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.

Isolated "Tillaux" fracture in adulthood: rarity where the key of success is not to miss it.

BACKGROUND: Fracture of the lateral border of the distal tibia is often referred as Tillaux fracture. It is an avulsion fracture due to the tension of the anteroinferior portion of the anterior tibiofibular ligament (1). This type of fracture is scarce in adulthood and can be easily overlooked. METHODS: From 2006 to the present day, 7 case reports describing the Tillaux fracture were found in the PubMed and Web of Science database, to which one case from our set of patients was added. Our goal was to focus on the diagnostic and a selected treatment described in each published case. RESULTS: We found no gender difference. The injury mechanism was mostly an external rotation. Treatment and diagnosis were, in all cases differentiated at specific points. Fixation and load reduction were indicated at least for six weeks in all of the patients. After three months, in almost all cases, a return to full function was achieved. CONCLUSION: Our assessments are not statistically significant, but our goal was to point out the existence of such a rare type of fracture. At the same time, based on previous publications, we developed an algorithm of diagnosis and treatment to facilitate the management of this type of fracture (Tab. 1, Fig. 5, Scheme 1, Ref. 21).

Short-term effect of the demonstration of human dissection and excursion on students´ ideas about the human organ systems: a Slovak experience.

BACKGROUND: Excursions in natural sciences education are justified in the current school system and within the scope of subject focus. The research aimed to observe the immediate impact of demonstration of the human dissection and excursion on the formation of ideas of students about the human body systems. METHODS: The experiment involved the first Slovak demonstration of a real human autopsy for students of the Biology Teaching Department (PdF TU in Trnava). Concepts of the structure of the human body were analyzed before and after experimental impact by a projective technique. The evaluation included the representation, description, and abundance of organs in the view of various systems. The subject of the research were also the questions focused on the use of illustrative procedures and tools in the educational process. RESULTS: Significant statistical differences were found in the group affected by human autopsy in understanding of the digestive, urinary, and locomotor systems. Conversely, the skin, endocrine glands, and lymphatic system were the least represented by the experimental groups. CONCLUSION: Considering the presented results, using different organizational forms and aids (real and virtual) is essential for bringing a positive educational effect on the studied problem (Tab. 1, Fig. 5, Ref. 20).

Fertility maintenance in male oncological patients: current state and future perspectives.

Growing cancer incidence in reproductive age goes hand in hand with a rising survival rate of patients who underwent anticancer therapy. This trend points to the necessity of discussion regarding the fertility maintenance. The patient´s future with respect to his reproductive ability has to be addressed properly to achieve a complex approach to cancer management. The germinal epithelium of the testes is highly susceptible to deleterious effects of chemotherapy. After the administration of gonadotoxic chemotherapeutic agents, a patient can develop oligospermia, or even azoospermia. Similarly, radiation exposure can damage spermatogenesis, while higher doses lead to azoospermia. This review brings an overview of the methods of assisted reproduction, which are currently in use for fertility maintenance in oncological patients, but also in those with non-malignant indications. Also, novel, yet still experimental, methods are discussed, which represent promising technologies applicable to prepubertal oncological patients. We also discuss historical milestones in the development of assisted reproduction, summarize the options of semen analysis, and we present a practical guide through the process of sperm cryopreservation and subsequent in vivo or in vitro fertilisation. We deem that fertility maintenance should be an integral part of the health care in oncological patients in reproductive age (Tab. 1, Ref. 85). Keywords: assisted reproduction technique, sperm cryopreservation, testicular tissue cryopreservation, spermatogenesis, sperm quality in oncological patients.

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).

Sentinel lymph node - historical background and current views on its significance in complex management of breast cancer patients.

Nowadays, breast cancer is the leading oncological diagnosis in women worldwide. On the other hand, breast cancer treatment can be considered one of the most progressive therapeutic approach in the medical field of oncology. The invasive types of breast cancer have a tendency to spread via lymphatic route, what brings in the issue of sentinel lymph node - the first node into which the lymph drains from a given anatomical location. This review paper discusses the historical background of the concept of sentinel lymph node and focuses on clinical significance of the positivity of sentinel lymph node(s) as well. Modern-day conservative therapeutic surgery of breast cancer should be in accordance with diagnostic and preventive interventions in the axilla, whose rate of invasiveness and morbidity must be also attenuated without worsening the patient´s prognosis and survival rate. Formerly, a complete axillary lymph node dissection was routinely performed for prophylactic and cancer staging purposes. The indiscriminate application of this approach was replaced by sentinel lymph node biopsy. Along with common histopathological examination, immunohistochemistry, as well as modern techniques of molecular biology are often employed. These state-of-the-art methods enabled the identification of micrometastases, or even nanometastases, though their real prognostic value is yet to be concluded (Ref. 52). Keywords: sentinel node, breast cancer, biopsy, historical background.

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.

The vascular patterns of the posterior part of the circulus arteriosus cerebri (Willisi).

BACKGROUND: Various authors defined three patterns of the posterior part of the circulus arteriosus cerebri Willisi (CW) according to the diameter of the posterior communicating artery (PCoA) and the precommunicating segment of the posterior cerebral artery (P1). In the adult pattern, the P1 has a diameter larger than the non-hypoplastic PCoA. In the transitional pattern, the diameter of the PCoA is equal to that of the P1. In the fetal pattern, the diameter of the P1 is smaller than the diameter of the PCoA. The study was aimed to evaluate the configurations and calibers of the posterior part of the CW. METHODS: The work was conducted on 185 adult post-mortem brains. The CW and its branches were photographed by a digital camera. We used the software Image J to evaluate and process the gained images. RESULTS: The fetal pattern was found unilaterally in 8.37 %, and bilaterally in 4.86 %. The transitional pattern was observed unilaterally in 6.47 %, and bilaterally in 1 %. The prevalence of the unilateral and bilateral adult patterns was equal (21.62 % for each configuration). The hypoplastic PCoA was found unilaterally in 17.57 %, and bilaterally in 16.76 %. CONCLUSION: Various factors including genetic and environmental may affect the development of the cerebral vessels and their dimensions. The distinguishing of the vascular dimensions in vivo can help in the expectation and may be the avoidance of possible cerebrovascular disturbances in the future. Correlation and interdisciplinary cooperation of the studies dealing with morphology, radiology, and hemodynamics of the cerebral vessels are becoming an urgent need. The assumed results of this cooperation can be used in tabulating the calibers of the cerebral vessels and determining the threshold dimensions under which failure of hemodynamics and collateral function may appear (Tab. 2, Fig. 5, Ref. 28).

Subserosal localization of myenteric ganglia in normal human appendix: immunostaining with neuronal and glial markers.

OBJECTIVES: Visualization of unexpected distribution of myenteric ganglia in normal human appendiceal wall by immunofluorescence. BACKGROUND: The myenteric plexus is located between the longitudinal and circular muscle layers of the GIT. However, recently the irregular distribution of myenteric ganglia was revealed in human appendix. METHODS: The cryosections prepared from normal human appendices were examined by immunofluorescence methods using antibodies to neurofilaments (NF) and glial fibrillary acidic protein (GFAP). RESULTS: Indirect immunofluorescence revealed the positive staining of myenteric ganglia with both neuronal and glial marker antibodies. Double labeling for NF/GFAP staining showed close assotiation between glia and neurons inside ganglia. GFAP-positive cells were often observed as the cells surrounding myenteric ganglia. The staining confirmed the irregular distribution of myenteric ganglia in human appendiceal wall and revealed the small ganglia in the subserosal area. CONCLUSION: Our results showed that localization of myenteric ganglia in human appendix differs from other parts of GIT. GFAP immunostaining is available for visualization of smaller myenteric ganglia located mainly in the subserosal area. Our studies may find application in current HIV research focused on enteric neuropathogenesis and in diagnostic laparoscopy for chronic abdominal pain: to detect the irritation of subserosal ganglia (Fig. 2, Ref. 26).

Use of graph algorithms in the processing and analysis of images with focus on the biomedical data.

INTRODUCTION: Image segmentation is a known problem in the field of image processing. A great number of methods based on different approaches to this issue was created. One of these approaches utilizes the findings of the graph theory. METHODS: Our work focuses on segmentation using shortest paths in a graph. Specifically, we deal with methods of "Intelligent Scissors," which use Dijkstra's algorithm to find the shortest paths. RESULTS: We created a new software in Microsoft Visual Studio 2013 integrated development environment Visual C++ in the language C++/CLI. We created a format application with a graphical users development environment for system Windows, with using the platform .Net (version 4.5). The program was used for handling and processing the original medical data. CONCLUSION: The major disadvantage of the method of "Intelligent Scissors" is the computational time length of Dijkstra's algorithm. However, after the implementation of a more efficient priority queue, this problem could be alleviated. The main advantage of this method we see in training that enables to adapt to a particular kind of edge, which we need to segment. The user involvement has a significant influence on the process of segmentation, which enormously aids to achieve high-quality results (Fig. 7, Ref. 13).

Biological aspects of appendix pathology: irregular distribution of myenteric ganglia in human appendiceal wall.

OBJECTIVES: Although appendicitis is a common disease, basic questions about risk factors and its etiology remain unexplained. BACKGROUND: An obstruction of the appendix lumen is usually considered to be the main cause of acute appendicitis. However, more studies are currently dealing with neuroimmune appendicitis. METHODS: We studied samples of human appendices with the histological diagnosis of chronic appendicitis. Fixed cryosections of appendiceal walls were examined by immunofluorescence methods using neuronal anti-neurofilament antibody markers and beta III tubulin. RESULTS: The immunostaining revealed an irregular distribution of myenteric ganglia in inflamed appendiceal walls and unexpected groups of large ganglia unequally distributed in the subserosal area. The comparative analysis of normal and inflamed appendix samples showed differences in the occurrence of myenteric ganglia in the subserosal area. They appeared more frequently on cryosections prepared from the inflamed appendiceal wall. CONCLUSION: We propose that the high variability and irregular location of myenteric ganglia in the appendiceal wall are due to an alteration in the motility which results in flaccid appendix emptying. In addition, superficially located myenteric ganglia are exposed to abdominal irritation and may explain the chronic abdominal pain which is often considered to be a sign of chronic appendicitis (Fig. 2, Ref. 23).

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).

The variable communicating branches between musculocutaneous and median nerves: a morphological study with clinical implications.

BACKGROUND: The course of the brachial plexus, its relations with surrounding structures and unique primary and secondary divisions result in its wide range of anatomical variations. Most of these variations were detected during anatomical dissections and studies. It has been found that 53% of studied brachial plexuses contained variations. The communication between musculocutaneous and median nerves is the most common variation of infraclavicular part of brachial plexus. METHODS: During gross anatomical dissections of peripheral nerves, we observed neuronatomical variations in upper limbs of four formalin embalmed adult cadavers. Musculocutaneous and median nerves were connected by a communicating branch at distinct level in each cadaver. The formation and relations of both nerves were noted in each case to exclude the existence of other anatomical variations. The connections were measured and documented by digital camera. RESULTS: The communicating fibers of variations 1 and 2 were located in the upper third of arm and proximally to musculocutaneous nerve penetration through coracobrachialis muscle. In variations 3 and 4, the communicating branch was situated in the lower third of arm and distal to the nerve penetration point. CONCLUSION: Variable interconnections between musculocutaneous and median nerve have to be considered in diagnosis of nerve lesions in axillary and arm regions. Compound musculocutaneous and median nerve neuropathy would occur in lesions of the interconnecting branches. Injuries of musculocutaneous nerve proximal to these branches can cause particular and unexpected symptoms, such as weakness of forearm flexors and thenar muscles (Fig. 6, Ref. 28).

Omphalocele with ectopic liver forming a mesodermal cyst.

BACKGROUND: Congenital anomalies of the abdominal wall are classified as anomalies with the abdominal wall defect (omphalocele, gastroschisis) and without the defect (umbilical hernia, persistent ductus omphaloentericus or urachus). Clinical presentations of these conditions are different, and so is the timing of surgical intervention and approach with or without the exploration of the peritoneal cavity. CASE: The presented case report refers to a rare finding of ectopic liver forming mesodermal cyst within the umbilical region. Full term neonate girl with 5 cm spheroid tumor in umbilicus was otherwise without problems. Ultrasonography (USG) of the lesion detected a tissue with good vascularization and a cystic cavity. There was no flow in the umbilical vessels and no evidence of intestinal loop in the sac. USG of the liver was normal. During surgery an additional narrow canaliculus was identified connecting the spheroid along with umbilical vessels to the liver. The whole spheroid together with the canaliculus was surgically removed without exploration of the abdominal cavity. Histological evaluation of the surgical specimen discovered liver tissue with a mesodermal cyst in the center. The sphere was connected to the liver by a bile duct. Six months after the operation the child is in a good clinical condition. CONCLUSION: In conclusion omphalocele may contain liver. Ectopic liver is an extremely rare condition. Surgical treatment in the presented case focused only on umbilicus without exploration of the abdominal cavity and appeared to be sufficient. Long-term postoperative follow up typical in pediatrics will be applied also in this patient (Fig. 3, Ref. 25).

The bases of the inflammatory acute abdomen.

Vermiform appendix is an anatomical structure, which due to its topographical relations usually causes many complications, especially in inflammation such as appendicitis. One of the manifestations of the inflammatory processes is pain, which may have different location. It could be probably a result of the neuron stimulation. The nerve formation and distribution of the vermiform appendix is still unknown in fine details and is a subject of further studies (Fig. 8, Ref.12).

[Position variability of the vermiform appendix and effect on diagnosis of appendicitis in children]. / Variabilita polôh apendix vermiformis a vplyv na diagnostiku apendicitídy u detí.

Vermiform appendix, seemingly inconspicious anatomical structure, may cause many complications. These result from its topographico-anatomical relations to surrounding structures in abdominal cavity, but also from potential position variability of the vermiform appendix. Variable position of the vermiform appendix can have effect on appendicitis diagnostic, as one of the most often cause of the acute abdomen in children.

The microscopical structure of the hippocampus in the rat.

The aim of this work was to study the rat's hippocampal formation by applying the light microscopic methods. The histological methods used to explore this region of the rat's brain were the Nissl technique, the Bielschowsky block impregnation method and the rapid Golgi technique. In the Nissl preparations, we identified only three fields of the hippocampus proprius (CA1, CA3 and CA4). CA2 was distinguished in the Bielschowsky impregnated blocks. The rapid Golgi technique, according the available literature, gives the best results by using the fresh samples. In this study, we reached good results by using formalin fixed sections. The layers of the hippocampal formation were differentiated. The pyramidal and granular cells were identified together with their axons and dendrites (Fig. 9, Ref. 22). Full Text (Free, PDF) www.bmj.sk.

A variable origin of the vertebral artery.

This study presents and describes the variation of the vertebral artery arising from the aortic arch as a case report. This variation was found in one of the cadavers at the Institute of Anatomy of Medical Faculty of Comenius University in Bratislava. Anomalous arising was discovered during the student's dissection of thorax and abdomen. Non standard arising of the vertebral artery is compared to the standard arising in the anatomical literature and atlases. The variable arising was confronted to the possible variations of the vertebral artery reported in literature (Fig. 4, Ref. 9). Full Text (Free, PDF) www.bmj.sk.

[Clinical and histopathologic picture of acute appendicitis in children]. / Klinický a histopatologický obraz detskej akútnej apendicitídy.

A retrospective study of the clinical and histopathological picture of acute appendicitis in children confirms agreement of the evaluation of the finding on the appendix made by the surgeon and pathologist in 72.2% and disagreement in 27.8%. The finding is overrated in 11.3% and underrated in 16.5%. Agreement is closest in phlegmonous appendicitis (88.1%) and lowest in gangrenous appendicitis (54.9%). The most frequently overrated finding is in catarrhal appendicitis (20.7%). The number of missed appendectomies is from the pathologist's point of view significantly lower (21.8%) than from the surgeon's point of view (34.7%). This indicates obviously that it is not possible to make an accurate macroscopic assessment of the progress of appendiceal inflammation. The number of "negative" appendectomies is according to the surgeon 15.8% and according to the pathologist 16.8%, i.e. almost equal.

[The clinical picture of acute appendicitis in children]. / Akútna apendicitída v klinickom obraze u detí.

The authors describe 3860 child patients operated on account of acute appendicitis and analyse the data with regard to age, sex, year of incidence and surgical finding. The highest incidence of acute appendicitis is between 8 and 11 years with a peak at the age of 10 years. It is more frequent in boys (58.3%) than in girls (43.7%), the ratio being 1.3:1. It occurs more often during the cold months (46.5%), in autumn (27.3%) and in winter (25.7%). The number of gangrenous appendicitis is 34.7%-56.7% in boys and 43.5% in girls. The highest rate of missed appendicitis is at the age of the highest incidence (15.2%) with a peak at the age of 10 years (17.8%) and in January (13.3%). The highest incidence of perforated appendicitis is also at the age of the highest incidence (14.9%) with the peak at the age of 8 years (15.6%) and in June (13.5%). Perforation is more frequent in boys (58.1%) than in girls (49.9%). The number of "negative" appendicitis is 15.8%. Prevention of acute appendicitis still remains open due to lack of knowledge of its etiopathogenesis.