Cytocompatibility and bioactive properties of the new dual-curing resin-modified calcium silicate-based material for vital pulp therapy.

OBJECTIVE: The aim of the present study was to evaluate the in vitro biocompatibility of Theracal PT, Theracal LC, and MTA Angelus, considered as bioactive materials used for vital pulp treatment, on human dental pulp stem cells (hDPSCs). MATERIALS AND METHODS: Human dental pulp stem cells (hDPSCs) were isolated from third molars, and material eluates were prepared (undiluted, 1:2, and 1:4 ratios). The hDPSC cytotoxicity, adhesion, morphology, viability, and cell migration were assessed. The mineralization nodule formation was determined by Alizarin red S staining (ARS). The odonto/osteogenic differentiation potential was assessed by osteo/odontogenic marker expression real-time qPCR. The chemical composition and ion release of the vital pulp materials were determined by energy dispersive X-ray (EDX) and inductively coupled plasma-mass spectrometry (ICP-MS), respectively. Statistical differences were assessed by ANOVA and Tukey's test (p < 0.05). RESULTS: The three vital pulp materials showed variable levels of calcium, tungsten, silicon, and zirconium release and in their chemical composition. Cytocompatibility assays revealed higher hDPSC viability and migration rates when treated with Theracal PT than with Theracal LC. The lowest cell adhesion and spreading were observed in all Theracal LC-treated groups, whereas the highest were observed when treated with MTA. Theracal PT and MTA promoted the upregulation of DSPP and RUNX2 gene expression (p < 0.05). After 21 days, both MTA Angelus and Theracal PT-treated cells exhibited a significantly higher mineralized nodule formation than the negative control (p < 0.05). CONCLUSIONS: This study demonstrates the favorable in vitro cytocompatibility and bioactive properties of the recently introduced Theracal PT and the well-established MTA Angelus on hDPSCs, as opposed to Theracal LC. More studies, including in vivo animal testing are suggested before these new formulations might be used in the clinical setting. CLINICAL RELEVANCE: Theracal PT is a new material that could be clinically suitable for vital pulp therapy. Further studies considering its biocompatibility and bioactivity are necessary.

Molecular progression in unusual recurrent non-pediatric intracranial clear cell meningioma.

We report a case of a recurrent clear cell meningioma (ccm) in the frontal lobe of the brain of a 67-year-old man. The patient developed three recurrences: at 3, 10, and 12 years after his initial surgery. Histopathology observations revealed a grade 2 ccm with positivity for vimentin and epithelial membrane antigen. Expression of E-cadherin was positive only in the primary tumour and in the first available recurrence. Fluorescence in situ hybridization analyses demonstrated 1p and 14q deletions within the last recurrence. Multiplex ligation-dependent probe amplification studies revealed a heterozygous partial NF2 gene deletion, which progressed to total loss in the last recurrence. The last recurrence showed homozygous deletions in CDKN2A and CDKN2B. The RASSF1 gene was hypermethylated during tumour evolution. In this report, we show the genetic alterations of a primary ccm and its recurrences to elucidate their relationships with the changes involved in the progression of this rare neoplasm.

Alteration of major vault protein in human glioblastoma and its relation with EGFR and PTEN status.

Glioblastoma (GBM) is the most frequent and malignant primary brain tumor. Conventional therapy of surgical removal, radiation and chemotherapy is largely palliative. Major vault protein (MVP), the main component of the vault organelle has been associated with multidrug resistance by reducing cellular accumulation of chemotherapeutic agents. With regard to cancer, MVP has been shown to be overexpressed in drug resistance development and malignant progression. The aim of the present study was to evaluate the MVP gene dosage levels in 113 archival samples from GBM and its correlation with patients' survival and epidermal growth factor receptor (EGFR) and phosphatase and tensin homolog (PTEN) gene dosages. Fluorescent in situ hybridization revealed polysomy of chromosome 7 in 76.1% of the GBMs and EGFR amplification in a 64.6% of the tumors. Genetic status of EGFR, PTEN and MVP copies was determined by multiplex ligation-dependent probe amplification (MLPA) technique. 31% of the tumors showed the EGFR is variant III mutation (EGFRvIII) mutation and 74.3% of them presented amplification of MVP gene. Amplification of EGFR and MVP was found in a 63.7% and 56.6% of the GBM, respectively. An inverse correlation between MVP and PTEN dosage values was observed. Besides, an inverse relationship between the survival of the patients treated with chemotherapy and the levels of MVP copies was determined. In conclusion, our study reveals an important role of MVP, together with EGFRvIII and PTEN, in the progression of GBM and proposes it as a novel and interesting target for new treatment approaches.

Demethylnobiletin inhibits delayed-type hypersensitivity reactions, human lymphocyte proliferation and cytokine production.

BACKGROUND AND PURPOSE: Our aim was to examine the effect of demethylnobiletin on various experimental models of delayed-type hypersensitivity (DTH) reactions and to determine its influence on the mediators and enzymes involved in these reactions. EXPERIMENTAL APPROACH: DTH was induced in mice by oxazolone, dinitrofluorobenzene (DNFB) and sheep red blood cells (SRBC). The effect of demethylnobiletin on the ensuing DTH was studied, especially in relation to oedema formation, cell infiltration and tissue damage. Its activity on different mediators implicated in DTH reactions was also determined and its effect on nitric oxide synthase (NOS)-2 analysed. Finally, its influence on T lymphocyte proliferation, apoptosis and caspase 3 activity was tested. KEY RESULTS: DTH reactions were all reduced by demethylnobiletin. The experimental results suggest that the compound may act by reducing cell infiltration and by suppressing mediators such as interleukin-2 (IC50=1.63 microM), interleukin-4 (IC50=2.76 microM), tumour necrosis factor-alpha (IC50=0.66 microM), interferon-gamma (IC50=1.35 microM), and interleukin-1 beta (46% at 2.5 microM) and by concomitantly increasing the production of the anti-inflammatory cytokine, interleukin-10. In addition, while demethylnobiletin affected nitric oxide production, it did not modify NOS-2 expression. Finally, demethylnobiletin inhibited proliferation of T cells and induced their apoptosis. CONCLUSIONS AND IMPLICATIONS: Demethylnobiletin decreased DTH reactions induced by various agents. This finding, along with the fact that the compound has a low toxicity and exhibits several other interesting properties, could pave the way for other structurally related citroflavonoids to be used as pharmacological agents in complementary therapies.

Concurrent EGFR amplification and TP-53 mutation in glioblastomas.

Glioblastoma multiforme is the most common and most aggressive of the primary brain tumors. The mean survival of patients is 10-12 months. Conventional therapy of surgery, radiation and chemotherapy is largely palliative. Cytogenetically, karyotypes of glioblastomas are very complex with trisomy 7 and monosomy 10 as the most frequent abnormalities. A genetic alteration that is significantly more frequent in primary than in secondary glioblastomas, the latter arising from preceding low-grade gliomas, is epidermal growth factor receptor gene (EGFR) amplification, whereas TP-53 mutations are significantly more frequent in low-grade gliomas and secondary glioblastomas derived there- from. We report the histological and genetic study of two glioblastomas, one case arising de novo and the other case arising 3 years after a previously diagnosed anaplastic astrocytoma, with concurrent EGFR amplification and TP-53 mutation. These anomalies were initially deemed as mutually exclusive. However, a small percentage of cases have been found with both anomalies although at a significantly lower level than could be expected. We have analyzed these two cases cytogenetically and by molecular studies in order to detect additional alterations associated with this phenotype. Cytogenetically, both cases showed in common the monosomy of chromosomes 10 and 17. At the molecular level, a rare mutation of TP-53 was found in the secondary glioblastoma and hypermethylation of the promoter region of p16(INK4a) and p14(ARF) genes were observed in the primary and secondary glioblastoma, respectively.

Primary glioblastoma with EGFR amplification and a ring chromosome 7 in a young patient.

Glioblastoma is the most common primary tumor of the central nervous system, but the underlying genetic changes that give rise to these tumors are still poorly understood. We report a primary glioblastoma with an unusual age of presentation. The patient was a 22-year-old man with a survival of 16 months. Morphological findings showed an increase of cellularity with positive GFAP and EGFR expression, increase of proliferate index, vascular hyperplasia with glomeruloid structures and necrosis. Molecular analysis showed EGFR amplification. No mutations of the TP53 or amplification of MDM2 and CDK4 were detected. Neither homozygous deletion of the 9p21 locus genes nor aberrant methylation were found. The cytogenetic study showed a clonal karyotype. The metaphases presented, among other anomalies, a small ring chromosome and double-minutes chromosomes. Using FISH and CGH techniques, it was found that the ring chromosome was a partial trisomy of chromosome 7, and the region implicated corresponded to 7p13-q21. Partial trisomies in glioblastoma could play an important role in defining those regions where genes implicated in this tumor process may be found. We studied the possible correlation of these findings with the tumoral phenotype.

Association of chromosome 7, chromosome 10 and EGFR gene amplification in glioblastoma multiforme.

Glioblastoma multiforme (GBM) is characterized by intratumoral heterogeneity in both histomorphological and genetic changes, displaying a wide variety of numerical chromosome aberrations, the most common of which are trisomy 7 and monosomy 10. The amplification of the epidermal growth factor receptor (EGFR) gene is the most frequently reported genetic abnormality. The associations between these parameters and their implication in the tumoral progression are poorly understood. We performed simultaneous fluorescence in situ hybridization (FISH) with centromeric DNA probes for chromosomes 7 and 10 in smear preparations, and EGFR gene amplification by PCR from 25 cases of GBM. Trisomy/ polysomy for chromosome 7 was present in 76% of cases and monosomy 10 in 68%. Both alterations were associated in 56% of cases. The EGFR gene was amplified in 52% of tumors; in 44% associated with trisomy/ polysomy 7, and in 36% with monosomy 10. The three parameters were associated together in 28% of cases. Kaplan-Meier survival rate analysis demonstrated lower survival rates in patients with monosomy 10, trisomy 7, and monosomy associated with trisomy 7. The other combinations were not different in frequency in relation to survival. In the present study, trisomy/polysomy 7 and monosomy 10 have been found to be frequently associated. The combination of both anomalies is probably important in the tumorigenesis of glioblastoma. Moreover, this association is apparently independent of EGFR gene amplification, which could be a later event in this process.

Meningioma: Un modelo de evolución citogenética en la iniciación y progresión tumoral / Meningioma: A model of cytogenetic evolution in tumoral initiation and progresion

Los meningiomas son tumores del sistema nervioso central con amplia heterogeneidad morfológica. Aunque son generalmente benignos, tienen la capacidad de evolucionar a un grado histológico mayor (atípico y anaplásico) que está relacionado con un incremento de su agresividad biológica y/o la capacidad de recidivar. Esta evolución se caracteriza a nivel citogenético por la monosomía total o parcial del cromosoma 22 en la etapa más temprana, seguida de cambios cromosómicos secundarios tanto numéricos como estructurales durante la progresión tumoral.En este trabajo presentamos una revisión sobre 85 casos de meningiomas, 43 benignos, 28 atípicos y 14 malignos, estudiando sus características clínicas, histopatológicas y citogenéticas, obteniéndose que la introducción de anomalías numéricas como la monosomía 10, 14 y 18, y anomalías estructurales como deleciones del cromosoma Ip están directamente relacionadas con los tumores de mayor agresividad, y especialmente, la combinación de alteraciones en el cromosoma Ip y 14 se presenta con mayor frecuencia en los meningiomas atípicos y anaplásicos. Estos hechos significan que la presencia de cariotipos complejos aumenta progresivamente desde los meningiomas de grado I a los meningiomas de grado III. Así mismo, estos cariotipos son los más habituales en los tumores recidivantes. (AU)

[Oligoastrocytoma with signet-ring cell differentiation. A morphological, ultrastructural and immunohistochemical study]. / Oligoastrocitoma con diferenciación en células en anillo de sello. Estudio morfológico, ultraestructrural e inmunohistoquímico.

We present a case of a mixed glial tumor (oligoastrocytoma) with signet-ring cells. This cellular feature is a rare differentiation in glial tumors of the central nervous system. Histological, immunohistochemical and ultrastructural findings have been analyzed. Signet-ring cells showed intense expression with GFAP, S-100 and vimentin. A differential diagnosis with other primary brain tumors and cerebral metástases with signet-ring cell differentiation was discussed.

Oligoastrocitoma con diferenciación en células en anillo de sello. Estudio morfológico, ultraestructural e inmunohistoquímico / Oligoastrocytoma with signet-ring cell differentiation. A morphological, ultrastructural and immunohistoche-mical study

Presentamos un caso de tumor glial mixto (oli-goastrocitoma) con células en anillo de sello. Esta diferenciación celular es rara en tumores gliales del sistema nervioso central. En este estudio analizamos las características morfológicas, ultraestructurales e inmunohistoquímicas del tumor. Las células neoplásicas con características morfológicas en anillo de sello mostraban expresión de GFAP, S-100 y vimentina. En la discusión consideramos el diagnóstico diferencial con otros tumores primarios del sistema nervioso central, así como con metástasis cerebrales de neoplasias con diferenciación en células en anillo de sello (AU)

[Meningioma: a model of cytogenetic evolution in tumoral initiation and progresion]. / Meningioma: un modelo de evolución citogenética en la iniciación y progresión tumoral.

Meningiomas are tumors of the central nervous system with a great morphological heterogeneity. They are generally benign, and have the capacity to progress to a higher histological grade (atypical and anaplastic), which is associated with an increase in biological aggressivity and/or capacity to recur. Citogenetically this evolution is characterized by total or partial monosomy 22 in the early phase, continued by numerical and structural changes during tumor progression. In this study, we present a review of 85 cases of meningiomas: 43 benign, 28 atypical and 14 anaplastic. We study the clinical and histopathological features, and their correlation with cytogenetie abnormalities present in these tumors. Numerical aberrations such as monosomy of chromosome 10, 14 and 18, and structural abnormalities such as deletions on 1p are directly associated with a higher agressivity of tumors. An association of aberatons on 1p and chromosome 14 are more commonly found in atypical and anaplastic meningiomas. These facts imply that the presence of complex karyotypes progressively increases from grade I to grade III meningiomas. Furthermore, these karyotypes are common in recurrent tumors.

Nasal glioma or nasal glial heterotopia? Morphological, immunohistochemical and ultrastructural study of two cases.

The term nasal glioma has been used to describe a congenital benign tumor of the nasal region containing neural tissue. The nature of these lesions remains open to controversy, because of the different locations of the heterotopic neural tissue involved, the deficient development of the bony structures and the persistence or not of the structural relations with the central nervous system. More recent terms define these lesions as ectopic nervous tissue. A clinical, morphological, ultrastructural and immunohistochemical study is made of two cases of nasal glioma, one associated with agenesis of the corpus callosum. In this case, the mother had been treated with clomiphene. In such cases, morphological and immunohistochemical findings support that "nasal glioma" remain valid as a descriptive term defining a congenital benign tumor composed of heterotopic neural tissue within the nasal region and covered by skin, that may recur following incomplete surgical resection.

Loss of 1p in recurrent meningiomas. a comparative study in successive recurrences by cytogenetics and fluorescence in situ hybridization.

Deletion of 1p is associated with histological progression to meningiomas. Detection of this alteration may be a predicting factor for recurrences in this tumor. We present 8 meningiomas from four patients: the original tumor and the first recurrence in one patient, and the first and second recurrences in the other three were studied. We compared results of monosomy 22 and deletion of chromosome 1p with cytogenetic methods and fluorescence in situ hybridization (FISH) analysis obtained from slides of direct preparations, of cultured cells and slides of touch preparations. The cytogenetic study showed normal chromosome 22 and deletion on 1p32 in both samples of one patient; only monosomy 22 in both recurrences in another patient, and normal karyotypes with different non-clonal anomalies in the other tumors. However, with FISH analysis, monosomy 22 in both recurrences of three patients was demonstrated, as well as the loss of 1p in all tumors. These results were more evident in the analysis of direct and touch preparations than in those of cultured cells.

Complex rearrangement of chromosomes 6 and 11 as the sole anomaly in atypical teratoid/rhabdoid tumors of the central nervous system.

Atypical teratoid/rhabdoid tumor of the central nervous system is a rare childhood tumor with a distinct histologic appearance and an aggressive clinical course. Few tumors have been analyzed cytogenetically. The only consistent chromosomal abnormality identified in some of these tumors has been monosomy or deletions of chromosome 22; in others, a normal chromosome 22 was present. The authors report an atypical teratoid/rhabdoid neoplasm of the central nervous system with a novel complex rearrangement affecting chromosomes 6 and 11 as the sole anomaly. The involvement of region 11p15 could be important in the pathogenesis of this entity.

Histopathological and cytogenetic findings in benign, atypical and anaplastic human meningiomas: a study of 60 tumors.

Meningiomas may display benign (grade I), atypical (grade II) and anaplastic (grade III) histopathological findings. The cytogenetic studies strongly suggest that secondary changes (beyond loss of chromosome 22) appear to be associated with more atypical features and with greater clinical aggressivity. We studied 60 tumors from 52 patients. Histopathological features such as nuclear pleomorphism, nucleolar prominence, mitosis, necrosis, cellular density, PCNA labeling index, and karyotype have been evaluated. The distribution in histological grades was: 50% benign, 33% atypical and 17% anaplastic meningiomas. Nuclear pleomorphism and nucleolar prominence showed a progressive increase in grades I, II and III. Multifocal micronecrosis was considered a criterion of malignancy. A significant correlation was observed between PCNA-LI, mitotic index and grades. Complex karyotypes increased progressively: benign (34% of cases), atypical (45% of cases) and anaplastic (70% of cases). The most common numerical alterations were losses of chromosomes 10, 14, 18 and 22. The chromosomes most often involved in structural anomalies were: 1, 4, 7, 14 and 22. Telomeric associations were present in four cases and double minutes in two cases. Prognostic criteria for these tumors have been analyzed on the basis of these data.

Histopathological and cytogenetic findings in benign, atypical and anaplastic human meningiomas: a study of 60 tumors.

Meningiomas may display benign (Grade I), atypical (Grade II) and anaplastic (Grade III) histopathological findings. The cytogenetic studies strongly suggest that secondary changes (moreover loss of chromosome 22) appear to be associated with more atypical features and with greater clinical aggressivity. We studied 60 tumors from 52 patients. Histopathological features such as nuclear pleomorphism, nucleolar prominence, mitosis, necrosis, cellular density, PCNA labeling index, and karyotype have been evaluated. Nuclear pleomorphism and nucleolar prominence showed a progressive increase in Grades I-III. Multifocal micronecrosis was considered a criterion of malignancy. A significant correlation was observed between PCNA-LI, mitotic index and grades. Complex karyotypes increased progressively: benign (34% of cases), atypical (45% of cases) and anaplastic (70% of cases). The most common numerical alterations were losses of chromosomes 10, 14, 18 and 22. The chromosomes most often involved in structural anomalies were: 1, 4, 7, 14 and 22. Telomeric associations was present in four cases and double minutes in two cases. Prognostic criteria for these tumors have been analyzed on the basis of these data.

Endometrial stromal sarcomas: immunohistochemical, electron microscopical and cytogenetic findings in two cases.

Uterine sarcomas are approximately 3% of all malignant uterine corpus tumours. Of these, the tumours that originate solely in the stromal elements of the uterine wall are infrequent and have not been well characterized cytogenetically. We report two cases of endometrial stromal sarcomas (ESS), one low grade and one high grade, diagnosed by conventional histology, immunocytochemistry, electron microscopy and cytogenetics. Morphologically clear-cut differential structures were seen at optical, immunohistochemical, and electron microscopic levels, permitting a clear differential diagnosis. The low-grade ESS expressed hormonal receptors and vimentin, whereas the high-grade ESS showed no hormone receptors, high Ki-67 activity, and occasional cytokeratin-positive cells. Ultrastructurally, no malignant epithelial differentiation was seen in the tumour cells, but cilia were found in both cases. Cytogenetic study of the low-grade ESS showed pseudodiploid karyotype with chromosomes 6 and 20 rearranged. The high-grade ESS showed a complex karyotype with clonal numerical and structural anomalies. The chromosomes involved in the structural rearrangements were 1, 3, 6, 7, 13, 14, 15, 17, 19, and 21.

New type of chimeric fusion product between the EWS and ATFI genes in clear cell sarcoma (malignant melanoma of soft parts).

We report a new case of clear cell sarcoma (CCS) harboring the t(12;22)(q13;q12). Reverse transcription-polymerase chain reaction (RT-PCR) analysis revealed the presence of a chimeric transcript between the EWS and ATFI genes, both in primary and metastatic tissue. Sequencing studies disclosed an in-frame fusion between EWS gene codon 265 and ATFI gene codon 110. This breakpoint has not been reported previously and indicates an important in vivo loss of EWS and ATFI gene domains, which could be associated with the unusually aggressive behavior of this tumor.