Mircea Leabu

Interstitial cells of Cajal (ICC) and gastrointestinal stromal tumor (GIST): facts, speculations, and myths.

Interstitial cells of Cajal (ICC) is a peculiar cell network composed of cells having processes described by the eminent Spanish neuroanatomist of the 19th century, S. Ramon y Cajal. ICC became a fascinating subject to many investigators and it is estimated that there are over 100 publications yearly on the subject related to ICC, in the last three years. Now it is widely accepted that ICC are pace maker cells of the gut and probable progenitor cells of gastrointestinal stromal tumors (GIST). Lately, interstitial Cajal‐like cells (ICLC) are being found in various organs and their physiological role is still to be defined. We have reviewed the literature trying to evaluate the validity of the current concept and found that there are a few salient points to be considered. 1) There has been some important departure in defining the identity of ICC from the original criteria of Cajal. In particular, ICC with myoid feafures in intestinal smooth muscle layers (ICC‐DPM) do not seem to fit to the original description of interstitial cell network by Cajal. We have also pointed out that the current reports assigning a pace maker role to ICC vastly depend on the scientific data on “ICC with myoid features”, not on “fibroblast‐like ICC”, which are more abundant and easier to identify. 2) There seem to be an overwhelming amount of data proving the relationship between ICC and GIST. Both are known to express c‐Kit and the ultrastructural characteristics seen in GIST roughly parallel those of ICC including minimal myoid differentiation seen in the majority of GIST, supporting the current concept that GIST are ICC tumors. 3) According to the original description of Cajal, ICC was not limited to the gut, suggesting an existence of ICC in other organs. The list of organs reported to contain ICC (currently identified by immunohistochemistry and electron microscopy) is ever growing and further studies are needed to define their identity and pathophysiologic role. 4). Recent data concerning gut development suggest that both c‐Kit expressing ICC (fibroblasts‐like as well as muscle‐like) and gut muscle cells derive from the common progenitor cells of the embryonic gut unifying the histogenetic concept of all GIST with heterogeneous cytomorphologic features. In this review we attempted to incorporate recent information on interstitial Cajal‐like cells (ICLC) found in other organs to broaden our understanding of ICC in general in terms of their ultrastructure, physiology, and neoplasia.

Synthesis, characterization antibacterial and antiproliferative activity of novel Cu(II) and Pd(II) complexes with 2-hydroxy-8-R-tricyclo[7.3.1.0.(2,7)]tridecane-13-one thiosemicarbazone.

Synthesis and biological activity investigation of complex compounds of Cu(II) are challenging issues because of the metal is not a xenobiotic one and the activity of ligands could be modulated by complexation. Complex combinations of Cu(II) and Pd(II) with thiosemicarbazone derivatives of 2-hydroxy-8-R-tricyclo[7.3.1.0.(2,7)]tridecane-13-one (where R=C(3)H(7), C(4)H(3)O) were synthesized. The characterization of the ligands and the newly formed compounds was done by (1)H NMR, (13)C NMR, UV-vis, IR, ESR spectroscopy, elemental analysis, molar electric conductibility and thermal studies. Experiments performed to identify the structures proved that the ligands coordinate to metal ions in different ways - neutral bidentate or mononegative bidentate. Also, if copper(II) acetate, copper(II) nitrate, copper(II) chloride and copper(II) thiocyanate were used, the ligands coordinated in a mononegative bidentate fashion. If copper(II) sulfate was used, the ligands coordinated in a neutral bidentate fashion. The biological activity for the copper(II) synthesized compounds was assessed in terms of antibacterial or antiproliferative activity. The antibacterial activity of the complexes against Staphylococcus aureus var. Oxford 6538, Escherichia coli ATCC 10536, Klebsielle pneumoniae ATCC 100131 and Candida albicans ATCC 10231 strains was studied and compared with that of free ligands. The effect of complex compounds on the proliferation of HeLa cells was tested. For all tested complexes an antiproliferative activity was noted at concentrations higher than 1 microM, but lower than 10 microM. Therefore, complex compounds of copper(II) were synthesized, structurally characterized and tested for biological activity, proving both antibacterial and antiproliferative activity.

Membrane fusion in cells: molecular machinery and mechanisms

Membrane fusion is a sine qua non process for cell physiology. It is critical for membrane biogenesis, intracellular traffic, and cell secretion. Although investigated for over a century, only in the last 15 years, the molecular machinery and mechanism of membrane fusion has been deciphered. The membrane fusion event elicits essentially three actors on stage: anionic phospholipids ‐ phosphatidylinositols, phosphatidyl serines, specific membrane proteins, and the calcium ions, all participating in a well orchestrated symphony. Three soluble N‐ethylmaleimide‐sensitive factor (NSF)‐attachment protein receptors (SNAREs) have been implicated in membrane fusion. Target membrane proteins, SNAP‐25 and syntaxin (t‐SNARE) and secretory vesicle‐associated membrane protein (v‐SNARE) or VAMP were discovered in the 1990s and suggested to be the minimal fusion machinery. Subsequently, the molecular mechanism of SNARE‐induced membrane fusion was discovered. It was demonstrated that when t‐SNARE‐associated lipid membrane is exposed to v‐SNARE‐associated vesicles in the presence of Ca2+, the SNARE proteins interact in a circular array to form conducting channels, thus establishing continuity between the opposing bilayers. Further it was proved that SNAREs bring opposing bilayers close to within a distance of 2‐3 Å, allowing Ca2+ to bridge them. The bridging of bilayers by Ca2+ then leads to the expulsion of water between the bilayers at the contact site, allowing lipid mixing and membrane fusion. Calcium bridging of opposing bilayers leads to the release of water, both from the water shell of hydrated Ca2+ ions, as well as the displacement of loosely coordinated water at the phosphate head groups in the lipid membrane. These discoveries provided for the first time, the molecular mechanism of SNARE‐induced membrane fusion in cells. Some of the seminal discoveries are briefly discussed in this minireview.

Synthesis and characterization of some new complexes of Cu(II), Ni(II) and V(IV) with Schiff base derived from indole-3-carboxaldehyde. Biological activity on prokaryotes and eukaryotes

Six new Cu(II), Ni(II), and VO(II) complexes (1-6) with Schiff base 1-phenyl-2,3-dimethyl-4-(1H-indole-3-carboxaldehyde)-3-pyrazolin-5-one (HL) were synthesized. The Schiff base was prepared through the condensation of 1-phenyl-2,3-dimethyl-4-amino-3-pyrazolin-5-one (antipyrine) with 1H-indole-3-carboxaldehyde. The new obtained compounds were characterized by (1)H NMR, (13)C NMR, UV-VIS, IR, EPR spectroscopy, elemental analysis, molar electric conductibility, magnetic susceptibility and thermal gravimetric analysis. In addition, the structure of the ligand HL has been determined by X-ray diffraction methods. The biological activity of complex compounds was investigated in terms of antibacterial effect on prokaryotic cells, by using paper disc diffusion technique, and for antiproliferative effect on eukaryotic cells, by monitoring mitotic activity in timelapse videomicroscopy experiments. The compounds were screened for their antibacterial activity against gram-positive bacteria (Staphylococcus aureus var. Oxford 6538, Klebsielle pneumoniae ATCC 100131 and Legionella monocytogenes ATCC 35182), gram-negative bacteria (Escherichia coli ATCC 10536, Pseudomonas aeruginosa ATCC 9027 and Salmonella typhimurium ATCC 14028) and anti-fungal activity (Candida albicans and Aspergillus flavus) using paper disc diffusion technique. The minimum inhibitory concentrations (MICs) of the compounds were also determined by agar streak dilution method. Compounds 3 and 4 proved to be the most effective as antibacterial agents. The antiproliferative activity was investigated by counting the number of mitoses for HeLa, and MCF7 cells. No significant antiproliferative effect was noted for HL and complex 2, for both used cell types. For complexes 1 and 3 complete inhibition of cell proliferation was observed in the case of HeLa cells, while the effects on MCF7 cell proliferation were lower. In conclusion, six new complex compounds were synthesized, and their biological activity investigated on both prokaryotic and eukaryotic cells, proving that some of them could be putative therapeutic substances.

CD117/c‐kit positive interstitial (Cajal‐like) cells in human pancreas

We provide evidence that interstitial Cajal‐like cells, previously described in human pancreas ‐ pICC (J Cel Mol Med, 9: 169, 2005), are positive for c‐kit irrespective of immunohistochemical procedures used. Various sample types (fresh cryosections or formalin‐fixed, paraffin‐embedded specimens), various slide pretreatments (with or without heat‐induced epitope retrieval) or different antibodies used (Dako polyclonal or Santa Cruz monoclonal), all showed CD117‐positive pICC.

Human topoisomerase II‐α is highly expressed in sinonasal‐inverted papilloma, but not in inflammatory polyp

Sinonasal‐inverted papilloma is a benign tumour with a high rate of recurrence, but possible malignant transformation. Therefore, inves tigation of predisposition to malignant transformation of sinonasal‐inverted papilloma gives clinicians the opportunity for adequate trea ment. Topoisomerase II‐α (topoII‐α) and Ki67 are markers of cell proliferation in both normal and neoplastic tissues and its level o expression could be used as a predictive parameter. Our goal was to investigate by immunochemistry the expression level of topoII‐in inverted papilloma, inflammatory nasal polyp and normal sinonasal epithelium and to compare it with expression level of Ki67. TopoI α nuclear immunostaining showed a differential positivity in the investigated cases. The topoII‐α index was 30.6 ± 12.8 in inverte papilloma, 10.7 ± 6.6 in the adjacent epithelium of inverted papilloma, but only 2.3 ± 2.0 in the normal sinonasal epithelium. The di ferences in topoII‐α expression between inverted papilloma and normal sinonasal epithelia were statistically significant. In inflammator nasal polyp group, topoII‐α index was 2.4 ± 2.1, and the difference in the topoII‐α index between inverted papilloma and inflammator polyp group was also statistically significant. Nuclear immunostaining for Ki67 followed a similar variation. The Ki67 index was 50.0 ± 20. in inverted papilloma, 9.0 ± 6.6 in the adjacent epithelium of inverted papilloma and 2.4 ± 0.9 in normal sinonasal epithelium. The di ferences in Ki67 expression between inverted papilloma and either adjacent or normal sinonasal epithelia were statistically significan Significant correlation coefficients were found between topoII‐α and epithelial thickness (r = 0.70, P > 0.0001), and between Ki67 inde and epithelial thickness (r = 0.71, P> 0.0001). In the inflammatory nasal polyp group Ki67 index was 5.9 ± 3.4. The difference in th Ki67 index between inverted papilloma and inflammatory nasal polyp groups was statistically significant. Significant correlation coeff cient was found between topoII‐α index and Ki67 index in inverted papilloma (r = 0.42, P > 0.05). These results suggest that the inverte papilloma contains a significantly higher cell population with proliferative activity by comparison with normal sinonasal and inflamma tory polyp epithelia, showing a significant correlation between topoII‐α and Ki67 expression, and indicating that topoII‐α could be a independent prognostic factor for a putative malignant transformation.

A new scoring system using multiple immunohistochemical markers for diagnosis of uterine smooth muscle tumors

The diagnosis of uterine smooth muscle neoplasms by light microscopy is difficult. Multiple classification schemes have been proposed based on mitotic rate, nuclear atypia, and the presence or absence of necrosis. None of these classification systems has been entirely successful. This study was undertaken to evaluate the use of selected immunohistochemical and histochemical markers in differentiating these tumors, in addition to accepted morphologic criteria. Ten cases of each of the following: leiomyosarcomas (LMS), atypical leiomyomas (AL), cellular leiomyomas (CL) and usual leiomyomas (UL), were classically evaluated for histological diagnosis and were stained for Ki‐67 (MIB‐1), bcl‐2 and p53 using monoclonal antibodies and the avidin‐biotin peroxidase method, and argyrophilic nucleolar organizer region (AgNORs). The number of stained cells was counted in the most positively stained region in a 4 mm2 square cover glass mounted on each slide. The mean value was calculated for each group of tumors. The data for Ki‐67 (MIB‐1), bcl‐2, p53 and AgNOR staining respectively, were significantly higher in LMS by comparison to UL, CL or AL. Because many singular cases had superimposed data being difficult to diagnose, a new scoring system for pathological evaluation was created. The results obtained by this scoring system suggest that immunohistochemical markers Ki‐67 (MIB‐1), bcl‐2, p53 together with the AgNOR staining could be useful, by the scoring system, as an adjunct to the current accepted morphologic criteria in differentiating smooth muscle tumors of the uterus.

UVA Irradiation of Dysplastic Keratinocytes: Oxidative Damage versus Antioxidant Defense

UVA affects epidermal cell physiology in a complex manner, but the harmful effects have been studied mainly in terms of DNA damage, mutagenesis and carcinogenesis. We investigated UVA effects on membrane integrity and antioxidant defense of dysplastic keratinocytes after one and two hours of irradiation, both immediately after exposure, and 24 h post-irradiation. To determine the UVA oxidative stress on cell membrane, lipid peroxidation was correlated with changes in fatty acid levels. Membrane permeability and integrity were assessed by propidium iodide staining and lactate dehydrogenase release. The effects on keratinocyte antioxidant protection were investigated in terms of catalase activity and expression. Lipid peroxidation increased in an exposure time-dependent manner. UVA exposure decreased the level of polyunsaturated fatty acids, which gradually returned to its initial value. Lactate dehydrogenase release showed a dramatic loss in membrane integrity after 2 h minimum of exposure. The cell ability to restore membrane permeability was noted at 24 h post-irradiation (for one hour exposure). Catalase activity decreased in an exposure time-dependent manner. UVA-irradiated dysplastic keratinocytes developed mechanisms leading to cell protection and survival, following a non-lethal exposure. The surviving cells gained an increased resistance to apoptosis, suggesting that their pre-malignant status harbors an abnormal ability to control their fate.

Keratinocyte Motility Is Affected by UVA Radiation—A Comparison between Normal and Dysplastic Cells

UVA radiation induces multiple and complex changes in the skin, affecting epidermal cell behavior. This study reports the effects of UVA exposure on normal (HaCaT) and dysplastic (DOK) keratinocytes. The adherence, spreading and proliferation were investigated by time-lapse measurement of cell layer impedance on different matrix proteins. Prior to UVA exposure, the time required for adherence and spreading did not differ significantly for HaCaT and DOK cells, while spreading areas were larger for HaCaT cells. Under UVA exposure, HaCaT and DOK cells behavior differed in terms of movement and proliferation. The cells’ ability to cover the denuded surface and individual cell trajectories were recorded by time-lapse videomicroscopy, during wound healing experiments. Dysplastic keratinocytes showed more sensitivity to UVA, exhibiting transient deficiencies in directionality of movement and a delay in re-coating the denuded area. The actin cytoskeleton displayed a cortical organization immediately after irradiation, in both cell lines, similar to mock-irradiated cells. Post-irradiation, DOK cells displayed a better organization of stress fibers, persistent filopodia, and new, stronger focal contacts. In conclusion, after UVA exposure HaCaT and DOK cells showed a different behavior in terms of adherence, spreading, motility, proliferation, and actin cytoskeleton dynamics, with the dyplastic keratinocytes being more sensitive.

Peroxidatic activity in heart effluent: a biochemical parameter for the assessment of experimental ischemia‐reperfusion injury

Peroxidatic activity in heart effluent was defined as a new biochemical parameter for the experimental study of myocardial ischemia. The peroxidatic reaction was determined by dot blot analysis with 3,3′‐diaminobenzidine as hydrogen donor. After ischemia, the level of peroxidatic activity in heart effluent was 2‐3 times higher than before. The effects in experimental modulation of ischemia, such as nicorandil or aprikalim protection, and the reversibility of protection by glibenclamide, could accurately be noted using the level of peroxidatic activity in heart effluent as a biochemical parameter. The results were in good agreement with those obtained for other enzymes used as biochemical parameters in experimental heart ischemia‐reperfusion studies.