Gluten enteropathy: clinical secrets.

Author: Dr. med.  Kilessa A.V., PhD, gastroenterologist-endoscopist, university clinic of Crimean State Medical University. Crimean state university by S.I.Georgiyevskiy. Professor assistant  of internal medicine dep. No. 2. Simferopol, Ukraine.

Co-author:  Dr.med. Jansen G.W., Prof., gastroenterologist, uniclinic, Internal med. dep.  Dortmund, Germany.





                                                                    GLUTEN ENTEROPATHY:

                                                CLINICAL SECRETS AND NEW DIAGNOSTICS ALGORITHM.



SUMMARY



    This article is the result of studying and diagnosing of patients (218 persons) with the gluten enteropathy diagnosis. It was done by two practicing gastroenterologists from Ukraine and Germany. This article describes atypical clinical implications of a gluten enteropathy and offers certain management helping practicing doctors with definition and specification of the diagnosis. Article contains the description of several cases of atypical clinical implications of gluten enteropathy (GEP) in patients from 0 to 54 years old observed in the central Dortmund's university clinic. Article gives classification of patients with GEP on three groups and two subgroups and list both classical, and atypical clinical implications of a disease. The algorithm of step-by-step diagnosing is offered by main author, based on endoscope studying of duodenal mucosa (n=1814), morphological and immunohistochemical researches of duodenal mucosa (n=324). There was found some interrelation between stomach intestinal metaplasia and gluten enteropathy.



Key words: Gluten enteropathy, atypical implications, diagnostics.



Introduction



The gluten associated enteropathy (GEP) – an autoimmune disease is expressed by a chronic inflammation of small intestine with various degree of a mucosal atrophy.

GEP represents an example of disease where the science and medicine reached considerable results in genetic and clinical diagnostics, but the real problem consists in recognizing asymptomatic and latent forms of disease which lead to potential complications.

Prevalence of GEP was sharply enlarged in the last 10 years due to development of accompanying diseases diagnostic, such as thyreoiditis, autoimmune hepatitis, primary cirrhosis, primary sclerosis cholangitis, a psoriasis, Edison's disease, a pseudo rheumatism, vitiligo, alopecia, and  genetic associated diseases, such as a Down syndrome, Williams's syndrome and Turner's syndrome. The risk of accompanying autoimmune diseases development increases at patients with Marsh II and Marsh III stages. Indicators of a case rate are about 0.5% to 1% in Europe, 0.53% to 1% in North Africa, 0.79% to 1% in the Central and South Africa, 0.6% to 1% in the countries of the Middle East, 0.88% to 1% in Japan, 0.6% to 1% in North America, and 0.7% to 1% in India.

Classical GEP implications begin in infancy and are expressed with diarrhea, an abdominal distention, acute malabsorbtion.  Serological researches show that most patients with gluten enteropathy (about 60% of all diagnosed) have oligosimptomatic, hidden and abenteric forms of implication.  Each misdiagnosed case of GEP is a certain person who remains unattended, that leads to such complications as infertility, osteoporosis, anemia, Diabetes, cancer.

According to the conclusions of many researches, GEP is one of the most widespread diseases based on a genetic factor. About 94% of the individuals with GEP have genetic markers on a chromosome 6p21, called the class II Human Leukocyte Antigen (HLA). HLA DQ2 prevails over HLA DQ8 and is observed in 90-95% of patients. Some researches also indicate correlation between DQ2 and a female and more frequent prevalence of classical clinical implications among the patients possessing DQ2.

Classical implications of a gluten enteropathy, such as the diarrhea, abdominal pain, fatigue are  small part of symptoms. Actually, the estimated ratio of the diagnosed cases to the undetectable varies between 1:8-1:10, because of atypical, minimum, or even absent complaints. Patients with atypical implications of a gluten enteropathy can be parted on 3 groups:

Group No1. Lack of gastrointestinal symptoms. Existence of abenteric implications: anemia, osteoporosis, dermatitis, bone formation disturbance, infertility.

Group No2. Total absence of symptoms. The diagnosis of gluten enteropathy is confirmed with histological or serological tests only. Disease is diagnosed casually during diagnostics of other diseases.

Group No. 3. Hidden form of GEP. Patients with the hidden form of GEP can be parted on 2 categories.

a) Asymptomatic disease. Patients with earlier established diagnosis of GEP and good answer to a GFD. Histological tests are in norm, but the quantity of intraepithelial lymphocytes is increased.

b)  Latent form of  disease. Mucosa of the intestine is not changed, patients use gluten contains nutrition. Further development of GEP is prognosticated.



Materials and methods:



      This article contains data of endoscopic, histopathological and genetic serological tests. Article is the result of 10th year studying, diagnosing and management of patients with the diagnosis of gluten enteropathy.

      The question about necessity of latent form treatment is discussed still. The difference between latent and asymptomatic gluten enteropathy is that asymptomatic form has deviations in biopsies of small intestines mucosa. Latent form of GEP has no any changes in biopsies. And the question about management of latent form remains open still. Opinions of experts are different. Some insist that latent GEP will have further implications and changes in biopsies. GFD is preferable to avoid these implications. The other opinion is different: the treatment of a latent form is unreasonable and unnecessary action which can bring to psycho- emotional disorders because life-style changes. And the third one opinion says that there is no latent GEP at all. But receiving of no pathological biopsies results can testify that the fields of lesion can be passed because of locality or lesion is too low. The best example is one unusual case of two male uniovular twins. At the age of 10 one of the twins was diagnosed with functional dyspepsia, Gastroezofageal Reflux disease and collagen microscopically colitis. Expected regression wasn't observed after strict GFD and treatment. The child was diagnosed on possible existence of GEP.  Antibodies serological screening was negative. There were no any pathological changes in small intestine mucosa according to the endoscopy and three target biopsies. Immunologic screening was carried out in two twins. The second twin had IgA TTG positive result, but he had no any gastro-intestinal symptoms. The genetic test was made for two twins. HLA DQ8 was positive in bough of them. The endoscopy research and multiple biopsies confirmed the diagnosis of a gluten enteropathy (Marsh I, 30-35 IEL).

      According to the opinion of our expert group, GFD is a necessary condition and best tactics of any GEP forms treatment.  The latent form is the most ambiguous and patients from this group need monitoring of doudenum, serological and histological tests.

    The GEP is diagnosed in any age and has very wide range of clinical implications that is a serious obstacle for correct diagnosis. It is considered that the disease is inherent to young age and there is a layer of patients aged from 45 till 80 years which are unattendant, because of atypical implications, such as loss of osteal mass which is associated with a menopause. Meanwhile, the diagnosing percent in this age group is 0.34% to 1% which is approximately peering to indicators of age group from 10 to 45 years (0.41% to 1%). There is the next case of atypical GEP's clinic in the female patient of 57 years old.  In 2004 she was diagnosed with Diabetum 1 type (T1DM). The reply on treatment was positive and the diagnosis was cancelled. But there was inexplicable and relapsing hypoglycemia within the next 4 years. Negative serological tests IgA tTG + EMA, IgA DGP, IgG DGP and lack of gastrointestinal symptoms postponed correct diagnosis for 2 years. The gluten enteropathy (Marsh II, hyperplasia of crypts) was diagnosed after the second endoscopy and morphological research of biopsies.    Abenteric implications of GEP such as hypoplasia, headache, an ataxia, encephalopathy, chorea, aphthous stomatitis, multiplex mononeuritis, and Huntington disease are more inherent in male patients of 92% (according to the research of 104 patients in the central university clinic). While gastrointestinal implications are most inherent in female patients: diarrhea, abdominal pain of 84%. 

Gastrointestinal implications of GEP are most inherent in female patients (n=86), especially children (n=90). Dermatologic implications are more often observed in male children (n=42) while neurological implications (n=34) are most widespread in (female) adults, in comparison with children. Nutritional implications (n=20) and autoimmune (n=43) meet approximately twice more often at female adult patients in comparison with male patients.

    The gold standards in diagnostic of GEP are the serological EMA and tTG tests and enteric biopsy. Here we want to warn practicing doctors. EMA and tTG have the highest sensitivity and specificity of 95%, but sensitivity of these tests remains disputable in patients with the asymptomatic and latent GEPS forms and in patients with infiltrative Marsh I. It is proved that sensitivity of EMA and tTG in this group of patients is no more than 56%. Serological tests can be absolutely not informative in infants till 2 years old.  In this group of patients the most sensitive is IgA AGA (78%) in comparison with IgA tTG (12%) and IgA EMA (21%). IEL augmentation, but normal architecture of duodenal mucosa can be observed in autoimmune diseases: pseudo rheumatism, Hashimoto's thyroiditis and in patients with asymptomatic and latent GEPS forms. In the same way the villous atrophy can be a consequence of other diseases, such as Crone’s disease, collagen sprue and an autoimmune enteropathy. The wide range of histological disorders, does interpreting of enteric biopsy problematic for a pathologist, particular after introduction of three-stage histopathological classification Corazza. We consider that Corraza classification concedes to six-step classification of Marsh. The next case of male patient 42 years old, who was diagnosed with Crone disease (L1, CDAI 200, abscesses of crypts), on the basis of colonoscopy and histological research. Primary and the subsequent kalprotektin tests are negative. The joint scheme of topical hormonal therapy and glucocorticoids was applied in treatment. In the second enteric biopsy were found additional clonal T-lymphocytes and subtotal villous atrophy that allowed us to assume the GEP diagnosis the (Marsh3). After two months of GFD the histological picture of biopsies remained invariable. The final diagnosis is refractory GEP ( II type). The clinical remission was received after the therapy with Imuranum, Cyclosporine A and Methylprednisolonum. There are many cases in medical practice when diagnosis of GEP is wrong because of clinical implications similarity that conducts to the wrong treatment tactic of such diseases as a post infectious diarrhea, protein-losing enteropathy, atypical tuberculosis, Whipple disease, ulcerative jejunitis, lymphocytic colitis, radiative enteritis, immunoproliferative disease of small intestines and an autoimmune enteropathy.

    There is a vicious circle, as we have a series of diseases which correlate with the GEP and lead to the wrong diagnosis and in turn undiagnosed GEP leads to the same diseases, such as lactase intolerance, bacterial overgrowth, functional dyspepsia, microscopically colitis, ulcerative colitis, Cron's disease and eosinophilic gastroenteritis. Enzyme and bacterial therapy is not completely enough and can not change GFD. Newly therapeutic alternatives are currently interested in the pathogenesis of the disease, focusing on engineering gluten-free grains, degradation of immunodominant gliadin peptides that resist intestinal proteases by exogenous endopeptidases, decrease in intestinal  permeability by blockage of the epithelial zonuline receptor, inhibition of  intestinal tTG2 activity by transglutaminase inhibitors, inhibition of gluten peptide presentation by HLA-DQ2 antagonists, modulation or inhibition of proinflammatory cytokines, and induction of oral tolerance to gluten. But, at this time, strict adherence to a GFD remains the only effective and safe therapy for GEP.

Thus complexity of GEP diagnostics is caused of variety of clinical implications of a disease and wrong decoding of endoscopic pathology symptoms which are the basis for the subsequent histological and immunohistochemical researches. Secondly, complexity of GEP diagnostics is caused by various degree of objectivity of diagnostic actions both non-invasive and invasive. Thirdly, the reasons of unsatisfactory diagnostics are casual character of biopsy in widespread endoscopic practice of conventional esophagogastroduodenoscopy. Fourthly, there is complexity of patohistologic and endoscopic differentiation GEP, chronic nonspecific duodenitis (CND) and their combined implications (Kilessa A.V., 2012).

    These questions were the basis for development of uniform complex morphological algorithm of GEP diagnostics, which is applied in Ukraine (Kiev, Simferopol) since 2011-2012.

    During (2008-2011) 1814 endoscopic researches were made by main author with use of Olympus Evis Exera II (series 180) station. Two levels of duodenum D2 and D3 (according S. Morzhatka 1996) were studied. At first survey was carried out with white light endoscopy without use of high resolution and NBI modes and then with use of NBI mode for best assessment of villous structure. The gradation of video endoscopic transformations of duodenal villous was carried out on a visual Z-scale (according R. Badreldin еt al. 2005) wit using author's modification (distribution of villous changes Z-2 in two subgroups: Z-2a and Z-2b). Target biopsy carried out after detection of endoscopic changes in duodenum. 324 patients with endoscopic duodenopathy signs were defined (17, 86% from total quantity of endoscopic researches). 324 target duodenobiopsies were taken from the changed parts of duodenum. Control biopsies of healthy patients were used after their written consent. The diagnosis "a gluten enteropathy" was verified in 114 cases (6, 28% from total quantity of endoscopic researches and 35, 18% from the duodenopathy).

Received biopsies were fixed in 10% of neutral Formalin, made paraffin blocks were made, serial sections 4-5mkm thickness were painted with hematoxylin and eosine.

Immunohistochemical reactions were carried out in paraffin sections with the corresponding primary antibodies ("DAKO", Denmark). System of visualization FLEX and an autosteyner DAKO were used. Nuclear were colored with hematoxylin additionally. Positive and negative samples, as standards, were used for control. Phenotype of lymphocytes defined with immunohistochemical markers of CD3+ (Clone F7.2.38) and CD8+ of lymphocytes (Clone C8/144B), proliferative activity – monoclonal antibodies of Ki-67 (Clone MIB-1).

Morphometric researches – calculation of cells with the Software DP-SOFT program is executed on tissue specimens. All primary medical information (cases, results of special researches) and results of all fragments of complex morphological research are checked, formalized and documented in a computer database. They were processed by standard methods of variation statistics (with use of didymous two-selective t-test for averages, comparison of average two independent selections by  Student criteria, definition of correlation indicators of iteration with Kendal coefficient) wit use Microsoft Office 2007 software.



Results and discussion.

   

    The diagnosis "a gluten enteropathy" was verified in 114 (35, 18%) patients from 324 with endoscopic signs of a duodenopathy. Video endoscopy of high resolution and NBI modes allowed us to identify differential and diagnostic criteria of GEP and CND in comparison with healthy duodenum, which are based on studying such villous characteristics as coloring, orientation, mobility, height, thickness, form of tops, existence of deformations, an assessment of a lymphatic capillary, width of intervillous intervals, identification of  centers of a gastric metaplasia centers and hyperplasia of  Brunner glands (Кilessa A.V. 2012).

Let's notice that in differential diagnostics of CND and GEP the assessment of villous lymphatic capillary has basic value. Inflammatory changes of duodenal mucosa in the GEP are seldom and are more often localized in distal part of duodenum. Thus, use digital video endoscopy of high resolution and NBI allows to make differential diagnostics of CND and GEP during duodenoscopy manipulations.

      Often, in diffuse villous Z-2b changes we found their local growths which haven't been described in scientific practical digital video endoscopy  guidance. They represented conglomerates of randomly located villous of various height and thickness. As a rule, such conglomerates were localized in descending part of duodenum. The lymphatic capillary wasn't defined in villous. Probably that is a sign of their functional incompetence (picture. 9). It would be important to notice, that in certain cases we found existence of large single lymphatic follicles 3 mm in diameter (picture. 10). The mucosa covering a follicle had the same villiferous structure (Z-3 type). The expressed blood staxis became perceptible, during biopsy from this part.

    During endoscopic research we observed focal changes of duodenal villous with a combination of various changes of Z-1 and Z-2a, Z-2a and Z-3.

Survey patohistological researches of duodenal biopsies in patients with CND identified a chronic inflammation in mucosa, where intensity and cellular structure of infiltrate showed activity of an inflammation and degree of illness expression. So, at the first degree of CND (according to R. Whitehead classification, 1990) the structure of villous remains invariable and  ratio of height  to the depth of crypts remains at the level of 3:1 and more, separate lymphocytes are visible intraepithelial, and in own duodenal plate – plasmocytes and lymphocytes in the ratio 1:4,5. In the second stage of CND, the changed structure of villous is visible with the wrong form and irregular border of absorptive enterocytes. The quantity of IEL and immunocompetent cells (plasmatic and lymphocytes with corresponding change of their ratio to 1:1, 6) in own duodenal plate is enlarge. In the third degree of CND – villous are short, crypts are deepened, the expressed infiltration of epithelial IEL lining and reduced quantity of goblet cells in lining. There are found a prismatic gastric epithelium (a zone of gastric metaplasia) in separate biopsies and large number of lymphocytes in own duodenal plate.

In similar duodenal biopsies of patients with suspicion on the GEP there is an infiltration of epithelium and own plate by lymphocytes, architectonics of villous is not changed. The ratio of villous height up to the depth of crypts doesn't differ from that in control group and in CND (the first degree) is 3:1 and above.  The lymphatic capillary is located the center of villous and blindly comes to the end in their apical part. Such pathohistological picture of duodenum is typical for an infiltrative stage, but has no authentically expressed difference with CND I degree.

In a certain quantity investigated biopsies we observed infiltration of intraepithelial intervals and own plate of mucosa and changes of a ratio between villous height to depth of crypts from 3:1 to 2:1.  The appearance of villous was a little changed (their thickness and a form); there was a narrowing of crypts and hyperplasia of their epithelium and partial obliteration of lymphatic capillary. These structural transformations are similar to changes in the hyperplasic stage of GEP, according to M.N. Marsh (1995) classification.  Reorganization of duodenal mucosa (in particular villous) which is described in survey pathohistological research has the qualitative characteristic and does not contain quantitative leading signs of pathology and is not only in the GEP, but also in CND. Thus, the surveyed method of research is not informative for verification early (infiltrative and hyperplasic) stages the GEP.

    In some analyzed biopsies with coloring by hematoxylin and eosine it was able to see changes of an epithelial lining, because of its damage and reparative neogenesis (augmentation of the cell sizes and their nuclear, a chromatin enlightenment and cytoplasma basophilic).  There is loss of basal nuclear orientation (epithelium pseudo-stratification) and illegibility of border contours. Changes in the ratio heights of villous to depth of crypts are 1:1 and even 0:1 which testifies the total villous atrophy, when the histological structure of duodenal mucosa is similar with a colon. There is a limfohistocytic infiltrate with impurity of plasmocytes, eosinocytes and  neutrophiles in own plate  of duodenal mucosa. The specified morphological changes are similar to described by M.N. Marsh (1995) and R. Whitehead (1990) for an atrophic stage of GEP and CND (third degree). The condition of a lymphatic capillary (its obliteration) is one of important differential signs of GEP.

Standard pathohistological research doesn't satisfy to requirements of a quantitative assessment of duodenal mucosa characteristics, which are important for GEP pathomorphogenesis, because it is impossible to count precisely quantity of IEL and various types of cells of an infiltrate in own plate of mucosa and it is impossible to define a phenotype of lymphocytes and to estimate proliferate activity of crypts enterocytes. Therefore it is impossible to make adequate idea about genesis of morphological changes in mucosa and to make differential diagnostics between GEP and CHD.

For patohistological criteria assessment of differential GEP diagnostics  we used immunohistochemical methods with marking of certain lymphocytes phenotypes and proliferating cells, which are corresponding  with antibodies (CD3+, CD8+ and Ki-67).

    In the research of CD3+ lymphocytes contents of CD3 in biopsies of duodenal mucosa it was identified that received results of IEL significantly differ from those in norm (2, 75±1, 13) and at CND (16, 29±5, 76). In own plate reliable difference is between CND (36, 29±7, 18) and GEP (30, 80±15, 30), but in infiltrative stage, the difference is impossible (р> 0, 05). Probable, the differences of these indicators GEP are found between infiltrative and final (atrophic) stage – 41,96±12,41 and 52,20±12,93. So, as screening differential and diagnostic criteria of GEP it is possible to use data about the maintenance of CD3+ IEL in duodenal mucosa. These criteria are unacceptable for staging of GEP.

    The quantitative analysis of CD8+ lymphocytes expression in CND (5,22±1,40) and  GEP (infiltrative stage 47,32 ±10,86) allows accurately identify these diseases (high reliability of a difference between IEL indicators  and lymphocytes of own plate, р.< 0,001).  Cells are cytotoxic, gliadine - sensitive and therefore, specific for GEP. Nevertheless, the maintenance of CD8+ IEL in duodenal mucosa can't be as criterion for definition of GEP stages, as staging difference of indicators is statistically doubtful (p> 0, 05).

    Studying of proliferate crypts activity was made with monoclonal antibodies Ki-67 and it was identified that in norm in CND and GEP (infiltrative stage), activity differs – 6,35±1, 79%, 7,32±1, 68% and 8,75±2, 18% respectively. In the GEP it is significantly higher in hyperplasic and atrophic stages. Terminative zones of reproduction in crypts are marked in an infiltrative stage only. In disease develop Ki-67 – positive enterocytes are localized on villous (up to 1/3-1/2 of its heights), accurately outlining the changed form of a lymphatic capillary and irregularly located goblet cells. Proliferate activity of duodenal epithelium reaches the maximum in atrophic stage of GEP when zones of reproduction cover almost all epithelial lining in parts of villous disappearance, and the index of proliferation reaches 59,25 ± 11,27%. We consider that the described phenomenon is pathognomonic for an atrophic GEP stage. Microphotograms in norm.

    We carried out a transmission sub microscopy and studied ultra structural changes of mucosa in the GEP. Unfortunately, this research method isn't used wide in clinical praxis.

    It was founded that in the earliest stage of GEP development the electron and microscopical research allows to identify some changes of absorptive enterocytes metastructures of duodenal villous which can't be identified in the light microscopy. Sometime it is able to see parts with disturbance of a border continuity and emergence of various on the extent "nonvillous" zones on an apical surface of enterocytes. But the next villous are partially extended with changed form covering the bared zones that indirectly testifies about partial compensation of the broken function (Picture. 21).There are changes of metastructures with multidirectional character in the hyperplastic stage of GEP in enterocytes of villous and other  cellular elements. Moderately expressed atrophy of enterocytes becomes perceptible during remaining and even a little amplifying lymphocytic infiltration of an epithelium and own plate of mucosa.  It is expressed by rising of cytoplasma density and appreciable profiles decreasing of a canaliculus of a granular and agranular cytoplasmatic network during decrease of free ribosomes quantity and the policy, accumulation of  protein granules and a lipofuscin, expansion of intercellular intervals. But, to our opinion, Pannet's cells are main in a hyperplastic stage  of the GEP. Their quantity  is sharply enlarged everywhere because of "juvenile" cells with finer granules emergence of "juvenile" cells with finer granules of a different osmiofilnity (picture. 22). In "mature"  Pannet's cells becomes perceptible a large number of highosmiofilnity large granules of a spherical form becomes perceptible and also the expressed hyperplasia of a cytoplasmic reticulum with expanded profiles of a canaliculus. It unambiguously testifies about the increased functional activity and high synthetic and secretory potential of Pannet's cells.

It is possible to assume that Panet's cells play an important role  in a hyperplastic stage of disease in its further pathology development, advance or attenuation.

    The mechanism of Panet's cells participation  in a  GEP morphgenesis should be study careful.

Analyzing the changes of duodenal mucosa in atrophic stage of the GEP, it would be necessary to emphasize that  this period includs inflammation implications and cellular infiltration own mucosa plate and an epithelium (that was established at immunohistochemical research with antibodies to CD8+ to lymphocytes).

The structure of an inflammatory cellular infiltrate changes towards sharp ascending of plasmocytes quantity  that testifies about participation of humoral immune mechanisms in pathology development due to the active immunoglobulins development by plasmocytes. However, the most essential, to our opinion,  is almost total disappearance of Panet's cells cellular  from an epithelial cellular pools and a hyperplasia of intestinal endocrineocysts. In norm the expressed prevalence of such cells is characteristic for an enteric epithelium, that is why it would be possible to assume that sharp change of substances duodenal structure in  atrophic stage  of the GEP. Apparently beginning from this moment, pathological GEP'S processes can be irreversible.

    The correlation analysis of received indicators showed that there are no any close interrelations between the maintenance of certain lymphocytes phenotypes in duodenal mucosa structures, proliferate activity of enterocytis and changes of villous in patients with GEP (infiltrative stage). In hyperplastic stages there are determined positive correlations between the levels CD3+ and CD + IEL (r=0, 63) and between indicators of proliferative crypts enterocytes activity Ki-67 and the point appropriated to each rank of a Z-scale (r=0, 61). In atrophic stage of GEP positive correlation interrelations are recorded between the maintenance of CD3+ and CD8 + IEL (r=0, 69) and quantity of CD3+ MEL and CD8+ of lymphocytes in own plate of mucosa (r=0, 51). Besides, in the research of dependence of villous restructurings from the maintenance of CD8+ MEL, it was established that in patients with infiltrative and hyperplastic GEP stages, endoscopic changes of villous were estimated as Z-2a and Z-2b, and the Z-3 type was in atrophic stage of disease only.

    Algorithm of GEP diagnostics allows to verify a disease in I and the II stages (according to M.N. Marsh classification) and includes consecutive performance of the following actions:  survey duodenoscopy without high resolution and NBI modes for the contents assessment of duodenum, cords,  elastance of walls, identification of inflammatory changes, erosive and ulcerative defects → a duodenoscopy with use of high resolution and NBI modes for an assessment of villous structure according to gradation of Z-scale, identification of focal pathological changes, receiving biopsies for pathohistological research → survey pathohistological research of correctly focused biopsies (coloring by hematoxylin and eosine) for identification of pathological changes in mucosa (augmentation of  IEL, deformation of villous) → immunohistochemical research with antibodies to CD3+  lymphocytes, for  IEL identification more than 30 on 100 epitheliocytes and expressions of lymphocytic infiltration of own  plate of mucosa → immunohistochemical research with antibodies to CD8+ to lymphocytes and definition  of proliferative activity index of  crypts epithelium with Ki-67 → the analysis of the data and differential diagnostics with CND, the formulation of the morphological diagnosis according to the M.N. Marsh classification.

    According to the data, the total quantity of patients with GEP disease is 63. The gluten enteropathy (infiltrative stage Marsh I) was diagnosed in 33 cases, a hyperplastic stage (Marsh II) – 17 cases, an atrophic stage (Marsh III) – 13 cases. Chronic nonspecific duodenitis II is revealed to 50 patients. The following pathomorphological ratios are defined after studying the histological material received from  patients with metaplastic changed mucosa of esophagus and a stomach: the chronic not atrophic gastritis (CNG) in patients with  GEP is revealed in 25 cases and CND in 31; chronic not atrophic gastritis with an enteric metaplasia (CNG with EM) – in the gluten enteropathy – 6, CND – 4; chronic not atrophic gastritis with a colic metaplasia (CNG with CM) – in the GEP – 9, CND – 2; the chronic atrophic gastritis (CAG) – in the GEP – 10, CND – 2; chronic atrophic gastritis with an enteric metaplasia (CAG with EM) – in the GEP – 2, CND – 2; chronic atrophic gastritis with a colic metaplasia (CAG with CM) – in the GEP – 5, CND – 1. The specialized мetaplasia of esophagus is revealed in 6 patients with GEP and 8 patients with CND. СNG with EM correlates with the GEP and CND in 6-9%. It is necessary to notice that the greatest frequency of CNG with CM (18%) occurrence is in the GEP (infiltrative Marsh I) and hyperplastic stages (Marsh II) whereas in atrophic stage the of the GEP (Marsh III) we didn't get diagnosis CNG with CM, but found 4% cases of CND. Patients with the GEP in infiltrative stage (Marsh I) had no CAG with EM but in hyperplastic and atrophic stages – 6% and 8% respectively and in CND – 2%. CAG with CM was found in patients with the GEP Marsh I stage – 6%, Marsh II – 6%, Marsh III – 8%. In CND the CAG with CM was found in 2% of patients only.  SME is found in patients with GEP and CND with equal occurrence, except Marsh II GEP – 6%. According to the results from our research it is able to claim that metaplastic changes of stomach mucosa are more inherit for GEP patients than CND. We consider that biopsies from distal part of a duodenum are necessary for patients with stomach metaplasia for GEP diagnostics. Various pathomorphologic changes of mucosa in esophagus and a stomach should be surveyed in a context with duodenal mucosa pathology. We believe that the primary source of these changes is localized in duodenal mucosa because of special regulatory characteristics of this gastrointestinal part.



CONCLUSION

   

      The high frequency and wide range of adverse reactions to gluten raise the question as to why this dietary protein is toxic for so many individuals in the world. One possible explanation is that the selection of wheat varieties with higher gluten content has been a continuous process during the last 2000 years, with changes dictated more by technological rather than nutritional reasons. Wheat varieties grown for thousands of years and mostly used for human nutrition up to the Middle Ages contain less quantities of the highly toxic 33-mer gluten peptide  Apparently the human organism is still largely vulnerable to the toxic effects of this protein complex, particularly due to a lack of adequate adaptation of the gastrointestinal and immunological responses.      Additionally, gluten is one of the most abundant and diffusely spread dietary components for most populations, particularly those of European origin. In Europe, the mean consumption of gluten is 10 g to 50 g per day. All individuals, even those with a low degree of risk, are therefore susceptible to some form of gluten reaction during their life span. Therefore, it is not surprising that during the past 50 years we have witnessed an «epidemic» of Gluten Enteropathy. Besides, today we didn't find any data about immediate type reaction (Artus's phenomenon) in the duodenal mucosa. Gluten Enteropathy diagnosing is difficult and ambiguous process with absence of the universal standard and algorithm and it would necessary to consider that the GEP is the disease where it is necessary to differentiate two terms "general practice" and "the best practice", which is the most desirable for management of patients.