Method for estimating treatment effectiveness in treating latent infantine kron disease cases of ileocecal localization

FIELD: medicine.

SUBSTANCE: method involves applying therapy with 5-aminosalicylic acid combine with anti-inflammatory means. Morphopmetric microcirculation bed examination is carried out before and during treatment but not earlier than in half year from the beginning of treatment course with this preparation. The number of blood vessels, microcirculation bed area, nucleoplasmatic ratio of endothelium cells of microcirculation bed and mean blood vessel lumen diameter is determined in ileal mucous membrane bioptate. Sharp angiogenesis reduction being observed as reduced blood vessels number by52±5%, microcirculation bed area reduction by 24±15%, two-fold increase in nucleoplasmatic ratio of endothelium cells of microcirculation bed witnessing their damage reduction and mean blood vessel lumen diameter increase by 40±8% in combination with reduction of perivascular space edema and infiltration, reduction of exudates in tissue, treatment is estimated as effective one.

EFFECT: high accuracy of estimate.

1 tbl

 

The invention relates to medicine, namely to a method of assessing the efficacy of treatment, including biopsy and subsequent morphometric study, and can be used to determine the adequacy of drug effects in latent form ileocecal Crohn's disease localization in children, correction of dosage or for preventive replacement regimens on more aggressive steroid, cytotoxic or online.

Crohn's disease is non-infectious inflammatory process affecting various parts of the gastrointestinal tract. The disease has not been elucidated the etiology and can occur in various forms. Most often affected small and large intestine. The disease can last for years, as a specific means of treatment is not found. The use of antibiotics, sulfonamides and steroid hormones can lead to recovery and chronicity of the process. Complications of chronic use of radical surgical treatment, consisting of resection of the significant areas of thick or thin intestines. However, after surgical treatment often (20-45%), relapse, which leads to the necessity of repeated interventions. Therefore, it is important to have a timely opportunity to assess the efficacy is ü conducted by conservative therapy. The development of such methods is now an actual problem.

A prototype of the invention is selected there is a method of monitoring the treatment of latent forms of ileocecal Crohn's disease localization in children, including common clinical and laboratory studies (see grebenyov A.L., Myagkova, L.P. bowel Disease current advances in diagnosis and therapy, M, Medicine, 1994, 400 S.).

Clinical trials include: assessment of subjective sensations and status preasens patient. Laboratory evaluation includes examination of such nonspecific factors as the increase in ROE, leukocytosis, thrombocytosis, anemia and dysproteinemia, C-reactive protein, α-1-glycoprotein (orosomucoid), chymotrypsin, antitripsin, haptoglobin. Altered levels of these indices is acute or exacerbation "in progress".

The disadvantage of this method is its inaccuracy, subjectivity and variability of many laboratory and clinical indicators, which does not allow to consider them as sufficient, and most importantly, objective to assess the effectiveness of treatment.

The objective of the proposed method is providing opportunities and improving the accuracy of estimation of efficiency of treatment of latent forms of Crohn's disease in children.

Tasked with the known method of evaluating the effectiveness of the barb treatment of latent forms of ileocecal Crohn's disease localization in children includes General clinical and laboratory examination is solved by the fact that extra spend the retrograde ileoscopy and morphometric study of the microvasculature, with biopsy of the mucosa of the ileum to determine the number of vessels and the area of the microvasculature, nuclear-cytoplasmic ratio of endothelial cells of the microvasculature and the average diameter of the lumen of the vessels before and during treatment, not earlier than after 0.5 years from the start of the medication and with a sharp reduction in angiogenesis in the form of a reduction in the number of vessels 52±5%reduction in the area of microcirculation on 24±15%, and increased nuclear-cytoplasmic ratio in the endothelial cells of the microvasculature in 2 times, which indicates a reduction in their damage and increasing the average diameter of the vascular lumen 40±8% together with reduction of edema and infiltration of perivascular spaces, reducing exudate in tissue judge reliable treatment.

The present invention meets the criteria of the invention of "novelty" and "inventive step", because in the process of conducting patent information research has not identified sources of information prejudicial to the novelty of the proposed method is the AK and technical solutions with the essential features of the proposed method.

A known method for diagnosis of ileocecal Crohn's disease localization in children, including assessment of correlations between clinical activity data retrograde ileoscopy and morphometric studies of the microcirculatory bed in biopsies of the mucosa of the ileum (Fadeev, M. Morphology and pathogenesis of disorders in the intestinal mucosa in Crohn's disease in children. // Abstract. Diss. CMN - SPb. - 2001).

Histological criteria for determining the degree of inflammatory activity are to determine the density of infiltration by lymphocytes, plasma cells, macrophages, mast cells, neutrophilic and eosinophilic granulocytes and fibroblasts per unit area of the drug. However, there was a relative increase in the density of the cellular infiltrate and absolute increase in the density of macrophages and fibroblasts in subscripting departments own plates of the mucous membrane of the ileum with a positive clinical dynamics and remission reached in the process of treatment of latent forms of Crohn's disease.

Thus, the current histological criteria of degree of inflammatory activity in the form of measuring the density of the infiltrate in Crohn's disease can even lead the researcher astray when assessing the effectiveness of treatment. Proceeding from the basic what about the mechanism of photomorphogenesis, that is, based on the degree of insufficiency of the layers of the intestinal wall, depending on the degree of productive vasculitis with pathological angiogenesis, not only and not so much on the number of infiltrating cell forms, as evidenced by the existence of a direct correlation dependence of the degree of damage to the epithelial layer of the ileum and cecum of the severity of angiogenesis (the Pearson correlation is +0,89).

The authors performed a morphometric examination of biopsy specimens of the proposed method from 17 patients children aged 2 to 17 years (boys and 10 for girls 7) latent ileocecal Crohn's disease localization with multiple endoscopic sighting biopetroleum and studied more than 700 slices in the 170-190 drugs. Only one boy was transferred to the steroid regimen, but this transition to a different treatment plan and contributed preceded migrated associated salmonellosis. Was not operated any sick child.

Basic drug therapy was mesalazine - 5-aminosalicylic acid (commodity form - Salofalk® made in Germany) in combination with other anti-inflammatory drugs. Steroid and cytotoxic drugs were not used. This treatment regimen was developed in Nizhny Novgorod research Institute of pediatric gastro is tarologie (RF patent No. 1826953).

It is established that in the course of treatment, but not earlier than after 0.5 years, decreased as the area of microcirculatory vessels and total vascularization, i.e. decreased phenomena of pathological angiogenesis. This, at first glance, the negative impact of therapy was accompanied by alternate changes in the form of the disappearance of perivascular edema and decrease the infiltration of perivascular spaces, decrease hydropic degeneration and swelling of endothelial cells, decreasing sliceupdown in the blood vessels, reducing plasma impregnation, reducing blood clots and microinvasive. There was an increase in the mean diameter of the lumen of the vessels at a constant diameter, as measured by their basal membranes and increased nuclear-cytoplasmic ratio in endothelial cells by reducing swelling of the cytoplasm, which indicates a decrease in the degree of damage to the endothelial cells. Under these conditions, improved supply of tissues with oxygen and nutrients, as evidenced by the improvement of the epithelial layer. The obtained data are presented in the form of a table.

Table
The condition of the EPITHELIAL LAYER AND microvasculature IN the PIC IS the TIME of the DIAGNOSIS of CROHN's DISEASE AND IN the PROCESS NONSTEROIDAL THERAPY
IndexAt diagnosis M±mEventually surveillance M±m
The height of the villi, mcm246±24378±30*
In crypt depth, µm204±28222±26
The height of the surface epithelium of, um26,5±2,232,8±3,2*
Height cryptologi epithelium, mcm17,7±4,423,3±2,5
Number of vessels per 1 mm2the area of medication462±20242±10*
Nuclear-cytoplasmic ratio, %17±435±3*
The diameter of the lumen of blood vessels100±7140±8*
The area of the vascular bed relative to the square of the drug (%0)177±12135±17*
*reliability of differences (P<0,05)

Thus, the proposed new objective histomorphometrical criteria to improve the accuracy and reliability of the assessment of the degree of the inflammatory process and the effectiveness of non-steroidal Altotting therapy, to determine the adequacy of the procedures.

The method is as follows./p>

After sampling using the colonoscope biopsy specimens fixed in 10% solution of buffered formalin and poured into paraffin blocks, slices prepare serially on a sledge microtome MS-2 and stained with hematoxylin and eosin for modification Rilli (Rilli. Pato-histological technique and practical histochemistry.//TRANS. from English. edited and with a Foreword by CL. - Corr. AMS Row, - M, WORLD, 1969).

The measuring grid mikrolimano re-photographed on the film (Avtandil - Morphometry. // M Medicine. - 1990. - 380 C.), is inserted into the eyepiece of the microscope. The measurement is made at the eyepiece 10 and the lens 100. Separately take into account the number of points 4 small grid squares, coincided with the structures of histological slice. To obtain more accurate data counts are 400 small squares (10,000 points in each observation). Up to, but no earlier than 1/2 of the year with the start of medication measure the height of the villi, the depth of the crypts, the height of top and cryptologi epithelium in micrometers (assessment of epithelial layer, characterizing the life expectancy and the level of differentiation of epithelial cells), to determine the number of vessels per 1 sq. mm histological slice and the area of the microvasculature in ppm relative to the square of the drug, as well as nuclear-cytoplasmic ratio is osenia in the endothelial cells and the average diameter of the lumen of the vessels of the microvasculature. Counts are outside of lymphoid, follicles, in large quantities resulting in Crohn's disease. With a sharp reduction in angiogenesis in the form of a reduction in the number of vessels 52±5%reduction in the area of microcirculation on 24±15%, and increased nuclear-cytoplasmic ratio in the endothelial cells of the microvasculature in 2 times, which indicates a reduction in their damage, and increase the mean diameter of the lumen of the vessels 40±8% together with reduction of edema and infiltration of perivascular spaces, reducing exudate in tissue judge reliable treatment.

A specific example of implementation is given in the form of extracts from the history.

1. The melikbekian IKE, age 7, case history No. 0960, diagnosis, first exhibited at endoscopy with biopetroleum, the histological conclusion - "first identified terminal REIT (Crohn's disease), in accordance with the developed in the Institute of pediatric gastroenterology scheme prescribed treatment. When you receive more than 1 year (history No. 1806) clinical deterioration has not occurred, retrograde ileocoecal the study found no negative dynamics of the disease. A comparative morphometric study of the biopsies obtained before and more than six months from the start the treatment, the following results are obtained: the height of the villi in the ileum increased from 232 to 400 μm; the depth of the crypts increased from 230 to 245 μm; the height of the covering epithelium was 27 and 35 μm, cryptologi 17 and 22 μm; the number of microvessels decreased 1.9 times the area of the vascular bed fell from 180 to 143 ppm; nuclear-cytoplasmic ratio in endothelial cells increased in 2 times, and the diameter of the lumen of blood vessels increased by 37% before and after treatment, respectively. Based on these data, the conclusion was made about the adequacy of treatment, scheme maintenance therapy unchanged. Further observation showed that the clinical, endoscopic and histological progression of the disease occurred.

2. Golikova Olga, 12 years old, medical history, No. 1630, the diagnosis is first exhibited endoscopically and confirmed morphologically 28.06.93 in the future were: history No. 1247 from 11.05.94, 2761 from 15.12.94, 1256 from 31.05.95, 0200 from 06.11.96, 1546//392 from 04.09.1998. Morphometric studies carried out on biopsies taken in 1994 and 1995, one or more parameters do not match the figures in the table so in may and December 1994 on the background of severe hydropic degeneration was revealed increased nuclear-cytoplasmic ratio of endothelial cells to only 28 and 31% comparedwith 19% before treatment, and the diameter of the lumen of the vessels of the microvasculature was 124 and 129% compared with that before treatment. Accordingly, although the expressed clinical deterioration of the patient was not observed, but satisfactory to call him, it was not - it was still lagging body weight, pain and mild dyspepsia. In 1994 he was made the correction of the treatment in the form of angioprotectors (prodektina), and 1995 was increased dose of basic drug (salofalk). When entering in 1996 morphometric study showed that the remission with normalization of the clinical picture, already adjusted treatment was saved, and the correctness of the chosen course was confirmed in another clinical-morphological observation in 1998.

A method of evaluating the effectiveness of treatment of latent forms of ileocecal Crohn's disease localization in children, including treatment using as a reference drug (salofalk) 5-aminosalicylic acid in combination with anti-inflammatory drugs, characterized in that is carried out before and during treatment not earlier than after 0.5 years from start of treatment morphometric study of the microvasculature, with biopsy of the mucosa of the ileum to determine the number of the vessels and the area of the microvasculature nuclear-cytoplasmic ratio microvascular endothelial cells and the average diameter of the vascular lumen and with a sharp reduction in angiogenesis in the form of a reduction in the number of vessels 52±5%, the reduction in the area of microcirculation on 24±15%, and increased nuclear-cytoplasmic ratio in the endothelial cells of the microvasculature in 2 times, which indicates a decrease damage and increase the mean diameter of the lumen of the vessels 40±8% together with reduction of edema and infiltration of perivascular spaces, reducing exudate in the tissue of the estimate of the treatment as effective.



 

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