Abstracts published in NLM Medline
Last updated: June 17, 2021
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32. Faecal immunochemical tests for haemoglobin: Analytical challenges and potential solutions.
Clin Chim Acta. 2021, 9; 517: 60-65
Benton SC, Symonds E, Djedovic N, Jones S, Deprez L, Kocna P, Maria Auge J.
International Federation of Clinical Chemistry Faecal Immunochemical Test Working Group (IFCC FIT-WG)
Quantitative faecal immunochemical tests for haemoglobin (FIT) are being used increasingly around the world in colorectal cancer screening programmes, and in patients presenting with lower bowel symptoms to determine who should proceed to further bowel visualisation investigations, usually colonoscopy. The clinical utility of FIT is well reported. There are a number of analytical challenges including pre-analytical variation, difficulty setting up external quality assessment schemes, access to third party internal quality control material and a lack of standardisation or harmonisation of FIT methods. Here we report the work of the International Federation of Clinical Chemistry FIT Working Group. We provide an overview of the main pre-analytical variables; discuss different approaches to external quality assurance of FIT; propose a solution to third party internal quality assurance materials and summarise the challenges of standardisation and harmonisation of FIT.
31. Specificities of the diagnostics and therapy of exocrine pancreatic insufficiency (Article in Czech)
Vnitr Lek. 2013 Jan;59(1):65 - 70
Díté P, Novotny I, Kocna P, Bojková M, Kupka T, Nechutová H, Kianicka B.
IV. interni klinika 1. LF UK a VFN, Praha. firstname.lastname@example.org
Abstract: Exocrine pancreatic insufficiency develops steadily; however, the initial reduction in secretion is practically not diagnosable. More advanced stages, which usually replicate morphological changes, can be determined with tests which asses the exocrine pancreatic capacity. Substantial damage of the pancreas and replacement of viable parenchyma with connective tissue is accompanied by the occurrence of steatorrhoea. This corresponds to a reduction in exocrine pancreatic secretion below 10% of physiological secretion. Exocrine pancreatic secretion tests are still not sufficiently sensitive for diagnosing early stages of pancreas defects and thus are not suitable for diagnostics. Furthermore, detecting reduced exocrine secretion does not provide any information about the aetiology of the disease, e.g. inflammation/tumor. The most precise test is a costly examination, including a stimulation of the gland with enterohormones; however, breath tests are usually recommended for the assessment of exocrine insufficiency therapy. Exocrine pancreatic insufficiency therapy consists of administering drugs containing pancreatin (amylase, lipase, and peptidase) to patients diagnosed with steatorrhoea, manifest pancreatic insufficiency. As standard, capsules containing microparticles of 1-2mm are recommended. They have a protective coating that prevents inactivation in the microparticles of the contained enzymes by gastric hydrochloric acid. The drug should be administered during each meal, i.e. several times a day. The most common mistake during pancreatic enzyme therapy is under dosage. The following rule applies to patients with digestive insufficiency: 40,000-50,000 UNT of lipase are to be administered during "main meals" and 25,000 UNT of lipase during morning or afternoon snacks. The drug should be taken during the meal; insufficient treatment and dosage are associated with insufficient digestion and absorption ofa number of substances and also with pancreatic malabsorption.
30. Laboratory screening markers in gastroenterology - state of the art
Biomed Pap 2013, 157:(2): 91-97
Kocna P, Vanickova Z, Zima T
Institute of Medical Biochemistry and Laboratory Diagnostics, 1st Faculty of Medicine, Charles University in Prague, Czech Republic. email@example.com
INTRODUCTION: Screening tests for gastrointestinal diseases acceptable for population with a high sensitivity and high specificity can now be offered by clinical laboratories. This paper summarizes major recent advances in this area of laboratory medicine.
METHODS: Relevant articles published within the last 5 years in the NLM (National Library of Medicine) PubMed - Medline database covering the three gastrointestinal diseases - colorectal cancer, coeliac disease, and atrophic gastritis were included for this overview.
RESULTS: In Europe, colorectal cancer (CRCA) is the second most frequent malignant disease. Quantitative immunochemical analysis of the stool for haemoglobin provides the best screening test to date, with both sensitivity and specificity approaching 95%. Even though coeliac disease (CD) affects approximately 1% of the general population, it remains largely unrecognised. Recommended methods for screening currently involve the detection of IgA and IgG antibodies against tissue transglutaminase and deamidated gliadin peptide. Evaluations of screening are now discussed for other diseases of the gastrointestinal tract - such as chronic atrophic gastritis (CAG), and inflammatory bowel disease (IBD). Detection of infection by Helicobacter pylori and stomach-specific plasmatic biomarkers, especially pepsinogen I/II ratio, could help with the prevention of gastric carcinomas. The use of faecal calprotectin as a screening test could substantially reduce the number of invasive methods necessary for the diagnostic work-up of patients with IBD.
CONCLUSIONS: Screening tests for CRCA and CD have been used worldwide for many years. Screening strategies for gastrointestinal diseases are suggested in the text, based on recent basic science, clinical papers as well as our own
29. Improvements in colorectal cancer screening programmes - quantitative immunochemical faecal occult blood testing - how to set the cut-off for a particular population..
Biomed Pap. 2012; 156(2):143-150
Kovarova JT, Zavoral M, Zima T, Zak A, Kocna P, Kohout P, Granatova J, Vanickova Z, Vranova J, Suchanek S, Benes Z, Celko MA, Povysil C.
4th Department of Internal Medicine, General Teaching Hospital, First Faculty of Medicine, Charles University Prague, Czech Republic. firstname.lastname@example.org.
Objective. The aim of the study was to determine the optimum cut-off value of the quantitative immunochemical test (q-FIT) OC-Sensor® for colorectal cancer and advanced adenomatous polyps in a particular population. Methods. 815 patients were referred for colonoscopy and were offered two q-FIT examinations at two different colonoscopy centers. The patients were classified according to the colonoscopic findings. Test sensitivity, specificity, and accuracy were statistically evaluated using one test and two tests at the levels of 50, 75, 100, 125, and 150 ng/mL of faecal hemoglobin in those patients with advanced polyps and colorectal cancer. The optimum cut-off test level for clinically significant neoplasia was determined using one test. Results. The optimum cut-off value of q-FIT OC-Sensor® for the detection of clinically significant neoplasia in our particular population was determined as 75 ng/mL using one test. This value provides an optimum proportion of 73% sensitivity (±95% CI 60.3% - 83.4%) and 90% specificity (±95% CI 86.8% - 92.8%), PPV and NPV were determined as 54.76% and 95.43% respectively. Conclusions. The first step in the implementation of q-FIT test in the screening program in our country is to determine the optimum cut-off level for a population, and to estimate the number of tests performed with respect to the optimum cost effectiveness and economical climate. Using one test, the optimum level of q-FIT OC-Sensor® in the Czech Republic was determined as 75 ng/mL. This study could serve as a model for further studies in other countries, where screening does not yet exist.
28. Monitoring of daily gliadin intake in patients on gluten-free diets.
Prague Med Rep. 2011; 112(1): 5 - 17
Gabrovská D, Kocna P, Rysová J, Borovská D, Tlaskalová-Hogenová H.
Food Research Institute Prague, Prague, Czech Republic. email@example.com
The aim of the study was to show patients suffering from the coeliac disease, their real gliadin daily intake, offer them very useful information concerning their diet and to find random possible mistakes. The monitoring was carried out within the context of their routine everyday diet regimen. The daily intake of gliadin in the diet was quantified on the basis of gliadin determination in their current daily food. The gluten-free diet was followed for 30 days. The patients were taking regular daily meals, drinks, and sometimes medicines or food supplements. The patients were provided with instructions, survey forms, digital scales, polyethylen bottles and sacks. The patients took out the stipulated amount, which served as a sample of each of their daily meals. The samples included both homemade meals as well as commercial products. The content of gliadin in daily meal was determined by the sandwich ELISA method. The daily gliadin intake was calculated on the base of the reported amount of meals ingested. 1,900 food samples were analyzed within the framework of this study. Several contaminated commercial foods were found; nevertheless this fact did not influence the otherwise satisfactory overall picture of the daily gliadin intake by the patients followed. The results in 14 patients revealed a satisfactory adherence to the gluten-free diet. It was proved that conscientiousness and awareness on the part of coeliac patients, or those taking care of them, is of paramount importance in determining the choice of foods comprising a gluten-free diet.
27. Faecal elastase I-its use in diagnosis of chronic pancreatitis (Article in Czech)
Cas Lek Cesk. 2006; 145 (6): 480 - 483
Krechler T, Kocna P, Vanickova Z, Svestka T, Lukas M, Dosedel J, Kohout P, Zak A.
IV. interni klinika 1. LF UK a VFN, Praha. firstname.lastname@example.org
BACKGROUND: The diagnosis of chronic pancreatitis is based on the imaging methods. These imaging methods show the main morphological changes in the pancreatic ducts and its parenchyma, but they do not define the function of the pancreas. The aim of our study was Faecal Elastase I. determination in patients with chronic pancreatitis. The test is a simple, non-invasive method of the investigation of the pancreatic exocrine insufficiency. The Faecal Elastase I occurring in the stool was correlated with the level of the damage of pancreatic tissue together with the control group of the patients with different diagnoses. METHODS AND RESULTS: Faecal Elastase I (mean values in ug/g of stool) detection is a simple, non-invasive method which correlates well with the damage of pancreatic tissue, stemming from chronic pancreatitis. This test is routinely used especially in the diagnosis of chronic pancreatitis. The classification of chronic pancreatitis currently depends on the morphological changes of the pancreatic duct system (the patho-morphological changes). We are currently missing the classification describing simultaneously the morphological changes of the gland and the function of the pancreas. In our studies we have used a newly proposed classification system, which was put together in Bern, 2000 (1). This new system encompasses morphological and functional changes. Faecal Elastase I was determined by a microplate ELISA method using monoclonal antibody to human pancreatic protein. The Faecal Elastase I. was tested in the stool of the 196 patients with chronic pancreatitis stemming from alcoholism. The occurrence of Faecal Elastase I. was classified according to the levels assigned by the classification system. The control group used in this study included 144 patients with different diagnoses. The results demonstrate a very good correlation of Faecal Elastase I. with the grading of the newly proposed classification system of chronic pancreatitis. Patients with the highest levels of the damage of the pancreas had a significantly lower occurrence of Faecal Elastase I. in comparison with the non-pancreatic control group and in patients with chronic pancreatitis who had no clinical complications or damage of endocrine and exocrine functions of the pancreas. CONCLUSIONS: Feacal Elastase I performance plays an important role in diagnosing of the severe cases of chronic pancreatitis and in the follow-up of the chronic pancreatitis in the patients with the intermediate damage of the pancreas.
26. Hyperamylasemia, laboratory and clinical aspects (Article in Czech)
Cas Lek Cesk. 2006; 145 (6): 449 - 452
Kocna P, Zima T.
Ustav klinicke biochemie a laboratorni diagnostiky 1. LF UK a VFN, Praha. email@example.com
Biochemical estimation of the total a-amylase represents in the Czech Republic almost two millions of assessments per year. Estimation of the total alpha-amylase for the diagnostics of pancreatic diseases has a very low specificity and it is therefore recommended to analyze specific pancreatic enzymes - pancreatic isoenzyme alpha-amylase and pancreatic lipase. Paper summarizes laboratory and clinical aspects aimed namely on hyperamylasamia.
25. Isotope Selective Nondispersive Infrared Spectrometry Can Compete with Isotope Ratio Mass Spectrometry in Cumulative
13CO2 Breath Tests: Assessment of Accuracy
Klin. Biochem. Metab. 2005, 13 (34): 2, 92–97
Chleboun J, Kocna P.
To measure a patient’s metabolic response to an administered 13C-enriched substrate, isotope selective nondispersive infrared spectrometry is used. Isotope abundance levels are relative, i. e., reported as differences between a tested sample and a reference sample. The reference 13C abundance is not known exactly. This uncertainty, uncertainty in CO2 production, and the inaccuracy of the measuring instrument contribute to the uncertainty in the results of breath tests. In this study, the particular impacts of uncertainty are estimated and expressed in a mathematical way by an uncomplicated formula illustrated by an example dealing with real-life data. It is shown that the uncertainty in the reference 13C abundance does not have severe consequences, so that the method can compete with the spectrometry methods that are able to deliver an absolute value of the 13C abundance. The inaccuracy of the measuring instrument is also
manageable, though its influence is greater than in the previous case. The analysis reveals that the uncertainty in CO2
production deserves great attention because it is difficult to estimate and its influence is rather strong. The problem of determination of a proper cut-off level is outlined.
24. IgA and IgG antigliadin, IgA anti-tissue transglutaminase and antiendomysial antibodies in patients with autoimmune thyroid diseases and their relationship to thyroidal replacement therapy.
Physiol Res. 2003; 52 (1): 79 - 88
Jiskra J, Limanova Z, Vanickova Z, Kocna P.
Third Medical Department, First Medical Faculty, Charles University, Prague, Czech Republic. firstname.lastname@example.org
Celiac disease is a chronic illness of the small bowel caused by gliadin intolerance in genetically predisposed subjects. The aim of this study was to investigate serum levels of IgA and IgG antigliadin antibodies, IgA antiendomysial antibodies, and IgA anti-tissue transglutaminase antibodies in 169 patients with autoimmune thyroid diseases, i.e. chronic thyroiditis and Graves' disease. Antiendomysial antibodies were positive in 2 out of 169 persons (1.18%), IgA antigliadin antibodies in 15.98%, IgG antigliadin antibodies in 51.48%, and IgA anti-tissue transglutaminase in 14.79%. The prevalence of positivity was higher compared to the 1312 control blood donors described in our previous study (Vancikova et al. 2002) (p<0.05). Patients with chronic thyroiditis treated with a high replacement dosage of levothyroxin (125-200 microg daily) had higher serum levels of IgA antigliadin antibodies in comparison with patients treated with a lower dosage (50-100 microg daily) (medians: 13.00 vs. 19.69, p=0.033). We found a negative correlation of IgA anti-tissue transglutaminase antibodies and total calcium serum levels (r = -0.480, p=0.0236, n=22). We can conclude that in persons with autoimmune thyropathy there is a high prevalence of positive antigliadin, anti-tissue transglutaminase and antiendomysial antibodies. Latent celiac disease may lead to impaired resorption of therapeutically administered levothyroxine, calcium, or other substances.
23. Tissue transglutaminase-serology markers for coeliac disease.
Clin Chem Lab Med. 2002 May; 40 (5): 485 - 492
Kocna P, Vanickova Z, Perusicova J, Dvorak M.
Institute of Clinical Biochemistry, 1st Faculty of Medicine & General Faculty Hospital, Charles University, Prague, Czech Republic. email@example.com
Serology markers of coeliac disease (CD) - antigliadin IgA/IgG antibodies (AGA/AGG) with purified alpha-gliadin, antiendomysium IgA antibodies (EmA) and anti-tissue transglutaminase (atTG) IgA/IgG antibodies--determined in 1451 serum samples, were analysed with respect to different screening algorithms. Determination of atTG using five ELISA methods was compared taking into account the impact of human recombinant antigen and IgG class of atTG. A subgroup of 119 patients undergoing small intestinal biopsy was used to calculate sensitivity and specificity of CD markers. The highest sensitivity (94%) was obtained for AGG, and the highest specificity (93.5%) was obtained for EmA. All coeliac disease patients were detected using the combination of all four CD markers, resulting in 100% sensitivity. CD and type 1 diabetes mellitus autoantigens were determined in 139 diabetic patients. The atTG IgA mean value (16.7 IU/ml) was higher in the antiglutamate dehydrogenase antibody (GAD)-positive subgroup, where at least one CD marker was positive in 83.6% subjects. In the GAD-negative subgroup atTG IgA was 8.73 lU/ml and at least one CD marker was positive in 57.4% subjects. atTG in IgA and IgG classes could be recommended as valuable serological markers of CD in the differential diagnosis of malabsorption as well as in various screening algorithms. ELISA determination of atTG with human antigen could increase the specificity, especially in patients with other autoimmune diseases.
22. Occult fecal blood loss--comparison of immunochemical and biochemical tests (Article in Czech)
Cas Lek Cesk. 2002 Apr 12;141 (7): 217 - 219
Dvorak M, Kocna P, Vanickova Z.
IV. interni klinika 1. LF UK a VFN, Praha.
BACKGROUND: Test of occult fecal blood loss belongs to the basic diagnostic procedures in gastroenterology. That examination is introductory method for screening, diagnosis and dispensarization of patients with colorectal cancer. Most frequently used tests in practice are biochemical and immunochemical tests. The aim of the study was to compare the results and clinical contribution of both kinds of tests. METHODS AND RESULTS: The results of immunochemical test Immocare (Care Diagnostica) and biochemical test Haemoccult (Rohm Pharma) were compared in a group of 253 patients (131 women, 122 men, average age 52.6 yr, range 19-88 yr). In all patients total coloscopy was subsequently performed. Sensitivity of immunochemical test was clearly higher (61.2%) than of biochemical test (29.4%), specificity was slight lower (95%, resp. 98%). Accuracy of Immocare test was also higher (82% than 72% of Haemoccult). CONCLUSIONS: Our results confirm advantages and competency of Haemoccult test for screening and dispensary programmes. More expensive and more difficult Immocare test is suitable for examination of symptomatic or risk patients.
21. The serologic screening for celiac disease in the general population (blood donors) and in some high-risk groups of adults (patients with autoimmune diseases, osteoporosis and infertility) in the Czech republic.
Folia Microbiol (Praha). 2002; 47 (6): 753 - 758
Vancikova Z, Chlumecky V, Sokol D, Horakova D, Hamsikova E, Fucikova T, Janatkova I, Ulcova-Gallova Z, Stepan J, Limanova Z, Dvorak M, Kocna P, Sanchez D, Tuckova L, Tlaskalova-Hogenova H.
1st Department of Pediatrics, University Hospital Motol, Prague, Czechia.
The prevalence of celiac disease (CD) was determined in healthy blood donors and in high-risk groups of adults (a total of 1835 adults--randomly selected 1312 healthy blood donors, 102 patients with primary osteoporosis, 58 patients with autoimmune diseases and 365 infertile women). It was calculated on the basis of a two-step serologic screening method--in the first step IgA and IgG antigliadin antibodies (AGA) and IgA anti-gamma-glutamyltransferase ('transglutaminase') antibodies (ATG) were estimated, in the second step sera positive for IgA AGA and/or IgA ATG were examined for antiendomysial IgA (AEA) antibodies. Immunoenzymic assay (ELISA) was used for determining of AGA and ATG antibodies; immunofluorescence method, performed on human umbilical cord tissue, was used for assaying of AEA antibodies. Total serum IgA level in only IgG AGA positive subjects was measured by routine turbidimetric method. 0.45% of healthy blood donors, 0.98% of osteoporotic patients, 2.7% of patients suffering from autoimmune disease and 1.13% of women with infertility considered as immunologically mediated were found to be positive in both steps of serologic screening (AGA and/or ATG and antiendomysium positive). The presumed high prevalence of seropositivity for CD in apparently healthy Czech adult population was confirmed. In the high-risk groups, the prevalence of seropositivity for CD was approximately 2-4 times higher than in healthy blood donors. The real prevalence of CD in the tested groups, however, can be estimated after performing small intestinal biopsy in the seropositive patients.
20. Digitalization, archival storage and use of image documentation in the GastroBase-II system (Article in Czech)
Cas Lek Cesk. 1997 May 14; 136 (10): 311 - 314
Laborator gastroenterologie I. LF UK a Odbor informatiky VFN, Praha. firstname.lastname@example.org
"GastroBase-II" is a module of the clinical information system "KIS-ComSyD"; The main part is represented by structured data-text with an expert system including on-line image digitalization in gastroenterology (incl. endoscopic, X-ray and endosonography pictures). The hardware and software of the GastroBase are described as well as six-years experiences with application of digitalized image data. An integration of a picture into text, reports, slides for a lecture or an electronic atlas is documented with examples. Briefly are reported out experiences with graphic editors (PhotoStyler), text editor (WordPerfect) and slide preparation for lecturing with the presentation software PowerPoint. The multimedia applications on the CD-ROM illustrate a modern trend using digitalized image documentation for pregradual and postgradual education.
19. Arginase activity determination. A marker of large bowel mucosa proliferation.
Eur J Clin Chem Clin Biochem. 1996 Aug; 34 (8): 619 - 623
Kocna P, Fric P, Zavoral M, Pelech T.
Department of Internal Medicine, 1st Medical Faculty, Charles University, Prague, Czech Republic.
Arginase activity of the intestinal mucosa was tested as a proliferative marker in the adenoma-carcinoma sequence. The enzyme activity was determined by an end-point colorimetric method with L-arginine as substrate. Arginase activity was evaluated in 430 biopsy samples of large bowel mucosa, polyps and cancer tissue. The activities (U/g protein, mean +/- SE; n) were: normal mucosa 83.2 +/- 7.3; 25, adenomas 199.4 +/- 19.1; 40, carcinomas 1269.7 +/- 174.9; 40, inflammatory bowel disease 1210.7 +/- 247.1; 34. The arginase activity differs significantly in the adenoma-carcinoma sequence according to the Duncan's test (p < 0.05).
18. The clinical information system GastroBase: integration of image processing and laboratory communication.
Medinfo. 1995; 8 Pt 1: 441
Lab. Gastroenterol, 1st Med. Faculty, Charles University, Prague, Czech Republic.
GastroBase, a clinical information system, incorporates patient identification, medical records, images, laboratory data, patient history, physical examination, and other patient-related information. Program modules are written in C; all data is processed using Novell-Btrieve data manager. Patient identification database represents the main core of this information systems. A graphic library developed in the past year and graphic modules with a special video-card enables the storing, archiving, and linking of different images to the electronic patient-medical-record. GastroBase has been running for more than four years in daily routine and the database contains more than 25,000 medical records and 1,500 images. This new version of GastroBase is now incorporated into the clinical information system of University Clinic in Prague.
17. Binding of gliadin to lymphoblastoid, myeloid and epithelial cell lines.
Folia Microbiol (Praha). 1995; 40 (4): 431 - 435
Farre Castany MA, Kocna P, Tlaskalova-Hogenova H.
Department of Immunology and Gnotobiology, Academy of Sciences of the Czech Republic, Prague, Czech Republic.
The aim of our work was to investigate the in vitro reactivity of gliadin peptides of natural and synthetic origin with various cell lines. We have found that all tested cell lines of human, mouse and rat origin were agglutinated by enzymically digested gliadin (peptic-tryptic- and peptic-tryptic pancreatic digest of alpha-gliadin) in a concentration dependent manner. In order to test the specificity of binding, inhibition studies were performed using a panel of sugars as well as natural and synthetic peptides derived from gliadin. We have found that among twelve tested sugars only fetuin and phosphomannan were able to inhibit the agglutination of K562 cells with peptic-tryptic- but not with peptic-tryptic pancreatic digest of alpha-gliadin. The lack of inhibition by gliadin peptides and most of the saccharides suggests that agglutinating activity of gliadin is the result of a nonspecific binding of gliadin to the cell membrane.
16. Characterization of human, mouse and rabbit anti-gliadin antibodies by ELISA and western blotting.
Folia Microbiol (Praha). 1995; 40 (6): 659 - 664
Vancikova Z, Kocna P, Tuckova L, Fric P, Dvorak M, Stoyanov S, Tlaskalova-Hogenova H.
Department of Clinical Immunology, Faculty Hospital Bulovka, Prague, Czech Republic.
Monoclonal, hyperimmune rabbit and human serum anti-gliadin antibodies were analyzed by ELISA and immunoblotting techniques. In Western blotting the difference in reactivity between monoclonal and human antibodies was quantitative rather than qualitative. Rabbit antisera differed in reactivity according to the protein used for immunization. The rabbits immunized by the peptic-tryptic pancreatic digest of gliadin reacted similarly to the patients. In ELISA, significantly higher reactivity with crude, A-, glyc-gli, alpha-, beta- and omega-gliadins was found in the patients' sera than in controls.
15. Molecular mimicry as a possible cause of autoimmune reactions in celiac disease? Antibodies to gliadin cross-react with epitopes on enterocytes.
Clin Immunol Immunopathol. 1995 Feb; 74 (2): 170 - 176
Tuckova L, Tlaskalova-Hogenova H, Farre MA, Karska K, Rossmann P, Kolinska J, Kocna P.
Department of Immunology and Gnotobiology, First Faculty of Medicine, Prague, Czech Republic.
Structural similarities between external antigen and self components are believed to be one of the possible causes of autoimmunity. This study describes the presence of similar structures shared by gliadin and enterocyte surface molecules recognized by antigliadin mAbs. The reactivity of mAbs to gliadin was followed by ELISA using fixed enterocytes, their brush-border membranes, or purified enterocyte antigen. The specificity of reaction was confirmed by ELISA inhibition studies and by immunohistochemical staining of rat tissue sections using biotin-avidin-peroxidase technique. Immunoprecipitation analysis of 125I-labeled intestinal epithelial cells using antigliadin mAb revealed the presence of two main cross-reactive molecules of 28 and 62 kDa. The 62-kDa and an associated 66-kDa protein were isolated by affinity chromatography. Immunoblotting analysis showed that a 28-kDa protein detected by immunoprecipitation also reacted with IgA of celiac disease patient sera.
14. Comparison of immunochemical and biochemical tests for occult hemorrhage in feces. (Article in Czech)
Cas Lek Cesk. 1993 Sep 13; 132 (17): 523 - 525
Benesova A, Fric P, Zavoral M, Kocna P, Matejovic F, Vokracka V, Mares K.
Interni oddeleni Fakultni polikliniky FN 2, Praha.
The authors compared in a group of 104 patients assessment of occult haemorrhage in faeces made by the immunochemical test HemeSelect (HS-SmithKline Diagnostics) and the biochemical Haemoccult test (HT-Rohm Pharma). In all patients subsequently total coloscopy was performed. In the whole group the sensitivity of HS was 49.3% and of HT 16.0% (p << 0.001) and the specificity 72.4% and 100% (p << 0.01). The predictive value in polyps, 10 mm in size or larger, was 65.2% (positive) and 60.0% (negative). The assessed sensitivity and predictive values of HS do not permit to recommend immunochemical tests of this type as an alternative procedure to repeated colonoscopic checks in the dispensarization of high risk groups of colorectal cancer.
13. Comparison of immunochemical and biochemical methods for determination of occult fecal blood. (Article in Czech)
Cas Lek Cesk. 1992 Mar 13; 131 (5): 149 - 151
Ferkl M, Kocna P, Fric P.
Interni odd. fakult. polikliniky fakultni nemocnice, Praha.
The authors compared two tests of occult Fecal Blood Loss--the immunochemical Hemolex test based on agglutination of latex particles lined with antibodies against human haemoglobin and the biochemical Haemoccult test. Both tests were used to examine 55 patients invited to the department for endoscopic polypectomy or coloscopy as part of dispensarization after resection of colorectal carcinoma. The sensitivity of the Hemolex test (48.6%) is statistically significantly higher, as compared with the Haemoccult test (17.1%), the specificity is insignificantly lower in the Hemolex test (90%), as compared with the Haemoccult test (100%). The results indicate some advantages of the immunochemical examination of occult Fecal Blood Loss and the assumed area of its application.
12. Effective peritoneal therapy of acute pancreatitis in the rat with glutaryl-trialanin-ethylamide: a novel inhibitor of pancreatic elastase.
Gut. 1992 May; 33 (5): 701 - 706
Fric P, Slaby J, Kasafirek E, Kocna P, Marek J.
Department of Medicine, Charles University, Prague, Czechoslovakia.
The six hour peritoneal lavage with glutaryl-trialanin-ethylamide, a low molecular competitive inhibitor of pancreatic elastase (IC50-8 mumol/l), effectively suppresses the evolution of taurocholate induced acute pancreatitis in the rat. The lavage alone is followed by a marked decrease of fat necrosis and amylase and lipase activity in serum. The area of pancreatic haemorrhage was significantly reduced only after the lavage solution was supplemented with Glt-Ala3-NHEt. The effect was not enhanced by a bolus injection of the inhibitor before starting the lavage. The combination of Glt-Ala3-NHEt with aprotinin or nafamstate mesilate produced only marginal greater benefit. The effect of Glt-Ala3-NHEt on pancreatic haemorrhage is time and dose related even with delayed onset of the lavage. Animals treated with peritoneal lavage without Get-Ala3-NHEt lived longer than controls (p less than 0.05), but by 60 hours the survival rate of both groups was almost the same (76 v 74%). All animals lavaged with Glt-Ala3-NHEt survived 120 hours and the difference in the survival rate between this and both remaining groups was significant (100% v 76% v 74% - p less than 0.05). The results were considered favourable and preliminary clinical trials of Glt-Ala3-NHEt in subjects with acute pancreatitis justified.
11. Relationship between gliadin peptide structure and their effect on the fetal chick duodenum.
Z Lebensm Unters Forsch. 1991 Feb; 192 (2): 116 - 119
Kocna P, Mothes T, Krchnak V, Fric P.
Laboratory of Gastroenterology, Faculty of General Medicine, Charles University, Prague, Czechoslovakia.
The tendency to form a beta-turn in alpha-gliadin was estimated using the B-cell determinant prediction program based on the Chou and Fasman probability of beta-turn formation. Six sequences possessing a high probability of beta-turn formation were found. A statistically high agreement was found between these six sequences and three areas in alpha-gliadin with the occurrence of Pro-Ser-Gln-Gln sequence which has recently been considered responsible for toxicity in coeliac disease. By means of solid-phase synthesis seven peptides were obtained covering the above-mentioned regions. Their toxicity was tested using the fetal chick duodenum. The results support the suggestion that peptides containing the sequences Pro-Ser-Gln-Gln and Gln-Gln-Gln-Pro may be involved in the pathogenesis of coeliac disease.
10. Continuous-flow synthesis of alpha-gliadin peptides in an ultrasonic field and assay of their inhibition of intestinal sucrase activity.
Pept Res. 1991 Sep-Oct; 4 (5): 284 - 288
Vagner J, Kocna P, Krchnak V.
Research Institute for Feed Supplements and Veterinary Drugs, Czechoslovakia.
Three dodecapeptide amides derived from the amino acid sequence of alpha-gliadin and four of their analogues were synthesized by continuous-flow solid-phase multiple peptide synthesis. Ultrasonic field conditions accelerated the coupling reaction without affecting purity. Biological tests of the synthetic fragments showed the relevance of toxicity prediction for preselection of immunogenic alpha-gliadin fragments.
9. Differentiation of pancreatic lipase and pseudolipase serum activity. (Article in Czech)
Sb Lek. 1989 Mar; 91 (2-3): 33 - 39
Kocna P, Slaby J.
The determination of the serum lipase activity by turbidimetric assay was modified and false-positive results due to pseudolipase presence were eliminated. The new method includes a preincubation with beta-mercaptoenthanol and its conditions are specified to use with kinetic analysers as LKB 2086. By using our new method we estimated the activity of the pseudolipase in 36.4% of the group of 85 patients with elevated serum lipase activity determined by standard turbidimetry. By introducing our differential method with beta-mercaptoethanol correction the increasing correlation about 27% between lipase activity and serum alpha-amylase activity was estimated.
8. The effect of Sinecal fiber on compensation in type 2 diabetics. (Article in Czech)
Vnitr Lek. 1989 Oct; 35 (10): 998 - 1005
Perusicova J, Fric P, Cacakova V, Mardesicova I, Stolba P, Kocna P.
The authors investigated in 28 type 2 diabetics the effect of addition of 21 g Sinecal fibre to the normal diet on the glucose and lipid metabolism. The value of C,peptide was significantly influenced after breakfast and a single dose of Sinecal. The other investigated parameters (blood sugar on fasting and after a meal, glycosuria, glycosylated proteins, C-peptide on fasting and after a meal, triacylglycerol and cholesterol) were not influenced by a single nor by long-term intake of Sinecal. Further development of fibre preparations with better tolerance and a greater effect on the glucose metabolism is desirable.
7. Monoclonal antibodies reacting with gliadin as tools for assessing antigenic structure responsible for exacerbation of celiac disease.
Immunol Lett. 1988 Apr; 17 (4): 335 - 338
Stoyanov S, Tlaskalova-Hogenova H, Kocna P, Kristofova H, Fric P, Hekkens WT.
Institute of Molecular Genetics, Czechoslovak Academy of Science, Prague.
Monoclonal antibodies reactive with gliadin were prepared by fusion of spleen cells isolated from gliadin-immunized BALB/c mice with myeloma cells. The reactivity of mAbs with different preparations of gliadin and their enzymatic digest were measured using ELISA method. The mAb produced by GL 1 clone was shown to react preferentially with alpha-gliadin and its enzymatic digest.
6. Isolation and analysis of peptidic fragments of alpha-gliadin using reversed-phase high-performance liquid chromatography.
J Chromatogr. 1988 Dec 30; 434 (2): 429 - 438
Kocna P, Fric P, Kocova-Holakova M, Slaby J, Kasafirek E, Hekkens WT.
Laboratory of Gastroenterology, Charles University, Faculty of General Medicine, Prague, Czechoslovakia.
Peptidic fragments of alpha-gliadin were obtained by peptic-tryptic-pancreatic (PTP) digestion of the alpha-gliadin fraction isolated by ion-exchange chromatography on a sulphopropyl-Sephadex C-50 column. The proteolytic digest was fractionated by ultrafiltration into three subfractions, PTPa1-PTPa3. The subfraction PTPa2 was then analysed and individual peaks were separated using reversed-phase high-performance liquid chromatography (RP-HPLC) using a gradient of acetonitrile in 0.1% trifluoroacetic acid and a Separon SGX-C18 sorbent. A 100-mg amount of the PTPa2 subfraction was separated in a single analysis by preparative RP-HPLC and twenty peaks were obtained for further characterization. The molecular mass in range 300-3000 was established for individual peptidic fragments by gel-permeation chromatography on a TSK-G2000 SW column.
5. Inhibitors of pancreatic and leukocyte elastase.
Collect. Czech. Chem. Commun. 1987, 52, 3034-3041
Kasafírek E., Frič P., Slabý J., Kocna P.
Research Institute for Pharmacy and Biochemistry, Prague; Laboratory of Gastroenterology, Charles University, Prague; Department of Medicine, Policlinic of Charles University, Prague
The following alkylamides of ?-carboxyalkanoyldi- and tripeptides of the Ala-Pro or Ala-Ala-Pro sequence have been prepared: ethylamide-, propylamide-, and isobutylamide of 3-carboxypropionylalanyl-proline, ethylamide of 3-carboxypropionylalanyl-alanyl-proline and ethylamide-, propylamide, and isobutylamide of 4-carboxybutyrylalanyl-alanyl-proline. The inhibitors were synthesized by fragment condensation in solution or by gradual construction. The inhibition constants Ki were determined by means of pancreatic elastase (substrates - p-nitroanilides of 4-carboxybutyrylalanyl-alanyl-alanyl-alanine and 3-carboxypropionylalanyl-alanyl-alanyl-alanine) and leukocyte elastase (substrate - p-nitroanilide of 4-carboxybutyrylalanyl-alanyl-alanyl-valine). The strongest inhibitions (Ki) were recorded in ethylamide of 4-carboxybutyrylalanyl-alanyl-proline, 2.0 and 1.6 µmol l-1 for pancreatic elastase, and in propylamide of 4-carboxybutyrylalanyl-alanyl-proline, 0.4 mmol l-1 for leukocyte elastase.
4. Modification of a Microprocessor-Controlled HPLC System (Perkin-Elmer).
Journal of Chromatographic Science, 1985, 23 (3), 132-136
Kocna P. Mittermüller B.J.
Laboratory of Gastroenterology, Faculty of Medicine, Charles University, Prague, Czechoslovakia; Perkin-Elmer Service Department, Vienna, Austria
Three modifications of a microprocessor-controlled high performance liquid chromatography system are described: initialization of peripheral devices by the injection valve enabling more precise estimation of retention constants, automatic return of the automatic switching valve to the RUN position after stop-flow spectroscopy, and solvent-saving by program cycling during overnight column stabilization.
3. Simple starch-gel electrophoresis of gliadin proteins using the LKB-multiphor system.
Zeitschrift für Lebensmitteluntersuchung Forschung A, 1983, 177 (6): 454 - 456
Kocna P, Holáková-Kocova M, Sasek A.
A simple modification of the horizontal starch-gel electrophoresis of gliadin proteins with the use of LKB Multiphor System has been developed. The thin-layer starch gel is prepared by using the original gel-moulding cassette and the procedure on the whole is easier. The sensitivity is comparable with that of the original method. Our modification moreover widens the field of application of the LKB Multiphor System.
2. Cleavage of p-nitroanilides of N-acylated tri- and tetrapeptides by alanine endopeptidase from the brush border membranes of rat enterocytes.
Experientia. 1983 Apr 15; 39 (4): 389 - 390
Kocna P, Kasafirek E, Fric P, Slaby J.
The activity of the alanine endopeptidase from the intestinal brush border was studied using chromogenic substrates of the general formula Sc-Ala2-X-pNA. Sc-Y-Z-Ala-pNA and W-Ala3-pNA respectively. Substrates with C-terminal Leu or Nle are hydrolyzed more readily than Ala-analogues. At least one Ala-residue in one of the positions adjacent to the C-terminus is necessary for the enzyme activity. An Na-substituent has no effect on the activity.
1. Endopeptidase of the brush border membrane of rat enterocyte. Separation from aminopeptidase and partial characterization.
Hoppe Seylers Z Physiol Chem. 1980 Sep; 361 (9): 1401 - 1412
Kocna P, Fric P, Slaby J, Kasafirek E.
The brush border of the enterocytes of the rat was isolated by the method of differential centrifugation with CaCl2 according to Schmitz. This material was solubilized with papain, trypsin and Triton X-100. The greatest amount of membrane enzymes was released to the supernatant (105 000 X g) with the use of Triton X-100. The tritonized supernatant was treated in the next step by papain, bromelain, ficin and trypsin (individually or in combinations). After simultaneous proteolysis with papain and bromelain a partial separation of the aminopeptidase from the endopeptidase by Sephadex G-200 chromatography was observed. These two enzyme activities were distinctly separated by isoelectric focusing at pH 4--6. Two enzymatically active bands (RF 0.13 and 0.24) in the aminopeptidase fraction and one single active band (RF 0.16) in the endopeptidase fraction using polyacrylamide gel electrophoresis were found. Co-migrating proteins to all of these activities were detected. Endopeptidase activity splits 3-carboxypropionyltrialanin-4-nitroanilide (SucAla3NAp) in the position P2-P1. Liberated aminoacyl-NAP may be further split to generate chromogenic 4-nitroaniline through aminopeptidase activity. Endopeptidase of the brush border of the rat enterocytes is characterized by the following properties: 1) molecular mass 130000 +/- 15 000 dalton; 2) Km value (substrate: SucAla3NAp) 1.1 X 10(-3) M; 3) pI 5.23; 4) ph optimum 8.5; 5) 50% activity remains after 15 min of preincubation at 50 degrees C; 6) activity is strongly inhibited by EDTA, p-chloromercuribenzoate, Mn2 and Co2.
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