Can permanent cell lines originate from low-grade astrocytomas: overgrowth of "glia-like" cells in pilocytic and fibrillary (diffuse) astrocytoma cultures.

OBJECTIVES: Due inter alia to wide-spread cell lines cross-contamination it is not clear, which kind of normal or tumoral tissue give rise to permanent cell lines. BACKROUND: Few permanent cell lines have been established from low-grade astrocytomas. However, recently some of these have been identified as being cross-contaminated with other cell lines. METHODS: Morphology, cell growth and GFAP immunophenotype of low-grade astrocytomas were examined on 9 pilocytic and 15 fibrillary (diffuse) tissue cultures. RESULTS: GFAP-positive process-bearing cells were present in all the cultures, mainly during the first days in vitro (DIV). In pilocytic cultures, cells with hairy (piloid) processes were present. GFAP-positive cells completely disappeared by passages 3 to 5 and all the cultures contained only GFAP-negative "glia-like" cells, which underwent cellular senescence within passages 8 to 15. CONCLUSION: Key differences in the morphology and GFAP expression between the neoplastic astrocytes and normal "glia-like" cells allow the observation of perceptibly more rapid growth of normal cells in astrocytoma cultures. We caution that cultures prepared from macroscopically tumoral brain tissue may contain rapidly proliferating normal cells. Based on this and our previous studies in relation to the high percentage of cross-contaminated cell lines, we conclude that cells in low-grade astrocytoma cultures lack the capacity for spontaneous immortalization (Fig. 14, Ref. 15). Text in PDF www.elis.sk Keywords: pilocytic astrocytoma, fibrillary astrocytoma, "glia-like" cells, glioma cell lines, GFAP.

High level EGFR amplification in a newly established glioblastoma cell line 170-MG-BA.

Glioblastoma multiforme is a highly invasive and incurable primary brain tumor. The most frequent genetic alteration therein is amplification of the epidermal growth factor receptor (EGFR) gene, the target of current clinical trials. However, EGFR amplification is poorly represented in glioblastoma cell lines. From the 30 cultures attempted herein, we were able to establish two glioblastoma permanent cell lines. The remaining cultures showed limited life span and underwent senescence between passage numbers (PN) 8 to 15. Our newly established glioblastoma cell lines, designated 170-MG-BA and 538-MG-BA, both originated between PN 3 and 5 when areas of smaller, more rapidly proliferating cells appeared. Both cell lines showed similar rates of growth, moderate morphological differences, cytoskeletal heterogeneity and multiple chromosome rearrangements. Analysis by molecular cytogenetics and comparative genomic hybridization (aCGH) revealed two copies of a stable marker chromosome in 170-MG-BA cells effecting focal amplification at 7q11 of the EGFR locus. Comparative RqPCR analysis confirmed that EGFR was uniquely highly expressed in 170-MG-BA cells. Combined targeted expression analysis and aCGH data excluded the recurrent EGFRvIII activating mutation. In contrast, EGFR expression in 538-MG-BA cells which lacked genomic EGFR amplification was not raised. Immunofluorescent staining showed high EGFR protein expression only in the 170-MG-BA cells. Cytogenetic, genomic and transcriptional analyses then confirmed high-level genomic amplification and transcriptional upregulation of wild type EGFR in 170-MG-BA; the first conventional cell line model for investigating the biology and targeted therapy of this key alteration in glioblastoma. Both cell lines are freely available from the DSMZ cell repository.

Short-term glioblastoma cultures may contain normal "glia-like" cells.

OBJECTIVES: Currently used glioblastoma cultures have many disadvantages and are being replaced by short-term cultures. However, these may include normal brain cells. BACKGROUND: A comparative model of normal and glioma cultures is lacking. A significant contributory factor is because cultures from adult human brain contain small amounts of cells with glial phenotypes. The predominant population of flat or spindle shaped cells does not express glial markers and are often termed as "glia-like". METHODS: Cryopreserved glioblastoma cultures from 28 bioptic samples were examined by immunofluorescence using antibodies to intermediate filaments (IF): glial fibrillary acidic protein (GFAP), cytokeratins (CK), nestin (Nes), vimentin (Vim) and neurofilaments (NF). RESULTS: In short-term glioblastoma cultures GFAP-positive cells occured at higher percentages in 3/28 cultures and in lower percentages in further 5 cultures. Subpopulation of nestin positive cells were observed in all cultures and CK-positive cells were found in 25/28 cultures. All cells in all cultures were positively stained only for vimentin and negatively for NF. Cells grew slowly in 5 cultures which showed early proliferation arrest between passages 7 to 8. A further 23 cultures showed growth arrest by passages 10 to 15. CONCLUSION: The presence of normal cells in short-term glioblastoma cultures may be caused by the infiltrative growth of these tumors. Our comparative analysis of morphological, growth and cytoskeletal properties revealed similarities between glioblastoma and normal brain cultures. In this study, the majority (28/30) of short-term glioblastoma showed limited life spans, similar to normal cells lacking spontaneous immortalization. The use of short-term glioblastoma cultures has two main problematic areas: cultures may contain a major subpopulation of normal "glia-like" cells; or they may contain the inital phases of spontaneously immortalized glioblastoma cells bearing properties of permanent cell lines (Tab. 1, Fig. 2, Ref. 19).

Unexpected presence of cytokeratins in human "glia-like" cells.

OBJECTIVES: To demonstrate the unexpected features of the predominant cell population in adult human brain tissue cultures usually termed "glia-like" cells. BACKGROUND: Cytokeratins (CK) are intermediate filaments (IF) specific for normal and neoplastic epithelial cell differentiation. METHODS: We examined adult human brain tissue cultures and cryosections prepared from ten biopsies. Immunofluorescence staining with monoclonal antibodies against IF proteins: anti-pan CK, glial fibrillary acidic protein (GFAP) and vimentin was performed on primary and secondary cultures up to passage 10. RESULTS: In primary cultures we detected only small numbers of immunocytochemically distinct astrocytes, oligodendrocytes and microglial cells. "Glia-like" cells were negatively stained with specific glial marker antibodies. They were positively stained with pan-CK antibodies in 8/10 cultures where 0.1 % to 70 % CK-positive cells were present in primary as well as in secondary cultures. Comparative immunofluorescence IF staining for CK, GFAP and vimentin showed differences in the cytoplasmatic distribution of IF fibres, numbers of positive cells and intensities of staining. Cryosections from brain biopsies stained negatively with pan-CK antibodies. CONCLUSION: These findings demonstrate the presence of CK in adult human brain cultures which is not caused by cross-reactivity of IF antibodies. Based on these results we propose that unexpected CK expression in human "glia-like" cells is due to cell dedifferentiation under culture conditions (Tab. 1, Fig. 2, Ref. 24).

Nitric oxide synthases in varicose vein wall.

INTRODUCTION: Venous wall weakness is supposed to be the most probable reason of primary varicosis. There are conflicting findings in literature about its structural changes. NO is potent vasodilatator due to the smooth muscle relaxation. It is synthesized by nitric oxide synthase (NOS). There are 3 known isoforms of NOS: nNOS (neuronal NOS), iNOS (inducible NOS), eNOS (endothelial NOS). MATERIAL AND METHODS: 10 varicose vein and 10 control vein samples were processed by standard light microscopy method. Sections were then processed by standard immuno-histochemic technique using rabbit polyclonal antibodies against all 3 NOS isoforms: iNOS, eNOS (SantaCruz, USA), nNOS (BioScience, USA). Antibodies expression was evaluated semiquantitatively and then proved morphometricaly by 2D image analysis (ImageJ 1.34n, National Institute of Health, USA). Total area of NOS isoforms expressions was determined by color analysis and color digital substraction. RESULTS: Histomorphological and semi quantitative evaluation of NOS isoforms showed discontinuous and significantly lower expression of all 3 NOS isoforms in tunica media of varicose veins compared with control group, where the expression of all 3 NOS isoforms was continuous. For the statistical analysis unpaired t-test was used. DISCUSSION AND CONCLUSION: Our results suppose lower NO levels in varicose vein wall, deducing that varicose vasodilatation is due to other mechanism, although the stage of chronic venous disease of varicose vein samples was undetermined. Our results are in contradiction with previously published results of Howlader et al., who observed raised total NO levels in patients with severe stages of chronic venous disease(Tab. 2, Fig. 13, Ref. 18).

Can obligate apomixis and more stable reproductive assurance explain the distributional successes of asexual triploids in Hieracium alpinum (Asteraceae)?

Although reproductive assurance has been suggested to be one of the most important factors shaping the differential distributional patterns between sexuals and asexuals (geographic parthenogenesis), it has only rarely been studied in natural populations of vascular plants with autonomous apomixis. Moreover, there are almost no data concerning the putative relationship between the level of apomictic versus sexual plant reproduction on one hand, and reproductive assurance on the other. We assessed the level of sexual versus apomictic reproduction in diploid and triploid plants of Hieracium alpinum across its distributional range using flow cytometric analyses of seeds, and compared the level of potential and realized seed set, i.e. reproductive assurance, between the two cytotypes under field and greenhouse conditions. Flow cytometric screening of embryos and endosperms of more than 4,100 seeds showed that diploids produced solely diploid progeny sexually, while triploids produced triploid progeny by obligate apomixis. Potential fruit set was much the same in diploids and triploids from the field and the greenhouse experiment. While in the pollination-limited environment in the greenhouse apomictic triploids had considerably higher realized fruit set than sexual diploids, there was no significant difference between cytotypes under natural conditions. In addition, sexuals varied to a significantly larger extent in realized fruit set than asexuals under both natural and greenhouse conditions. Our results indicate that triploid plants reproduce by obligate apomixis, assuring more stable and predictable fruit reproduction when compared to sexual diploids. This advantage could provide apomictic triploids with a superior colonisation ability, mirrored in a strong geographic parthenogenesis pattern observed in this species.

Comparison of collagen subtype I and III presence in varicose and non-varicose vein walls.

STUDY OBJECTIVES: The connective tissue alterations in varicose vein wall are supposed to be one of the main causes of primary varicose vein (main sign of human lower limbs chronic venous insufficency). METHODS: 5 varicose vein samples from 5 patients undergoing stripping surgery of long saphenous vein were compared with 5 control samples of healthy (non-dilated) long saphenous veins from necroptic material (with no history of varicosis). They were fixed in a Baker solution, processed by use of light microscopic method, cut to ultra-thin sections (4-5 microm) and stained with PicroSirius Red for collagen. Sections were scanned with light microscope (Leica, Germany) and camera Canon S50 (Germany) and analysed by morphometric programme Image J v.1.38g (National Institute of Health, USA). RESULTS: In the group of healthy (non-dilated) veins the mean collagen I/III ratio value was 31.40 and in the group of varicose veins the mean collagen I/III ratio was 12.35; the difference is statistically significant: healthy veins contain significantly more of collagen subtype I and varicose veins contain significantly more of collagen subtype III in their walls. CONCLUSION: The statistically significant difference in the collagen I/III ratio between the groups of healthy (non-dilated) and varicose (dilated) vein walls is worthy of further following (Tab. 2, Fig. 7, Ref. 12). Full Text (Free, PDF) www.bmj.sk.

Against abuse of digital photography techniques in morphology--Ethical Code of Slovak Anatomical Society.

Digital graphical methods allow extensive manipulation of pictures literally beyond any limits. By such methods, it is possible to change original information obtained by morphological observation, even to fraudulently produce distorted results or forgeries. For this reason, basic rules were proposed defining what is allowed and what is not accepted during picture processing. These rules were discussed and approved by a plenary meeting of Slovak Anatomical Society on September 9th, 2007 under the name Ethical Code of Slovak Anatomical Society. We call on all potential authors of publications and dissertation works to obey the rules of Ethical Code of Slovak Anatomical Society and thus to prevent any doubts which may arise about the faithfulness of published materials in morphological or other disciplines, which use a picture as an evidence or illustration means (Fig. 3). Full Text (Free, PDF) www.bmj.sk.

Both iso- and hyperosmotic ethanol stimulate release of hypothalamic thyrotropin-releasing hormone despite opposite effect on neuron volume.

Previous studies have indicated that isosmolar, but not hyperosmolar, ethanol induces in vitro gonadotropin-releasing hormone secretion from the basal hypothalamus, presumably by causing cell swelling. Moreover, ethanol reduces secretion of another hypothalamic neuropeptide vasopressin. We have studied the acute effect of ethanol on specific hypophysiotropic basal and K+-stimulated thyrotropin-releasing hormone secretion in vitro especially in relation to cell swelling. Isosmotic 40-160 mM ethanol increased thyrotropin-releasing hormone release from the hypothalamic paraventricular nucleus and median eminence in a dose-dependent manner. Both a 30% decrease of osmolarity and isosmotic 80 mM ethanol induced 12% swelling of hypothalamic neurons. Hyperosmotic 80 mM or 160 mM ethanol induced release of thyrotropin-releasing hormone from both hypothalamic structures but did not cause cell swelling (80 mM) or even induced cell shrinkage (160 mM). Depletion of medium Ca2+ did not affect thyrotropin-releasing hormone secretion caused by either isosmotic or hyperosmotic ethanol. Our data indicate that both iso- and hyperosmotic ethanol stimulated release of hypophysiotropic thyrotropin-releasing hormone despite opposite effects on neuron volume. The mechanism of ethanol action appears complex and variable depending on the type of cell and neuropeptide affected.

Vaginal leukocyte characteristics in urogenital trichomoniasis.

Morphological and functional characteristics of vaginal exudate leukocytes were examined in 47 patients with urogenital trichomoniasis. Electron microscopic morphology, viability, phagocytosis of Candida albicans blastospores and ability to undergo respiratory burst in the iodonitrotetrazolium reductase test were evaluated in these cells. Vaginal inflammatory leukocytes were almost exclusively polymorphonuclear neutrophils, and their concentration was positively correlated (r = 0.58; p less than 0.001) with the number of trichomonads in the exudate. Median leukocyte viability reached 39% and both phagocytic and tetrazolium reductase activities of these cells were significantly reduced in comparison with those of circulating polymorphonuclear leukocytes. Patients with a clinical picture of severe mucosal inflammation had significantly higher vaginal exudate leukocyte concentrations and viability than those without inflammatory signs. The possible role of vaginal leukocytes in the pathogenesis of urogenital trichomoniasis is discussed.

Co-expression of GFAP, vimentin and cytokeratins in GL-15 glioblastoma cell line.

Glial fibrillary acidic protein (GFAP), vimentin (Vi) and cytokeratin (CK) intermediate filament (IF) proteins were studied in glioblastoma cell line GL-15. The immunofluorescence staining revealed strong positive staining for vimentin in all cultured cells. Approximately 20% of analyzed cells showed strong and 50% moderate intensity of staining for GFAP. About 3% of all cells were positively stained with a mixture of anti-CK monoclonal antibodies. The expression of all IF was not in relation to the cell density or days in vitro after passage. The double immunofluoresce revealed that all CK-positive cells express GFAP and vimentin. This study demonstrates the heterogeneity of the clonal GL-15 glioma cell line which consists in three immunocytochemically distinct cell types: Vi+/GFAP-/CK-, Vi+/GFAP+/CK-, and Vi+/GFAP+/CK+. These findings give further evidence about the expression of non-glial IF in cultured glioma cells.

Characterization of two new permanent glioma cell lines 8-MG-BA and 42-MG-BA.

The establishment and characterization of two permanent glioma cell lines (8-MG-BA and 42-MG-BA) are described. Both cell lines were derived from the human glioblastoma multiforme. Analyzed cells were within the passage 200 to 220. The cells in both cultures showed similar morphology. In majority they consisted from flat polygonal cells. Growth kinetic studies demonstrated a population doubling time of 20 to 24 h in cell line 8-MG-BA and 48 to 54 h in cell line 42-MG-BA. The cell lines showed different hyperdiploid karyotypes. The immunofluorescence staining was performed for glial fibrillary acidic protein (GFAP) and vimentin. In the culture 8-MG-BA only a small amount of cells showed the GFAP-positive staining. At confluent 42-MG-BA culture the GFAP-positive cells reached 50 to 70% of all cells. Vimentin was found in all glioma cells in both cultures.

Heterogeneity of keratin intermediate filaments expression in human glioma cell lines.

Keratin intermediate filaments (Ifs) are specific for epithelial cell differentiation. This study demonstrates the presence of keratin in two recently established human glioblastoma cell lines 8-MG-BA and 42-MG-BA. Immunofluorescence staining was performed on cells within passage 230 to 235 using monoclonal pan-cytokeratin antibodies. The cells were analyzed during several DIV at different cell density. Keratin-positive stained cells reached 5 to 7% in 8-MG-BA and less than 0.1% in 42-MG-BA cell line. The presence of keratin-positive cells was independent on cell density and days in vitro. Keratin-positive cells appeared unevenly distributed in both cell lines. They were observed as single or areas of keratin-positive cells. The morphological features of keratin-positive and keratin-negative cells were similar. The results are discussed with respect to previous studies on glial fibrillary acidic protein (GFAP) and vimentin to show the heterogeneity of IFs expression in glioma cell lines.

Establishment, morphological, growth and cytoskeletal properties of 135-BCA carcinoma cell line derived from lung brain metastasis.

Many cell lines have been established from lung cancer but carcinoma cell lines derived from brain metastases occur rarely. The carcinoma cells growth relatively slowly in comparison with brain cells which often overgrow the tumor cells in early passages. The origin of these rapidly dividing brain cells in carcinoma cultures is discussed with respect to the previous studies on adult human brain tissue cultures. It was found that the majority of cells in adult human brain cultures derived from brain biopsies of patients with non-cancer diseases do not express glial markers. Based on the previous studies we suggest that they are glial precursor cells. The high proliferative capacity and non-glial phenotype of these brain cells may lead to the suggestion that they are of cancer origin. In this study the establishment and characterization of a new carcinoma cell line 135-BCA is described. The tissue cultures were derived from brain metastasis of lung large cell carcinoma. The cell line is specific by the epithelial cell morphology and evident cytokeratins expression during the whole subcultivation. All tumor cells were strongly immunoreactive for vimentin and negative stained for glial fibrillary acidic protein (GFAP). The new cell line may prove of value in biological and therapeutic studies of lung cancer. In addition, the further comparative analysis may reveal the environmental influence of brain tissue on carcinoma cells.

Detection of ischemic changes in the cytoplasm of neurocytes from rat brain and spinal cord by densitometric measurement of methylene blue binding.

Ischemic changes in neurocytes from brain and spinal cord of rats were studied by densitometric measurement of bound basic stain--methylene blue. Statistically significant differences in integrated optical density (I.O.D.) of cytoplasm near to cell nucleus in brain and spinal cord neurocytes were detected after ischemia. After 10 minutes of ischemia, the average values of I.O.D. decreased to 65% and to 69.9% of I.O.D. values of controls. After 2 hours of ischemia, the average values of I.O.D. in brain cell cytoplasm reached only 43.6% and in the spinal cord cells they fell to 54.5% of control values.

[Procedure for multiorgan procurement (heart, liver, kidneys)]. / Postup pri multiorgánových odberoch (srdce, pecen, oblicky).

A significant growth in transplantation activity has been recorded in the past decade, namely in transplantations of the heart and liver. The numbers of transplantations of the lungs, and of the combination of pancreas with kidneys have increased to a smaller extent. The number of transplanted kidneys has become stabilized during the past several years. The development of organ transplantations led to a change of conception in the procurement of cadaveric organs from the until now performed procurement of a single organ to multiorgan procurements where in addition to the kidneys at least one other organ is procured. In countries with the highest number of multiorgan procurements the latter form as many as 80% of the total number of cadaveric procurements. Since January 1, 1995 a principle has been officially recognized in the Slovak Republic where an agreement with organ donation is presumed. Procurements of organs do not take place in cases when the donors have made during their lives a written or any other kind of declaration disapproving any intervention into their corporal integrity. Organs are also not procured in cases of foreigners, persons under legal sentence and those whose identities cannot be proved. The authors describe the technique of multiorgan procurement (heart, liver, kidneys) as well as contraindications to procurements of individual organs. (Fig. 2, Tab. 1, Ref. 12.).

Professor Julius Alexander Ledenyi-Ladziansky--centenary of a personality.

Professor J. A. Ledenyi-Ladziansky, academician, anatomist, researcher, scientist, author, beloved and disliked by others at the same time, a personality in a true sense, was born 100-years ago. (Fig. 3, Ref. 8.)

[Primary ciliary dyskinesia--importance of early diagnosis]. / Primárna dyskinéza riasiniek--význam vcasnej diagnózy.

BACKGROUND: Primary ciliary dyskinesia (PCD) is an inherited disease characterized by specific ultrastructural defects of cilia and sperms. The impairment of mucociliary clearance (MCC) results in chronic respiratory infections and subsequently in bronchiectasis. MAIN PURPOSE: The evaluate rational decisions in early diagnosis of PCD. METHODS: Samples of nasal mucosa or tissue of tonsilla pharyngea after adenotomy were studied by transmission electron microscopy (TEM) in 47 patients aged 1-15 years, suffering from recurrent or chronic respiratory infections. RESULTS: Congenital ultrastructural ciliary defects specific for PCD--the lack of dynein arms, radial spokes defects and microtubular transposition--were observed in 13 patients. TEM investigation is an expansive, time consuming method not available in routine practice. Therefore we have evaluated a diagnostic procedure which uses available examination methods focused on the diagnoses of PCD. TEM of respiratory cilia is indicated in patients with situs viscerum solitus if chronic respiratory disease develops and after more frequent causes--asthma, cystic fibrosis, congenital anomalies of respiratory system and immunodeficiency had been excluded. CONCLUSIONS: The correct and early diagnosis is important for effective therapy in order to improve MCC. This approach can prevent the development of bronchiectasis during childhood.

[Morphometric analysis of the spleen]. / Skúsenosti s morfometrickou analýzou sleziny.

Present possibilities of morphometric analysis of the spleen have been studied. We have compared possibilities provided by stereological calculation and by computer image analysators a) Telemet II (Tesla, Piestany) and b) CUE-2 GALAI (Israel). In both latter cases equidensitometric evaluation and image processing methods were used. Stereological calculations are not technically demanding but time consuming. Computer image processing on the base of equidensitometric measurements is more effective that stereological calculations. The difference between used equipments is based on access to appropriate microscopic and computer technique and software equipment.

[Quo vadis the azygo-portal disconnection]. / Quo vadis azygoportálna dekonexia.

Based on experience at their department since 1983 the authors analyze the position of the azygoportal disconnection in the treatment of complications of portal hypertension. During the observation period the number of operations declined due to extensive use of endoscopic diagnosis and treatment. However the azygoportal disconnection still remains part of surgical intervention in indicated cases, in particular in secondary hypersplenism and profuse haemorrhage.