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FOLIA HISTOCHEMICA
ET CYTOBIOLOGICA
Vol. 44, No. 1, 2006
Jarosław Całka
Department of Functional Morphology, Division of Animal Anatomy, University of Warmia and Mazury, Olsztyn, Poland
Abstract: Nitric oxide (NO) affects reproductive processes both at the level of the brain and reproductive tract and this review is focused on its role as an essential regulator of the hypothalamic control of reproduction. The data gathered indicate that glutamate stimulates noradrenergic neurons which subsequently activate NO-ergic cells via α1-adrenergic receptors. The released NO diffuses into luteinizing hormone-releasing hormone (LHRH) terminals where it triggers LHRH secretion by activation of guanylyl cyclase and cyclooxygenase. The NO released by estrogen-stimulated NO-ergic ventromedial neurons plays a crucial role in the regulation of sexual behavior. Furthermore, an increased expression of inducible nitric oxide synthase in the LHRH and oxytocin neurons underlies the destructive action of NO on the aging of the hypothalamic neuroendocrine pathways. Within the hypothalamo-hypophyseal system, NO exerts an inhibitory effect in the control of oxytocin secretion. This action seems to employ an indirect mechanism by which NO may modulate the release of GABA. This review provides an overview of the role of NO in hypothalamic control of LHRH and oxytocin release, aging of the LHRH and oxytocin neurons and sexual behavior.
Author’s e-mail: calkaj@uwm.edu.pl
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Expression of NPY-immunoreactive neurons in the hypothalamus of the cycling ewe
Jolanta Polkowska, Marta Wańkowska and Anna Wójcik-Gładysz
The Kielanowski Institute of Animal Physiology and Nutrition, Polish Academy of
Sciences, Jabłonna, Poland
Abstract: The purpose of the study was to localise neuropeptide Y (NPY) immunoreactive (ir) neurons in the hypothalamus during two phases of the oestrous cycle in the ewe. Hypothalamic tissue was collected from Polish Merino ewes (n=8) in the follicular (15th day) and preovulatory (17th day) phases of the oestrous cycle. NPY-ir neurons were detected in the hypothalamus using immuohistochemistry followed by image analysis; positive staining was expressed as the percentage of stained area and optical density. Two populations of the NPY-positive neurons were detected and evaluated in the infundibular and periventricular nuclei of the hypothalamus. The population of NPY-ir neurons located in the infundibular nucleus exhibited a prominent expression of NPY immunoreactivity in the perikarya and fibres only during the preovulatory phase. Both, percent area and the optical density of NPY immunostaining measured in this area were higher (P<0.01) in the preovulatory than in the follicular phase. Another population of NPY-ir neurons was localised in the periventricular nucleus and did not show any changes during the two phases of the cycle. The present study suggests that NPY-ir neurons present in the infundibular nucleus can play a role in the preovulatory GnRH discharge from the median eminence.
Author’s e-mail: j.polkowska@ifzz.pan,pl
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VEGF, VEGFR-1 and VEGFR-2 immunoreactivity in the porcine arteries of vascular subovarian plexus (VSP) during the estrous cycle
A. Postek, A. Andronowska, T. Doboszyńska, H. Niewęgłowski and K. Jankowska
Department of Reproductive Histophysiology, Division of Reproductive
Endocrinology and Pathophysiology, Institute of Animal Reproduction and
Food Research, Polish Academy of Sciences, Olsztyn, Poland
Abstract: Vascular endothelial growth factor (VEGF) is an important angiogenic factor in the female reproductive tract. It binds to cell surface through ligand-stimulatable tyrosine kinase receptors, the most important being VEGFR-1 (flt-1) and VEGFR-2 (flk-1). The broad ligament of the uterus is a dynamic organ consisting of specialized complexes of blood vessels connected functionally to the uterus, oviduct and ovary. Endothelial cells form an inner coating of the vessel walls and thus they stay under the influence of various modulators circulating in blood including ovarian steriods involved in developmental changes in the female reproductive system. The aim of the present study was to immunolocalize VEGF and its two receptors: VEGFR-1 and VEGFR-2 in the broad ligament of the uterus in the area of vascular subovarian plexus during different phases of the estrous cycle in pig and to determine the correlation between immunoreactivity of the investigated factors and phases of the estrous cycle. The study was performed on cryostat sections of vascular subovarian plexus stained immunohistochemically by ABC method. Specific polyclonal antibodies: anti-VEGF, anti-VEGFR-1 and anti-VEGFR-2 were used. Data were subjected to one-way analysis of variance. Our study revealed the presence of VEGF and its receptors in endothelial and smooth muscle cells of VSP arteries. All agents displayed phase-related differences in immunoreactivity suggesting the modulatory effect of VEGF, VEGFR-1 and VEGFR-2 on the arteries of the VSP in the porcine broad ligament of the uterus.
Author’s e-mail: teresa@pan.olsztyn.pl
Morphology of the epithelial cells and expression of androgen receptor in rat prostate dorsal lobe in experimental hyperprolactinemia
Sylwia Słuczanowska-Głąbowska1, Maria Laszczyńska2, Wojciech Głąbowski3 and Marcin Wylot3
1Department of Physiology, 2Laboratory of
Embryology, and 3Department of Histology and Embryology,
Pomeranian Medical University, Szczecin, Poland
Abstract: The effect of hyperprolactinemia on the prostate has not been well investigated. Since androgens play an important role in prostate development, growth and function, the goal of the present study was to estimate the influence of hyperprolactinemia on expression of the androgen receptor (AR) in rat epithelial cells of prostate dorsal lobe and on morphology of these cells. Studies were performed on sexually mature male Wistar rats. The experimental group rats received metoclopramide (MCP) intraperitoneally to provoke hyperprolactinemia. The control group animals were given saline in the same way. For light and electron microscopy the prostate dorsal lobes were obtained routinely. To evaluate the intensity of immunohistochemical reaction for AR in epithelial cells, the optical density was measured and computer-assisted image analysis system was used. Morphological observations of the dorsal lobe epithelial cells were carried out in transmission electron microscope. MCP caused over twofold increase in prolactin (PRL) serum levels. In rats with hyperprolactinemia, the testosterone levels (T) were twofold decreased. The intensity of immunohistochemical reaction for AR in epithelial cells of dorsal lobe in the experimental group was significantly lower than in the control group. In the dorsal lobe epithelial cells of experimental group animals, the transmission electron microscopy (TEM) revealed highly dilated RER cisternae and reduced number of microvilli on the cellular surface when compared to the control group. The results show that hyperprolactinemia in male rats causes morphological abnormalities in the dorsal lobe of prostate. The abnormalities are caused by elevated prolactin either directly or indirectly through decreased level of testosterone. Decreased expression of AR in epithelial cells of prostate dorsal lobe is likely to be caused by decreased testosterone level.
Author’s e-mail: wojtekgl@sci.pam.szczecin.pl
Spontaneous apoptosis of melanotic and amelanotic melanoma cells in different phases of cell cycle: relation to tumor growth
Mirosława Cichorek, Krystyna Kozłowska, Małgorzata Wachulska and Katarzyna Zielińska
Department of Embryology, Medical University, Gdańsk, Poland
Abstract: Since the spontaneous alteration of native melanotic (Ma) into amelanotic (Ab) transplantable melanoma line it has been observed that this alteration is accompanied by the acceleration of growth of Ab line. The aim of the present study was to check and estimate spontaneous apoptosis of cells from cell cycle phases.Cytometric cell cycle analysis was performed by staining cells with propidium iodide (PI). Apoptosis estimated by the TUNEL method, alterations in the plasma membrane structure (annexin V staining), changes in the mitochondrial transmembrane potential - ΔΨm (JC-1 staining) showed that amelanotic melanoma cells have decreased ability to undergo spontaneous apoptosis. The obtained results showing that in the native melanotic line about 30% of cells are in S+G2/M phases and that 33% of these cells undergo apoptosis could lead to the conclusion that the slower growth of this melanoma line is the result of lower proliferation activity and higher rate of apoptosis of these tumor cells. The number of cells in S+G2/M phases in amelanotic melanoma line increases up to 40% and only 7% of them undergo apoptosis. This observation seems to suggest that the expansive growth of this melanoma line depends mainly on the decreased ability to undergo spontaneous apoptosis, especially in case of cells from S+G2/M phases. Moreover, the obtained results indicate that alteration of melanotic line into amelanotic one, accompanied by differences in many biological features also concerns basic cell processes such as cell cycle and cell death.
Author’s e-mail: cichorek@.amg.gda.pl
Proliferating cell nuclear antigen (PCNA) expression in pituitary adenomas: relationship to the endocrine phenotype of adenoma
Marek Pawlikowski1, Anna Gruszka1, Ilona Kurnatowska3, Katarzyna Winczyk1, Jolanta Kunert-Radek2 and Andrzej Radek4
1Department of Neuroendocrinology and 2Department of Clinical Endocrinology, Chair of Endocrinology, 3Department of Nephrology, 4Department of Neurosurgery and Surgery of Peripheral Nerves, Medical University, Łódź, Poland
Abstract: The expression of proliferating cell nuclear antigen (PCNA) correlates to cell proliferation and for this reason it is commonly considered as one of proliferation markers. Since proliferation rate is an important factor determining the tumor aggressiveness, the evaluation of PCNA index (the percentage of PCNA-immunopositive nuclei in the investigated tumor sample) is suggested as useful in predicting pituitary adenoma outcome. Seventy three unselected, surgically removed pituitary adenomas were immunostained with antibodies against the pituitary hormones or their subunits and against the proliferating cell nuclear antigen (PCNA).The highest PCNA index was found in ACTH-immunopositive tumors without the manifestation of the Cushing’s disease (“silent” corticotropinomas). This value was significantly different in comparison to other adenoma subtypes including corticotropinomas manifesting themselves by Cushing’s disease.The lowest PCNA index was noticed in monohormonal GH-secreting tumors. The adenomas which express more than one hormone (plurihormonal adenomas) seem to have a higher PCNA indices than monohormonal ones; the difference was significant in the case of mono- and plurihormonal prolactinomas. The recurrent tumors presented a higher mean PCNA index as compared to the primary tumors, although the difference was significant only in the case of prolactinomas.These findings suggest that the proliferative potential of pituitary adenomas is related to the tumor recurrence and hormone expression.
Author’s e-mail: pawlikowski.m@wp.pl
Density of intranodal lymphatics and VEGF-C expression in B-cell lymphoma and reactive lymph nodes
Tomasz Wróbel1, Grzegorz Mazur1, Piotr Dzięgiel2, Michał Jeleń3, Andrzej Szuba4, Kazimierz Kuliczkowski1 and Maciej Zabel2
1Department of Haematology, Blood Neoplasms and Bone Marrow Transplantation, 2Department of Histology and Embryology, 3Department of Pathology, and 4Department of Internal Medicine and Occupational Diseases, Medical University, Wrocław, Poland
Abstract: Lymphatic vasculature in solid tumors may serve as the pathway for metastatic spread of the cancer to the regional lymph nodes and to distant organs. Controversy still exists whether tumors metastasize through existing lymphatics or through newly formed vessels (lymphangiogenesis). The role of lymphangiogenesis in lymphoma spread and proliferation is not clearly established. VEGF-C is the most potent inducer of lymphangiogenesis. LYVE-1 was shown to be a specific marker for lymphatic vessels in normal and tumor tissue. The aim of the present study was the evaluation of lymph node LYVE-1-positive lymphatic sinus density (LSD) and VEGF-C expression in patients with non-Hodgkin's lymphoma (nHL) and in reactive lymph nodes. Sixty paraffin-embedded lymph nodes from newly diagnosed patients with B-cell nHL were evaluated. Twelve lymph node biopsy specimens from adult patients with reactive lymphonodulitis were used as controls. Sections of lymph nodes were stained immunohistochemically for LYVE-1 and VEGF-C. VEGF-C expression in lymph nodes of nHL patients was low and not significantly different from that in the control (p=0.6). Moreover, VEGF-C expression did not differ significantly between aggressive and indolent lymphomas (p=0.53). Similarly we did not find differences in LSD in aggressive nHL and in indolent nHL (p=0.49). The mean LSD in reactive lymph nodes was higher than in nHL (p=0.03). Only in 2 out of 12 reactive lymph nodes LYVE-1-positive vessels were absent. In all groups we demonstrated a strong positive correlation between VEGF-C and LYVE-1 expression (p= 0.0001). Higher LSD in reactive lymph nodes as compared to those of nHL patients suggests that lymphoma proliferation leads to the destruction of the existing lymphatics rather than to lymphangiogenesis within lymph nodes. NHL are not associated with increased expression of VEGF-C nor increased LYVE-1-positive lymphatic sinuses density within lymph nodes.
Author’s e-mail: wrobelt@hemat.am.wroc.pl
Prognostic significance of Ki67-negative blast cell clone in the high risk group of children treated for acute myeloid leukaemia
Michał Nowicki1, Danuta Ostalska-Nowicka2 and Bogdan Miśkowiak1,3
Departments of 1Histology and Embryology, 2Pediatric Cardiology and Nephrology and 3Optometry and Biology of the Visual System, University of Medical Sciences, Poznań, Poland
Abstract: The aim of this study was to demonstrate the value of immunocytochemical staining of Ki67 antigen expression in blast cells of children with acute myeloid leukemia (AML) and to evaluate its correlation with treatment failure. The material included bone marrow specimens obtained during induction treatment from 46 children treated for AML between 1998–2003. Immunocytochemical staining for Ki67 was based on the ABC technique. Expression of Ki67 antigen on day 0 of induction treatment was confirmed in all patients. The percentage of immunopositive blasts ranged from 88.4% to 99.8% (mean 91.8%). On day 15, according to chemotherapy response, patients were divided into two groups: G1 – 36 children who responded to induction treatment and reached remission (blast level < 5%, low risk group) and G2 – 10 patients who did not meet remission criterion (blast level > 5%) and were assigned to the high risk (HR) group. Out of 10 children assigned to this group, Ki67 expression in blast cells was confirmed in 4 cases. The fraction of immunopositive blasts ranged from 78.4% to 88.6%. In the other 6 cases, blasts were Ki67-negative. In 12-month period after beginning the treatment, 18 cases of treatment failure (including 7 deceases) were observed in both groups. Five deaths, observed in the HR group, concerned the patients characterized by Ki67-negative blasts.The results indicate a possible correlation between the Ki67-immunonegative blast pattern on day 15 of treatment induction and early decease of AML children assigned to HR group.
Author’s e-mail: mnowicki@amp.edu.pl
Flow cytometric enumeration of CD34+ hematopoietic stem and progenitor cells in leukapheresis product and bone marrow for clinical transplantation: a comparison of three methods
A Gajkowska1, T Oldak1,2, M Jastrzewska1, EK Machaj1,2, J Walewski3, E Kraszewska4 and Z Pojda1,2
1Department of Experimental Hematology, M. Sklodowska-Curie Memorial Cancer Center and Institute of Oncology, 2Department of Radiology, WIHiE Institute of Hygiene and Epidemiology, 3Department of Lymphoproliferative Disease Hematooncology Intensive Care Unit, and 4Department of Biostatistics, M. Sklodowska-Curie Memorial Cancer Center and Institute of Oncology, Warsaw, Poland
Abstract: Flow cytometric enumeration of CD34+ hematopoietic stem and progenitor cells (HSCs) is widely used for evaluation of graft adequacy of peripheral blood and bone marrow stem cell grafts. In the present study, we review and compare the major counting techniques of stem and progenitor cells. The methods are: the Milan/Mullhouse protocol, two-platform ISHAGE (International Society of Hematotherapy and Graft Engineering) and single-platform ISHAGE analysis system. According to the Milan/Mulhouse protocol, HSCs are identified by CD34 antibody staining and easy gating strategy. The ISHAGE guidelines for detection of CD34+ cells are based on a four-parameter flow cytometry method (CD34PE/CD45PerCP staining, side and forward angle light scatter) thus employing multiparameter gating strategy. With two-platform ISHAGE protocol, an absolute CD34+ count is generated by incorporating the leukocyte count from an automated hematology analyser. The single-platform ISHAGE method to determine the absolute CD34+ count directly from a flow cytometer includes the use of Trucount tubes (Becton Dickinson) with a known number of fluorescent beads. CD34+ cells were quantified in mobilized peripheral blood, collected by leukapheresis, and bone marrow from 42 samples from patients with hematological malignancies. The differences against the means display low disagreement between the Milan/Mulhouse and ISHAGE protocols, with discrepancies of up to 2.5% (two-platform ISHAGE) – 2.6% (single-platform ISHAGE) in enumeration of CD34+ cells in leukapheresis product and 4.8% (two-platform ISHAGE) – 4.9% (single-platform ISHAGE) in bone marrow. Our results show high correlation among all three methods. Since the three protocols are compatible, choosing the most convenient in terms of costs, simplicity and compliance with clinical results appears to be a logical consequence.
Author’s e-mail: agnigajk@coi.waw.pl
The number and distribution of blood dendritic cells in the epidermis and dermis of healthy human subjects
Joanna Narbutt1, Aleksandra Lesiak1, Anna Sysa-Jedrzejowska1, Piotr Smolewski2, Tadeusz Robak2 and Anna Zalewska1
1Department of Dermatology and Venereology and 2Department of Haematology, Medical University, Łódź, Poland
Abstract: Human blood dendritic cells (BDC) can be divided into three subsets: plasmacytoid DC (PDC) and two myeloid subsets - MDC1 and MDC2. Several studies revealed the presence of both MDC and PDC in blood of healthy subjects, however no precise literature data exist on the number and distribution of BDC in the skin. The aim of our study was to assess the number and distribution of BDC and their subtypes in the healthy skin. The study included 30 healthy volunteers (age 18-51). Punch biopsies were taken from the buttock skin from each subject, and immunofluorescent staining was performed using monoclonal mouse IgG1 antibodies directed against BDCA-1, BDCA-2, BDCA-3 and BDC-4. The BDC were present both in the epidermis and dermis. PDC were detected mainly in the dermis (mean 1.2 cells per field). Myeloid subtypes were observed mainly in the middle layers of the epidermis and in the upper part of the dermis (mean 1.8 cells per field). The detection of blood dendritic cells in the skin proves their role in immune cutaneous surveillance.
Author’s e-mail: joanna.narbutt@onet.pl
Autofluorescence in eleocytes of some earthworm species
Justyna Cholewa1, Graham P. Feeney2, Michael O’Reilly2, Stephen R. Stürzenbaum2, A. John Morgan2 and Barbara Płytycz1
1Department of Evolutionary Immunobiology, Institute of
Zoology, Jagiellonian University, Kraków, Poland
2Cardiff School of Biosciences, Cardiff University,
Wales, UK
Abstract: Immunocompetent cells of earthworms, coelomocytes, comprise adherent amoebocytes and granular eleocytes (chloragocytes). Both cell populations can be expelled via dorsal pores of adult earthworms by exposure to an electric current (4.5 V) for 1 min. Analysis by phase contrast/fluorescence microscopy and flow cytometry demonstrated that eleocyte population of several species exhibits a strong autofluorescence. A high percentage (11-35%) of autofluorescent eleocytes was recorded in Allolobophora chlorotica, Dendrodrilus rubidus, Eisenia fetida, and Octolasion sp. (O. cyaneum, O. tyrtaeum tyrtaeum and O. tyrtaeum lacteum). In contrast, autofluorescent coelomocytes were exceptionally scarce (less than 1%) in representative Aporrectodea sp. (A. caliginosa and A. longa) and Lumbricus sp. (L. castaneus, L. festivus, L. rubellus, L. terrestris). Thus, this paper for the first time describes profound intrinsic fluorescence of eleocytes in some – but not all – earthworm species. The function (if any) and inter-species differences of the autofluorescent coelomocytes still remain elusive.
Author’s e-mail: plyt@zuk.iz.uj.edu.pl