The impact of different molecular weights hyaluronans on adult smooth muscle cells and asthmatic smooth muscle cells
Astapenko D.1,2,3, Ravix J.4, Wicher S.4, Roos B.4, Pabelick Ch.4,5, and Prakash Y. S.4,5 Student: David Astapenko, M.D.
Mentor: prof. Vladimir Cerny, M.D., Ph.D., FCCM
1 Dept. of Anesthesiology, Resuscitation and Intensive Care Medicine, University Hospital Hradec Kralove, CZE
2 Medical Faculty in Hradec Kralove, Charles University, CZE
3 Centre for Research and Development, University Hospital Hradec Kralove, CZE
4 Dept. of Physiology and Biomedical Engineering, Mayo Clinic, Rochester, MN, USA
5 Dept. of Anesthesiology and Perioperative Medicine, Mayo Clinic, Rochester, MN, USA
Hyaluronic acid (HA) is among the most abundant polysaccharides in an extracellular matrix. A distinct biological effect of different molecular weights HA on human cells has been demonstrated in vitro. Low molecular weight HA (LMWHA) exerts inflammatory effect whereas high molecular weight HA (HMWHA) anti-inflammatory effect. We intended to investigate a biological effect of LMWHA and HMWHA on adult bronchial smooth muscle cells as a potential new treatment of bronchial asthma which is a common chronic disease of the respiratory system that affects approximately 300 million people worldwide. We also planned to investigate HA signaling pathways and regulatory enzymes.
Adult non-asthmatic smooth muscle cells (SMCs) and asthmatic smooth muscle cells (ASMCs) were incubated with HMWHA(>800 kDa) and LMWHA (<60 kDa) in 10M concentration for 30 minutes and examined by calcium imaging system. The amount of intracellular calcium concentration was visualized in fluorescent microscopy using a Fura2am staining after adding an agonist histamine in 10M concentration. For protein analysis, the SMCs and ASMCs were harvested after 48 hours incubation with HMWHA and LMWHA in 10M concentration. The protein expressions of hyaluronan synthase 1, 2, 3, hyaluronidase 1, CD 44 and TLR 4 were visualized by western blot. All data were tested for normality and are shown as mean ( standard deviation). Group analysis was done by unpaired t-test. The level of significance was at p=0,05.
The amplitude of intracellular calcium concentration was significantly higher after stimulation with agonist in SMCs and ASMCs incubated with LMWHA: 1,61mol ( 1,70), 1,68 mol ( 1,51) respectively, p= 0,004 and p= 0,0006 respectively compared to controls:
0,49 mol ( 0,28), 0,22 mol ( 0,06) respectively and also in ASMCs incubated with HMWHA: 0,98 mol ( 0,72) compared to controls, p= 0,0003. As for protein expression we did not find any remarkable change in HA signaling pathways and regulatory enzymes between SMCs and ASMCs.
We have demonstrated that LMWHA and HMWHA induce the different biological response of SMCs and ASMCs to the agonist by the higher intracellular concentration of calcium. Our findings deserve further investigation whether HMWHA could by targeted as an anti-inflammatory agent in bronchial asthma treatment.
A mouse model of BRAF V600E mutated papillary thyroid cancer, imitating sporadic conditions
Iva Jakubíková1,2, Elin Schoultz1, Ellen Johansson1, Shawn Liang1, Konrad Patyra3, Jukka Kero3, Pavel Žák2, Mikael Nilsson1
1Sahlgrenska Cancer Center, Institute of Biomedicine, University of Gothenburg, Göteborg, Sweden
24th Department of Internal Medicine – Haematology, University Hospital Hradec Králové, Charles University, Faculty of Medicine in Hradec Králové, Czech Republic
3Department of Physiology, Institute of Biomedicine, University of Turku , Turku, Finland Introduction:
Worldwide constantly rising incidence of thyroid cancer promotes research activities. Nowadays the most speculated issue is the clinical significance of BRAF mutation. For sure BRAF
mutation is a cancer-specific somatic mutation consistent with papillary thyroid cancer
phenotype, otherwise, its role in tumor aggressiveness, progression, and the overall poor outcome is controversial. As far as several clinical studies claim opposing opinions, the learning aspect of mouse models comes to its point.
Objective:
To evaluate the effect of BRAF V600E mutation on follicular cells of the thyroid gland in a mouse model, which imitates sporadic oncogenic pathway.
Methods:
A transgenic mouse model of a spontaneous Cre activation in the absence of tamoxifen leading to focal instead of global BRAF V600E activation, under the Thyroglobulin promoter, was observed at several time points. Thyroid glands of these mice underwent different immunohistochemical stainings (IHCS), that were compared with wild-type controls.
Results:
BRAF V600E mutation was gradually activated in follicular cells and some areas of the thyroid gland revealed after 6 months papillary formations with typical nuclear characteristics for
carcinoma. From the very beginning, the follicles and so the whole thyroid gland was growing in size, without causing breathing impairment. Blood measurements were performed, confirming normal serum levels of T4 and TSH. The oldest sacrificed mouse, aged 18 months, shown in each of its thyroid lobes several foci of papillary thyroid carcinoma (PTC). There were mainly areas of classical PTC, then solid PTC as well as the hobnail variant pattern. The proliferative rate
(including Ki67 IHCS) was overall very low. Loss of thyroglobulin expression occurred early after mutant Braf activation i.e. before overt tumor formation. In larger tumor formations further
loss of dedifferentiation was documented by the loss of E-cadherin and weaker expression of Nkx2-1 (TTF-1) in 12 months or older mice. The tumor niche did not show any rapid
involvement of inflammatory cells.
Conclusion:
This mouse model imitates a sporadic oncogenic tumor initiation under the activation of BRAF V600E point mutation solely expressed in follicular cells of mice thyroid gland. The model recapitulates BRAF V600E-mediated tumor initiation, development, and progression that may be used for further investigation such as drug treatment with tyrosine kinase inhibitors or solely Braf kinase inhibitors in different phases of papillary cancer growth.
References:
Chakravarty D, Santos E, Ryder M, et al. Small-molecule MAPK inhibitors restore radioiodine incorporation in mouse thyroid cancers with conditional BRAF activation. J Clin Invest.
2011;121(12):4700–4711.
Charles RP, Iezza G, Amendola E, et al. Mutationally Activated BRAFV600E Elicits Papillary Thyroid Cancer in the Adult Mouse. Cancer Res 2011;71:3863-3871.
Knauf JA, Sartor MA, Medvedovic M. Progression of BRAF-induced thyroid cancer is
associated with epithelial–mesenchymal transition requiring concomitant MAP kinase and TGFb signaling. Oncogene 2011;30:3153–3162.
Knauf JA, Xiaolan M, Smith EP, et al. Targeted Expression of BRAFV600E in Thyroid Cells of Transgenic Mice Results in Papillary Thyroid Cancers that Undergo Dedifferentiation. Cancer Res 2005;65:4238-4245.
Kim CS, Zhu X. Lessons from Mouse Models of Thyroid Cancer. Thyroid 2009;19(12):1317- 1331.
Knostman KAB, Jhiang SM, Capen CC. Genetic Alterations in Thyroid Cancer: The Role of Mouse Models. Vet Pathol 2007;44:1–14.
Kusakabe T, Kawaguchi A, Kawaguchi R. Thyrocyte-Specific Expression of Cre Recombinase in Transgenic Mice. Genesis 2004;39:212–216.
SERRATED AND OTHER MUCOSAL LESIONS IN INFLAMMATORY BOWEL DISEASE – SINGLE INSTITUTION MOLECULAR AND IMMUNOHISTOCHEMICAL STUDY
Kamarádová Kateřina1, MUDr.
Co-authors: Vošmiková H1, Rozkošová K1, Tachecí I2, Laco J1
1 The Fingerland Department of Pathology, Charles University Faculty of Medicine and University Hospital Hradec Králové, Czech Republic
2 2nd Department of Internal Medicine-Gastroenterology, Charles University Faculty of Medicine and University Hospital Hradec Králové, Czech Republic
Tutor: Prof. Jan Laco, Ph.D.
Introduction:
Chronically inflamed mucosa in inflammatory bowel diseases (IBD) as ulcerative colitis (UC) and Crohn’s disease (CD) is associated with an increased risk of developing IBD-related dysplasia and adenocarcinoma.
[1] Besides conventional IBD-associated dyplasia, the attention is currently drawn to other epithelial changes including goblet cell depletion, serrated change/dysplasia, NOS (not otherwise specified) and villous hypermucinous change and their role as potential new precursors of IBD-associated cancer. [2, 3]
Aim of the study:
The aim of the study was to review surgical and endoscopical specimen of IBD patients with a focus on detection of serrated and other non-conventional mucosal changes and evaluation of their
immunohistochemical and molecular properties.
Methods:
Surgical specimens and/or endoscopical biopsy samples of IBD patients examined between January 2006 – December 2016 at The Fingerland department of pathology were reviewed. Samples revealing any type of serrated change, goblet cell depletion, villous hypermucinous change, IBD-related dysplasia and/or
colorectal carcinoma (CRC) were selected for immunohistochemical analysis (IHC) with antibodies against MLH1, p53 and MGMT and for testing of the presence of KRAS, NRAS and BRAF mutation.
Results:
A total number of 309 consecutive specimens of IBD patients were reviewed. Totally 86 lesions were found in 55 pts (17,7 % from all patients reviewed). Multiple lesion in the same and/or additional specimen were found in 19 patients. There were 25 males and 30 females with 29 cases of CD and 25 cases of UC. Types of lesions and their frequency in the studied set is seen in Figure 1.
Loss of MLH1 and MGMT expression and aberrant p53 expression was evaluated in all lesions.
Most of the lesions showed a peculiar pattern of MLH1 staining with retained basal expression and
superficial loss of expression. Complete loss of MGMT expression was prominent in goblet cell depletion, IBD-associated dysplasia, TSA and colorectal carcinoma. Similarly, IBD-associated dysplasia and CRC and TSA showed p53 overexpression. Interestingly, lesions with serrated morphology showed superficial loss of MGMT expression and only basal p53 overexpression.
Figure 1: Types of mucosal lesions and their frequency in the study set.
Regarding molecular results, almost half of the evaluated lesions (46,5 %) showed KRAS mutations and 7,2
% BRAF mutations. We have also discovered cases with concurrent mutation of KRAS and BRAF and NRAS and BRAF, totally in 7 cases (8,1 %) presented in cases of serrated change/dysplasia, NOS, SSA and HP.
.
Discussion:
We have shown in our study that nearly one fifth (55 out of 309, 17,7 %) of IBD patients have various IBD- related mucosal changes. These mucosal lesions were found more commonly in cases of ulcerative colitis (25 out of 44 cases, 56,8 %) than in Crohn’s disease (29 out of 263 cases, 11 %). Most common change was serrated change/dysplasia, NOS and villous hypermucinous change or combined lesions. These lesions were more common than conventional IBD-associated dysplasia. Nevertheless, they did not show significant aberrant expression in studied markers. Overall, we have shown that almost half of lesions (46,5 %) were mutated in KRAS including true serrated lesions as SSA which contrasts with sporadic non-IBD-associated serrated pathway showing mostly mutations in BRAF. Moreover, seven lesions (from HP, SSA and serrated change/dysplasia, NOS group) showed concurrent KRAS/NRAS/BRAF mutations despite the widely believed mutual exclusivity in KRAS and BRAF mutations.
Conclusion/summary:
Serrated change/dysplasia, NOS and villous hypermucinous change in mucosa of IBD patients show certain immunohistochemical and molecular changes and further characterization of these lesions is needed.
Recognition of these changes is necessary since they may represent another type of potentially preneoplastic or precursor lesion in CRC carcinogenesis in IBD patients. Further study of cases with both KRAS (NRAS) and BRAF mutations is also desirable.
References:
[1] Harpaz N et al. Precancerous lesions in inflammatory bowel disease. Best Pract Res Clin Gastroenterol. 2013 Apr;27(2):257-267.
[2] Rubio CA. Serrated neoplasias and de novo carcinomas in ulcerative colitis: a histological study in colectomy specimens. J Gastroenterol Hepatol. 2007 Jul;22(7):1024-31.
[3] Kilgore SP et al. Hyperplastic-Like Mucosal Change in Crohn’s Disease: An Unusual Form of Dysplasia? Mod Pathol 2000;13(7):797–80.
The study was supported by the project MH CZ – DRO (UHHK, 00179906), University Hospital Hradec Kralove and by the Charles University Research Development Program PROGRES Q40/11, by the project BBMRI-CZ LM2015089 (Ministry of Education, Youth and Sports, Czech Republic), and by European Regional Development Fund-Project BBMRI-CZ.: Biobank network – a versatile platform for the research of the etiopathogenesis of diseases, No: EF16 013/0001674.
DIAGNOSTIC OF CLOSTRIDIUM DIFFICILE: A COMPARISION BETWEEN TWO IMMUNOASSAYS WITH PCR CONFIRMATION
Martin Kracík, MSc.
Tutor: Associate Professor Helena Žemličková, M.D., Ph.D.
Introduction:
Clostridium difficile (Clostridioides difficile) is an anaerobic bacteria which could colonized colon of patient on antibiotic treatment. Spores of C. difficile contaminate surroundings of patient and could infect another person by fecal-oral route. Colonisation in itself has no clinical symptoms until the toxins (A, B) are released. (1) The toxins destroy epitelial layer of colon and cause diarrhea, pseudomembranous colitis or toxic megacolon (mortality 30-80 %).
C. difficile cause hospital acquired infections (HAI) due to chemical, radiation and drying resistant spores. (2, 3) Diagnostic of C. difficile is based on detection of glutamate dehydrogenase (GDH) and toxins by enzyme immunoassays. In Czech Republic chromatographic immunoassay C. diff QuikChek Complete is widely used. This test is quick and easy to use but has quite low sensitivity. We tested Liaison C. difficile GDH and toxins A/B assays and compared it with chromatographic test, cultivation and in-house PCR.
Aims:
The aim of this study was to determinate analytical performance of chemiluminescence kits Liaison C. difficile GDH and Liaison C. difficile toxins A/B compare to imunoassay C. diff QuikChek Complete, cultivation and in-house multiplex PCR.
Methods:
164 samples of native stool from patients with diarrhea were tested on presence of GDH and toxins during January – March 2018. C. diff QuikChek Complete (TechLab, U. S. A.), Liaison C. difficile GDH and Liaison C. difficile Toxins A/B (Diasorin, Italy) were performed on all of samples. GDH positive results were confirmed by anaerobic cultivation (48 hours, 37
°C) on Brazier’s Clostridium Difficile Selective Agar (Oxoid, Czech Republic). PathogenFree DNA Isolation Kit (Geneproof, Czech Republic) was used to isolate DNA from cultivated strains. Samples with discrepant results of immunoassays were tested by multiplex in-house polymerase chain reaction (PCR). PCR was designed to detect genes for toxin A (tcdA) and toxin B (tcdB). Reaction mix consists of 12 µL HotStarTaq MasterMix (Qiagen, Netherlands), 5 µl DNA free H2O, 5 µl primer mix (4) and 5 µl isolated DNA. PCR conditions were 15 min at 94 °C, followed by 35 cycles of 45 s at 94 °C, 45 s at 50 °C and 1 min at 72 °C, with final extension of 30 min at 72 °C. Products of PCR was visualised by capillary electrophoresis on MultiNA (Shimadzu, Japan).
Results:
On both systems (QuikChek, Liaison) in each marker (GDH, toxin A/B) were 106 (64,6 %) samples negative (GDH-toxA/B-) and 19 (11,6 %) samples positive (GDH+toxA/B+). Eight (4,9 %) samples were GDH positive and toxin negative (GDH+toxA/B-). Thirty one (18,9 %) samples have discrepant result in at least one marker. Positivity of GDH was confirmed by cultivation in 41 cases from 58 suspect samples (GDH+toxA/B+, GDH+toxA/B-, discrepant groups). Genes for toxin A or B were detected by PCR in 19 cases from 39 GDH+toxA/B- and discrepant samples. The positive/negative results consensus of 164 samples was 41/123 for GDH and 38/126 for toxins. Comparison of sensitivity, specificity, negative predictive value (NPV) and positive predictive value (PPV) of immunoassays shows table 1.
GDH (41+, 123-) Toxins A/B (38+, 126-) CDI Interpretation (38+, 126 -) Liaison QuikChek Liaison QuikChek Liaison QuikChek
True positive 40 34 29 20 29 20
False positive 7 1 10 0 0 0
True negative 116 122 116 126 126 126
False negative 1 7 9 18 9 18
PPV 85,1 % 97,1 % 74,4 % 100,0 % 100,0 % 100,0 %
NPV 99,1 % 94,6 % 92,8 % 87,5 % 93,3 % 87,5 %
Sensitivity 97,6 % 82,9 % 76,3 % 52,6 % 76,3 % 52,6 % Specificity 94,3 % 99,2 % 92,1 % 100,0 % 100,0 % 100,0 % Table 1: Comparison of immunoassays
Discussion:
It is known that ezyme immunoassays have low sensitivity to C. difficile toxins. (5) We showed that Liaison C. difficile toxins A/B sensitivity is higher then sensitivity of C. diff QuickChek Complete. Low PPV of Liaison toxin test (74,4 %) was caused by 10 false positive samples. These samples were GDH negative by Liaison and were interpreted as C. difficile negative. Because of this PPV of Liaison`s CDI (Clostridium difficile infection) interpretation reached 100 %. NPV of QuikChek GDH was 94,6 %, NPV of Liaison`s GDH reached 99,1 %. Higher NPV is very important for screening methods.
Conclusion / Summary:
In this study we tested 164 samples of native stool by two types of immunoenzymatic assays, cultivation and PCR. We made results consensus and determinated analytical performance of enzyme immunoassays. Liaison C. difficile GDH and Liaison C. difficile toxins A/B reached higher sensitivity comparing to C. difficile QuickChek assay in CDI testing.
References:
1. Kazanowski, M., et al. Clostridium difficile: epidemiology, diagnostic and therapeutic.
Techniques in Coloproctology. 2014, 18, pages 223-232.
2. Vedantam, G., et al. Clostridium difficile infection: Toxins and non-toxin virulence factors, and their contributions to disease establishment and host response. Gut Microbes. 2012, 3, pages 121-134.
3. Hall, J. A., Keul, R. R. and Shanks, J. D. Dipping into the Clostridium difficile pool: Are alcohol-based dispensers fomites for C difficile? American Journal of Infection Control. 2017, 45, pages 1279-1280.
4. Persson, S., Torpdahl, M. and Olsen, K. E. P. New multiplex PCR method for the detection of Clostridium difficile toxin A (tcdA) and toxin B (tcdB) and the binary toxin (cdtA/cdtB) genes applied to a Danish strain collection. Clinical Microbiology and Infection, 2008, 14, pages 1057–1064.
5. Crobach, M. J. T., et al. European Society of Clinical Microbiology and Infectious Diseases: update of the diagnostic guidance document for Clostridium difficile infection.
Clinical Microbiology and Infection. 2016, 22, pages 63-81.
THERAPEUTIC POTENTIAL OF NOVEL QUINAZOLINE DERIVATIVES AS A CHEMOSENSITIZER FOR CANCER THERAPY
Monika Kulhavá, Ing.
Department of Medical Biochemistry, Faculty of Medicine in Hradec Králové, Charles University in Prague
Co-authors: M. Andrš, M. Seifrtová, R. Havelek, D. Jun, P. Tomšík, L. Prchal, R. Doležal, A.
Tichý, T. Kučera, M. Řezáčová, J. Korábečný Tutor: prof. MUDr. Martina Řezáčová, Ph.D.
Introduction
DNA-dependent protein kinase (DNA-PK) is a member of the phosphatidylinositol 3-kinase (PI3K) related protein kinase family (PIKK). With two other members of this group, ataxia telangiectasia mutated (ATM) and ataxia telangiectasia and Rad3-related (ATR), they take care of DNA maintenance and manage DNA damage response (DDR) [1]. DNA-PK is the major regulator of the cellular answers to DNA double strand breaks (DSBs), where both strands of the DNA helix are severed. DSBs, often caused by ionizing radiation (IR) or chemotherapeutic drugs, are the most lethal type of DNA lesion and if unrepaired, they can lead to cell death or to genome rearrangements with subsequent malignant transformation. DNA-PK helps the cells to deal with DSBs through activation of non-homologous end joining (NHEJ) [2–4].
DDR is a highly studied area of current cancer research. Modulation of these complex signalling pathways brings promise as radio- or chemo-potentiating agents (IR, chemotherapeutic drugs) and for selective killing of cancer cells through ‘synthetic lethality’ [5,6].
Aims
The aim of this study was biological evaluation of 17 novel quinazoline derivatives with potent chemosensitizing activity in colorectal adenocarcinoma cells.
Methods
At first, single dose testing of growth inhibition on the screening panel of 16 human cancer and one healthy cell lines was performed with inhibitors at a concentration of 10 μmol/L by WST- 1 test after 48 hours treatment. In the next step, the ability to potentiate the effects of the standard chemotherapeutic agent doxorubicin was tested. HT-29 cells were exposed to 10 μmol/L of the quinazoline derivatives in combination with 0.5 μmol/L doxorubicin for 48 hours.
The Caspase-Glo Assay was used for assessment of mode of the cell death induced by combination therapy. The phases of cell cycle were evaluated by flow cytometry analysis.
Finally, the specific proteins were observed by electrophoresis and Western blotting.
Results
From the screening panel, the colorectal adenocarcinoma cell line HT-29 was chosen as the most suitable model for further evaluation, as none of the tested inhibitors significantly affected proliferation of this cell line. Combination therapy of derivatives with 0,5 μmol/L doxorubicin caused significant chemosensitizing effect in HT-29 cell by 13 out of 17 tested compounds, especially by 14c and 14d. The derivative 14d was chosen for further testing. The Caspase-Glo Assay showed significant increase (P ≤ 0,05) in the activities of caspase 3/7 and caspase 9 when 14d or the standard DNA-PK inhibitor NU7441 was combined with doxorubicin. The activity of caspase 8 was significantly increased only in the combination of inhibitor 14d with doxorubicin. Cell cycle was affected when the combination therapy caused accumulation in G2
phase. Western blotting showed up-regulation of p53 at Ser15 in cells treated with doxorubicin and combination therapy with 14d or NU7026 after 4 and 24 hours. Doxorubicin alone and in combination with both inhibitors (14d or NU7026) provoked phosphorylation of Chk2 on Thr68 and Chk1 on Ser345.
Discussion
Inhibitors of PIKK are still at an early stage of their development. Recently two ATR inhibitors and two DNA-PK inhibitors have entered into phase I clinical trials. We observed statistically significant decrease in cell proliferation which corresponded with other studies [7, 8] of DNA- PK inhibitors in cancer cells. Combination therapy caused accumulation in G2 phase of cell cycle that was in consistent with the observation made by Zhao et al. who tested DNA-PK inhibition in colon cancer cell line SW620 [9]. In 2014, Pastwa et al. also noticed G2/M arrest after application of wortmannin and cisplatin/etoposide in human glioblastoma cell line M059 [10]. We proved that effect of compound 14d was not induced by inhibition of other PIKKs (ATM and ATR) because of phosphorylation p53 on Ser15 [11] and Chk2 on Thr68 and Chk1 on Ser345 that is known to be activated in ATM/ATR-dependent manner [12].
Conclusion
Biological assessment of novel quinazoline derivatives showed a chemosensitizing effect of compound 14d with the standard anticancer agent doxorubicin, with a strong effect on the proliferation of human colorectal carcinoma HT-29 cells. This effect was caused by caspase- dependent apoptosis. We confirmed that this process was not initiated by the ATM/ATR pathway. The chemosensitizing effect of 14d seems to be the synergistic action of DNA-PK inhibition. This work is very promising step for searching of further chemosensitising ligands.
Funding
This work was supported by Charles University project GAUK 932516 and programs Progres Q40/01 and SVV-260397/2017.
Literature
[1] Y. Shiloh, ATM and related protein kinases: safeguarding genome integrity, Nat. Rev. Cancer.3 (2003) 155–168.
[2] K.K. Khanna, S.P. Jackson, DNA double-strand breaks: signaling, repair and the cancer connection, Nat. Genet. 27 (2001) 247–254.
[3] A.A. Goodarzi, P.A. Jeggo, The repair and signaling responses to DNA double-strand breaks, Adv. Genet. 82 (2013) 1–
45.
[4] A.J. Davis, B.P.C. Chen, D.J. Chen, DNA-PK: a dynamic enzyme in a versatile DSB repair pathway, DNA Repair. 17 (2014) 21–29.
[5] S.P. Jackson, J. Bartek, The DNA-damage response in human biology and disease, Nature. 461 (2009) 1071–1078.
[6] W.G. Kaelin, The concept of synthetic lethality in the context of anticancer therapy, Nat. Rev. Cancer. 5 (2005) 689–698.
[7] Y. Zhao, H.D. Thomas, M.A. Batey, I.G. Cowell, C.J. Richardson, R.J. Griffin, A.H. Calvert, D.R. Newell, G.C.M. Smith, N.J. Curtin, Preclinical evaluation of a potent novel DNAdependent protein kinase inhibitor NU7441, Cancer Res. 66 (2006) 5354–5362.
[8] W.M. Ciszewski, M. Tavecchio, J. Dastych, N.J. Curtin, DNA-PK inhibition by NU7441 sensitizes breast cancer cells to ionizing radiation and doxorubicin, Breast Cancer Res. Treat. 143 (2014) 47–55.
[9] Y. Zhao, H.D. Thomas, M.A. Batey, I.G. Cowell, C.J. Richardson, R.J. Griffin, A.H. Calvert, D.R. Newell, G.C.M.
Smith, N.J. Curtin, Preclinical evaluation of a potent novel DNAdependent protein kinase inhibitor NU7441, Cancer Res. 66 (2006) 5354–5362.
[10] E. Pastwa, T. Poplawski, U. Lewandowska, S.B. Somiari, J. Blasiak, R.I. Somiari, Wortmannin potentiates the combined effect of etoposide and cisplatin in human glioma cells, Int. J. Biochem. Cell Biol. 53 (2014) 423–431.
[11] P.J. McKinnon, ATM and ataxia telangiectasia, EMBO Rep. 5 (2004) 772–776.
[12] L. Huang, H. Miao, Y. Sun, F. Meng, X. Li, Discovery of indanone derivatives as multi-targetdirected ligands against Alzheimer’s disease, Eur. J. Med. Chem. 87 (2014) 429–439. doi:10.1016/j.ejmech.2014.09.081.
GENOTOXIC POTENTIAL OF COMBINED DERMAL EXPOSURE TO POLYCYCLIC AROMATIC HYDROCARBONS AND ULTRAVIOLET RADIATION
Andrea Málková, M.D.
Department of Hygiene and Preventive Medicine, Faculty of Medicine in Hradec Králové, Charles University
Co-authors: J. Šmejkalová, K. Hamáková, J. Kremláček, T. Švadláková, L. Borská, Z. Fiala Tutor: prof. Zdeněk Fiala, M.Sc, Ph.D.
Introduction
Combined dermal exposure to crude coal tar (CCT) and ultraviolet radiation (UVR) represents one of the most effective therapies of psoriasis (Goeckerman therapy, GT) [1, 2]. On the other hand, however, the CCT contains a mixture of polycyclic aromatic hydrocarbons (PAHs) some of them have proven immunosuppressive, mutagenic, genotoxic and carcinogenic effects [3, 4].
According to International Agency for Research on Cancer (IARC) [5] both components of GT (CCT and UVR wavelengths 100-400 nm, encompassing UVA, UVB, and UVC) were included into group 1 (Carcinogenic to humans). In addition, the results of some studies show that UVR alone or in combination with other common environmental pollutants (including PAHs) could increase photo-damage and risk of skin cancer [6]. For these reasons, it is currently a debate about the extent of genotoxic hazard/risks of GT.
Aim
The aim of this study was to contribute to a better understanding of the genotoxic hazard of GT.
Methods
The study group of patients suffering from chronic stable plaque psoriasis (n=32, male n=15, smokers n=13, 18 to 69 years, median 53.5, Q1-Q3 34.17-60 years) was treated by GT (4% crude coal tar ointment applied topically and UVB 311 nm whole body irradiation). The heparinized peripheral blood samples were collected into vacuum tubes before the first and immediately after the last procedure. We used „Cytokinesis-block” micronucleus test (cultivation 72 hours, Cytochalasin B in 44th hour, hypotonic 0.55% KCl solution) and chromosomal aberration assay (cultivation 50 hours, Colcemid in 48th hour, hypotonic 0.55%
KCl solution) stained by Giemsa solution. We assessed (1) number and distribution of micronuclei (MN), nuclear buds (gen amplification) and nucleoplasmic bridges (dicentric chromosome) in 1 000 binucleated lymphocytes (BNL) and (2) structural aberration (SAL) and numerical (NAL) aberration in 100 metaphasical lymphocytes.
Results
Total number of BNL with MN (p<0.001), BNL with 1 MN (p<0.001), BNL with 2 MN (p<0.05), BNL with ≥3 MN (p<0.01), total number of aberrated cells (SAL+NAL; p<0.001) and SAL (p<0.001) were significantly increased after GT. Smokers had significantly higher total number of aberrated cells (p<0.05) and number of smoked cigarettes correlated with total number of aberrated cells before GT (rho=0.38, p<0.05). We found correlation between age
and BNL with MN before GT (rho=0.55, p<0.001) and correlation between range of psoriatic lesions and total number of aberrated cells after GT (r=0.37, p<0.05). PASI score (Psoriasis Area Severity Index) decreased significantly (p < 0.001) after GT.
Discussion
According to the literature, no one has yet evaluated the genotoxic potential of GT using by CBMN (MN counts, including nucleoplasmic bridges and nuclear buds). The total number of BNL with MN (mean±standard deviation; 21.16±13.84 ‰ after GT) was similar as in the group of Turkish asphalt workers (21.6±2.5 ‰) [7], lower than in the group of sewage workers in Paris (38.02±7.16 ‰) [8] and higher than in the group of Prague city policemen (7.32±3.42 ‰) [9]. After GT there was increase in BNL with 2 or more MN especially in women. The number of SAL after GT (5.56±1.92 %) was similar as in the group of Indian coke oven workers (4.6±1.4%) [10] and Roma airport personnel (4.13 %) [11] but higher than in the group of Prague city policemen (2.07±1.48 %) [12].
Conclusions/Summary
Combined therapeutic exposure to PAHs and UVB induced an increase in the level of the micronuclei and chromosomal aberrations in lymphocytes of psoriatic patients and thus increased the hazard of malignant transformation of blood cells.It can be assumed that a similar situation also occurs in tissues that are intensively supplied with blood.
Supported by projects PROGRES Q40/09 and SVV-260397/2017.
References
1. Goeckerman WH. The Treatment of Psoriasis. Northwest Med. 1925;24(May):229-231.
2. Gupta R, Debbaneh M, Butler D, et al. The Goeckerman regimen for the treatment of moderate to severe psoriasis. J Vis Exp. 2013(77):e50509.
3. Kotingová L, Voříšek V, Borská L, Čermáková E, Fiala Z. Vliv rozpouštědla na dermální absorpci pyrenu in vitro. Hygiena. 2012;57(2):50-55.
4. Abdel-Shafy HI, Mansour MSM. A review on polycyclic aromatic hydrocarbons: Source, environmental impact, effect on human health and remediation. Egyptian Journal of Petroleum.
2016;25(1):107-123.
5. IARC, Agents Classified by the IARC Monographs, Volumes 1–120. 2018.
6. Fu PP, Xia Q, Sun X, Yu H. Phototoxicity and environmental transformation of polycyclic aromatic hydrocarbons (PAHs)-light-induced reactive oxygen species, lipid peroxidation, and DNA damage. J Environ Sci Health C Environ Carcinog Ecotoxicol Rev. 2012;30(1):1-41.
7. Karaman A, Pirim I. Exposure to bitumen fumes and genotoxic effects on Turkish asphalt workers. Clin Toxicol (Phila). 2009;47(4):321-6.
8. Al Zabadi H, Ferrari L, Sari-Minodier I, et al. Integrated exposure assessment of sewage workers to genotoxicants: an urinary biomarker approach and oxidative stress evaluation.
Environ Health. 2011;10:23.
9. Rossnerova A, Spatova M, Rossner P, Solansky I, Sram RJ. The impact of air pollution on the levels of micronuclei measured by automated image analysis. Mutat Res. 2009;669(1-2):42-7.
10. Sureshkumar S, Balachandar V, Devi SM, et al. Estimation of cytogenetic risk among coke oven workers exposed to polycyclic aromatic hydrocarbons. Acta Biochim Pol. 2013;60(3):375-9.
11. Cavallo D, Ursini CL, Carelli G, et al. Occupational exposure in airport personnel:
characterization and evaluation of genotoxic and oxidative effects. Toxicology. 2006;223(1- 2):26-35.
12. Sram RJ, Beskid O, Rossnerova A, et al. Environmental exposure to carcinogenic polycyclic aromatic hydrocarbons--the interpretation of cytogenetic analysis by FISH. Toxicol Lett.
2007;172(1-2):12-20.
Vitamin D status of very low birth weight infants at birth and the effects of generally recommended supplementation on their vitamin D levels at discharge.
Tomas Matejek M.D.1
Co-Authors: Martina Navratilova1, Lenka Zaloudkova2, Jana Malakova2, Jan Maly1 Tutors: Doc. MUDr. Sylva Skalova, PhD. 1, prof. MUDr. Vladimir Palicka, CSc., dr.h.c.2
1 Department of Paediatrics, Charles University in Prague, Faculty of Medicine Hradec Kralove, University Hospital Hradec Kralove, Hradec Kralove, Czech Republic
2 Institute of Clinical Biochemistry and Diagnostics, University Hospital Hradec Kralove, Hradec Kralove, Czech Republic
Abstract
Background: Although the major function of vitamin D is on bone metabolism, in recent years other effects of vitamin D have attracted attention (e.g. lung and central nervous system development, maturation of immune system) [1]. Very low birth weight infants are likely to be at risk of low vitamin D status because of the high prevalence of vitamin D deficiency in pregnancy, the lack of sunlight exposure during hospitalization, the minimal fat mass (vitamin D and metabolites are stored there), and the difficulty in achieving adequate enteral nutrition [2]. The European Society for Paediatric Gastroenterology Hepatology and Nutrition recommends 800-1000 IU of vitamin D per day [3]. A serum level of 25(OH)D > 50 nmol/L is taken to indicate sufficiency, a serum concentration < 50 nmol/L indicates
deficiency, and a serum concentration < 25 nmol/L indicates severe deficiency for the entire paediatric population [4, 5, 6]
Purpose: To evaluate vitamin D status in mothers and their very low birth weight infants (VLBW) at birth (umbilical cord blood) and at discharge with current recommended supplementation of vitamin D.
Methods: 94 infants with birth weight less than 1500 g completed the study. The total daily vitamin D intake was 800 to1000 IU. 25-hydroxyvitamin D [25(OH)D] was determined using isotope-dilution liquid chromatography-tandem mass spectrometry ID-LC MS/MS. We examined 25(OH)D levels in maternal serum before labor, in cord blood, and in infants’
serum at discharge.
Results: The median (IQR) vitamin D [25(OH)D] level of maternal serum samples before delivery was 32 (22 – 53) nmol/L. Median (IQR) serum 25(OH)D was 21 (14-36) nmol/L in cord blood, and 46 (37-60) nmol/L at discharge. Serum 25(OH)D was < 50 nmol/L in 71.3 % of mothers, in 91.5 % of cord blood samples, and in almost 60 % of preterm
newborns at discharge (after 8 weeks of supplementation).
Conclusions: In our cohort we found that due to the very high prevalence of 25(OH)D deficiency among mothers, the current generally recommended dose of vitamin D (800-1000 IU per day) for VLBW infants was unable to improve vitamin D levels above the desired 50 or even 75 nmol/L before discharge. This information may have influence on the future nutritional standards concerning vitamin D supplementation in preterm infants.
Funding: This work was supported by University Hospital Hradec Kralove, Czech Republic project MH CZ - DRO (UHHK, 00179906).
Literature:
1. Matejek T, Navratilova M, Zaloudkova L, et al. Parathyroid hormone - reference values and association with other bone metabolism markers in very low birth weight infants - pilot study. J Matern Fetal Neonatal Med. 2018 Mar 21:1-8. doi:
10.1080/14767058.2018.1450858. PubMed PMID: 29562766; eng.
2. Abrams SA, Nutrition Co. Calcium and vitamin d requirements of enterally fed
preterm infants. Pediatrics. 2013 May;131(5):e1676-83. doi: 10.1542/peds.2013-0420.
PubMed PMID: 23629620; eng.
3. Agostoni C, Buonocore G, Carnielli VP, et al. Enteral nutrient supply for preterm infants: commentary from the European Society of Paediatric Gastroenterology, Hepatology and Nutrition Committee on Nutrition. J Pediatr Gastroenterol Nutr. 2010 Jan;50(1):85-91. doi: 10.1097/MPG.0b013e3181adaee0. PubMed PMID: 19881390;
eng.
4. Braegger C, Campoy C, Colomb V, et al. Vitamin D in the healthy European paediatric population. J Pediatr Gastroenterol Nutr. 2013 Jun;56(6):692-701. doi:
10.1097/MPG.0b013e31828f3c05. PubMed PMID: 23708639; eng.
5. Misra M, Pacaud D, Petryk A, et al. Vitamin D deficiency in children and its management: review of current knowledge and recommendations. Pediatrics. 2008 Aug;122(2):398-417. doi: 10.1542/peds.2007-1894. PubMed PMID: 18676559; eng.
6. Saraf R, Morton SM, Camargo CA, Jr., et al. Global summary of maternal and newborn vitamin D status - a systematic review. Matern Child Nutr. 2016 Oct;12(4):647-68. doi: 10.1111/mcn.12210. PubMed PMID: 26373311; eng.
SOFTWARE TRUS/MRI FUSION PROSTATE BIOPSY – OUR EXPERIENCE WITH 83 PROCEDURES
Jakub Musil
Department of Urology, Regional Hospital Kolín a.s.
Faculty of Medicine Hradec Králové, Charles University Co-author: Jandejsek J.
Tutor: Assoc. Prof. Miloš Broďák PhD.
Introduction:
Prostate cancer is nowadays most frequent malignancy in adult men. Diagnosis of prostate cancer is based on digital rectal examination (DRE), prostate specific antigen level (PSA) and random transrectal ultrasound guided prostate biopsy (TRUS). Unfortunately, PSA has low specificity for prostate cancer. Only 20-30% of patients with PSA level 4-10ng/ml have prostate cancer, remaining 70-80% will undergo unnecessarily prostate biopsy. There are 2 ways how increase low specificity of PSA. First possibility is use of tumor marker with higher specificity, such as free PSA, fPSA/tPSA, prostate health index PHI or PCA3. Second way is use of imaging method, which is able to image prostate cancer. For this objective is used multiparametric MRI of prostate.
Objective:
Evaluate our group of 83 patients with increased PSA level, who underwent targeted TRUS/MRI fusion biopsy.
Patients and methods:
We prospectively analyzed group of 83 patients with increased PSA level, who underwent targeted TRUS/MRI fusion prostate biopsy between September 2017 and September 2018 in Regional Hospital Kolín, Czech republic. In 83 patients we biopted 119 suspicious lesions reported by multiparametric MRI of prostate.
TRUS guided random biopsies were performed using ultrasound device BK FlexFocus 400 with side-fire endorectal probe 8808e with biplane imaging (BK Medical, Harlev, Denmark).
TRUS imaging includes standard B-mode imaging in transversal and sagital sections and power doppler imaging of prostate perfusion.
MpMRI examinations were performed using 1,5T Toshiba MRI machine. Protocol of mpMRI imaging included T1 and T2 weighted imaging, diffusion weighted imaging (DWI) with apparent diffusion coefficient (ADC) mapping and dynamic contrast enhancement (DCE) imaging. MRI findings were reported using PI-RADS v2 classification system.
MRI-targeted biopsies were performed using external navigation system Biopsee 3.0 (Medcom GmbH, Germany). This device is compatible with BK ultrasound device and with standard endorectal probe. Protocol of TRUS/MRI prostate biopsy consists of 2-4 bioptic cores per lesion and then random TRUS guided prostate biopsy – 10 cores from peripheral zone and 2 cores from transitional zone. Periprostatic trimecaine infiltration nerve blockade was used.Antibiotic prophylaxis with cotrimoxazol was administered.
Average age in our group was 64.27 years (42-80). Mean PSA level was 8.21 (0.4-39.08).
From 83 patients, 33 patients were biopsy-naive, remaining 50 patients undergo 1-4 previous negative prostate biopsies.
In 83 patients we biopted 119 lesions with PI RADS score 3-5. Mean volume of lesions is 0.75ml.
Results:
Overall prostate cancer detection is 40.9% (34/83). In biopsy-naive men detection rate was 36.4% (12/33). In PI-RADS 3 subgroup prostate cancer was detected in 8.51% of cases (4/47). Prostatitis was reported in PI RADS 3 subgroup in 5 cases. Detection rate in PI-RADS 4 subgroup was 15.09% (15/53), in this subgroup reported prostatitis was in 5 cases, ASAP in 1 case and HG PIN in 1 case. Highest detection of prostate cancer was in subgroup PI-RADS 5 – 89.7% (17/19).
Both, random and targeted biopsies were negative in 49 patients, positive in 17 cases.
Positive TRUS guided biopsy and negative MRI targeted biopsies were reported in 8 cases.
Positive MRI targeted biopsy alone was seen in 9 cases.
Conclusion:
TRUS/MRI targeted biopsy is useful tool in prostate cancer detection. MRI-guided biopsy is necessary in detection of prostate cancer in atypical localization e.g. in anterior fibromuscular stroma. Our results validates, that MRI targeted biopsy increase detection rate of prostate cancer for 10.8% patients. On the other hand, there was 8 patients with negative targeted cores and positive random TRUS biopsy. It is the reason to still perform systematic TRUS biopsy in standard protocol of TRUS/MRI biopsy.
References:
[1.]Tonttila, Panu P. et al.: Prebiopsy Multiparametric Magnetic Resonance Imaging for Prostate Cancer Diagnosis in Biopsy-naive Men with Suspected Prostate Cancer Based on Elevated Prostate-specific Antigen Values: Results from a Randomized Prospective Blinded Controlled Trial, European Urology , Volume 69 , Issue 3 , 419 - 425
[2.] Venderink, Wulphert et al.: Results of Targeted Biopsy in Men with Magnetic Resonance Imaging Lesions Classified Equivocal, Likely or Highly Likely to Be Clinically Significant Prostate Cancer, European Urology , Volume 73 , Issue 3 , 353 - 360
[3.] Musil, J. et al.: Cognitive TRUS-MRI fusion prostate biopsy – useful tool for prostate cancer detection, European Urology Supplements , Volume 16 , Issue 11 , e2878
Lymphocytes in the treatment with interferon beta-1 b
MUDr. Zbyšek Paveleka
Co-authors: Oldřich Vyšataa, Blanka Klímováa, Ctirad Andrýsb, Doris Vokurkováb, Martin Vališa
a Department of Neurology, Faculty of Medicine and University Hospital Hradec Králové, Charles University, Hradec Králové, Sokolská 581, 500 05, Czech Republic b Department of Clinical Immunology and Allergology, University Hospital Hradec Králové, Hradec Králové, Sokolská 581, 500 05, Czech Republic
Tutor: Assoc. Prof. Martin Vališ, Ph.D.
Background: Multiple sclerosis (MS) is a chronic inflammatory demyelinating and neurodegenerative disease affecting the central nervous system. One of the basic medications for the treatment of a clinically isolated syndrome (CIS) or relapsing- remitting MS is interferon beta (INFβ). Although the exact mechanism of its effects is unknown, the medication has an anti-inflammatory and immunomodulatory effect. It dampens the activity of Th1 subset of T lymphocytes by induction of IL- 10 production.
It decreases the production of proinflammatory cytokine. IL-17. It leads to the reduction of antigen presentation and T lympho- cytes proliferation. It decreases the permeability of the blood-brain barrier by blocking the adhesive interactions, dampening the effect of matrix metalloproteinases and leukocytes migration (Anderton and Liblau, 2008; Chabot et al., 1997; Chen et al., 2009; Markowitz, 2007).
Methods: A total of 97 patients (25 males and 72 females) were included into the study.
Patients were treated by INFβ 1-b (subcutaneous injection, 250 μg, each other day).
Clinical evaluations were performed by an attending neurologist. Peripheral blood
samples were obtained just prior to treatment and 5 years after INFβ 1-b. Statistical analysis and processing of the obtained data were performed by using a comprehensive statistical software package MATLAB®.
Results: A significant decrease of the observed parameters after 5 years’ of treatment (significant at the 1% significance level) was found in the absolute and relative CD69 count, absolute cytotoxic/suppressor T lymphocyte count, absolute total leukocyte count, absolute natural killer cells count. A significant decrease of the observed parameters after 5 years’ of treatment (significant at the 5% significance level) was found in the absolute lymphocyte count, relative cytotoxic/suppressor T lymphocyte count, relative
CD3+CD69+ count and absolute CD8+CD38+ count.
Discussion: The goal in this study was to determine which immunological parameters from peripheral blood are affected by the long-term INFβ 1- b treatment. A significant decrease of the parameter CD69 was found. CD69, one of the earliest specific antigens acquired during the lymphoid activation, acts as a signal-transducing receptor involved in cellular activation events, including proliferation and induction of specific genes (Llera et al., 2001). The recent discovery of a ligand for CD69 expressed on dendritic cells,
Galectin-1, has confirmed the immunoregulatory role of CD69 mainly by the inhibition of Th17 differentiation and function in mice and humans. In this regard, the expression of CD69, both in Th17 lymphocytes and by a subset of regulatory T cells, has an important role in the control of the immune response and the inflammatory phenomenon (González- Amaro et al., 2013). A significant decrease was found in Tc lymphocytes. Cytotoxic T lymphocytes present in lesions have a regulatory function in relation to the disease progression. They mediate suppression of Th lymphocytes through secretion of perforin, which is cytotoxic for Th lymphocytes. They also cause axonal transection and increased vascular permeability (Kasper and Shoemaker, 2010). Zafranskaya et al. (2007)
demonstrated that INFβ therapy reduces CD8+ T lymphocytes reactivity in MS. NK – phenotype CD3-/CD16+56+ - natural killer cells were originally defined as effector lymphocytes of innate immunity endowed with constitutive cytolytic functions. The untreated patients with MS showed impaired regulatory NK cell responses which could be attributed to the abundant NK cells in secondary lymphoid tissues, specifically,
CD56brightCD16–NK cells (Lünemann et al., 2011). Although the mechanism of action of INFβ is not known exactly, INFβ activated leukocyte migration through human brain microvascular endothelial cell monolayer (Lou et al., 1999). During the observation, a decrease in the absolute lymphocytes’ count was proven. Many of the other examined parameters play a role in MS pathogenesis. However, the findings did not confirm that they were affected by long-term NFβ treatment.
Conclusion: The treatment with interferon beta reduces clinical exacerbations in multiple sclerosis (MS) through several known immunomodulatory mechanisms. However, the exact mechanism of effect of this medication is not known. This study presents some parameters that were affected by the long-term INFβ treatment.
References:
1. Anderton, SM, Liblau RS. Regulatory T cells in the control of inflammatory demyelinating diseases of the central nervous system. Curr Opin Neurol 2008;21:248-54.
2. Chabot S, Williams G, Yong VW. Microglial production of TNF-alpha is induced by activated T lymphocytes. Involvement of VLA-4 and inhibition by
interferonbeta-1b. J Clin Invest. 1997 Aug 1;100(3):604-12.
3. Chen M, Chen G, Nie H, Zhang X, Niu X, Zang YC, Skinner SM, Zhang JZ, Killian JM, Hong J. Regulatory effects of IFN-beta on production of osteopontin and IL-17 by CD4+ T Cells in MS. Eur J Immunol. 2009 Sep;39(9):2525-36.
4. González-Amaro R, Cortés JR, Sánchez-Madrid F, Martín P. Is CD69 an effective brake to control inflammatory diseases? Trends Mol Med. 2013 Oct; 19(10): 625–
632.
5. Kasper L, Shoemaker J. Multiple sclerosis immunology: The healthy immune system vs. the MS immune system. Neurology.2010;74, S2–S8.
6. Llera AS, Viedma F, Sánchez-Madrid F, Tormo J. Crystal structure of the C-type lectin-like domain from the human hematopoietic cell receptor CD69. J Biol Chem. 2001 Mar 9;276(10):7312-9.
7. Lou J, Gasche Y, Zheng L, Giroud C, Morel P, Clements J, Ythier A, Grau GE.
Interferon-beta inhibits activated leukocyte migration through human brain microvascular endothelial cell monolayer. Lab Invest. 1999 Aug;79(8):1015-25.
8. Lünemann A, Tackenberg , DeAngelis T, Barreira da Silva R, Messmer B,
Vanoaica L, Miller A, Apatoff B, Lublin F, Lünemann J, and Münzcorresponding C. Impaired IFN-γ production and proliferation of NK cells in Multiple Sclerosis.
Int Immunol. 2011 Feb; 23(2): 139–148.
9. Markowitz CE. Interferon-beta: mechanism of action and dosing issues.
Neurology. 2007 Jun 12;68(24 Suppl 4):S8-11.
10. Zafranskaya M, Oschmann P, Engel R, et al. Interferon-beta therapy reduces CD4+ and CD8+ T-cell reactivity in multiple sclerosis. Immunology.
2007;121(1):29–39.
FASTER AND EQUALLY EFFECTIVE ISOLATION PROTOCOL FOR DENTAL STEM CELLS
Nela Pilbauerová
Department of Dentistry, Faculty of Medicine and University Hospital in Hradec Králové
Tutor: MUDr. Jakub Suchánek, Ph.D.
Introduction: A dental pulp represents an easily accessible source of adult dental pulp stem cells (DPSCs). They were isolated for the first time in 2000[1] and since then many
researchers have investigated and analyzed their biological characteristics. A tooth extraction is a common surgical procedure performed in local anesthesia and can be leveraged
throughout the entire adult life. The pulp tissues from human wisdom teeth or premolars represent the most common source for DPSC harvesting. [2] Nowadays the preferred approach for DPSC isolation is an enzymatic digestion method using enzymes in order to obtain single cell suspensions from the solid tissues. [1,2] However, the length of enzymatic activity is crucial, because prolonged time can cause a cell damage [3].
Aim: The purpose of this study was to optimize the isolation protocol for DPSCs, namely to shorten the time of enzymatic digestion of the dental pulp, and to cultivate isolated DPSCs using this new approach, investigate their proliferation, phenotype, cell viability and determine their ability to differentiate into mature cells, chondroblasts, osteoblasts, and adipocytes.
Materials and methods: The sources of pulp tissues for DPSCs harvesting were permanent teeth in different stages of root development. After pulp tissue collection, we used a homogenization method using a mini tissue grinder (Rondoti) in order to obtain single cell suspensions from the solid pulp tissue and 0.05 % trypsin for 10 minutes for the enzymatic digestion. The isolated populations of DPSCs were cultivated in a modified cultivation media (Alpha-MEM) for mesenchymal adult progenitor cells containing 2 % fetal bovine serum (FBS) and supplemented with growth factors and Insulin-Transferrin-Selenium supplement (ITS). The cell viability, cell count and other properties were examined using a Vi-Cell analyzer and Z2-Counter. The phenotype analysis was performed using a flow cytometer Cell Lab Quanta. For differentiation in chondroblasts, osteoblasts and adipocytes, we used
commercially available differentiation media. The evidence of differentiation was proved by the histological staining (blue Masson's trichrome, von Kossa stain, alcian blue and oil red staining).
Results: We were able to isolate eight DPSC populations using this method and cultivate them over 43.4 (SD = 3.2) population doublings (PD). The average population doubling time (DT) was 50 hours (SD = 14.9 hours). The average cell viability was 92.9 % (SD = 1.9%) in the second passage and 93.2 % (SD = 2.3 %) in the eighth passage. DPSCs showed high positivity for mesenchymal stem cell markers CD29, CD44, CD90 and for stromal associated markers CD13, CD73, CD166, and negative expression or low positivity for hematopoietic markers CD34, CD45 and for CD31 and HLA II. DPSCs differentiated into osteoblasts and chondroblasts. Even after the exposition of the strong adipogenic medium they did not show any signs of differentiation into adipocytes.
Discussion: The enzymatic digestion is a multistep procedure, therefore very technically demanding. A solution of collagenase type I and dispase in ratio 1:1 [2] is the most commonly used today. However, the activity time of this solution is 30 - 60 minutes, according to the size of the solid tissue. Generally, there has been a desire to shorten the length of the enzymatic reaction to the minimum due to a risk of developing a cell toxicity.
There are two approaches, how to achieve that. One is to grind the solid pulp tissue into homogenous pieces to facilitate the enzyme effect, and the other to use a different enzyme.
Conclusion: We have successfully optimized the isolation protocol for DPSCs by shortening the time of enzymatic digestion of the dental pulp using the homogenization method and trypsin instead of using the solution of collagenase type I and dispase in ratio 1:1. DPSCs isolated using the new method demonstrated a high proliferation and differentiation potential.
We did not observe any signs of spontaneous differentiation or cell degeneration throughout long-term cultivation.
References
[1] Gronthos S, Mankani M, Brahim J et al (2000) Postnatal human dental pulp stem cells (DPSCs) in vitro and in vivo. Proc Natl Acad Sci USA. 2000;97(25):136
[2] Ferrúa CP, Centeno EGZ, Rosa LC, Amaral, Severo CC, Sarkis-Onofre RF et al. How has dental pulp stem cells isolation been conducted? A scoping review. Braz. Oral Res.
2017;(31):e87
[3] Lindner U, Kramer J, Rohwedel J, Schlenke P. Mesenchymal Stem or Stromal Cells:
Toward a Better Understanding of Their Biology? Transfus Med Hemother. (2010);37: 75–83
Pic. 1 DPSC primary culture (the spindle shape of stem cells) after reaching 70 % of confluence. Phase contrast microscopy, direct magnification 200x
USE OF OPTICAL COHERENCE TOMOGRAPHY AND VISUAL EVOKED POTENTIALS IN PREOPERATIVE AND POSTOPERATIVE MONITORING OF
PATIENTS WITH OPTIC CHIASM COMPRESSION
Pavel Póczoš, M.D.
Department of Neurosurgery - Faculty of Medicine Hradec Králové, University Hospital Hradec Králové
Co-authors: T. Česák, J. Kremláček, M. Macháčková Tutor: Prof. Naďa Jirásková, M.D., Ph.D., FEBO
Introduction
Pituitary tumors, presenting as the most common cause of optic chiasm (OC) compression, make up to 12-15% of all intracranial tumors. When indicated, surgical resection represents the most effective tumor reduction method [1]. Visual acuity (VA) and visual field examination (VF), stratum opticum (RNFL) thickness measurement by optical coherence tomography (OCT), and tumors morphological assessment in magnetic resonance imaging help predict the severity of compression of the visual pathway. Indications for OC decompression are based on patient’s complaints, imaging findings and the results of neuroophthalmological examination. Surgery is often postponed when VA impairment, VF loss and hormonal disturbance are absent. In such cases, visual evoked potentials (VEPs), used as a novel diagnostic technique in our conditions, could bring a significant contribution to the therapeutic approach.
Aims
We have several aims. Firstly, to verify that OCT and VEP can detect a disorder of the optical path sooner than conventional examination methods (VA, VF) in patients without subjective visual difficulties and minimal OC compression. Secondly, to objectivize the OC decompression by using OCT and VEP. Another aim is to find if visual outcomes correlate to the degree of chiasm compression, VF failure and the results of OCT and VEP measurements.
Finally, we are interested if preoperative monitored parameters may predict postoperative changes.
Methods
Patients with OC compression were involved in the study on a voluntary basis. The exclusion criterion was another serious illness of the visual pathway and the inability to cooperate in VEP measurement. The project did not contain a control group of healthy volunteers because we compared patients’ data before and after the surgery. One preoperative and three postoperative (1 week, 3 and 6 months) sessions were performed. We used two types of VEPs: pattern-reversal VEP (P-VEP), and motion-onset VEP (M-VEP) induced by monocular stimulation of vertical hemifields. For P-VEP, a black and white checkerboard (contrast 85%
according to Michelson), with element size of 60′, reversed at a frequency of 2 rev/s, and we evaluated the P100 peak time and inter-peak amplitudes in the Oz derivation. For M-VEP, a low contrast pattern, consisting of grey concentric circles, randomly expanded/contracted in a periodical radial motion, and the N160 peak time and inter-peak amplitudes were read from Pz derivation. The stimuli subtended 11°× 14° of the left or right visual field. The VEPs parameters were compared with the OCT morphological parameters using correlation analysis. All parameters were used as predictors in a multifactorial regression analysis to
estimate the patient's clinical outcomes (VA and VF impairments). We also determined the degree of OC compression on preoperative MRI studies.
Results
We bring the results of a primary, exploratory, analysis that was performed on a group of 10 patients. The median age of ten patients (6 females and 4 males) was 50.7 years. Seven of them had OC suppression caused by adenoma presence, two patients had meningioma and in one case compression was caused by apoplexy. The most common degree of OC oppression was grade IV. Median of VA (logMAR), determined by Landolt-Cs method, was pre- operatively 0.40, postoperatively 0.40, 0.30, respectively 0.30. Concerning the VF examination, preoperatively the median mean deviation was -10.25 dB, post-operatively -3.62 dB, -0.90 dB, respectively -1.43 dB. The comparison of the preoperative RNFL values (median: 85.0 m) with the results of the 2nd (82.5 m) and 3rd (82.5 m) control measurements indicates statistically significant changes. The amplitude or peak time of the dominant P-VEPs and M-VEPs have not improved in all patients consistently.
Discussion
The majority of patients show an improvement in their VF defects and of their VA following surgery, even those with a thin pre-operative RNFL. Our results did not show any postoperative RNFL increase contrary to Danesh-Meyer et al. study [2]. They demonstrated that average RNFL thickness showed initially a slight decrease then increased. Such inconsistency might be caused by a longer follow-up in their study (up to 15 months). Several VEP amplitude parameters were reduced mainly in the temporal hemifield preoperatively, as proclaimed in other works as well [3]. Improvement in amplitudes of the dominant peaks during pattern-reversal stimulation was statistically significant only in some cases.
Conclusions
Our pilot results confirm that there is a relationship between the degree of OC and VF depression. Median RNFL thickness 6 months post-treatment significantly decreased. VEPs have an improving trend, but it is non-consistent among patients and between P-VEPs and M- VEPs. Evoked potentials represent a sensitive and noninvasive tool to objectivize state of the visual function and exemplify already used psychophysical methods evaluating subjective patients’ responses.
References:
1. Ezzat S, Asa SL, Couldwell WT, et al. The prevalence of pituitary adenomas : a systematic review. Cancer 2004;101:613-619.
2. Danesh-Meyer HV, Wong A, Papchenko T, et al. Optical coherence tomography predicts visual outcome for pituitary tumors. J Clin Neurosci 2015 Jul;22(7):1098-104 3. Sousa RM, Oyamada MK, Cunha LP, et al. Multifocal visual evoked potential in eyes with temporal hemianopia from chiasmal compression: correlation with standard automated perimetry and OCT findings. Invest Ophthalmol Vis Sci. 2017;58:4436–
4446.
ALTERNATIONOF BILE ACID HOMEOSTASIS BY IRON OVERLOAD IN RATS
Alena Prasnicka1,4, Hana Lastuvkova1, Jolana Cermanova1, Jitka Hájková1, Milos Hroch2, Jaroslav Mokry3, Eva Dolezelova4, Fatemeh Alaei Faradonbeh1, Petr Pavek5, Martin Lenicek6, Libor Vitek6, Petr Nachtigal4, Stanislav Micuda1
1Department of Pharmacology, 2Department of Medical Biochemistry, 3Department of Histology and Embryology, Charles University, Faculty of Medicine in Hradec Kralove, Czech Republic; 4Department of Biological and Medical Sciences, 5Department of
Pharmacology and Toxicology, Charles University, Faculty of Pharmacy in Hradec Kralove, Czech Republic; 6Department of Medical Biochemistry and Laboratory Diagnostics, 1st Faculty of Medicine, Charles University in Prague, Prague, Czech Republic
E-mail: prasnickaa@lfhk.cuni.cz
Excessive iron accumulation in the liver may affect bile acid metabolism but this effect has not been comprehensively studied. Thus, we evaluated the effect of iron overload on bile acid synthesis, biliary secretion and output through feces in rats. Male Wistar rats fed were administered either with eight dosses of saline every second day i.p., or with eight doses of iron (100 mg/kg) in the same timing. Iron-overloaded (IO) rats developed significant iron deposition in the liver. Comparison with control rats showed decrease in bile flow in IO group as a result of decreased biliary excretion of bile acids. The molecular mechanisms responsible for these changes in IO group were decreased protein expression of Cyp7a1, the rate limiting enzyme in the conversion of cholesterol to bile acid, and decreased expressions of Bsep, the transporter responsible for bile acids efflux into bile. IO did not change net BA content in feces reflecting increased conversion of BA into HDCA and reduced intestinal reabsorption. Plasma concentration of BA were therefore not modified in comparison with saline group. IO increased plasma cholesterol concentrations. It corresponded with reduced Cyp7a1 expression but also with increased protein expression of HmgCR, the rate limiting enzyme in liver de novo cholesterol synthesis. In summary, this study reports mechanisms modifying metabolism and enterohepatic kinetics of BA during IO. Altered elimination pathways for BA and cholesterol may potentiate liver damage accompanying excessive iron deposition. The study was supported by the Charles University Progres Q40, and the Grant Agency of the Czech Republic project No. 17-068415.
CARBON-BASED NANOMATERIALS AND IMMUNE SYSTEM: EFFECT OF CARBON NANOTUBES AND GRAPHENE PLATELETS ON NLRP3 ACTIVATION
Tereza Švadláková, M.Sc.
Department of Clinical Immunology and Allergology, Faculty of Medicine in Hradec Kralove, Charles University and University Hospital in Hradec Kralove
Co-authors: J. Turánek, J. Mašek, P. Kulich, F. Hubatka, P. Turánek Knotigová, A. Málková, M. Ledvina, M.
Koláčková, P. Vicherková, Z Fiala Tutor: prof. RNDr. Jan Krejsek, CSc.
Introduction:
Carbon-based nanomaterials (CNMs) represent unique type of nanomaterials whose extraordinary physicochemical properties like thermal and chemical stability, electrical conductivity and large surface areas make these materials appropriate candidates for a wide range of High-technology applications as well as for biomedical applications. However, still incomplete toxicology data together with increasing production of these materials, leads to severe questions about their safety [1]. An important concern relates to interaction with immune system and the ability to induce inflammation. Both carbon nanotubes (CNT) and graphene have been extensively studied during past years, however results are still inconsistent and often contradictory. Sharp and asbestos-like shape of CNTs leads to the conclusion that both single and multi-walled carbon nanotubes (SWCNTs and MWCNTs) have the capacity to induce acute and chronic inflammation [2]. Depending on shape, length, functionalization and presence of impurities, mechanical disruption is considered to be main mechanism of cytotoxicity [3, 4].
Analogously, a similar situation could arise for graphene, a 2D sheet-shape nanomaterial which can also be expected to cause damage via flat and sharp edges [5]. Unlike graphene oxide, less is known about biological effects of other derivatives such as graphene platelets and sheets (GPs) which workers are exposed during manufacturing. These non-biodegradable materials could pose a risk especially for respiratory system after exposure by inhalation. It has been proved that GPs of a certain size can be delivered beyond the ciliated airways and deposit in alveoli [6]. There are internalized mostly by alveoli macrophages or simply persists in intercellular spaces, where can physically interact with other cells. This may lead to inflammation, disruption off cell metabolism and tissue damage.
Aims:
In our study, we focus on comparison between several types of MWCNTs and GPs depending on their internalization into cells and their intracellular distribution. All these nanomaterials are assumed to cause chronic inflammation, so we focus on their ability to induce NLRP3 inflammasome as a key mediator of inflammation.
Methods:
Two different types of graphene platelets (GPs) and two different types of carboxylate multi-walled carbon nanotubes (MWCNTs) were used for experiments. Stock solutions (0.25 mg/ml) of GPs were prepared by sonication in 0.02% sodium cholate (30 min, 75% amplitude). Stock solutions of MWCNTs (0.5 mg/ml) were prepared by sonication in Milli-Q water (5 min, 65% amplitude). All CNMs were characterized using Zetasizer Nano-ZSP (hydrodynamic diameter and zeta potential) and Transmission Electron Microscopy (TEM). Human monocyte cell line THP-1 was used for viability assay (LDH; 24h and 48h) and intracellular localization of CNMs (TEM, confocal microscopy). Human reporter cell line THP1-null was used for study of cathepsin B release and NLRP3 activation (24h - 48h) as the cells express high levels of NLRP3, ASC and pro-caspase 1. As a negative control was used another human reporter cell line THP1-defNLRP3 with reduced NLRP3 activity. IL-1β, product of NLRP3, was determined using human reporter HEK-Blue™ IL-1β cells.
