George Gatzounis

Neurotrophin receptors expression and JNK pathway activation in human astrocytomas

BackgroundNeurotrophins are growth factors that regulate cell growth, differentiation and apoptosis in the nervous system. Their diverse actions are mediated through two different transmembrane – receptor signaling systems: Trk receptor tyrosine kinases (TrkA, TrkB, TrkC) and p75NTR neurotrophin receptor. Trk receptors promote cell survival and differentiation while p75NTR induces, in most cases, the activity of JNK-p53-Bax apoptosis pathway or suppresses intracellular survival signaling cascades. Robust Trk activation blocks p75NTR -induced apoptosis by suppressing the JNK-p53-Bax pathway. The aim of this exploratory study was to investigate the expression levels of neurotrophin receptors, Trks and p75NTR, and the activation of JNK pathway in human astrocytomas and in adjacent non-neoplastic brain tissue.MethodsFormalin-fixed paraffin-embedded serial sections from 33 supratentorial astrocytomas (5 diffuse fibrillary astrocytomas, WHO grade II; 6 anaplastic astrocytomas, WHO grade III; 22 glioblastomas multiforme, WHO grade IV) were immunostained following microwave pretreatment. Polyclonal antibodies against TrkA, TrkB, TrkC and monoclonal antibodies against p75NTR and phosphorylated forms of JNK (pJNK) and c-Jun (pc-Jun) were used. The labeling index (LI), defined as the percentage of positive (labeled) cells out of the total number of tumor cells counted, was determined.ResultsModerate to strong, granular cytoplasmic immunoreactivity for TrkA, TrkB and TrkC receptors was detected in greater than or equal to 10% of tumor cells in the majority of tumors independently of grade; on the contrary, p75NTR receptor expression was found in a small percentage of tumor cells (~1%) in some tumors. The endothelium of tumor capillaries showed conspicuous immunoreactivity for TrkB receptor. Trk immunoreactivity seemed to be localized in some neurons and astrocytes in non-neoplastic tissue. Phosphorylated forms of JNK (pJNK) and c-Jun (pc-Jun) were significantly co-expressed in a tumor grade-dependent manner (p < 0.05). Interestingly, a statistically significant (p < 0.05) reverse relationship between Trk receptors LIs and pc-Jun/pJNK LIs was noted in some glioblastomas multiforme.ConclusionIn the context of astrocytomas, Trk receptors (TrkA, TrkB, TrkC) expression may promote tumor growth independently of grade. Furthermore, activation of JNK pathway may contribute to progression towards malignancy. Considering the fact that regional tumor heterogeneity may be a limiting factor for immunohistochemical studies, the significance of the reverse relationship between Trk receptors and pc-Jun/pJNK LIs with respect to biological behavior of human astrocytomas requires further evaluation.

A comparative biochemical analysis of glycosaminoglycans and proteoglycans in human orthotopic and heterotopic bone

Glycosaminoglycans and proteoglycans are macromolecules of the bone and are involved in the assembly, maturation, mineralization, and maintenance of the extracellular matrix. Heterotopic ossification is the rapid development of calcified bone tissue at ectopic sites of the body, mainly in soft tissues that normally do not ossify. The aim of this study was to characterize the molecular profiles of glycosaminoglycans and proteoglycans in normal and heterotopic bone samples to assess whether differences exist between orthotopic and heterotopic bone. Heterotopic bone tissues contained lower amounts of glycosaminoglycans compared to normal femoral bone. Structural analysis of chondroitin sulfate (CS) revealed that both heterotopic and normal femoral bones were composed mainly of 6‐sulfated disaccharides. Quantitative differences in the disaccharide composition of CS, such as the decrease of 6‐sulfated disaccharides in heterotopic bone with a concurrent increase of 4‐sulfated and nonsulfated disaccharides, were found between ectopic bone and normal femoral bone. The proteoglycans decorin and aggrecan were both detected in all bone samples using specific antibodies. The detection of minor amounts of aggrecan in mature human bone, such as femoral bone, as well as in ectopic bone is described for the first time. These results may elucidate the phenomenon of ectopic bone formation and assist in early detection.

Loss of inhibitor of growth (ING-4) is implicated in the pathogenesis and progression of human astrocytomas.

Inhibitor of growth 4 (ING‐4) is a tumor suppressor gene that interacts with nuclear factor‐kappaB (NF‐κB) and represses its transcriptional activity. Several lines of evidence suggest that the tumor suppressor gene ING‐4, the transcription factor NF‐κB and its target genes matrix metalloproteases MMP‐2, MMP‐9 and urokinase plasminogen activator (u‐PA) are critically involved in tumor invasion. The aim of the present study was to investigate immunohistochemically the expression pattern of ING‐4, NF‐κB and the NF‐κB downstream targets MMP‐2, MMP‐9 and u‐PA in human astrocytomas from 101 patients. We found that ING‐4 expression was significantly decreased in astrocytomas, and ING‐4 loss was associated with tumor grade progression. Expression of p65, a NF‐κB subunit, was significantly higher in grade IV than in grade III and grade I/II tumors, and a statistical significant negative correlation between expression of ING‐4 and expression of nuclear p65 was noticed. MMP‐9, MMP‐2 and u‐PA were overexpressed in human astrocytomas. Of note, astrocytomas of advanced histologic grades (grade III, IV) displayed significantly higher expression levels of these proteins compared to tumors of lower grades (grade I, II). Collectively, our data suggest an essential role for ING‐4 in human astrocytoma development and progression possibly through regulation of the NF‐κB‐dependent expression of genes involved in tumor invasion.

New experimental rabbit animal model for cervical spondylotic myelopathy

Study design:Cervical spondylotic myelopathy (CSM) represents the most commonly acquired cause of spinal cord dysfunction among individuals over 55 years old. The pathophysiology of the disease involves static and dynamic mechanical factors, which are the result of chronic degeneration. The clinical course of the disease remains unpredictable. In the past, many experimental animal models have been developed to study the cellular and molecular mechanisms underlining the pathophysiology of the disease.Objectives:To create a new animal model of CSM, which will reproduce the temporal course of the disease and the local microenvironment at the site of spinal cord compression.Methods:We performed posterior laminectomy to New Zealand rabbits at the level of C7, and a thin sheet (5–7 μm) of aromatic polyether was implanted with microsurgical technique at the epidural space underneath C5–C6 laminae. Motor function evaluation was performed after the operation and once a week thereafter.Results:After 20 weeks, the animals were killed, and the histological evaluation of spinal cord at the site of compression above and below it, using eosin hematoxylin, immonohistochemistry and Kluver–Barrera techniques reveals axonal swelling and demyelination, interstitial edema and myelin sheet fragmentation. Moreover, histological evaluation of C5 and C6 laminae reveals osteophyte formation.Conclusion:We believe that this CSM model reproduces the temporal evolution of the disease and creates a local microenvironment at the site of spinal cord compression, which shares the same characteristics with that of human disease.

The Role of Oligodendrocytes in the Molecular Pathobiology and Potential Molecular Treatment of Cervical Spondylotic Myelopathy

Cervical spondylotic myelopathy (CSM) is a very common and debilitating disease; however, its underlying pathocellular process remains uncertain. Attempts have been made to reproduce CSM in experimental animal models in order to deepen the knowledge on the molecular pathobiology of this disease. The up-to-date observations have established the apoptosis of oligodendrocytes (OLGs) as the principal pathocellular process of CSM. Since favorable neurological recovery cannot be obtained in afflicted patients, even after the decompression surgery, elucidation of the apoptotic cascade in OLGs may unveil possible molecular treatments which could inhibit demyelination and ameliorate the neurological deficits. Moreover, additional therapeutic benefits may include improvement of myelin self-repair capability by stimulating OLG progenitor cells to become mature and finally, myelinating OLGs. This review focuses on the factors and mechanisms of crucial importance for developing antiapoptotic treatments. Critical evaluations of the role of OLGs in molecular pathobiology of CSM as well as strategies for potential remyelination of CSM are also provided. The analyses and evaluations of the experimental findings can possibly lead to treatment of CSM as well as to development of novel biopharmacenticals.

Immunohistochemical profile of NF-κB/p50, NF-κB/p65, MMP-9, MMP-2, and u-PA in experimental cervical spondylotic myelopathy.

Study Design. The immunohistochemical profile of nuclear factor–&kgr; B (NF-&kgr;B)/p50, NF-&kgr;B/p65, matrix metalloproteinase (MMP)–9, MMP-2, and urokinase-type plasminogen activator (u-PA) proteins was examined in spinal cord tissues coming from rabbits, which underwent chronic cervical spinal cord compression. Objective. To study the potential role of NF-&kgr;B and extracellular matrix proteins under the chronic mechanical compression of the cervical spinal cord. Summary of Background Data. Cervical spondylotic myelopathy (CSM) is the most common cause of spinal cord dysfunction among adults older than 55 years. Neuronal loss, myelin destruction, axonal degeneration, and glial scar formation are the principal neuropathological features of CSM. However, the biologic pathways that lead to these features remain unclear. Methods. In this study, we used a new animal experimental model of CSM developed in our laboratory. Briefly, after posterior cervical laminectomy, gradual and progressive compression (during 20 weeks) was achieved by introducing a piece of aromatic polyether (0.07 mm thick) under the C6 lamina in 15 New Zealand rabbits. In control animals (n = 15), the aromatic polyether was implanted and then removed after 60 seconds (sham operation). The immunoreactivity of p50 and p65 subunits of NF-kB, as well as that of MMP-2, MMP-9, and u-PA, was evaluated in paraffin-embedded spinal cord sections coming from both groups. The evaluation was performed using immunohistochemistry technique and the results were analyzed using SPSS for Windows, release 12.0 (SPSS Inc., Chicago, IL). Results. Increased immunoreactivity of both NF-&kgr;B subunits, p50 and p65, as well as MMP-2, MMP-9, and u-PA was demonstrated in animals with CSM in comparison with controls. Statistical analysis of the results revealed strong positive correlation between NF-&kgr;B subunits immunoreactivity and that of MMP-9, MMP-2, and u-PA. Conclusion. There is a strong correlation between the immunoexpression of NF-&kgr;B/p50, NF-&kgr;B/p65, MMP-2, MMP-9, u-PA, and CSM.

Immunohistochemical detection of phosphorylated JAK-2 and STAT-5 proteins and correlation with erythropoietin receptor (EpoR) expression status in human brain tumors

Phosphorylated (activated) forms of Janus Kinase 2 (pJAK-2) and STAT-5 transcription factor (pSTAT-5), which are preferentially expressed after binding of erythropoietin (Epo) to its receptor EpoR, are known to be implicated in the molecular mechanisms controlling brain development. The purpose of this study was to investigate the expression of these proteins (pJAK-2, pSTAT-5, and EpoR) in human brain tumors compared with normal brain. Using specific antibodies and immunohistochemistry on formalin-fixed, paraffin-embedded semi-serial tissue sections a total of 87 human brain tumors and samples from normal brain tissue were studied. pJAK-2/pSTAT-5 nuclear co-expression was detected in 39% of astrocytomas, 43% of oligodendrogliomas, 50% of ependymomas, and in all (100%) of the medulloblastomas examined. In contrast, most of the meningiomas showed weak or no immunoreactivity for pJAK-2/pSTAT-5 proteins. A significant percentage of tumors exhibited pSTAT-5 immunoreactivity, being pJAK-2 immunonegative. EpoR/pJAK-2/pSTAT-5 co-expression was detected in a small percentage of astrocytomas (18%) and ependymomas (33%). Oligodendrogliomas and medulloblastomas were EpoR immunonegative. Tumor vessels exhibited EpoR, pJAK-2, and pSTAT-5 immunoreactivity. In normal brain tissue, EpoR immunoreactivity was detected in neurons and vessels whereas pSTAT-5 and pJAK-2 immunoreactivity was limited to some neurons and a few glial cells, respectively. These results indicate the existence of ligand (other than Epo)-dependent or independent JAK-2 activation that leads to constitutive activation of STAT-5 in most human brain tumors. Given the oncogenic potential of the JAK/STAT pathway, detection of different pJAK-2 and pSTAT-5 expression profiles between groups of tumors may reflect differences in the biological behavior of the various human brain tumors.

Phonological fluency strategy of switching differentiates relapsing-remitting and secondary progressive multiple sclerosis patients.

The strategies used to perform a verbal fluency task appear to be reflective of cognitive abilities necessary for successful daily functioning. In the present study, we explored potential differences in verbal fluency strategies (switching and clustering) used to maximize word production by patients with relapsing-remitting multiple sclerosis (RRMS) versus patients with secondary progressive multiple sclerosis (SPMS). We further assessed impairment rates and potential differences in the sensitivity and specificity of phonological versus semantic verbal fluency tasks in discriminating between those with a diagnosis of MS and healthy adults. We found that the overall rate of impaired verbal fluency in our MS sample was consistent with that in other studies. However, we found no differences between types of MS (SPMS, RRMS), on semantic or phonological fluency word production, or the strategies used to maximize semantic fluency. In contrast, we found that the number of switches differed significantly in the phonological fluency task between the SPMS and RRMS subtypes. The clinical utility of semantic versus phonological fluency in discriminating MS patients from healthy controls did not indicate any significant differences. Further, the strategies used to maximize performance did not differentiate MS subgroups or MS patients from healthy controls.

p75NTR and TrkC Neurotrophin Receptors Demonstrate a Different Immunoreactivity Profile in Comparison to TrkA and TrkB Receptors in Human Normal Pituitary Gland and Adenomas

Background/Aims: Recent knowledge indicates that neurotrophins play a significant role in neuroendocrine systems through their specific receptors TrkA, TrkB, TrkC and low-affinity p75NTR receptor. TrkA and TrkB receptors have been previously detected in numerous endocrine cells in human anterior pituitary and adenomas. In the present study, the localization of p75NTR and TrkC along with TrkA and TrkB receptors was investigated. Methods: Semi-serial paraffin-embedded sections of 5 human normal pituitaries and 30 adenomas were immunostained using specific antibodies. Results: Expression of p75NTR receptor was demonstrated in the intricate capillary and reticulin network in the anterior pituitary and in the pericapillary tissue and pituicytes in the posterior lobe. p75NTR immunoreactivity was absent from all adenomas. In normal anterior pituitary, a few scattered cells showed weak TrkC immunoreactivity in contrast to a high percentage of endocrine cells distributed throughout the pars distalis and pars intermedia which exhibited strong TrkA and/or intermediate TrkB immunoreactivity. Double immunohistochemistry demonstrated TrkA immunoreactivity in more than 80% of lactotropes and 70% of corticotropes and to a lesser extent in other cell types. Furthermore, in the majority of adenomas, independently of type, sex and age, a high percentage of TrkA- and/or TrkB-positive cells was detected. Interestingly, TrkC expression appeared to be increased in some adenomas compared to normal pituitary. Endothelial cells and perivascular connective tissue were always TrkB-immunostained. Conclusion: The above findings support a potential role of all neurotrophins, through their different receptors, in pituitary functions.

Brain Gray – White Matter Discrimination in Dual Energy CT Imaging: A Simulation Feasibility Study

The aim of this study is to investigate the feasibility of using dual-energy (DE) imaging for discrimination of gray-white matter with brain CT. Initially, an optimization study was accomplished that aimed to find the optimal low and high beam energy spectra for the DE application. Simulations were carried out with 181 noise free brain dual-energy CT (DECT) images, simulated with an in-house developed software simulator for several monochromatic and polychromatic spectra within a full gantry acquisition arc of 3600. Ten monochromatic beams with energy from 20 keV to 110 keV and four polychromatic beam spectra 80, 100, 100, 140 kVp were simulated. The software brain phantom was the Digital Brain Phantom II, that is voxel-based model of the brain with no tumor insertion. To obtain DE projections, a non-linear algorithm for combining low and high energy images was exploited. Further, the optimal parameters of this algorithm were determined. The optimization study showed that the maximum contrast in brain DECT is achieved for the low/high energy combination of 100/110 keV in case of monochromatic beams and 80/100 kVp for polychromatic beams. For these cases, the contrast improvement was of the order of 12 and 7 times fold, respectively, compared to brain CT images acquired at high energies. The visual observation demonstrated superiority of the brain DECT compared to single energy brain CT images in terms of improvement in brain tissue differentiations. Evaluation of line profiles taken through different regions of interest on single and brain DECT images showed an excellent gray and white matter tissue discrimination.