1. Identification and quantitative analysis of various advanced glycation end products
(AGEs) in serum of patient with atherosclerosis or diabetes oratherosclerosis with
key researcher: Agnieszka Bronowicka-Szydełko, PhD
Financial support:Research grant from National Science Center #2012/05/N/NZ5/00836:
- The methods for the preparation of the MAGE antigen analogue and the LMW-AGE nickel-cross-linked molecule were designed and required to set a standard curve, and the conditions of immunoenzymatic tests allowing the quantitative evaluation of MAGE DG in blood serum were optimized
- The conditions of the SLOT DOT-BLOT method for semi-quantitative assessment of AGE-1, AGE-3, AGE-4 and 3-DG in blood serum were developed.
- Statistical analyzes showed that the occurrence of AGE-1, AGE-4 and MAGE has diagnostic value:
1) The presence of MAGE is associated with the occurrence of microangiopathy
2) the level of AGE-1 is dependent on the occurrence of metabolic syndrome (MetS), obesity, hyperlipidemia and alcohol-free fatty liver steatosis and is more strongly associated with lipid metabolism abnormalities than glucose;
3) In patients with abnormal glucose metabolism, metformin treatment was associated with a higher level of AGE4. The AGE4-positive result was significantly lower in patients with diabetes treated with glyphine. Multivariate analysis showed that polyneuropathy and obesity are independently associated with a positive AGE4 result.Logistic regression showed that AGE4 and HbA1c are independent indicators of polyneuropathy. Consideration of both indicators allows a correct classification of 70.4% of cases with a general accuracy of 76%.
The project involved studies under the glycation process – a cascade of non-enzymatic reactions between sugars or oxo-aldehydes, and amino groups of proteins, nucleic acids and lipids, leading to the formation of final products of advanced glycation end-products AGEs. Glycation is an endogenous process (physiological glycation progresses with age, whereas pathological – intensifies during metabolic disorders especially in diabetes and atherosclerosis) as well as exogenous (occurs during thermal food processing). Due to the fact that AGE are persistent compounds that are not sensitive to enzymes action, they are considered to be potential diagnostic biomarkers of diabetes and its complications, atherosclerosis, neurodegenerative diseases, cardiovascular diseases, and cancer. Although glycation products are a group of different compounds with different physical and biological properties, only a few AGE are known and diagnostic tests are developed only for them. Determination of the diagnostic value of other AGEs would allow better monitoring of the disease progression and effectiveness of therapy.
The aim of the project was to develop immunoenzymatic methods that enable the detection of 5 different advanced glycation end-products: MAGE (unknown in the literature yet), AGE-1 (BSA-glucose), AGE-3 (BSA-glycoaldehyde), AGE-4 (BSA-methylglyoxal) and 3DG (BSA-imidazolone) in the blood serum of patients with type 2 diabetes, atherosclerosis and atherosclerosis complications of diabetes. As part of the project, the conditions of one quantitative competitive ELISA test for the MAGE assessment were developed. The obtained results indicate the existence of a relationship between the MAGE level in the blood serum of patients with type 2 diabetes and the occurrence of microangiopathy. To develop the test, it was necessary to elaborate conditions for the synthesis and purification of high molecular mass MAGE (antigen analogue), neo-epitope (low molecular weight) LMW-AGE, and to obtain monoclonal anti-MAGE antibodies. The molecular weight and structure of the low molecular weight LMW-AGE were also determined and the physical and biological properties of MAGE and LMW-AGE were studied. Currently, the patent application is being prepared. The conducted studies provide knowledge on optimizing the synthesis, purification and testing of physical and biological properties of compounds that are glycation products. This knowledge is fundamental in conducting further research aimed at determining the structures of individual AGEs. This will allow to receive complete information on the physical and biological properties of the AGEs under investigation and their diagnostic value.
It has also been demonstrated that the detection of AGE-1 (BSA-glucose), AGE-3 (BSA-glycaldehyde), AGE-4 (BSA-methylglyoxal) and 3DG (BSA-imidazolone) in human serum is practically impossible by means of standard immunoenzymatic methods ( ELISA, western blot), because these compounds are present in the blood serum in trace amounts. It has been shown, however, that the use of appropriate concentration conditions for anti-AGE1, anti-AGE3, anti-AGE4 and anti-3DG and serum antibodies allows for detection and semi-quantitative assessment of these compounds using a different immunoenzymatic method – SLOT DOT-BLOT. This method allows testing of much more biological material than ELISA or western blot. The diagnostic values of AGE-1 and AGE-4 have been demonstrated. The level of AGE-1 is related to the occurrence of metabolic syndrome (MetS), obesity, hyperlipidemia and non-alcoholic steatosis of the liver and is more strongly associated with irregularities of lipid metabolism than glucose. It was also shown that treatment with metformin was associated with a higher level of AGE-4 in patients with abnormal glucose metabolism. Moreover, metformin treatment was associated with a higher level of AGE-4 in patients with abnormal glucose metabolism. However, the AGE-4 level was significantly lower in patients with diabetes treated with glyptine. It has been found that polyneuropathy and obesity are independently associated with AGE-4 and AGE-4 and HbA1c are independent indicators of polyneuropathy.
The research carried out in project allowed for a better understanding of the properties of glycation products and for determining the diagnostic value of AGEs formed during protein glycation by three types of glycating agents: monosaccharides (AGE-1), disaccharides (MAGE) and oxo-aldehydes (AGE-3 and AGE-4). These studies provide an introduction to further work on learning about other glycation products - the development of methods for the synthesis and purification of AGE, learning about their biological and physical properties, checking their diagnostic value and developing methods for detecting individual AGE in biological material. Research on learning about AGE compounds is consistent with the assumptions of both: The National Smart Specialization 1 (KIS1) “Healthy Society” (division II “Diagnostics and disease therapy”, section II “Markers/tests”) and The National Smart Specialization 2 (KIS2) “Innovative technologies, processes and products of agri-food and forest-wood sector” (Division IX “Food and consumer”, section II “The use of innovative technologies to create tools supporting better nutrition planning and the assessment of diet at individual and collective level", section V “Developing tools and modern research techniques and food quality markers, including the bioavailability of ingredients, for the purpose of assessing the impact of food products on human health” and section VI: “Development of methods of analysis and selection of food dedicated at the population and individual level"). Conducting research on the phenomenon of glycation contributes not only to the development of modern diagnostics, but also to public awareness that avoiding the preparation of meals at high temperatures under anhydrous conditions or in microwaves is an effective prevention against the progress of metabolic disorders. Further research is also planned to develop methods for determining AGE in food.
1. Bronowicka-Szydełko A, Kuzan A, Serek P, Gostomska-Pampuch K: Comparison of the effectiveness of slot dot-blot and ELISA methods in marking compounds occurring in blood serum in trace amounts. W: Interdyscyplinamość badań naukowych 2017: praca zbiorowa pod red. Jarosława Szreka; Wrocław: oficyna Wydawnicza Politechniki Wrocławskiej, 2017; s. 13-18
3 publications in publishing processes
1. 26-27.05.2016r., Budapeszt, Węgry. 3rd World Congress on Maillard Reaction & Glycation "Targeting glycation in health & diseases" Agnieszka Bronowicka-Szydełko, Aleksandra Kuzan, Kinga Gostomska-Pampuch, Małgorzata Gacka, Jadwiga Pietkiewicz, Urszula Jakobsche-Policht, Rajmund Adamiec, Andrzej Gamian.: Slot dot-blot as an effective method for detection of AGEs present in sera in trace amounts. Abstracts book & scientific agenda; s.64
2. 13-16.09.2016r. Wrocław (Poland), BIO 2016 Congress. Agnieszka Bronowicka-Szydełko, Aleksandra Kuzan, Kinga Gostomska-Pampuch, Małgorzata Krzystek-Korpacka, Jadwiga Pietkiewicz, Andrzej Gamian.: Detecting of the presence of advanced glycation end products AGE 1 and AGE3 in the serum blood of diabetic patients by the slot dot-blot method. Acta Biochim.Pol. 2016 Vol.63 suppl.2; s.211 poz.LA9. Late abstracts
3. 29.06.2017 - 1.07.2017r., Szklarska Poręba, Polska - Interdyscyplinarna Konferencja dla Młodych Naukowców "Kooper field 2017" Agnieszka Bronowicka-Szydełko, Aleksandra Kuzan, Paweł Serek, Kinga Gostomska-Pampuch. Comparison of the effectiveness of slot dot-blot and ELISA methods in marking compounds occurring in blood serum in trace amounts.
2. Biomechanical determinants of the mechanical load-bearing process of the walls of abdominal aortic aneurysm
National Science Centre grant no. DEC-2013/09/D/ST8/04007
Researcher: Agnieszka Chwiłkowska, PhD
The abdominal aortic aneurysm (AAA) is a permanent and progressive, local enlargement of the diameter of infrarenal abdominal aorta by at least 50% compared to its proper diameter. Formation and development of the aneurysm is a threat to the patient’s life, as it may lead to an interruption of the continuity of the aneurysm wall, i.e. to its rupture. As the latest research shows, the degree of aneurysm growth is not determined by its size, but rather by disturbances in the structure of AAA walls, which relate mainly to the elements transferring mechanical loads, both passivelly, such as elastin and collagen fibers, and actively, i.e. smooth muscle cells. The degradation of these structural components leads to disturbances in the process of transfer of mechanical loads through the degenerated blood vessel wall. The ability of each material, including biological materials, to transfer loads can be assessed on the basis of its mechanical properties. The complex structure of blood vessel walls, i.e. three layers with multi-phase and composite structure of each layer, required investigation of mechanical properties taking into account the different levels in the hierarchical structure of the vessel wall. Hence, the main objective of the project is to assess: the ability of degenerated aneurysm wall structure to transfer mechanical loads, and the contribution of elastin and collagen fibers and smooth muscle cells in the process of transferring mechanical loads through the walls of abdominal aortic aneurysms.
Parametry mechaniczne jako wskaźniki w ocenie stopnia rozwoju tętniaka aorty brzusznej oraz prawdopodobieństwa jego pęknięcia. Marta Kozuń, Magdalena Kobielarz, W Interdisciplinary of Scientific Research (PL: Interdyscyplinarność Badań Naukowych), Wrocław, Poland, 2014, s. 205-208.
Abdominal aortic aneurysm from mechanical point of view. Magdalena Kobielarz, Marta Kozuń, Agnieszka Chwiłkowska, 22nd Congress of the European Society of Biomechanics, 2016, Lyon, France
Experimental verification of abdominal aortic aneurysm incompresibility, Magdalena Kobielarz, Marta Kozuń, 22nd Congress of the European Society of Biomechanics, 2016, Lyon, France
The effect of smooth muscle cells relaxation on mechanical properties of abdominal aortic aneurysm wall. Marta Kozuń, Agnieszka Chwiłkowska, Krzysztof Maksymowicz, Magdalena Kobielarz, Biomechanics 2016, International Conference of the Polish Society of Biomechanics
3. Midkine status and role in inflammatory bowel disease and colorectal cancer
Key researcher: Małgorzata Krzystek-Korpacka, PhD
1. Research grant from National Science Center #2011/01/D/NZ5/02835: “Midkine, a multipotential cytokine, in inflammatory bowel disease (Crohn’s disease and ulcerative colitis) – its role in pathogenesis and regeneration, diagnostic and therapeutic potential”
2. EIT+: “Biotechnologies and advanced medical technologies – BioMed” (POIG 01.01.02-02-003/08-00) financed from the European Regional Development Fund (Operational Programme Innovative Economy, 1.1.2)
Inflammatory bowel disease (IBD), encompassing Crohn's disease (CD) and ulcerative colitis (UC), are incurable chronic inflammatory conditions of uncertain etiopathology. It is presumed, however, that disease results from excessive immune response to pathogenic or functionally altered commensal enteric bacteria in genetically susceptible subjects. Increasing incidence, chronic relapsing character, high activity of inflammatory processes, extraintestinal manifestations, and frequently poor response to treatment, make IBD a serious clinical/public health problem. Moreover, patients with IBD are at greater risk for the development of colorectal cancer (CRC) and as such require constant surveillance.
A more thorough understanding of IBD pathogenesis is needed to provide new therapeutic options for biological therapies and potential biomarkers. Currently, IBD diagnosis and CRC surveillance is based almost entirely on the endoscopy/biopsy-based approach - invasive, expensive, and non-objective. The only, yet unsatisfactory, blood-based biomarkers of clinical relevance in IBD and CRC are, respectively, C-reactive protein (CRP) and carcinoembryonic antigen (CEA).
Midkine is a multifunctional cytokine highly expressed during mid-gestation and re-induced during carcinogenesis, inflammation and tissue repair. It displays a number of properties potentially relevant for IBD and CRC induction and progression. Midkine shows chemotactic activity towards neutrophils and monocytes, is a mitogenic, antiapoptotic and proangiogenic factor, T-regulatory cell suppressor, inductor of expression of proinflammatory cytokines, extracellular matrix components, and metaloproteinases. As such, midkine may constitute a new target for both IBD and CRC therapies and/or serve as disease biomarker. Yet, its status in IBD and CRC remains largely unknown.
We were first to demonstrate an elevation in circulating midkine in IBD that corresponded with disease clinical and endoscopic activity, implying its involvement in disease pathogenesis and/or progression. We also found midkine to be elevated in patients with CRC, in whom an increase in midkine levels was associated mainly with a metastatic disease. Midkine expression is up-regulated both in transformed and inflamed tissue compare to normal one. Contrary to circulating mdkine, its tissue expression was the highest in less advanced cancers. Interestingly, we found it to increase along with CRC advancement also in untransformed bowel tissue, demonstrating a field effect in cancer. In IBD, inflammation-induced up-regulation can be observed in large but not small bowel where, solely in ulcerative colitis, it paralleles the disease clinical activity. This phenomenon, together with a less pronounced elevation in Crohn’s lesions, implies a tighter midkine association with ulcerative colitis. Midkine discriminated early colorectal cancer (CRC) from IBD more accurately than CEA. As such, midkine might constitute a valuable component of multimarker panels devised for CRC surveillance. In vitro, midkine modulated angiogenesis and inflammatory response by regulating the expression/secretion of VEGF-A, IL8, MCP1, and TGFb among others. Yet, it did not affect proliferation of endothelial cells. Contrary to gastric cells, midkine exerted an inhibitory effect on SW620, SW480 and HT29 lines, which was accompanied by down-regulation of proliferative indices Ki67 and PCNA and cell cycle regulators, mainly CDK6, and up-regulation of its inhibitors CDKN1A and TP53. Midkine also attenuated the growth inhibition caused by endoplasmatic reticulum (ER) stress inductor, thapsigargin.
1. Krzystek-Korpacka M, Neubauer K, Matusiewicz M. Clinical relevance of circulating midkine in ulcerative colitis. Clin Chem Lab Med. 2009;47(9):1085-90.
Pkt. MNiSW: 27.000
2. Krzystek-Korpacka M, Neubauer K, Matusiewicz M. Circulating midkine in Crohn's disease: clinical implications. Inflamm Bowel Dis. 2010 Feb;16(2):208-15.
Pkt. MNiSW: 32.000
3. Krzystek-Korpacka M, Diakowska D, Grabowski K, Gamian A. Tumor location determines midkine level and its association with the disease progression in colorectal cancer patients: a pilot study. Int J Colorectal Dis. 2012 Oct;27(10):1319-24.
4. Krzystek-Korpacka M, Diakowska D, Neubauer K, Gamian A. Circulating midkine in malignant and non-malignant colorectal diseases. Cytokine. 2013 Oct;64(1):158-64. doi: 10.1016/j.cyto.2013.07.008. Epub 2013 Jul 27.
5. Krzystek-Korpacka M, Diakowska D, Kapturkiewicz B, Bębenek M, Gamian A. Profiles of circulating inflammatory cytokines in colorectal cancer (CRC), high cancer risk conditions, and health are distinct. Possible implications for CRC screening and surveillance. Cancer Lett. 2013 Aug 28;337(1):107-14. doi: 10.1016/j.canlet.2013.05.033. Epub 2013 May 29
6. Krzystek-Korpacka M, Diakowska D, Bania J, Gamian A. Expression stability of common housekeeping genes is differently affected by bowel inflammation and cancer: implications for finding suitable normalizers for inflammatory bowel disease studies. Inflamm Bowel Dis. 2014 Jul;20(7):1147-56.
7. Krzystek-Korpacka M, Matusiewicz M. Circulating midkine in malignant, inflammatory, and infectious diseases: a systematic review. In: Midkine: from embryogenesis to pathogenesis and therapy ; eds Mine Erguven, Takashi Muramatsu, Ayhan Bilir; Dordrecht [etc.] : Springer, 2012; pp69-85; ISBN 978-94-007-4233-8