Waldemar Uszyński, Mieczysław Uszyński, Ewa Żekanowska, Krzysztof Góralczyk: Thrombin activatable fibrinolysis inhibitor (TAFI) in cord blood pp. 33-36
Immune-mediated bone marrow failure syndromes of progenitor and stem cells: molecular analysis of cytotoxic T cell clones
Jaroslaw P. Maciejewski, Christine O'Keefe, Lukasz Gondek, Ramon Tiu
Experimental Hematology and Hematopoiesis Section, Taussig Cancer Center,
Cleveland Clinic Foundation, Cleveland, OH, USA
Abstract: The unique structure of the T cell receptor (TCR) enables molecular identification of individual T cell clones and
provides an unique opportunity for the design of molecular diagnostic tests based on the structure of the rearranged TCR
chain e.g., using the TCR CDR3 region. Initially, clonal T cell malignancies, including T cell large granular lymphocyte
leukemia (T-LGL), mucosis fungoides and peripheral T cell lymphoma were targets for the TCR-based analytic assays such
as detection of clonality by T-gamma rearrangement using gamma-chain-specific PCR or Southern Blotting. Study of these disorders
facilitated further analytic concepts and application of rational methods of TCR analysis to investigations of polyclonal T
cell-mediated diseases. In hematology, such conditions include graft versus host disease (GvHD) and immune-mediated
bone marrow failure syndromes. In aplastic anemia (AA), myelodysplastic syndrome (MDS) or paroxysmal nocturnal
hemoglobinuria (PNH), cytotoxic T cell responses may be directed against certain antigens located on stem or more lineage-
restricted progenitor cells in single lineage cytopenias. The nature of the antigenic targets driving polyclonal CTL
responses remains unclear. Novel methods of TCR repertoire analysis, include VB flow cytometry, peptide-specific tetramer
staining, in vitro stimulation assays and TCR CDR3-specific PCR. Such PCR assay can be either VB family-specific or multiplexed
for all VB families. Amplified products can be characterized and quantitated to facilitate detection of the most
immunodominant clonotypes. Such clonotypes may serve as markers for the global polyclonal T cell response. Identification
of these clonotypes can be performed in blood and tissue biopsy material by various methods. Once immunodominant
clonotypes corresponding to pathogenic CTL clones are identified they can serve as surrogate markers for the activity of the
pathophysiologic process or even indicate the presence of specific antigens. The relevance of the individual clonotypes can
be ascertained from clinical correlations with the activity of the disease. Quantitative clonotypic assays such as sequencing
of multiple CDR3 clones or clonotypic Taqman PCR can be applied for the monitoring of the immunosuppressive therapy
and prediction of relapse. Future technologies may allow for the design of clonotypic microarrays or other more clinically
applicable methods of clonotypic diagnostics. Similarly, identification of immunodominant clonotypes may facilitate targeting
of autoimmune or malignant clones with vaccination and induction of anti-idiotypic responses.
Author’s e-mail: maciejj@cc.ccf.org
Full text
Loss and recovery of androgen receptor protein expression in the adult rat testis following androgen withdrawal by ethane dimethanesulfonate
Włodzimierz Łuczyński1, Oksana Kowalczuk2, Elżbieta Iłendo3, Anna Stasiak-Barmuta4, Maryna Krawczuk-Rybak1, Iwona Malinowska5, Andrzej Kołtan6, Tomasz Szczepański7, Igor Olejnik8, Radosław Jaworowski3, Lech Chyczewski2, Michał Matysiak5, Mariusz Wysocki6, Danuta Sońta-Jakimczyk7, Maria Wieczorek8
Departments of Pediatric Hematology and Oncology in: 1Białystok, 5Warsaw, 6Bydgoszcz, 7Zabrze, 8Chorzów;
2Department of Clinical Molecular Biology, 3Department of Cytogenetics,
4Department of Flow Cytometry, Medical University in Białystok, Poland
Abstract: The use of cancer vaccines based on dendritic cells (DC) presenting tumor antigens can be a promising tool in
the treatment of leukemia. The functional characteristics of leukemia derived DC is still to be elucidated. CD40 promotes
survival, proliferation and differentiation of normal B cells. CD40 triggering was used to enhance the poor antigen-presenting
capacity of leukemic B-cells. Since it is still unclear whether CD40 ligation drives neoplastic B-cells to apoptosis
or not, we assessed the mRNA expression of FLICE, FAS, FADD and TRADD - important components of apoptosis machinery,
using real-time PCR in acute lymphoblastic leukemia cells before and after CD40 and IL-4 stimulation. ALL cells stimulated
with CD40L/IL-4 expressed dendritic cell phenotype at mRNA and protein levels (upregulation of main costimulatory
and adhesion molecules noted in real-time RT PCR and flow cytometry); they also expressed higher amounts of mRNA
for FLICE, TRADD and FADD after CD40L/IL-4 stimulation. However differences statistically significant comparing cells
cultured with CD40L/IL-4 and medium alone regarded only FLICE. Concluding, we showed upregulation of important elements
of apoptosis at mRNA level in ALL cells after CD40 ligation.
Author’s e-mail: w.luczynski@wp.pl
Coexpression of CD1a,langerin and Birbeck's granules in Langerhans cell histiocytoses (LCH) in children: ultrastructural and immunocytochemical studies
Piotr Dzięgiel1, Barbara Dolińska-Krajewska1, Małgorzata Dumańska1, Jadwiga Węcławek2, Michał Jeleń2, Marzena Podhorska-Okołów1, Ewa Jagoda1, Magdalena Fic1, Maciej Zabel1,4
1Department of Histology and Embryology, University School of Medicine, Wrocław;
2Department of Bone Marrow Transplantation, Pediatric Oncology and Hematology,
University School of Medicine, Wrocław;
3Department of Pathomorphology, University School of Medicine, Wrocław;
4Department of Histology and Embryology, University of Medical Sciences, Poznań, Poland
Abstract: Langerhans cell histiocytoses (LCH) represent rare diseases of unclear etiology and pathogenesis. Most of the
cases include children, 1 to 15 years of age, and various organs are involved (bones, skin, liver, lymph nodes, bone marrow
and other). The diagnosis of LCH used to be established by biopsy of the inflamed tissue and demonstration of expression of
markers specific for Langerhans cells: CD1a and langerin. The diagnosis can be ultimately confirmed by demonstration of Birbeck's
granules in the electron microscopy. The present study was aimed at immunocytochemical demonstration, in the examined
LCH material (skin, bones, lymph nodes), of the specific antigen expression and at comparing it with the presence of Birbeck's
granules. In the examined 11 cases co-expression of CD1a with langerin and with the presence of Birbeck's granules was
noted. Also in all examined biopsies the expression of S-100 protein on inflammatory cells was found. The results corroborate
the usefulness of immunocytochemical studies on CD1a and langerin expression in diagnosis of LCH.
Author’s e-mail: piotr@hist.am.wroc.pl
Evidence that platelet-derived microvesicles may transfer platelet-specific immunoreactive antigens to the surface of endothelial cells and CD34+ hematopoietic stem/ progenitor cells - implication for the pathogenesis of immune thrombocytopenias
Marcin Majka1, Jacek Kijowski1, Ewa Lesko1, Jolanta Goździk1, Barbara Zupanska3
and Mariusz Z. Ratajczak2
1Department of Transplantation, Polish-American Institute of Pediatrics, Jagiellonian University Medical
College, Cracow, Poland
2Stem Cell Biology Program, James Graham Brown Cancer Center, University of Louisville, KY USA
3Institute of Hematology, Warsaw, Poland
Abstract: The pathogenesis and tissue damage that accompanies destruction of platelets in immune thrombocytopenias (IT)
is still not understood very well and in addition to platelets, other cells (e.g. endothelial cells, CD34+ hematopoietic
stem/progenitors) may also become affected. Based on our previous work that platelet antigens (e.g., CD41) may be transferred
by platelet-derived microvesicles (PMV) to the surface of other cells, we asked if platelet derived-antigens, especially
those that are involved in the formation of anti-platelet antibodies in IT (e.g., against antigen HPA1a) could be also transferred
by similar mechanism. To address this issue normal human CD34+ cells, human umbilical vein-endothelial cells
(HUVEC) and monocytic cell line THP-1 were incubated with PMV derived from HPA1a+ donors. We noticed that the
HPA1a antigen is highly expressed on PMV-derived from the HPA1a positive platelets and is transferred in PMV-dependent
manner to the surface of CD34+ cells, HUVEC and monocytic THP-1 cells. These cells covered with HPA1a positive
PMV but not by PMV derived from HPA1a negative platelets reacted with anti-HPA1a antibodies derived from the alloimmunized
pregnant women. More importantly, human hematopoietic cells that were preincubated with HPA1a+ PMV and
subsequently exposed to anti-HPA1a serum and human NK cells, become subject to elimination by antibody dependent cell
cytotoxicity ADCC. Thus, we postulate that PMV-dependent transfer of antigens may playing an important role in "expanding"
the population of target cells that may be affected by anti-platelet antibodies and explain several pathologies that
accompany IT (e.g. damage of endothelium, cytopenias).
Author’s e-mail: mmajka@cm-uj.krakow.pl
Thrombin activatable fibrinolysis inhibitor (TAFI) in cord blood
Waldemar Uszyński1, Mieczysław Uszyński2, Ewa Żekanowska3, Krzysztof Góralczyk3
1Obstetric-Gynecological Ward of the Regional Hospital in Włocławek,
2Department of Propedeutics of Medicine, Nicolaus Copernicus University in Torun -
Collegium Medicum in Bydgoszcz, and
3Department of Pathophysiology, Nicolaus Copernicus University in Torun - Collegium Medicum
in Bydgoszcz, Poland
Abstract: Thrombin activatable fibrinolysis inhibitor (TAFI) is a plasma zymogene (procarboxypeptidase B) which can
decrease fibrinolysis and thus act as a haemostatic factor. TAFI is now extensively studied in many complications as well
as in physiological and complicated pregnancy. The question we posed in the present study was whether TAFI antigen is
present in cord blood plasma. The study group consisted of 38 parturient women, 26 primiparous and 12 multiparous with
normal course of pregnancy and delivery. The cord blood was sampled from the cord vein, and the mother's blood from the
antecubital vein. 3.2% sodium citrate was used as an anticoagulant. TAFIa/ai antigen was measured by ELISA method.
TAFIa/ai antigen was identified in all samples of cord blood plasma. Its level was 91.50 ng/ml (range: 71.76 - 160.77 ng/ml)
vs. 55.46 ng/ml (range: 39.77 - 68.54 ng/ml ) in the mother's blood, which means that the level of TAFIa/ai antigen was significantly
higher in fetal blood than in maternal blood (p<0.00001). TAFIa/ai antigen is an integral component of cord blood
plasma. The concentration of TAFIa/ai antigen is about two times higher in fetal blood than in maternal blood.
Author’s e-mail: kizproped@cm.umk.pl
Expression of matrix metalloproteinase 9 in pancreatic ductal carcinoma is associated with tumor metastasis formation
Anna Pryczynicz1, Katarzyna Guzińska-Ustymowicz1, Violetta Dymicka-Piekarska2,
Jolanta Czyżewska2 and Andrzej Kemona1
1Department of General Pathomorphology and
2Department of Clinical Laboratory Diagnostics, Medical University of Białystok, Białystok, Poland
Abstract: The objective of the current study was to assess the expression of matrix metalloproteinase 9 (MMP-9) in pancreatic
ductal carcinoma and to examine its correlation with chosen clinico-anatomical parameters. The study group consisted
of 36 patients with pancreatic ductal carcinoma. Tumors were stained using immunohistochemical method (NCL -
MMP-9, Novocastra). No correlation was found between tumor MMP-9 expression and age, gender or grade of histological
malignancy. However, statistical analysis revealed a relationship between tumor MMP-9 expression and histological
type (adenocarcinoma mucinosum) of pancreatic carcinoma. The expression was strongly correlated with lymph node
involvement and occurrence of distant metastases (p<0.00001). The results indicate a correlation between the expression of
MMP-9 in pancreatic ductal carcinoma and worse prognosis (shown by lymph node involvement and distant metastases).
Author’s e-mail: kguzinska@poczta.onet.pl
Expression of class III ?-tubulin in malignant epithelial
tumours: an immunohistochemical study using TU-20
and TuJ-1 antibodies
Tomáš Jirásek1, Eva Písaoíková1, Vladimír Viklický2 and Václav Mandys1
1Department of Pathology, 3rd Faculty of Medicine, Charles University, and
2Institute of Molecular Genetic, Academy of Sciences of the Czech Republic, Prague, Czech Republic
Abstract: Class III ?-tubulin has been discovered as a marker of early phases of neuronal differentiation in developmental
conditions, as well as in different tumours of neuronal origin. More recently, the expression of class III ?-tubulin molecule
has been described as a marker of different types of malignant epithelial tumours. This study attempts to compare the
immunostaining features of two different mouse monoclonal antibodies TU-20 and TuJ-1, both detecting class III ?-tubulin,
in a group of twenty bioptically evaluated carcinomas of various sites. The proposal that class III ?-tubulin expression
can correlate with the degree of tumour differentiation and thus could be potentially used as predictive marker of prognosis
has been previously done; one of aims of our study was to confirm this hypothesis. Our results showed that both TuJ-1 and
TU-20 antibodies displayed similar immunostaining profile and pattern within individual tumours. Surprisingly, we discovered
that only 50% of tumours included in our group showed expression of class III ?-tubulin, however, positive immunoreaction
did not correspond with the degree of differentiation of individual tumours. In our group of carcinomas, the class III
?-tubulin positivity was not related to the tumour site, histologic type of tumour or its grade.
Author’s e-mail: tjirasek@fnkv.cz
Rare genotype del2,3/2184insA in a cystic fibrosis patient
Alina Minarowska1, Oksana Kowalczuk2, Maciej Kaczmarski1, Danuta Kowalczuk3,
Łukasz Minarowski2 and Lech Chyczewski2
13rd Department of Children's Diseases, 2Department of Clinical Molecular Biology and
3Department of Children's Radiology, Medical University of Białystok, Białystok, Poland
Abstract: In this paper we present an interesting case of cystic fibrosis patient with rare genotype del2,3/2184insA and
atypical clinical image including: mild symptoms in an early phase of disease, quick progress of lung disease, complicated
with pneumothorax after Bordetella pertussis infection and very good response to systemic and inhaled steroid
therapy.
Influence of maternal dexamethasone treatment
on morphometric characteristics of pituitary GH cells
and body weight in near-term rat fetuses
M. Manojlovia-Stojanoski, N. Nestorovia, N. Negia, B. Filipovia, B. Šošia-Jurjevia,
M. Sekulia and V. Miloševia
Institute for Biological Research "Siniša Stankovia", Belgrade, Serbia and Montenegro
Abstract: Growth hormone (GH) and glucocorticoids have a powerful influence on controlling fetal growth, differentiation
and maturation of numerous tissues. In the present study, the effect of maternal dexamethasone (Dx) treatment on GH cells
and body weight in 19- and 21-day-old rat fetuses was investigated using immunocytochemical and morphometric methods.
Pregnant female rats received daily injections of 1.0-0.5-0.5 mg Dx/kg b.w. on days 16-18 of pregnancy (experimental
group), while the control group received an equal volume of saline. Dx treatment of pregnant rats enhanced immunostaining
intensity and significantly increased (p<0.05) GH nuclear and cell volume, as well as volume density and number of GH
cells per square millimeter in 19-day-old fetuses compared to the controls. In 21-day-old fetuses after maternal Dx administration,
immunoreactivity, volume density and number of GH cells remained significantly increased (p<0.05). Dx treatment
of pregnant rats resulted in marked body weight reduction of 21-day-old but not 19 days old fetuses in comparison
with the corresponding controls. The presented results demonstrate that maternal Dx application has pronounced effect on
morphometric parameters of GH cells of 19- and 21-day-old fetuses. Also, in near-term rat fetuses body weight was largely
independent of pituitary GH cell activity.
Author’s e-mail: manojlo@ibiss.bg.ac.yu
Preparation of rat synovial membrane for studies of
cytokine secretion
Anna Hyc, Anna Osiecka-Iwan, Piotr Dziunycz, Stanisław Moskalewski
Department of Histology and Embryology, Medical University of Warsaw, Warsaw, Poland
Abstract: The objective of this work was to devise an in vitro system for studies on cytokine secretion by synovial membrane
treated as a whole organ with various synoviocyte populations. Synovial membrane from knee joints of WAG rats was
dissected and incubated in culture medium without serum for 4 - 48 h. The level of IL-1? was determined in synovial lysates
and IL-6 in culture medium. The synovial membrane from left and right knee joint of the same rat produced similar amount
of cytokines both in lysates and in the medium. Synovial membrane stimulated by LPS for 4 or 24 h gave significantly
stronger cytokine response than the membrane from the opposite (control) knee. After 48 h incubation of synovial membrane
drastic drop in cytokine level was noted, which indicated on deterioration of the membranes. The test may be useful
in studies on factors affecting cytokine secretion by synoviocytes.