Perzelová A

Effects of a novel synthetic retinoid on malignant glioma in vitro: inhibition of cell proliferation, induction of apoptosis and differentiation.

Among six synthetic retinoids tested, the retinoid 6-[3-(1-adamantyl)-4-hydroxyphenyl]-2-naphthalene carboxylic acid (CD437) was highly efficient in inducing growth inhibition of 8MG-BA and GL-15 human glioblastoma cell lines, with growth arrest at the S phase of the cell cycle. CD 437 also induced apoptosis in these cells, with 8MG-BA being the most sensitive. In these cells, induction of apoptosis by CD437 has been related to the downregulation of Bcl-2 expression and to CPP32 activation, but not to p53 expression. The remaining non-apoptotic cells presented a morphological pattern of astroglial differentiation with overexpression of glial fibrillary acidic protein (GFAP) and glutamine synthetase (GS). The mechanism of action of CD437, originally developed as a RARgamma agonist, is not yet elucidated. However, our results suggest that it acts through an increase of the expression of retinoid-inducible genes, such as RARbeta2 and/or RARalpha2.

Differential expression of laminin and fibronectin and of their related metalloproteinases in human glioma cell lines: relation to invasion.

In the present work, we analyzed the expression of two major components of the extracellular matrix (ECM), laminin and fibronectin and of two related matrix-metalloproteinases, MMP-2 and MMP-9, in three human glioma cell lines (8 MG, 42 Mg and GL-15) in relation with their differential invasive properties. Immunocytochemistry and Western-blots assays indicated the presence of a 200 kDa laminin, similarly expressed in the three cell lines but undetectable in their ECM. In the opposite, a 230 kDa fibronectin, detected in the three cell lines was differently expressed and only observed in the ECM of the less invasive 8 and 42 MG cells. MMP-2 mRNA analyzed by Northern blots and proMMP-2, evaluated by zymography, were found in the three cell lines but were both ten times higher in the most invasive GL-15 cells. In addition, the active form of MMP-2 was only found in the GL-15 cells. In the opposite, the expression of specific tissular inhibitor (TIMP)-2, an endogenous MMP-2 inhibitor, was restricted to the less invasive cells. MMP-9 activity was detected only in the 8 and 42 MG cells and may not be directly involved in invasion. Taken together, these results indicate that a high MMP-2/TIMP-2 ratio may be responsible for the absence of extracellular fibronectin, underlining the participation of tumour cells in the proteolytic degradation of the ECM. An unbalanced MMP-2/TIMP-2 ratio in the micro-environment of malignant cells may contribute to their invasive properties.

GFAP-positive astrocytes are rare or absent in primary adult human brain tissue cultures

Traditionally, astrocytes are divided into fibrous and protoplasmic types based on their morphologic appearance. Here the cultures were prepared separately from the adult human cortical gray and white matter of brain biopsies. Both cultures differed only in the number of glial fibrillary acidic protein (GFAP)-positive cells. In the gray matter these were absent or rare, whereas in confluent cultures from the white matter they reached 0.1% of all cells. Three main morphologic types of GFAP-positive cells were found in this study: stellate, bipolar and large flat cells. GFAP-positive cells with two or three long processes mimic a neuron-like morphology. We did not find process-bearing cells expressing neuronal markers (MAP-2, NF, and N-CAM). The conflicting reports concerning GFAP immunostaining and the study dealing with the presence of putative neurons in adult human brain cultures are discussed with respect to these findings. The latter classification of astrocytes into type 1 and type 2 is based on immunostaining to A2B5 antigen: type 1 (GFAP+/A2B5−) and type 2 (GFAP+/A2B5+) astrocytes are proposed to be analogous to protoplasmic and fibrous astrocytes, respectively. In adult human brain cultures we found only small amount of A2B5-positive cells. Double immunofluorescence revealed that astroglial cells of similar fibrous or bipolar shape grown on one coverslip were either GFAP+/A2B5+ or GFAP+/A2B5−. On the other hand, the A2B5+/GFAP− immunophenotype was not observed. These results indicate that in general the cell phenotype from adult human brain tissue is not well established when they are in culture.

Subpopulation of nestin positive glial precursor cells occur in primary adult human brain cultures

Glial fibrillary acidic protein (GFAP) is an intermediate filament protein considered to be the best astroglial marker. However, the predominant cell population in adult human brain tissue cultures does not express GFAP; these cells have been termed “glia-like” cells. The basic question about histological origin of adult human brain cultures remains unanswered. Some authors showed that “glia-like” cells in adult human brain cultures might be of non-glial origin. We examined primary explant tissue cultures derived from 70 adult human brain biopsies. Within first 5–10 days approximately 5–10% of the small explants became attached. Outgrowing cells were mostly flat cells. These cells formed confluent layer over 3–6 weeks in culture. At confluence the cultures contained 2–5% of microglial cells, 0.1% GFAP-positive astrocytes, less than 0.01% oligodendrocytes and 95–98% GFAP-negative “glia-like” cells. This population of flat “glia-like” cells was positively stained for vimentin, fibronectin, and 20–30% of these cells stained for nestin. Our findings revealed that 1 mM dibutyryl-cAMP addition, in serum free conditions, induced a reversible stellation in 5-10% of the flat “glia-like” cells but did not induce the expression of GFAP or nestin in morphologically changed stellate cells. These results demonstrate that “glia-like” cells in primary adult human brain cultures constitute heterogeneous cell populations albeit with similar morphological features. Two distinct subpopulations have been shown: (i) the one immunostained for nestin; and (ii) the other reactive for dibutyryl-cAMP treatment.

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).

Atypical localization of myenteric ganglia in the human appendical wall: a comparative study with animal appendix

The presence of well developed appendices in some animals when compared to humans has led to speculation that appendix is a vestigial organ. Increasing number of studies have revealed that the appendix serves as an important organ in humans. The function of animal appendix, and the differences between species remain poorly understood. In this study we examined human myenteric plexus and compared them with animal studies. Appendices were obtained from five young adults in which the appendix was found to be normal after removal. Fixed appendix cryosections were examined by immunofluorescence methods using neuronal marker antibodies to neurofilaments and beta III tubulin. Both antibodies stained myenteric ganglia which were arranged in an apparently irregular pattern in human appendix wall. We observed unexpected localization of myenteric ganglia in the subserosa often accompanied by rarely occurring ganglia in the longitudinal muscle layer. These ganglia were of different sizes and shapes and unequally distributed under a thin layer of serosa. Our findings raise many questions about the possible role of irregular and atypical myenteric ganglia localization in relation to altered motility and subsequent pathogenesis of the appendix in inflammatory disease in humans. On the other hand, studies of the literature have revealed simplicity in the organization of myenteric plexus, e.g., in well-developed rabbit appendix. In addition, appendicitis in animals is restricted to in apes with similarly shaped appendix to humans.