS, valutando, quale cemochina, la CXCL10; - Figure 1 is a diagram illustrating the results of tests conducted on peripheral blood samples from healthy patients and GS patients, evaluating CXCL10 as a cemoquine;
- la Figura 2 mostra risultati di prova condotti su un paziente non GS trattato con bustine contenente farine con glutine o placebo; - Figure 2 shows test results conducted on a non-GS patient treated with sachets containing flours with gluten or placebo;
- la Figura 3 mostra i risultati ottenuti per un paziente border-line trattato con bustine contenente farine con glutine o placebo; - Figure 3 shows the results obtained for a border-line patient treated with sachets containing flours with gluten or placebo;
- la Figura 4 mostra i risultati ottenuti in un paziente GS trattato con bustine contenente farine con glutine o placebo; - Figure 4 shows the results obtained in a GS patient treated with sachets containing flours with gluten or placebo;
- la Figura 5 mostra schematicamente il metodo o sequenza operativa mediante un kit secondo la presente invenzione; e Figure 5 schematically shows the method or operating sequence by means of a kit according to the present invention; And
- la Figura 6 illustra lo schema di caricamento di una piastra multiwell fornita in un kit secondo la presente invenzione. Figure 6 illustrates the loading scheme of a multiwell plate supplied in a kit according to the present invention.
Un metodo secondo la presente invenzione à ̈ un metodo extracorporeo che si basa sulla valutazione delle risposte immunologiche di cellule mononucleate di sangue periferico (PBMC) esposte in vitro al contatto con estratti proteici, di preferenza estratti proteici totali, di alimenti di cui si suppone sussista sensibilità od intolleranza. Gli stessi estratti vengono messi in contatto diretto con le PBMC solubilizzando le proteine totali estratte nel mezzo di coltura in cui vengono tenute le PBMC (proteine in sospensione) oppure facendo aderire le proteine totali ad un substrato, ad esempio un substrato di PVDF (polivinildenfluoruro) o nitrocellulosa o materiali plastici capaci di trattenere le proteine, che si trovano sul fondo dei pozzetti in cui vengono successivamente tenute le PBMC. A method according to the present invention is an extracorporeal method which is based on the evaluation of the immunological responses of peripheral blood mononuclear cells (PBMCs) exposed in vitro to contact with protein extracts, preferably total protein extracts, of foods of which it is supposed to exist. sensitivity or intolerance. The same extracts are put in direct contact with the PBMCs by solubilizing the total proteins extracted in the culture medium in which the PBMCs (proteins in suspension) are kept or by adhering the total proteins to a substrate, for example a substrate of PVDF (polyvinyldenfluoride). o nitrocellulose or plastic materials capable of retaining proteins, which are found at the bottom of the wells in which the PBMCs are subsequently kept.
Si fanno quindi incubare per 4 - 24 ore le PBMC in presenza degli estratti proteici. Il tempo di incubazione dipende dalla sensibilità della metodica di dosaggio delle chemochine di cui si dispone. Infatti le PBMC di pazienti GS, a contatto con gli estratti proteici, producono chemochine che si accumulano nel terreno di coltura delle PBMC. Dopo l’incubazione con questi estratti, si valutano le risposte immunologiche delle PBMC attraverso il dosaggio delle chemochine secrete da parte delle PBMC esposte agli estratti proteici totali degli alimenti oggetto di analisi. The PBMCs are then incubated for 4 - 24 hours in the presence of the protein extracts. The incubation time depends on the sensitivity of the chemokine assay method available. In fact, the PBMCs of GS patients, in contact with the protein extracts, produce chemokines that accumulate in the PBMC culture medium. After incubation with these extracts, the immunological responses of the PBMCs are evaluated by measuring the secreted chemokines by the PBMCs exposed to the total protein extracts of the foods under analysis.
L’Esempio 1 riportato in seguito fa riferimento ad un tempo di incubazione di 16 ore, ossia un valore intermedio tra i due valori limite, tempo dopo il quale la quantità di chemochine che si accumulano nel terreno di coltura à ̈ dosabile con sistemi Elisa o Luminex<TM>, che hanno sensibilità dell'ordine dei 10 pg/ml. Tempi più lunghi possono essere utilizzati per dosaggi con metodiche meno sensibili, tempi più brevi possono essere utilizzati abbinati a metodiche di dosaggio più sensibili. Example 1 below refers to an incubation time of 16 hours, that is an intermediate value between the two limit values, time after which the quantity of chemokines that accumulate in the culture medium can be measured with Elisa systems o Luminex <TM>, which have a sensitivity of the order of 10 pg / ml. Longer times can be used for dosages with less sensitive methods, shorter times can be used combined with more sensitive dosage methods.
Questo metodo à ̈ stato testato e messo a punto per la sensibilità al glutine, utilizzando estratti proteici di farine contenenti glutine, ma può essere facilmente adottato per qualunque alimento, (quali farine, carni, frutta, verdura, latte e loro derivati), da cui si possano ottenere estratti proteici totali. This method has been tested and developed for gluten sensitivity, using protein extracts of gluten-containing flours, but it can be easily adopted for any food (such as flours, meats, fruit, vegetables, milk and their derivatives), from which can be obtained total protein extracts.
Con elevato grado di probabilità , anche perché non si vedono controindicazioni di sorta, il metodo sopra descritto à ̈ applicabile pure ad altri tipi di sensibilità (ad esempio: la sensibilità al pomodoro, alla soya, ecc.) o intolleranze alimentari. With a high degree of probability, also because there are no contraindications whatsoever, the method described above is also applicable to other types of sensitivity (for example: sensitivity to tomato, soya, etc.) or food intolerances.
Attualmente, non à ̈ stato finora possibile testare il metodo di cui sopra per altri tipi di sensibilità , perché non sono ancora stati definiti i criteri diagnostici per sensibilità diverse dalla sensibilità al glutine. Currently, it has not so far been possible to test the above method for other types of sensitivities, because diagnostic criteria for sensitivities other than gluten sensitivity have not yet been defined.
Il metodo secondo la presente invenzione à ̈ volto ad identificare alcuni marcatori della sensibilità o delle intolleranze alla componente proteica degli alimenti (tipicamente la sensibilità al glutine o la celiachia), nonché a mettere a punto un test in forma di kit, per la diagnosi e la terapia di queste sensibilità . The method according to the present invention is aimed at identifying some markers of sensitivity or intolerance to the protein component of foods (typically sensitivity to gluten or celiac disease), as well as to develop a test in the form of a kit, for the diagnosis and the therapy of these sensitivities.
Il metodo secondo la presente invenzione si basa sulla purificazione delle cellule mononucleate (PBMC) ottenute da un prelievo di sangue periferico del paziente. Le cellule PBMC così ottenute vengono messe a diretto contatto, in vitro, con estratti proteici totali di alimenti, quali farine ad elevato tenore di glutine (come la farina Manitoba, farina Claudio) ed estratti totali di alimenti privi di glutine (come la farina di riso e la farina di mais). Indi si misurano le variazioni di secrezione differenziale di chemochine, che vengono prodotte dalle cellule PBMC come conseguenza del loro contatto diretto con le proteine alimentari contenenti il glutine. Le chemochine, la cui secrezione può risultare alterata in seguito al contatto delle cellule PBMC con le proteine alimentari come il glutine, sono le seguenti: The method according to the present invention is based on the purification of the mononuclear cells (PBMC) obtained from a peripheral blood sample from the patient. The PBMC cells thus obtained are put in direct contact, in vitro, with total protein extracts of foods, such as flours with a high gluten content (such as Manitoba flour, Claudio flour) and total extracts of gluten-free foods (such as flour of rice and corn flour). The variations in the differential secretion of chemokines, which are produced by PBMC cells as a consequence of their direct contact with food proteins containing gluten, are then measured. The chemokines, whose secretion can be altered following the contact of PBMC cells with food proteins such as gluten, are the following:
- CXCL10 (IP10); - CXCL10 (IP10);
- CXCL9 (MIG); e - CXCL9 (MIG); And
- CXCL11 (I-TAC). - CXCL11 (I-TAC).
Tra queste, per quanto riguarda la sensibilità al glutine, la chemochina CXCL10 si à ̈ dimostrata essere molto sensibile, mentre per quanto riguarda la CXCL9 e la CXCL11, esse hanno un’elevata omologia con la CXCL10 e legano lo stesso identico recettore cellulare. E’ quindi molto probabile e verosimile che anche la loro secrezione risulti alterata nelle PBMC dei pazienti sensibili ad un particolare alimento (come il grano) esposti alle proteine estratte da quell’alimento (come il glutine). Among these, as regards the sensitivity to gluten, the chemokine CXCL10 has proved to be very sensitive, while as regards the CXCL9 and CXCL11, they have a high homology with the CXCL10 and bind the exact same cell receptor. It is therefore very probable and likely that their secretion is also altered in the PBMCs of patients sensitive to a particular food (such as wheat) exposed to the proteins extracted from that food (such as gluten).
Il prelievo di sangue, di preferenza, venoso periferico per la realizzazione del metodo extracorporeo secondo la presente invenzione à ̈ stato condotto estraendo, ad esempio, 5 ml di sangue venoso periferico da ciascun paziente che si sottopone al test, in tubi contenenti un anticoagulante, come per esempio EDTA (acido etilendiamminicotetracetico). The blood sampling, preferably peripheral venous blood for carrying out the extracorporeal method according to the present invention was carried out by extracting, for example, 5 ml of peripheral venous blood from each patient who undergoes the test, in tubes containing an anticoagulant, such as EDTA (ethylenediaminicotetraacetic acid).
Per la realizzazione del metodo secondo la presente invenzione, potrebbe essere utilizzato anche sangue arterioso o di altro tipo, purché non contaminato (ad esempio sangue fecale). For carrying out the method according to the present invention, arterial or other blood could also be used, provided it is not contaminated (for example fecal blood).
Si estraggono le proteine totali da farine commerciali secondo il metodo “Osborne fractionation†un metodo ben noto ad un esperto del ramo, modificato secondo van den Broeck et al. per l'estrazione di gliadine e glutenine (si veda “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). Le farine di partenza ad elevato tenore di glutine sono la farina Manitoba (grano tenero) e la farina di grano Claudio (grano duro). Le farine di partenza prive di glutine sono farine di riso e di mais certificate come alimenti per celiaci. Gli estratti proteici così ottenuti vengono conservati sotto forma di pellet essiccati in freezer a -20°C. Total proteins are extracted from commercial flours according to the Osborne fractionation method, a method well known to an expert in the art, modified according to van den Broeck et al. for the extraction of gliadins and glutenins (see â € œA modified extraction protocol enables detection and quantification of celiac disease-related gluten proteins from wheatâ € By van den Broeck et al. - Journal of Chromatography B, 2009; 877: 975â € "982). The starting flours with a high gluten content are Manitoba flour (soft wheat) and Claudio wheat flour (durum wheat). The gluten-free starting flours are rice and corn flours certified as gluten-free foods. The protein extracts thus obtained are stored in the form of pellets dried in the freezer at -20 ° C.
Le cellule mononucleate di sangue periferico (PBMC) si ottengono da una normale estrazione su gradiente di densità , ad esempio in gradiente di Ficoll del sangue prelevato. Per ottenere le PBMC dal sangue periferico si possono utilizzare kit commerciali quali il SepMate<TM>di Voden Spa, o gradienti di Ficoll-Paqueâ„¢, ma non si escludono altre adatte procedure. Come à ̈ noto il Ficoll à ̈ un copolimero sintetico ramificato di alto peso molecolare molto idrosolubile sintetizzato a partire da saccarosio e epicloridrina ed utilizzato per preparare gradienti di densità per la separazione cellulare. Peripheral blood mononuclear cells (PBMCs) are obtained by a normal density gradient extraction, for example in Ficoll gradient of the collected blood. Commercial kits such as SepMate <TM> from Voden Spa, or Ficoll-Paqueâ „¢ gradients can be used to obtain PBMCs from peripheral blood, but other suitable procedures are not excluded. As is known, Ficoll is a highly water-soluble, high molecular weight, synthetic, branched copolymer synthesized starting from sucrose and epichlorohydrin and used to prepare density gradients for cell separation.
Esempio 1 Example 1
Si misero in cultura cellule PBMC provenienti da ogni singolo prelievo utilizzando terreno commerciale RPMI 1640 (Sigma, USA) con aggiunta del 10% di siero bovino fetale scomplementato (un reagente ben noto agli esperti del ramo e comunemente usato per le colture cellulari) ed antibiotici (quale la penicillina 100 U/ml e la streptomicina 100 mg/ml). PBMC cells from each individual sample were cultured using commercial RPMI 1640 medium (Sigma, USA) with the addition of 10% of decomplemented fetal 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).
Le PBMC furono distribuite in 5 pozzetti identici in piastre da 24 pozzetti, ciascun pozzetto contenendo 300.000 cellule e 1,5 ml di terreno completo (RPMI 1640 siero fetale antibiotici). PBMCs were distributed in 5 identical wells in 24-well plates, each well containing 300,000 cells and 1.5 mL of complete medium (RPMI 1640 fetal serum antibiotics).
Al primo pozzetto non si aggiunse nulla (bianco), mentre ai restanti 4 pozzetti furono aggiunti 40 microgrammi di proteine totali, rispettivamente, di Riso, Mais, Manitoba e Claudio, disciolte in 500 microlitri di terreno completo. Nothing (white) was added to the first well, while the remaining 4 wells were added 40 micrograms of total protein, respectively, of Rice, Corn, Manitoba and Claudio, dissolved in 500 microliters of complete medium.
Le piastre da 24 pozzetti furono poste per 16 ore in un incubatore termostatato a CO2(Heraeus-b-5060-ek) in atmosfera controllata, (37°C, 6% di CO2) in leggera agitazione su piatto oscillante. The 24-well plates were placed for 16 hours in a thermostated CO2 incubator (Heraeus-b-5060-ek) in a controlled atmosphere (37 ° C, 6% CO2) under gentle agitation on a rocking plate.
Al termine dell’incubazione il contenuto dei pozzetti venne passato in tubi tipo eppendorf da 2 ml. Le provette eppendorf furono quindi centrifugate a 500 g/min per 10 minuti a 4°C. Al termine della centrifugazione, si prelevò il surnatante o terreno condizionato (1 ml) per trasferirlo in un nuovo tubo tipo eppendorf. Il pellet, ossia l’ammasso semisolido di cellule che si addensano sul fondo delle provette dopo la centrifugazione, contenente le PBMC fu scartato. At the end of the incubation, the contents of the wells were passed into 2 ml eppendorf tubes. The eppendorf tubes were then centrifuged at 500 g / min for 10 minutes at 4 ° C. At the end of the centrifugation, the supernatant or conditioned medium (1 ml) was withdrawn and transferred to a new eppendorf tube. The pellet, ie the semisolid mass of cells that thicken at the bottom of the tubes after centrifugation, containing the PBMCs was discarded.
L’esempio di realizzazione sopra descritto à ̈ stato sviluppato utilizzando piastre da 24 pozzetti e gli estratti proteici sono stati aggiunti in sospensione. E’ altrettanto possibile adottare micropiastre da 96 pozzetti con fondo in PVDF (polivinilidenfluoruro), dove vengono preventivamente adsorbiti ed adesi gli stimoli al fondo del pozzetto. Il test eseguito in questa modalità ha mostrato di fornire identici risultati a quello descritto in precedenza. The embodiment example described above was developed using 24-well plates and the protein extracts were added in suspension. It is also possible to use 96-well microplates with a PVDF (polyvinylidene fluoride) bottom, where the stimuli are previously adsorbed and adhered to the bottom of the well. The test performed in this mode has been shown to give identical results to the one described above.
La quantificazione delle citochine infiammatorie nei terreni condizionati può essere effettuata con ben note tecnologie, quali la tecnologia ELISA, la tecnologia Luminex<TM>, i metodi radioimmunologici (RIA) od altre adatte metodiche quantitative con analoga sensibilità . Gli inventori della presente invenzione hanno utilizzato la tecnologia Luminex<TM>con reagenti forniti dalla BioRad. The quantification of inflammatory cytokines in conditioned media can be performed with well-known technologies, such as ELISA technology, Luminex <TM> technology, radioimmunological methods (RIA) or other suitable quantitative methods with similar sensitivity. The inventors of the present invention used the Luminex <TM> technology with reagents supplied by BioRad.
Il bianco, contenente PBMC in assenza di estratti proteici fornisce il valore basale di secrezione di chemochine da parte delle PBMC, in assenza di estratti proteici contenente glutine. The blank, containing PBMC in the absence of protein extracts provides the basal value of chemokine secretion by PBMCs, in the absence of protein extracts containing gluten.
I pozzetti in cui vengono aggiunti estratti proteici di Riso e Mais costituirono i controlli negativi (stimoli privi di glutine). I pozzetti con Manitoba e Claudio costituirono la produzione di chemochine in presenza di glutine. Il valore di riferimento adottato à ̈ dato dalla formula sottostante: The wells in which Rice and Corn protein extracts are added constituted the negative controls (gluten-free stimuli). The wells with Manitoba and Claudio constituted the production of chemokines in the presence of gluten. The reference value adopted is given by the formula below:
PG = P(manitoba)-P(riso) PG = P (manitoba) -P (rice)
PG = produzione di chemochina causata dal glutine PG = chemokine production caused by gluten
P(manitoba)= produzione di chemochina in presenza di proteine estratte da manitoba P (manitoba) = chemokine production in the presence of proteins extracted from manitoba
P(riso) = produzione di chemochina in presenza di proteine estratte da riso. P (rice) = production of chemokine in the presence of proteins extracted from rice.
Per la chemochina CXCL10 con riferimento alla sensibilità al glutine, in base ai valori ottenuti dai campioni che furono analizzati, si stabilì, sulla base dell'analisi dei risultati che: For the chemokine CXCL10 with reference to the sensitivity to gluten, based on the values obtained from the samples that were analyzed, it was established, on the basis of the analysis of the results that:
PG < 50 pg/ml = paziente negativo per la gluten sensitive (sano) PG <50 pg / ml = patient negative for gluten sensitive (healthy)
PG > 100 = paziente positivo per la gluten sensitive (affetto da GS) PG> 100 = patient positive for gluten sensitive (suffering from GS)
50 ≤ PG ≤100 = paziente border line (al limite) 50 â ‰ ¤ PG â ‰ ¤100 = border line patient (at the limit)
I valori ottenuti per Mais e Claudio furono usati da controlli interni dell’esperimento e devono risultare il primo simile al valore del riso, il secondo simile al valore della manitoba. The values obtained for Mais and Claudio were used by internal controls of the experiment and must be the first similar to the value of the rice, the second similar to the value of the manitoba.
Per un campione di 19 individui sani e 22 pazienti già diagnosticati come GS in base a criteri di esclusione, si realizzò il metodo secondo la presente invenzione. Dosando la chemochina CXCL10 (si veda Figura 1), i risultati mostrano che, mentre le PBMC ottenute dal sangue di controlli sani ed esposti ad estratti proteici contenenti glutine hanno valori di secrezione inferiori a 100 pg/ml e nella maggior parte dei casi inferiori a 50 pg/ml, le PBMC di pazienti affetti da GS esposti al contatto con estratti proteici contenenti glutine hanno mostrato valori di secrezione molto più elevati, con picchi che spesso superano i 250 pg/ml. Inoltre, i dati ottenuti dai dosaggi di CXCL10, applicando un T-test ben noto agli esperti del ramo tra i due gruppi, si sono dimostrati statisticamente molto significativi con valori di P (probabilità di errore) inferiori allo 0,001, come riportato nella sottostante Tabella 1. For a sample of 19 healthy individuals and 22 patients already diagnosed as GS on the basis of exclusion criteria, the method according to the present invention was carried out. By assaying the chemokine CXCL10 (see Figure 1), the results show that, while PBMCs obtained from the blood of healthy controls and exposed to gluten-containing protein extracts have secretion values lower than 100 pg / ml and in most cases lower than 50 pg / mL, PBMCs of GS patients exposed to contact with gluten-containing protein extracts showed much higher secretion values, with peaks often exceeding 250 pg / mL. Furthermore, the data obtained from the assays of CXCL10, applying a T-test well known to those skilled in the art between the two groups, proved to be statistically very significant with P values (probability of error) lower than 0.001, as reported in the table below. 1.
Con riferimento alla Figura 1, le indicazioni “paziente sano†e “paziente GS†erano state stabilite sulla base dell’esito negativo o positivo di altri criteri diagnostici tradizionali. In particolare, se i pazienti lamentavano disturbi a seguito di assunzione di alimenti includenti, tra l’altro, glutine, ma risultavano sani rispetto ad altre malattie o disturbi differenti dalla sensibilità al glutine, allora questi venivano indicati come pazienti GS e viceversa. Sugli stessi soggetti à ̈ stato condotto un test diagnostico secondo la presente invenzione, a seguito del quale sono stati rilevati i valori di chemochina CXCL10 indicati in Figura 1 e quindi si à ̈ valutato che erano: With reference to Figure 1, the indications “healthy patient” and “GS patient” were established on the basis of the negative or positive outcome of other traditional diagnostic criteria. In particular, if patients complained of complaints following the intake of foods including, among other things, gluten, but were healthy compared to other diseases or disorders other than gluten sensitivity, then these were referred to as GS patients and vice versa. A diagnostic test was conducted on the same subjects according to the present invention, following which the CXCL10 chemokine values indicated in Figure 1 were detected and therefore it was estimated that they were:
- sani, vale a dire non sensibili al glutine, i pazienti 1, 3, 4 e da 6 a 19 anche precedentemente ritenuti sani, nonché i pazienti da 4 a 7, 16, 18, 19 e 22 precedentemente ritenuti sensibili al glutine, - healthy, i.e. not sensitive to gluten, patients 1, 3, 4 and 6 to 19 also previously considered healthy, as well as patients 4 to 7, 16, 18, 19 and 22 previously considered sensitive to gluten,
- border line, il paziente 2 precedentemente ritenuto sano, nonché il paziente 14 precedentemente ritenuto sensibile al glutine, e - border line, patient 2 previously considered healthy, as well as patient 14 previously considered sensitive to gluten, e
- sensibili al glutine, il paziente 5 precedentemente ritenuto sano, nonché i pazienti da 1 a 3, da 8 a 13, 15, 17, 20 e 21 precedentemente ritenuti sensibili al glutine. - sensitive to gluten, patient 5 previously considered healthy, as well as patients 1 to 3, 8 to 13, 15, 17, 20 and 21 previously considered sensitive to gluten.
Sugli stessi pazienti si effettuò successivamente una sperimentazione basata sull'assunzione di glutine oppure placebo in doppio cieco, la quale confermò in toto i risultati ottenuti con il metodo secondo la presente invenzione e smentì in buona parte le conclusioni raggiunte sulla base della semplice diagnosi di sensibilità al glutine per esclusione di altre malattie/disturbi. An experiment based on the assumption of gluten or placebo in double blind was subsequently carried out on the same patients, which fully confirmed the results obtained with the method according to the present invention and largely denied the conclusions reached on the basis of the simple diagnosis of sensitivity to gluten by excluding other diseases / disorders.
Tabella 1 Table 1
Analisi statistica (Unpaired T-test) applicata ai valori ottenuti di CXCL10, nel gruppo “sani†confrontati con il gruppo dei pazienti confermati come GS Statistical analysis (Unpaired T-test) applied to the obtained values of CXCL10, in the â € œhealthyâ € group compared with the group of patients confirmed as GS
Il valore di P ottenuto (two-tailed P value) Ã ̈ inferiore a 0.001. The two-tailed P value obtained is less than 0.001.
Gruppo Controlli Sani Pazienti GS GS Patient Health Checks Group
Media 18.30 pg/ml 269,64 pg/ml Average 18.30 pg / ml 269.64 pg / ml
SD 28.12 140,15 SD 28.12 140.15
SEM 6.45 37.46 SEM 6.45 37.46
N 19 14 N 19 14
Per validare ulteriormente il test oggetto della presente invenzione, à ̈ stato effettuato uno studio in cieco, dove i pazienti classificati come GS e controlli sani furono trattati con bustine contenenti o farine con glutine o placebo (farine senza glutine), da aggiungere agli alimenti ai pasti. Lo studio à ̈ stato effettuato in collaborazione con l’Unità di Gastroenterologia (SVD), Prof. Alberto Lanzini e Dott.ssa Chiara RICCI - A. O. Spedali Civili di Brescia, Università degli Studi -Brescia. To further validate the test object of the present invention, a blinded study was carried out, where patients classified as GS and healthy controls were treated with sachets containing either gluten or placebo flours (gluten-free flours), to be added to foods meals. The study was carried out in collaboration with the Gastroenterology Unit (SVD), Prof. Alberto Lanzini and Dr. Chiara RICCI - A. O. Spedali Civili of Brescia, University of Brescia.
I risultati dei campioni analizzati, una volta aperto il cieco, hanno mostrato che il test basato su CXCL10 à ̈ in grado di discriminare con precisione tra controlli sani e pazienti GS e mostra valori fortemente positivi in corrispondenza dell’assunzione di glutine e della comparsa di sintomatologia dichiarata dai pazienti. Hanno mostrato inoltre che le risposte alle diverse farine utilizzate risentono del tenore di glutine presente nelle stesse. The results of the analyzed samples, once the blind was opened, showed that the test based on CXCL10 is able to accurately discriminate between healthy controls and GS patients and shows strongly positive values in correspondence with gluten intake and appearance of symptoms declared by patients. They also showed that the responses to the different flours used are affected by the gluten content present in them.
In Figura 2 si sono illustrati i risultati di prova condotti su un paziente non affetto da GS, il quale ha dichiarato sintomi moderati durante l’assunzione di placebo e sintomi assenti durante l’assunzione di glutine. Più in particolare, nella stessa Figura à ̈ riportata l’analisi della secrezione differenziale di CXCL10 (farina con glutine-riso, in pg/ml) da parte delle PBMC di un paziente possibile GS, che in seguito à ̈ stato classificato non GS. Tale paziente à ̈ stato stimolato con estratti proteici di farine ad elevato tenore di glutine (Manitoba e Claudio), basso tenore di glutine (Kamut e Senatore Cappelli) o prive di glutine (mais). La farina di riso (gluten free) à ̈ stata utilizzata come valore di controllo. Al tempo T1 il soggetto era a dieta senza glutine da almeno 90 giorni, al tempo T2 ha assunto placebo (dichiarando sintomi moderati), al tempo T1a era nuovamente a dieta senza glutine ed al tempo T1b assunse un carico di glutine quotidiano pari a 2 piatti di pasta da 100 g ciascun (dichiarando la scomparsa dei sintomi). Figure 2 shows the results of tests conducted on a patient not suffering from GS, who declared moderate symptoms while taking placebo and absent symptoms while taking gluten. More specifically, the same Figure shows the analysis of the differential secretion of CXCL10 (flour with gluten-rice, in pg / ml) by the PBMCs of a possible GS patient, who was later classified as not GS . This patient was stimulated with protein extracts of flours with a high gluten content (Manitoba and Claudio), low gluten content (Kamut and Senatore Cappelli) or gluten-free (corn). Rice flour (gluten free) was used as a control value. At time T1 the subject had been on a gluten-free diet for at least 90 days, at time T2 he took placebo (declaring moderate symptoms), at time T1a he was again on a gluten-free diet and at time T1b he assumed a daily gluten load equal to 2 dishes of pasta of 100 g each (declaring the disappearance of symptoms).
In Figura 3, sono riportati i risultati ottenuti per un paziente border-line, che ha dichiarato sintomi moderati durante l’assunzione di glutine. Più in particolare, in tale Figure si à ̈ mostrata l’analisi della secrezione differenziale di CXCL10 (manitoba-riso, in pg/ml) da parte delle PBMC di paziente border line GS stimolati con estratti proteici di farine ad elevato tenore di glutine (Manitoba e Claudio), basso tenore di glutine (Kamut e Senatore Cappelli) o prive di glutine (mais). Anche in tale caso, la farina di riso gluten free à ̈ stata utilizzata come valore di controllo. Al tempo T1 il soggetto era a dieta senza glutine da almeno 90 giorni, al tempo T2 assunse placebo (senza sintomi), al tempo T1a era nuovamente a dieta senza glutine ed al tempo T1b assunse un carico di glutine quotidiano pari a 2 piatti di pasta da 100 g ciascuno (lieve sintomatologia). Figure 3 shows the results obtained for a border-line patient, who declared moderate symptoms while taking gluten. More specifically, this Figure shows the analysis of the differential secretion of CXCL10 (manitoba-rice, in pg / ml) by PBMCs of border line GS patients stimulated with protein extracts of high gluten content flours (Manitoba and Claudio), low gluten content (Kamut and Senatore Cappelli) or gluten-free (corn). Again, gluten free rice flour was used as a control value. At time T1 the subject had been on a gluten-free diet for at least 90 days, at time T2 he took placebo (without symptoms), at time T1a he was again on a gluten-free diet and at time T1b he assumed a daily gluten load equal to 2 pasta dishes of 100 g each (mild symptoms).
In Figura 4 sono riportati i risultati ottenuti per un paziente affetto da GS, che ha dichiarato una forte sintomatologia in corrispondenza dell’assunzione di glutine. Anche per questo paziente si à ̈ analizzata la secrezione differenziale di CXCL10 (manitoba-riso, in pg/ml) da parte delle PBMC del paziente stimolato con estratti proteici di farine ad elevato tenore di glutine (Manitoba e Claudio), basso tenore di glutine (Kamut e Senatore Cappelli) o prive di glutine (mais), mentre la farina di riso gluten free à ̈ stata utilizzata come valore di controllo. Come si può osservare, al tempo T1 il soggetto era a dieta senza glutine da almeno 90 giorni, al tempo T2 assunse placebo (senza sintomi), al tempo T1a era nuovamente a dieta senza glutine ed al tempo T1b assunse un carico di glutine quotidiano pari a 2 piatti di pasta da 100 g ciascuno (forte sintomatologia). In quest’ultimo paziente, si nota bene come grani ad elevato tenore di glutine (Manitoba) diano risposte maggiormente positive, mentre grani a minor tenore di glutine (senatore cappelli) danno risposte meno positive. Figure 4 shows the results obtained for a patient suffering from GS, who declared a strong symptomatology in correspondence with gluten intake. Also for this patient the differential secretion of CXCL10 (manitoba-rice, in pg / ml) by the PBMC of the patient stimulated with protein extracts of flours with a high gluten content (Manitoba and Claudio), low gluten content was analyzed (Kamut and Senatore Cappelli) or gluten-free (corn), while gluten-free rice flour was used as a control value. As it can be observed, at time T1 the subject had been on a gluten-free diet for at least 90 days, at time T2 he took placebo (without symptoms), at time T1a he was again on a gluten-free diet and at time T1b he assumed a daily gluten load equal to to 2 pasta dishes of 100 g each (strong symptoms). In this last patient, it is well known that grains with a high gluten content (Manitoba) give more positive responses, while grains with a lower gluten content (Senatore Cappelli) give less positive responses.
Il mais non fornisce alcuna risposta, a conferma del fatto che le risposte ottenute sono glutine-dipendenti. Corn does not provide any answers, confirming that the answers obtained are gluten-dependent.
Rispetto al metodo insegnato da WO2011163258, il metodo oggetto della presente invenzione si differenzia sia per la popolazione di partenza, che per lo stimolo che viene utilizzato e, soprattutto, per ciò che viene misurato come risposta. Le chemochine che si dosano secondo la presente invenzione sono infatti prodotte essenzialmente da monociti e da granulociti (si veda: Molecular mechanisms underlying the synergistic induction of CXCL10 by LPS and IFN-gamma in human neutrophils. di Tamassia N, Calzetti F, Ear T, Cloutier A, Gasperini S, Bazzoni F, McDonald PP, Cassatella MA, Eur J Immunol. 2007 Sep;37(9):2627-34) mentre secondo WO2011163258 le citochine dosate vengono ottenute da linfociti. Inoltre, nella presente invenzione si utilizzano estratti proteici totali da alimenti, mentre secondo WO2011163258 si utilizzano peptidi sintetici a sequenze note. Compared to the method taught by WO2011163258, the method object of the present invention differs both for the starting population, for the stimulus that is used and, above all, for what is measured as a response. The chemokines that are determined according to the present invention are in fact produced essentially by monocytes and granulocytes (see: Molecular mechanisms underlying the synergistic induction of CXCL10 by LPS and IFN-gamma in human neutrophils. Di Tamassia N, Calzetti F, Ear T, Cloutier A, Gasperini S, Bazzoni F, McDonald PP, Cassatella MA, Eur J Immunol. 2007 Sep; 37 (9): 2627-34) while according to WO2011163258 the dosed cytokines are obtained from lymphocytes. Furthermore, in the present invention total protein extracts from foods are used, while according to WO2011163258 synthetic peptides with known sequences are used.
Un kit secondo la presente invenzione comprende, di preferenza: A kit according to the present invention preferably comprises:
- una piastra a più pozzetti (multiwell) da 96 pozzetti con fondo in PVDF e proteine totali estratte da farina con e senza glutine pre-adsorbite sul fondo (come da schema di Figura 5); - a 96-well multiwell plate with PVDF bottom and total proteins extracted from gluten-free and gluten-free flour pre-adsorbed on the bottom (as shown in Figure 5);
- una prima confezione di soluzione di polimero per estrazione di PMBC in gradiente di densità , ad esempio una bottiglia di Ficoll® da 100 ml; - a first package of polymer solution for PMBC extraction in density gradient, for example a 100 ml bottle of Ficoll®;
- una seconda confezione, ad esempio una bottiglia di terreno RPMI 1640 contenente FBS al 10% ed antibiotici; e - a second package, for example a bottle of RPMI 1640 medium containing 10% FBS and antibiotics; And
- 200 provette tipo eppendorf da 0,5 ml. - 200 0.5 ml eppendorf type tubes.
Per la realizzazione di un metodo di diagnosi secondo la presente invenzione con il kit sopra indicato si procede come segue. For the realization of a diagnosis method according to the present invention with the kit indicated above, the procedure is as follows.
Fase 1: Estrazione delle PBMC Phase 1: Extraction of PBMCs
Si diluiscono 5 ml di sangue intero con 5 ml di PBS, come illustrato nella prima immagine a sinistra in Figura 5. 5 ml of whole blood are diluted with 5 ml of PBS, as shown in the first image on the left in Figure 5.
Si stratifica il sangue diluito in PBS su 5 ml di Ficoll® (seconda immagine da sinistra in Figura 5). Si centrifuga per 30 minuti a 900 g/min con centrifuga settata in modalità "senza freno". The blood diluted in PBS is stratified on 5 ml of Ficoll® (second image from the left in Figure 5). It is centrifuged for 30 minutes at 900 g / min with the centrifuge set in "no brake" mode.
Si preleva l’anello di PBMC come illustrato nella terza immagine da sinistra in Figura 5 e lo si trasferisce in una nuova provetta contenente 10 ml di PBS per lavare le PBMC, agitando. Si centrifuga a 500 g/min per 15 minuti senza freno. The PBMC ring is removed as shown in the third image from the left in Figure 5 and transferred to a new tube containing 10 ml of PBS to wash the PBMCs, shaking. It is centrifuged at 500 g / min for 15 minutes without brake.
Si ripetere il passaggio precedente scartando il surnatante e lavando nuovamente il pellet in 10 ml di PBS. Si centrifuga a 500 g/min per 15 minuti con centrifuga settata in modalità "senza freno". Repeat the previous step discarding the supernatant and washing the pellet again in 10 ml of PBS. Spin at 500 g / min for 15 minutes with spin set in "no brake" mode.
Si risospende il pellet in 1 ml di terreno completo RPMI1640 (fornito nel kit). The pellet is resuspended in 1 ml of complete RPMI1640 medium (provided in the kit).
Fase 2: Semina dei PBMC Phase 2: Planting of PBMCs
Si contano le cellule con camera contaglobuli. Si aggiunge RPMI 1640 fino a raggiungere per ogni paziente la concentrazione di 300.000 cellule per ml. Mantenendo in agitazione la sospensione cellulare ottenuta nella fase precedente, si semina per ogni paziente 200µl di sospensione di PBMC in ognuno dei 6 pozzetti relativi al paziente, secondo lo schema indicato in Figura 6. The cells are counted with a blood cell counting chamber. RPMI 1640 is added until the concentration of 300,000 cells per ml is reached for each patient. While stirring the cell suspension obtained in the previous phase, 200µl of PBMC suspension is sown for each patient in each of the 6 wells relating to the patient, according to the scheme shown in Figure 6.
In Figura 6 si à ̈ riportato lo schema di caricamento di una piastra multiwell da 96 pozzetti in dotazione nel kit. I pozzetti contrassegnati con M hanno adsorbite sul fondo proteine estratte da farina con glutine (Manitoba), quelli contrassegnati con R hanno adsorbite sul fondo proteine estratte da farina di riso gluten-free, mentre quelli contrassegnati con B non hanno proteine adsorbite sul fondo del pozzetto. Il kit permette di caricare PBMC provenienti da 16 pazienti (indicati con i numeri 1-16). Figure 6 shows the loading diagram of a 96-well multiwell plate supplied in the kit. The wells marked with M have adsorbed on the bottom proteins extracted from flour with gluten (Manitoba), those marked with R have adsorbed on the bottom proteins extracted from gluten-free rice flour, while those marked with B have no proteins adsorbed on the bottom of the well . The kit allows to load PBMCs from 16 patients (indicated with numbers 1-16).
Fase 3: Incubazione e dosaggio di CXCL10 Step 3: Incubation and assay of CXCL10
Terminata la semina, si incuba per 16 ore in un incubatore umidificato con 5% CO2. After sowing, it is incubated for 16 hours in a humidified incubator with 5% CO2.
si preleva il contenuto dei pozzetti e lo si trasferisce in tubi tipo eppendorf da 0,5 ml. Sii centrifuga a 100 g/min per 10 minuti per eliminare le PBMC. Si raccoglie il surnatante e lo si conserva a -80°C fino al momento del dosaggio (massimo 120 giorni) oppure si procede immediatamente al dosaggio. the contents of the wells are taken and transferred into 0.5 ml eppendorf tubes. Be centrifuged at 100 g / min for 10 minutes to eliminate PBMCs. The supernatant is collected and stored at -80 ° C until the moment of the assay (maximum 120 days) or the assay is carried out immediately.
Si dosa con metodica ad elevata sensibilità (Elisa, Luminex, RIA) il contenuto in CXCL10 dei singoli surnatanti raccolti. The content in CXCL10 of the individual supernatants collected is measured using a highly sensitive method (Elisa, Luminex, RIA).
Fase 4: Analisi dei risultati Phase 4: Analysis of the results
Stimolazione di CXCL10 da estratti proteici contenenti glutine: Stimulation of CXCL10 by gluten-containing protein extracts:
Paziente 1 = (media valori M1)-(media valori R1) Patient 1 = (mean M1 values) - (mean R1 values)
I valori si intendono The values are meant
positivi, se il risultato supera i 100 pg/ml; positive, if the result exceeds 100 pg / ml;
border line per valori tra i 50 ed i 100 pg/ml, border line for values between 50 and 100 pg / ml,
negativi, se il risultato à ̈ inferiore a 50 pg/ml. negative, if the result is less than 50 pg / mL.
I valori di B fungono da controlli e devono risultare inferiori a 50 pg/ml. Valori più elevati di B indicano che la produzione di CXCL10 in quel paziente à ̈ elevata anche in assenza di estratti proteici e rendono, quindi, inattendibile il risultato del test per quel paziente. The B values serve as controls and should be less than 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 therefore make the test result unreliable for that patient.
L’invenzione sopra descritta à ̈ suscettibile di numerose modifiche e varianti entro l’ambito di protezione definito dalle rivendicazioni. The invention described above is susceptible of numerous modifications and variations within the scope of protection defined by the claims.
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| PCT/IB2013/058148 WO2014037858A1 (en) | 2012-09-04 | 2013-08-30 | Method and kit for the diagnosis and monitoring of sensitivity and intolerance to foods |
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| JP6943479B2 (en) * | 2016-07-08 | 2021-09-29 | バイオメリカ・インコーポレイテッドＢｉｏｍｅｒｉｃａ， Ｉｎｃ． | Compositions, devices and methods for depression susceptibility testing |
| CA3057421C (en) * | 2017-05-12 | 2022-10-25 | Laboratory Corporation Of America Holdings | Compositions and methods to detect non-coeliac gluten sensitivity |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| WO2005016954A2 (en) * | 2003-08-14 | 2005-02-24 | Boston Medical Center Corporation | Immunologic activity measure(s) for t “helper” on-associated conditions |
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| US20130089879A1 (en) | 2010-06-21 | 2013-04-11 | Pharmasan Labs, Inc. | Compositions and methods for diagnosing and monitoring disease and treatment via antigen-specific molecules |
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| WO2005016954A2 (en) * | 2003-08-14 | 2005-02-24 | Boston Medical Center Corporation | Immunologic activity measure(s) for t “helper” on-associated conditions |
Non-Patent Citations (3)
| Title |
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| ENZO SPISNI: "Analisi dei meccanismi fisiopatologici alla base della sensibilità al glutine", 19 June 2012 (2012-06-19), XP055059159, Retrieved from the Internet <URL:https://www.aricweb.unibo.it/AssegniRicerca_Richieste/_doc/2012/ID%5B9958%5DRichiesta_Nuovo_Assegno_Spisni_2012.pdf> [retrieved on 20130411] * |
| FOR THE CANADIAN INSTITUTES OF HEALTH RESEARCH NATIONAL TRAINING PROGRAM IN ALLERGY AND ASTHMA RESEARCH THOTTINGAL ET AL: "Human subjects without peanut allergy demonstrate T cell-dependent, TH2-biased, peanut-specific cytokine and chemokine responses independent of TH1 expression", JOURNAL OF ALLERGY AND CLINICAL IMMUNOLOGY, ELSEVIER, AMSTERDAM, NL, vol. 118, no. 4, 1 October 2006 (2006-10-01), pages 905 - 914, XP005685911, ISSN: 0091-6749 * |
| RASHTAK S ET AL: "Gliadin Stimulation of Monocytes Leads to Increased Expression of Multiple T Cell Recruiting Chemokines: A Novel Innate Immune Response", CLINICAL IMMUNOLOGY, ACADEMIC PRESS, US, vol. 135, 1 January 2010 (2010-01-01), pages S47, XP027048943, ISSN: 1521-6616, [retrieved on 20100101] * |
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| WO2014037858A1 (en) | 2014-03-13 |
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