Search

Forty-seven patients with Glioblastoma (42) and Anaplastic Astrocytoma (5) were studied with MR 24 hrs after surgery. In order to evaluate the role of early MR in defining the extent of surgical resection and its relation with the prognosis of malignant glioma patients, three categories of surgical resection were considered: gross total, sub-total and partial resection. The results were correlated with progression-free survival (PFS) and overall survival (ST). As demonstrated by early-MR, gross total resection was performed in 17 patients, sub-total and partial resection in 19 and 11 patients, respectively. The PFS was 6 months in gross total resection, 6 and 3 months in sub-total and in partial resection, respectively. The median survival time was 16 months in total resection patients, 13 months and 7 months in sub-total resection and partial resection patients, respectively. The study confirms that early-MR has to be considered an accurate technique for monitoring the extension of malignant glioma surgical resection and shows a good correlation between early-MR findings, PFS and ST.

The activity of the p53 gene product is regulated by a plethora of posttranslational modifications. An open question is whether such posttranslational changes act redundantly or dependently upon one another. We show that a functional interference between specific acetylated and phosphorylated residues of p53 influences cell fate. Acetylation of lysine 320 (K320) prevents phosphorylation of crucial serines in the NH(2)-terminal region of p53; only allows activation of genes containing high-affinity p53 binding sites, such as p21/WAF; and promotes cell survival after DNA damage. In contrast, acetylation of K373 leads to hyperphosphorylation of p53 NH(2)-terminal residues and enhances the interaction with promoters for which p53 possesses low DNA binding affinity, such as those contained in proapoptotic genes, leading to cell death. Further, acetylation of each of these two lysine clusters differentially regulates the interaction of p53 with coactivators and corepressors and produces distinct gene-expression profiles. By analogy with the "histone code" hypothesis, we propose that the multiple biological activities of p53 are orchestrated and deciphered by different "p53 cassettes," each containing combination patterns of posttranslational modifications and protein-protein interactions.

The dual role of microglia in cytotoxicity and neuroprotection is believed to depend on the specific, temporal expression of microglial-related genes. To better clarify this issue, we used high-density oligonucleotide microarrays to examine microglial gene expression after spinal cord injury (SCI) in rats. We compared expression changes at the lesion site, as well as in rostral and caudal regions after mild, moderate, or severe SCI. Using microglial-associated anchor genes, we identified two clusters with different temporal profiles. The first, induced by 4 h postinjury to peak between 4 and 24 h, included interleukin-1beta, interleukin-6, osteopontin, and calgranulin, among others. The second was induced 24 h after SCI, and peaked between 72 h and 7 days; it included C1qB, Galectin-3, and p22(phox). These two clusters showed similar expression profiles regardless of injury severity, albeit with slight decreases in expression in mild or severe injury vs. moderate injury. Expression was also decreased rostral and caudal to the lesion site. We validated the expression of selected cluster members at the mRNA and protein levels. In addition, we demonstrated that stimulation of purified microglia in culture induces expression of C1qB, Galectin-3, and p22(phox). Finally, inhibition of p22(phox) activity within microglial cultures significantly suppressed proliferation in response to stimulation, confirming that this gene is involved in microglial activation. Because microglial-related factors have been implicated both in secondary injury and recovery, identification of temporally distinct clusters of genes related to microglial activation may suggest distinct roles for these groups of factors.

Clinical variability is common in inherited gene defects of the central nervous system in humans and in animal models of human disorders. Here, we used the homozygous spastic (spa) mutant mice, which resemble human hereditary hyperekplexia, to determine the molecular remodeling of the spinal cord through the course of the disease, and develop a model for clinical disparity between littermates. The spa mutation is an insertion of a LINE-1 element in the gene for the beta subunit of the glycine receptor, Glrb. The insertion causes aberrant splicing in the beta subunit of glycine receptor gene with a consequent important reduction of glycine receptors. At young ages, all homozygous spa animals were spastic, showed loss of glycine receptors, increased expression of vesicular glycine/GABA transporter and NMDA receptors, induction of activated caspase3, and preferential loss of glycinergic interneurons consistent with neurotransmitter toxicity model. Those littermates that recovered from symptoms showed strong over-expression of the glycine receptor alpha 1 subunit (Glra1), and increased myelination and synaptic plasticity. Littermates that showed a deteriorating clinical course failed to over-express Glra1, and also showed relative loss of gephyrin (receptor clustering). These molecular changes were associated with a preferential loss of GABAergic interneurons, and extensive motorneuron loss. These data suggest that functional recovery is likely due to expression of homomeric glycine receptors, rescue from excitotoxicity, and subsequent neuronal remodeling. We propose that human patients with hyperekplexia show remodeling similar to that of the recovering spa mice, as human patients also show a lessening of symptoms as a function of age.

Upregulation of cell cycle proteins occurs in both mitotic and post-mitotic neural cells after central nervous system (CNS) injury in adult animals. In mitotic cells, such as astroglia and microglia, they induce proliferation, whereas in post-mitotic cells such as neurons they initiate caspase-related apoptosis. We recently reported that early central administration of the cell cycle inhibitor flavopiridol after experimental traumatic brain injury (TBI) significantly reduced lesion volume, scar formation and neuronal cell death, while promoting near complete behavioral recovery. Here we show that in primary neuronal or astrocyte cultures structurally different cell cycle inhibitors (flavopiridol, roscovitine, and olomoucine) significantly reduce upregulation of cell cycle proteins, attenuate neuronal cell death induced by etoposide, and decrease astrocyte proliferation. Flavopiridol, in a concentration dependent manner, also attenuates proliferation/activation of microglia. In addition, we demonstrate that central administration of flavopiridol improves functional outcome in dose-dependent manner after fluid percussion induced brain injury in rats. Moreover, delayed systemic administration of flavopiridol significantly reduces brain lesion volume and edema development after TBI. These data provide further support for the therapeutic potential of cell cycle inhibitors for the treatment of clinical CNS injury and that protective mechanisms likely include reduction of neuronal cell death, inhibition of glial proliferation and attenuation of microglial activation.

Changes in gene expression contribute to pathophysiological alterations following spinal cord injury (SCI). We examined gene expression over time (4 h, 24 h, 7 days) at the impact site, as well as rostral and caudal regions, following mild, moderate, or severe contusion SCI in rats. High-density oligonucleotide microarrays were used that included approximately 27,000 genes/ESTs (Affymetrix RG-U34; A, B and C arrays), together with multiple analyses (MAS 5.0, dChip). Alterations after mild injury were relatively rapid (4 and 24 h), whereas they were delayed and prolonged after severe injury (24 h and 7 days). The number and magnitude of gene expression changes were greatest at the injury site after moderate injury and increased in rostral and caudal regions as a function of injury severity. Sham surgery resulted in expression changes that were similar to mild injury, suggesting the importance of using time-linked surgical controls as well as naive animals for these kinds of studies. Expression of many genes and ESTs was altered; these were classified functionally based on ontology. Overall representation of these functional classes varied with distance from the site of injury and injury severity, as did the individual genes that contributed to each functional class. Different clustering approaches were used to identify changes in neuronal-specific genes and several transcription factors that have not previously been associated with SCI. This study represents the most comprehensive evaluation of gene expression changes after SCI to date. The results underscore the power of microarray approaches to reveal global genomic responses as well as changes in particular gene clusters and/or families that may be important in the secondary injury cascade.

Traumatic brain injury (TBI) causes neuronal apoptosis, inflammation, and reactive astrogliosis, which contribute to secondary tissue loss, impaired regeneration, and associated functional disabilities. Here, we show that up-regulation of cell cycle components is associated with caspase-mediated neuronal apoptosis and glial proliferation after TBI in rats. In primary neuronal and astrocyte cultures, cell cycle inhibition (including the cyclin-dependent kinase inhibitors flavopiridol, roscovitine, and olomoucine) reduced up-regulation of cell cycle proteins, limited neuronal cell death after etoposide-induced DNA damage, and attenuated astrocyte proliferation. After TBI in rats, flavopiridol reduced cyclin D1 expression in neurons and glia in ipsilateral cortex and hippocampus. Treatment also decreased neuronal cell death and lesion volume, reduced astroglial scar formation and microglial activation, and improved motor and cognitive recovery. The ability of cell cycle inhibition to decrease both neuronal cell death and reactive gliosis after experimental TBI suggests that this treatment approach may be useful clinically.

Following spinal cord injury, there are numerous changes in gene expression that appear to contribute to either neurodegeneration or reparative processes. We utilized high density oligonucleotide microarrays to examine temporal gene profile changes after spinal cord injury in rats with the goal of identifying novel factors involved in neural plasticity. By comparing mRNA changes that were coordinately regulated over time with genes previously implicated in nerve regeneration or plasticity, we found a gene cluster whose members are involved in cell adhesion processes, synaptic plasticity, and/or cytoskeleton remodeling. This group, which included the small GTPase Rab13 and actin-binding protein Coronin 1b, showed significantly increased mRNA expression from 7-28 days after trauma. Overexpression in vitro using PC-12, neuroblastoma, and DRG neurons demonstrated that these genes enhance neurite outgrowth. Moreover, RNAi gene silencing for Coronin 1b or Rab13 in NGF-treated PC-12 cells markedly reduced neurite outgrowth. Coronin 1b and Rab13 proteins were expressed in cultured DRG neurons at the cortical cytoskeleton, and at growth cones along with the pro-plasticity/regeneration protein GAP-43. Finally, Coronin 1b and Rab13 were induced in the injured spinal cord, where they were also co-expressed with GAP-43 in neurons and axons. Modulation of these proteins may provide novel targets for facilitating restorative processes after spinal cord injury.

Functional recovery after spinal cord injury (SCI) may result in part from axon outgrowth and related plasticity through coordinated changes at the molecular level. We employed microarray analysis to identify a subset of genes the expression patterns of which were temporally coregulated and correlated to functional recovery after SCI. Steady-state mRNA levels of this synchronously regulated gene cluster were depressed in both ventral and dorsal horn neurons within 24 h after injury, followed by strong re-induction during the following 2 wk, which paralleled functional recovery. The identified cluster includes neuritin, attractin, microtubule-associated protein 1a, and myelin oligodendrocyte protein genes. Transcriptional and protein regulation of this novel gene cluster was also evaluated in spinal cord tissue and in single neurons and was shown to play a role in axonal plasticity. Finally, in vitro transfection experiments in primary dorsal root ganglion cells showed that cluster members act synergistically to drive neurite outgrowth.

Cellular stress leads to DNA damage and activation of the intrinsic apoptotic pathway in which translocation of mitochondrial cytochrome c to the cytosol plays a critical role. Previous studies have suggested alternative mechanisms responsible for this process. We examined initiation mechanisms of the intrinsic apoptotic pathway using human neuroblastoma and breast cancer cells. Results indicated that translocation of cytochrome c does not require prior activation of caspases but rather depends on activation of specific BCL-2 family members, depending upon the type of death signal. Thus, DNA damage-induced apoptosis requires new protein synthesis, accumulation of p53 tumor suppressor protein, and p53-dependent induction of BOK and NOXA genes, while a role for BAX in this pathway is not essential. In contrast, apoptosis induced by staurosporine does not require protein synthesis but is characterized by translocation of BAX. Based on these findings, we propose a model of the intrinsic apoptotic cascade induced by DNA damage where proapoptotic BOK substitutes for a function of BAX.

Acute quadriplegic myopathy (AQM; also called "critical illness myopathy") shows acute muscle wasting and weakness and is experienced by some patients with severe systemic illness, often associated with administration of corticosteroids and/or neuroblocking agents. Key aspects of AQM include muscle atrophy and myofilament loss. Although these features are shared with neurogenic atrophy, myogenic atrophy in AQM appears mechanistically distinct from neurogenic atrophy. Using muscle biopsies from AQM, neurogenic atrophy, and normal controls, we show that both myogenic and neurogenic atrophy share induction of myofiber-specific ubiquitin/proteosome pathways (eg, atrogin-1). However, AQM patient muscle showed a specific strong induction of transforming growth factor (TGF)-beta/MAPK pathways. Atrophic AQM myofibers showed coexpression of TGF-beta receptors, p38 MAPK, c-jun, and c-myc, including phosphorylated active forms, and these same fibers showed apoptotic features. Our data suggest a model of AQM pathogenesis in which stress stimuli (sepsis, corticosteroids, pH imbalance, osmotic imbalance) converge on the TGF-beta pathway in myofibers. The acute stimulation of the TGF-beta/MAPK pathway, coupled with the inactivity-induced atrogin-1/proteosome pathway, leads to the acute muscle loss seen in AQM patients.

Spinal cord injury causes secondary biochemical changes leading to neuronal cell death. To clarify the molecular basis of this delayed injury, we subjected rats to spinal cord injury and identified gene expression patterns by high-density oligonucleotide arrays (8,800 genes studied) at 30 minutes, 4 hours, 24 hours, or 7 days after injury (total of 26 U34A profiles). Detailed analyses were limited to 4,300 genes consistently expressed above background. Temporal clustering showed rapid expression of immediate early genes (30 minutes), followed by genes associated with inflammation, oxidative stress, DNA damage, and cell cycle (4 and 24 hours). Functional clustering showed a novel pattern of cell cycle mRNAs at 4 and 24 hours after trauma. Quantitative reverse transcription polymerase chain reaction verified mRNA changes in this group, which included gadd45a, c-myc, cyclin D1 and cdk4, pcna, cyclin G, Rb, and E2F5. Changes in their protein products were quantified by Western blot, and cell-specific expression was determined by immunocytochemistry. Cell cycle proteins showed an increased expression 24 hours after injury and were, in part, colocalized in neurons showing morphological evidence of apoptosis. These findings suggest that cell cycle-related genes, induced after spinal cord injury, are involved in neuronal damage and subsequent cell death.

The aim of this work was to investigate in muscle the role of apoptosis and of oxidative stress in mitochondrial disorders with dysfunction of respiratory chain. In patients with cytochrome c oxidase deficiency (COX) we found a variable number of myofibers with apoptotic nuclei that matched with the level of enzymatic reduction and roughly correlated with muscle weakness. In parallel, a positive immunostaining for apoptosis-related proteins and Mn and Cu/Zn superoxide dismutase (SOD) were mostly localized in COX-negative fibers. Moreover, glutathione peroxidase activity was increased in muscles with high number of SOD-positive myofibers and prominent apoptotic features. No signs of apoptosis were observed in patients with deficiencies of complexes I and II and without muscle weakness. These data suggest that apoptosis along with increased ROS production, revealed by anti-oxidant enzymes overexpression, may play an important role in the pathophysiology of mitochondrial diseases associated with COX deficiency., (Copyright 2001 Academic Press.)

Mitochondrial encephalomyopathies caused by mitochondrial DNA (mtDNA) defects are a genetically and phenotypically heterogeneous group of disorders. The site, percentage and distribution of mutations do not explain the overall clinical heterogeneity that is found. Apoptosis (programmed cell death) is an evolutionarily conserved mechanism that is essential for tissue development and homeostasis. Dysregulation of apoptosis has been implicated in the pathogenesis of various human diseases, such as cancer and autoimmune and neurodegenerative disorders. Recent in vitro evidence has indicated the central role of mitochondria in the apoptotic process. We investigated the occurrence of apoptosis in muscle biopsies of 36 patients carrying different mtDNA mutations and four patients with inclusion body myositis and mitochondrial abnormalities. Apoptotic features, mainly localized in cytochrome c oxidase-negative fibres, were observed in muscle fibres of patients carrying a high percentage of single mtDNA deletions (>40%) and of tRNA point mutations (>70%). By contrast, no apoptotic changes were observed in inclusion body myositis and in patients carrying mutations of mtDNA structural genes. Our study suggests that apoptosis is not simply a means whereby cells with dysfunctional mitochondria are eliminated, but that it seems to play a role in the pathogenesis of mitochondrial disorders associated with mtDNA defects affecting mitochondrial protein synthesis. The imbalance and relative abundances of nuclear-encoded and mtDNA-encoded subunits may favour cytochrome c inactivation and release. Cytochrome c, together with respiratory chain dysfunction, could activate apoptotic pathways that, in turn, inhibit the rate of mitochondrial translation and the importation of nuclear-encoded mitochondrial protein precursors. This vicious circle may amplify the biochemical defects and tissue damage and contribute to the modulation of clinical features.

The authors retrospectively reviewed the MR examinations of 46 patients with clinical and laboratory findings of monoclonal gammopathies (MG). All cases had been submitted to radiographic examination which had shown skeletal involvement in 22 cases and osteoporosis in 11, with rupture of the vertebral body in 3 patients. Scintigraphy had been performed on all patients and CT on 12; 36 patients were subsequently submitted to follow-up (at 6, 12 and 24 months). MR examinations were performed with dedicated coils and standard sequences for the subjects with skeletal localizations on X-ray images. The extant cases, with no radiographic evidence of skeletal involvement, were submitted to MRI of the spine, skull and pelvis. In agreement with clinical and laboratory findings and with follow-up results (in 36 patients), MRI diagnosed MG with no skeletal involvement in 13 cases, osteoporosis in 8 (with rupture of the vertebral body in 2), asymptomatic non-progressive myeloma in 4, solitary myeloma in 3, and multiple myeloma in 18 cases. The good identification of bone marrow and its multiplanarity make MRI the method of choice in the study of patients with suspected or known gammopathies. If compared with other modalities, MRI is more sensitive and accurate in depicting the tumor, its size and relationship to periskeletal tissues, and its possible multifocality. Moreover, the technique has proven to be a valid tool during the follow-up, showing tumor response to therapy.

We evaluated the frequency and the functional activity of peripheral blood mononuclear cells (PBMCs) with natural killer (NK) cell phenotype in patients with monoclonal gammopathies. CD16+ and CD56+ PBMCs were strongly increased in monoclonal gammopathies of undetermined significance (MGUS) and multiple myeloma (MM). Furthermore, increased frequency of CD16+/CD3+ PBMCs was found in 7/15 patients with MGUS, indicating that T lymphocytes with NK-like phenotype are expanded in at least a subset of these patients. However, despite the increased frequency of PBMCs with natural killer phenotype, the functional NK activity was as comparable in both MGUS and MM patients as in normal individuals. The discrepancy between the expansion of circulating NK cells and the normal NK activity in patients with monoclonal gammopathies requires further investigation. However, at least in some MGUS patients, this discrepancy could be accounted for by the expansion of PBMCs with the rare phenotype CD16/CD3 which have been reported not to mediate significant NK activity.

Increased interleukin-6 levels in culture media of mitogen-driven non adherent peripheral blood mononuclear cells were measurable in patients with monoclonal gammapathies of unknown significance but not in patients with multiple myeloma, indicating that in the former circulating mononuclear cells other than monocytes are involved in producing interleukin-6. Increased interleukin-4 levels were detected in supernatants of mitogen-driven peripheral blood mononuclear cells from patients with monoclonal gammapathies of unknown significance and from patients with multiple myeloma. The further increased interleukin-4 content in supernatants of non adherent cell cultures of multiple myeloma patients only suggests a somewhat inhibitory role of monocytes on interleukin-4 production, at least in multiple myeloma. Undetectable interleukin-2 levels in culture media were found in patients with monoclonal gammapathies of unknown significance and in patients with multiple myeloma. Serum levels of interleukin-6 and interleukin-2 were not measurable in either group, and interleukin-4 was detected only in a few patients. Our study suggests that in monoclonal gammapathies peripheral blood mononuclear cells could participate in producing cytokines involved in the regulation of B lymphocyte proliferation and differentiation. However, the pathophysiologic role in these patients of IL-6 and IL-4 in vitro, and possibly in vivo, produced by circulating lymphocytes remains to be established.

Beta-2-microglobulin concentrations were determined in serum samples from 45 patients with benign and malignant monoclonal gammopathies. In the group of patients suffering from multiple myeloma or Waldenström macroglobulinemia the mean beta 2-microglobulin level was significantly higher than in the group with monoclonal gammopathy of undetermined significance. Values above 3 mg/L were highly indicative of a neoplastic process and were observed in all the Waldenström patients and in greater than 90% of myeloma patients. No significant correlation was noticed between beta 2-microglobulin and monoclonal protein levels in any of the groups examined.

Serum beta 2-microglobulin (beta 2M) was measured in 22 patients with B chronic lymphocytic leukemia (CLL) and in 15 healthy age-matched control subjects. The patients were allocated in stages in accordance with the Rai and Binet systems. The beta 2M mean value in the CLL group was significantly higher than in the control group. There was a positive correlation between beta 2M and the clinical stage in both staging systems. The mean serum levels of beta 2M in stages A, B and C of Binet's system showed statistical differences while in stage A they did not differ from those found in the control group. No correlation was demonstrated between beta 2M and the peripheral lymphocyte concentration. A slight correlation was found between beta 2M and the degree of bone marrow lymphocyte infiltration, whereas a stronger relationship was observed when the type of infiltration (non-diffuse versus diffuse) was considered. Patients with bulky disease had mean beta 2M values much higher than the others.

Pseudothrombocytosis and pseudoleukocytosis occurred in a patient with essential mixed cryoglobulinemia (EMC) and atypical cutaneous ulcers, when the blood cell counts were estimated by the Model S Plus Coulter Counter. The spurious cell counts were found in serum as well as in plasma and whole blood, so the involvement of fibrinogen in this phenomenon is questioned. When timed serial counts were performed on whole blood at room temperature the highest value of WBCs was detected one hour after sample collection and that of platelets occurred at 6 hours, when leukocytosis had disappeared. Possible explanations for this phenomenon are offered.