Chromosome instability predicts the progression of premalignant oral lesions.

OBJECTIVES: One of the main problems in reducing the incidence of oral squamous cell carcinoma (OSCC) is the inability to appropriately deal with leukoplakia. Accurately identifying lesions which will progress to malignancy is currently not possible. The present study aims to establish the value of chromosome instability (CI) detection by DNA image cytometry and FISH analysis for prognosis and monitoring of oral leukoplakia. MATERIALS AND METHODS: For this purpose, we included from our archives 102 oral leukoplakia cases, which had been diagnosed between 1991 and 2008. Patient follow-up data were collected and the histopathological diagnosis was revised. CI assessment was carried out on paraffin-embedded tissue sections using both DNA image cytometry (ICM) and dual target FISH for chromosomes 1 and 7. RESULTS: 16 of 102 Patients developed carcinoma in situ or OSCC. Both detection methods were found to yield prognostic information independent of the histopathological diagnosis. CI was a strong individual marker of progression, with hazard ratios (HRs) of 7.2 and 6.8 for ICM and FISH respectively. Moreover, this approach seems suitable for monitoring lesions over time (especially ICM). Combining histopathology and CI enables subdivision of patients into three risk groups, with different probabilities of malignant progression. CONCLUSION: CI detection seems a reliable method for risk assessment of oral premalignancies and its application may contribute to a better risk-counselling and appropriate treatment regimen or watchfull-waiting approach of patients.

Targeted therapy for Ewing's sarcoma: significance of heterogeneity.

BACKGROUND: Survival in Ewing's sarcoma (ES) is limited. Experience with insulin-like growth factor targeting drugs, which require specific molecular tumour alterations, herald a major breakthrough. We screened for tumour heterogeneity within patients by DNA quantification. MATERIALS AND METHODS: DNA image cytometry (IC) was performed on 41 samples from 21 patients, evaluating if ploidy state remained constant over time and between different lesions within patients and the prognostic value of ploidy was assessed. RESULTS: DNA content varied over time and different ploidy states were found to coexist at a single timepoint. Non-diploid DNA content was associated with shorter overall survival (median, 19 vs. 84 months, p=0.047). CONCLUSION: We encountered a change and heterogeneity of ploidy state. This implies that screening for targets on a single tumour sample is insufficient and may lead to under- or overtreatment. The fact that non-diploid DNA content was associated with an adverse outcome confirms that this technique discriminates biologically different tumour clones.

The prediction of mandibular invasion by squamous cell carcinomas with the expression of osteoclast-related cytokines in biopsy specimens.

Destruction of bone by tumour is caused by osteoclasts rather than by tumour cells directly. Tumour cells of invasive oral squamous cell carcinomas (SCC) release osteoclast-related cytokines and cytokines activate osteoclasts. The purpose of this study was to investigate the possibility of predicting mandibular invasion by SCC by analysis of the expression of osteoclast-related cytokines in biopsy specimens of SCC, adjacent or fixed to the mandible. Thirty-five biopsy specimens from the pathology archives were examined from patients who had been treated for SCC, adjacent or fixed to the mandible. The patients were divided into those with and without medullary invasion. The expression of tumour necrosis factor (TNF)-alpha, interleukin (IL)-6 and IL-11 was studied by immunohistochemical analysis. No significant differences were found in expression of TNF-alpha, IL-6 and IL-11 between biopsy specimens with or without medullary invasion. Quantification of the density of tumour-infiltrating lymphocytes was not reproducible. In conclusion, the expression of TNF-alpha, IL-6 and IL-11 in biopsy specimens of SCC, adjacent or fixed to the mandible, is not an appropriate method for predicting the presence of medullary invasion of the mandible.

Specific association of small heat shock proteins with the pathological hallmarks of Alzheimer's disease brains.

The small heat shock protein family (sHsp) comprises molecular chaperones able to interact with incorrectly folded proteins. Alzheimer's disease (AD) is characterized by pathological lesions such as senile plaques (SPs), cerebral amyloid angiopathy (CAA) and neurofibrillary tangles (NFTs), predominantly consisting of the incorrectly folded proteins amyloid-beta (Abeta) and tau respectively. The aim of this study was to investigate the association of the chaperones Hsp20, HspB2, alphaB-crystallin and Hsp27 with the pathological lesions of AD brains. For this purpose, a panel of well-characterized antibodies directed against these sHsps was used in immunohistochemistry and immunoblotting. We observed extracellular expression of Hsp20, Hsp27 and HspB2 in classic SPs, and Hsp20 expression in diffuse SPs. In addition, extracellular expression of HspB2 was observed in CAA. Both Hsp27 and alphaB-crystallin were also observed in astrocytes associated with both SPs and CAA. Furthermore, none of the sHsps were observed in NFTs in AD brains. We conclude that specific sHsp species may be involved in the pathogenesis of either SPs or CAA in AD.

Differential gene expression in human brain pericytes induced by amyloid-beta protein.

Cerebral amyloid angiopathy is one of the characteristics of Alzheimer's disease (AD) and this accumulation of fibrillar amyloid-beta (Alphabeta) in the vascular wall is accompanied by marked vascular damage. In vitro, Abeta1-40 carrying the 'Dutch' mutation (DAbeta1-40) induces degeneration of cultured human brain pericytes (HBP). To identify possible intracellular mediators of Abeta-induced cell death, a comparative cDNA expression array was performed to detect differential gene expression of Abeta-treated vs. untreated HBP. Messenger RNA expression of cyclin D1, integrin beta4, defender against cell death-1, neuroleukin, thymosin beta10, and integrin alpha5 were increased in DAbeta1-40-treated HBP, whereas insulin-like growth factor binding protein-2 mRNA expression was decreased. Corresponding protein expression was investigated in AD and control brains to explore a potential role for these proteins in pathological lesions of the AD brain. Cyclin D1 expression was increased in cerebral amyloid angiopathy and cells in a perivascular position, suggesting that the cell cycle may be disturbed during Abeta-mediated degeneration of cerebrovascular cells. Moreover, cyclin D1 expression, but also that of integrin beta4, defender against cell death-1, neuroleukin and thymosin beta10 was found in a subset of senile plaques, suggesting a role for these proteins in the pathogenesis of senile plaques.

Heparan sulfate proteoglycan expression in cerebrovascular amyloid beta deposits in Alzheimer's disease and hereditary cerebral hemorrhage with amyloidosis (Dutch) brains.

Cerebrovascular deposition of amyloid beta protein (A beta) is a characteristic lesion of Alzheimer's disease (AD) and hereditary cerebral hemorrhage with amyloidosis of the Dutch type (HCHWA-D). Besides A beta, several other proteins and proteoglycans accumulate in cerebral amyloid angiopathy (CAA). We have now analyzed the expression of the heparan sulfate proteoglycan (HSPG) subtypes agrin, perlecan, glypican-1, syndecans 1-3 and HS glycosaminoglycan (GAG) side chains in CAA in brains of patients with AD and HCHWA-D. Hereto, specific well-characterized antibodies directed against the core protein of these HSPGs and against the GAG side chains were used for immunostaining. Glypican-1 was abundantly expressed in CAA both in AD and HCHWA-D brains, whereas perlecan and syndecans-1 and -3 were absent in both. Colocalization of agrin with vascular A beta was clearly observed in CAA in HCHWA-D brains, but only in a minority of the AD cases. Conversely, syndecan-2 was frequently associated with vascular A beta in AD, but did not colocalize with vascular A beta deposits in HCHWA-D. The three different syndecans, agrin, glypican-1 and HS GAG, but not perlecan, were associated with the majority of senile plaques (SPs) in all brains. Our results suggest a role for agrin in the formation of SPs and of CAA in HCHWA-D, but not in the pathogenesis of CAA in AD. Both syndecan-2 and glypican, but not perlecan, may be involved in the formation of CAA. We conclude that specific HSPG species may be involved in the pathogenesis of CAA in both AD and HCHWA-D, and that the pathogenesis of CAA and SPs may differ with regard to the involvement of HSPG species.

Amyloid-beta-induced degeneration of human brain pericytes is dependent on the apolipoprotein E genotype.

Amyloid-beta (A beta) deposition in cerebral vessels (cerebral amyloid angiopathy, CAA) is accompanied by degeneration of vascular cells, including pericytes and smooth muscle cells. Previous studies indicated that specific A beta protein isoforms are toxic for cultured human brain pericytes and smooth muscle cells. In particular, A beta 1-40 carrying the E22Q mutation, as in hereditary cerebral hemorrhage with amyloidosis of the Dutch type (HCHWA-D), is toxic. We investigated the effects of the A beta-binding protein apolipoprotein E (ApoE) on the toxicity of A beta for cultured human brain pericytes. We compared the toxicity of HCHWA-D A beta 1-40 for pericyte cultures with different ApoE genotypes, studied the accumulation of A beta and ApoE in these different cell cultures, and investigated the effects of exogenous ApoE. Pericyte cultures with an ApoE epsilon 2/epsilon 3 genotype were more resistant to HCHWA-D A beta 1-40 treatment than cultures with a epsilon 3/epsilon 3 or epsilon 3/epsilon 4 genotype. Cell death was highest in cultures homozygous for ApoE epsilon 4. The extent to which both A beta ApoE accumulated at the cell surface was parallel to the degree of toxicity. The addition of purified ApoE resulted in a decrease in cell death. These data suggest that ApoE4 may direct A beta more efficiently than other ApoE isoforms into a pathological interaction with the HBP cell surface. The results of this study are in line with the observations that inheritance of the ApoE epsilon 4 allele increases the risk of developing Alzheimer's disease, and that the ApoE epsilon 2 allele has a relatively protective effect.

Agrin is a major heparan sulfate proteoglycan accumulating in Alzheimer's disease brain.

Heparan sulfate proteoglycans (HSPGs) have been suggested to play an important role in the formation and persistence of senile plaques and neurofibrillary tangles in dementia of the Alzheimer's type (DAT). We performed a comparative immunohistochemical analysis of the expression of the HSPGs agrin, perlecan, glypican-1, and syndecans 1-3 in the lesions of DAT brain neocortex and hippocampus. Using a panel of specific antibodies directed against the protein backbone of the various HSPG species and against the glycosaminoglycan (GAG) side-chains, we demonstrated the following. The basement membrane-associated HSPG, agrin, is widely expressed in senile plaques, neurofibrillary tangles and cerebral blood vessels, whereas the expression of the other basement membrane-associated HSPG, perlecan, is lacking in senile plaques and neurofibrillary tangles and is restricted to the cerebral vasculature. Glypican and three different syndecans, all cell membrane-associated HSPG species, are also expressed in senile plaques and neurofibrillary tangles, albeit at a lower frequency than agrin. Heparan sulfate GAG side chains are also associated with both senile plaques and neurofibrillary tangles. Our results suggest that glycosaminoglycan side chains of the HSPGs agrin, syndecan, and glypican, but not perlecan, may play an important role in the formation of both senile plaques and neurofibrillary tangles. In addition, we speculate that agrin, because it contains nine protease-inhibiting domains, may protect the protein aggregates in senile plaques and neurofibrillary tangles against extracellular proteolytic degradation, leading to the persistence of these deposits.

Human brain pericytes as a model system to study the pathogenesis of cerebrovascular amyloidosis in Alzheimer's disease.

Cerebrovascular amyloidosis belongs to the pathological hallmarks of Alzheimer's disease brains. Although definite proof is still lacking, it is very well possible that the amyloid and its associated proteins are produced locally in the brain. In this paper we describe the development of a model system of cultured human brain pericytes to study the mechanisms of microvascular amyloid formation in vitro. These cultured cells may serve to study several aspects of cerebrovascular amyloidosis, which include the production of the amyloid precursor protein and of amyloid beta-protein-associated proteins as well as cytotoxic effects of amyloid beta-protein on perivascular cells. We demonstrated that pericytes produce and metabolize the amyloid precursor protein, and that they produce amyloid beta-protein-associated proteins, such as heparan sulfate proteoglycans, apolipoprotein E, and complement factor C1q. They are also prone to cellular degeneration after treatment with amyloid beta-protein, which is accompanied by increased expression of a number of amyloid beta-protein-associated proteins. This may be an important mechanism to explain the cell death observed in vivo. Our data indicate that this cell culture model of human brain pericytes provides a useful and pathophysiologically relevant tool to study cerebrovascular amyloidosis.

Distribution of A beta-associated proteins in cerebrovascular amyloid of Alzheimer's disease.

Senile plaques and cerebrovascular amyloidosis (CA) are two of the major neuropathological lesions in brains of patients with dementia of the Alzheimer type. We studied the expression of a number of amyloid beta (A beta)-associated proteins in CA, which have previously been identified in senile plaques and which were suggested to play an important role in the pathogenesis of these lesions. Our findings show that involvement of inflammatory components in CA is restricted to activation of the complement system, resulting in deposition of the complement factors C1q, C3c, C4d and the membrane attack complex C5b-9 as well as of the complement inhibitor clusterin. Furthermore, we observed expression of apolipoprotein E, amyloid P component and heparan sulfate proteoglycans in CA, whereas expression of lactoferrin was almost absent. Other inflammatory proteins, known to be present in senile plaques, such as alpha1-antichymotrypsin, alpha2-macroglobulin and intercellular adhesion molecule-1, were absent or detectable only in small amounts. These data suggest that an incomplete inflammatory response occurs in CA as compared to senile plaques. This was confirmed by the finding that the number of cells of the monocyte/macrophage lineage around CA was not increased compared to unaffected vessels. Based on their expression patterns, complement factors, apolipoprotein E and heparan sulfate proteoglycans may be produced early in the process of CA formation and may play an important role in the formation of A beta fibrils in CA. The absence of a number of A beta-associated proteins in CA in comparison to senile plaques is in support of a different pathogenesis for these two lesions.

[Inflammatory mechanisms in the pathogenesis of Alzheimer's disease]. / Ontstekingsmechanismen in de pathogenese van de ziekte van Alzheimer.

Senile plaques belong to the pathological hallmarks of the brains of patients with Alzheimer's disease. There is an increasing amount of evidence that the formation of senile plaques is accompanied by an acute phase reaction, involving the production of several inflammation-associated proteins and the activation of microglial cells. The products of these inflammatory reactions may contribute to the fibrillogenesis of the amyloid beta protein, the major constituent of senile plaques. Both fibrils of the amyloid beta protein and products of activated microglial cells may be neurotoxic, leading to neuronal degeneration and to clinical symptoms of dementia. Recent epidemiological findings have drawn attention to the possibility of therapy with anti-inflammatory agents. Although the results of these studies suggest a beneficial effect of such therapy, further study is warranted to gain more insight into the fundamental aspects of such treatment as well as to develop specific drugs that have little side-effects.

Differential expression of intercellular adhesion molecule-1 (ICAM-1) in the A beta-containing lesions in brains of patients with dementia of the Alzheimer type.

Inflammatory processes have been implicated in the formation of senile plaques in the cerebral cortex of patients with dementia of the Alzheimer type (DAT), since several inflammation-induced proteins are present within these plaques. The relation between inflammatory components and other amyloid beta protein (A beta)-containing lesions of the DAT brain [cerebrovascular amyloidosis (CA) and cerebellar senile plaques] is unclear. We studied the distribution of the inflammation-inducible protein intercellular adhesion molecule-1 (ICAM-1) in CA and in senile plaques of the cerebellum, using an immunohistochemical approach. We observed striking differences in ICAM-1 reactivity between the different types of A beta-containing lesions. ICAM-1 was only expressed in classic senile plaques in the granular and Purkinje cell layer of the cerebellum, and not in diffuse senile plaques of the molecular layer. Also, ICAM-1 was not associated with CA; only when the vascular amyloid extended into the neuropil (dyshoric angiopathy) was perivascular ICAM-1 reactivity observed. This is in contrast to the putative primary involvement of inflammation in the formation of cerebrocortical classic and diffuse senile plaques. Our findings indicate that ICAM-1 expression, which may be an indicator of an inflammatory reaction, is induced in the neuropil depending on the specific site of A beta production.

A lysosomal marker for activated microglial cells involved in Alzheimer classic senile plaques.

One of the major histopathological lesions in brains of patients with dementia of the Alzheimer type (DAT) is the senile plaque. Although previous studies have shown that senile plaques are often accompanied by microglial cells, the role of these cells in DAT pathology is still unclear. In an immunohistochemical and immunoelectron microscopical analysis of DAT and control brain tissues we addressed this issue using two monoclonal antibodies (mAbs KP1 and 25F9) directed against lysosomal antigens in monocytes and macrophages. Whereas KP1 stained lysosomes in both resting and activated microglial cells, 25F9-staining was predominantly found in lysosomes of activated microglial cells in classic senile plaques. The number and size of 25F9-positive lysosomes in activated microglial cells was increased compared to 25F9-staining in unaffected areas in DAT and control sections. We conclude that mAb 25F9 is a unique and useful lysosomal marker, with a higher specificity than other known markers, for activated microglial cells associated with classic, but not with diffuse, senile plaques.

Induction of alpha-smooth muscle actin expression in cultured human brain pericytes by transforming growth factor-beta 1.

Pericytes are cells localized at the abluminal side of the microvascular endothelium and completely enveloped by a basement membrane. Pericytes have close contact with endothelial cells and are probably involved in the regulation of endothelial cell functions. Previous studies suggested a role for pericytes in microvascular proliferation in tumors. To study this cell type, we isolated human brain pericytes from microvessel segments derived from autopsy brain tissue. These cells were characterized in vitro using a panel of monoclonal antibodies. Human brain pericytes were reactive with monoclonal antibodies directed against the high molecular weight-melanoma associated antigen and intercellular adhesion molecule-1, but only a minority of the cells expressed alpha-smooth muscle actin (alpha-SMA, 0 to 10%) or vascular cell adhesion molecule-1 (10 to 50%). In histologically normal human brain microvessels in situ, pericytes consistently lacked staining for these four markers. Tissue with microvascular proliferation, however, showed a marked pericyte staining for both alpha-SMA and high molecular weight-melanoma associated antigen. The expression of alpha-SMA in vitro could be slightly up-regulated by incubation with serum-containing medium. An increase in alpha-SMA expression up to 40% of the total cell population was seen when pericytes were treated with transforming growth factor-beta 1, whereas basic fibroblast growth factor slightly inhibited alpha-SMA expression. Incubation with other factors (platelet-derived growth factor-AA, heparin, interferon-gamma, tumor necrosis factor-alpha) had no effect on the alpha-SMA expression at all. Transforming growth factor-beta 1 thus induces smooth muscle-like differentiation in pericytes in vitro and might play a role in the activation of pericytes during angiogenesis in vivo.

Accumulation of intercellular adhesion molecule-1 in senile plaques in brain tissue of patients with Alzheimer's disease.

The still unsolved pathogenesis of Alzheimer's disease (AD) has been the subject of extensive speculation. Some years ago, a local acute phase reaction involving production of interleukin-1 (IL-1) and IL-6 was proposed as the triggering event in AD. Since it has been reported that these cytokines induce expression of intercellular adhesion molecule-1 (ICAM-1), we analyzed AD brain tissue cryosections for the presence of ICAM-1 by immunostaining and for ICAM-2 expression as a control. In senile plaques a marked diffuse or granular staining for the ICAM-1 domains 1, 4, and 5 was observed, whereas ICAM-2 expression was observed in microglial cells. Immunoprecipitation analysis demonstrated the presence of a 85 kd ICAM-1 molecule in AD frontal cortex. Our findings indicate that ICAM-1 accumulates in senile plaques as a complete 5-domain molecule at a relatively early stage of senile plaque formation. Our results are in support of a cytokine-mediated pathogenesis of senile plaque formation.

Exogenous aluminum accumulates in the lysosomes of cultured rat cortical neurons.

In order to study the intracellular localization of aluminum, 0.01% AlCl3 was added to rat cerebral organotypic cultures following 14 days incubation in a standard medium. The cultures were maintained in the aluminum (Al)-containing medium for 1 or 3 days. Subsequently, electron probe X-ray micro-analysis (EPXMA), was used to localize aluminum in the neurons. The Al was found in the cells as early as after 1 day of AlCl3 exposure. The Al was detected exclusively in the neuronal lysosomes, in 66% (1 day exposure) and 97% (3 days) of the measured lysosomes. This localization was confirmed by laser microprobe mass analysis (LAMMA) measurements. Our results demonstrate an Al localization in the neurons, exposed to exogenous Al, different from that in the brains of patients with Alzheimer's disease.