In vitro assessment of radiobiology of meningioma: A pilot study.

BACKGROUND: Meningioma are the second most common brain tumors in adults and can cause significant morbidity and mortality. The scarcity of in vitro and in vivo models represents the major obstacle to understand the molecular basis of meningioma tumorigenesis. The main aim of this study was to assess a method for radiobiology of meningioma cells colture by means of well-known meningioma lines. NEW METHOD: We carried out a protocol of cells culture for irradiation of meningioma cells. We used the immortalized cell lines IOMM-Lee and CH-157 to study their radiation-reponse by means of clonogenic assays and to evaluate their proliferation and apoptosis. We irradiated the cells with different total doses using two different linear accelerators. RESULTS: We observed a more radiation resistance of the IOMM-Lee than the CH-157. Indeed, the cellular death of CH-157 was obtained at a very low dose irradiation. Moreover, we showed a dose-response effect due to the early and late apoptosis, in fact the rate of apoptotic cells is greater than that of the necrotic cells at any dose of irradiation and at any time of analysis. COMPARISON WITH EXISTING METHODS: There is not a standardized method for radiobiology of meningioma experiments. CONCLUSIONS: Our method of cells culture appears suitable for radiosensitivity studies on meningioma. We can confirm that the response to radiotherapy depends not only on irradiation features, but also on tumor radiosensitivity.

Loss of CD45 cell surface expression in canine T-zone lymphoma results from reduced gene expression.

Canine T-zone lymphoma (TZL) is a peculiar lymphoma subtype characterized by an indolent clinical course and aberrant CD45-negative phenotype, commonly recognized by flow cytometry (FC). Recent studies have described clinical presentation and behavior, but to date the mechanisms behind the loss of CD45 protein expression have never been investigated. The aims of this study were: 1) to confirm the absence of CD45 in canine TZL via the concomitant use of FC and immunohistochemistry with two different sources of antibody; and 2) to investigate the amount of CD45 transcript and the presence of CD45 gene in the neoplastic cells of dogs affected by TZL. 57 lymph node aspirates were included in the present study: 40 (70.2%) TZLs, 7 (12.3%) high grade T-cell lymphomas and 10 (17.5%) reactive lymph nodes. Neoplastic cells and normal T-cells were isolated from TZL and reactive lymph nodes, respectively, via cell sorting. Immunohistochemistry was performed on 2 TZL, 2 reactive lymph nodes and 2 Peripheral T-cell Lymphomas. Total RNA and genomic DNA were extracted from lymph-nodes aspirates. Two different quantitative real-time PCR experiments were designed, to determine the amount of the CD45 transcript and of the corresponding gene fragment. All TZL cases were negative for CD45 at immunohistochemistry. CD45 transcript amount was significantly lower in TZL compared to controls (p<0.001). This difference was not significant (p=0.584) for CD45 DNA load, that was similar between TZL and controls. Moreover, CD45 transcript amount was inversely correlated with the percentage of neoplastic cells in each TZL sample (p=0.010). These results confirm that CD45 protein is lacking on cell surface irrespective of the technique and antibody source adopted. This phenotypic aberrancy is apparently due to the absence of gene transcription, as CD45 DNA was present, whereas CD45 transcript was virtually absent in the neoplastic cells. The data here reported support further studies investigating possible factors impairing CD45 gene transcription.

Human skin-derived fibroblasts used as a 'Trojan horse' for drug delivery.

BACKGROUND: Drug toxicity currently represents the main challenge of tumour chemotherapy. Our group recently developed a new method for drug delivery inspired by the 'Trojan Horse' concept. Human mesenchymal stem cells (hMSCs) have been shown to play the role of new 'horses' in delivering anti-tumour agents, without involving any genetic manipulation. As human stromal dermal fibroblasts (hSDFs) represent an interesting alternative to hMSCs, being easy to isolate, they could be an ideal candidate for this kind of procedure. AIM: To investigate whether hSDFs can take up and deliver paclitaxel (PTX) in sufficient concentrations to inhibit a very aggressive melanoma tumour (IgR39) in vitro. METHODS: hSDFs were primed with high doses of PTX, and then the effect of drug delivery on IgR39 melanoma proliferation in vitro was evaluated using several assays (antiproliferation, transwell cocultures, rosette assays and colony growth assays). Furthermore, the cell cycle and PTX uptake/release mechanism of hSDFs were studied both under both normal and hypoxic conditions. RESULTS: hSDFs incorporated PTX and then released it with unaffected pharmacological activity, inhibiting human IgR39 melanoma growth in vitro. The hypoxic conditions did not induce changes in cell cycle pattern and the uptake-release mechanism with PTX was not affected. CONCLUSIONS: hSDFs can be used as a Trojan horse, as the released drug was functionally active. These results indicated that these cells could be used for clinical treatment as the drug was released into the cellular environment and the primed cells underwent apoptosis.

Melanoma cells homing to the brain: an in vitro model.

We developed an in vitro contact through-feet blood brain barrier (BBB) model built using type IV collagen, rat astrocytes, and human umbilical vein endothelial cells (HUVECs) cocultured through Transwell porous polycarbonate membrane. The contact between astrocytes and HUVECs was demonstrated by electron microscopy: astrocytes endfeet pass through the 8.0 µm pores inducing HUVECs to assume a cerebral phenotype. Using this model we evaluated transmigration of melanoma cells from two different patients (M1 and M2) selected among seven melanoma primary cultures. M2 cells showed a statistically significant higher capability to pass across the in vitro BBB model, compared to M1. Expression of adhesion molecules was evaluated by flow cytometry: a statistically significant increased expression of MCAM, αvß3, and CD49b was detected in M1. PCR array data showed that M2 had a higher expression of several matrix metalloproteinase proteins (MMPs) compared to M1. Specifically, data suggest that MMP2 and MMP9 could be directly involved in BBB permeability and that brain invasion by melanoma cells could be related to the overexpression of many MMPs. Future studies will be necessary to deepen the mechanisms of central nervous system invasion.

Adipose tissue-derived stromal cells primed in vitro with paclitaxel acquire anti-tumor activity.

Many strategies, including those based on genetically modified Mesenchymal Stromal Cells (MSCs), have been developed in recent years in order to obtain high concentrations of anticancer drugs effective on tumor mass. In previous studies, we showed that human and murine bone marrow-derived MSCs (BM-MSCs) and human skin-derived stromal fibroblasts (hSDFs) acquired strong anti-tumor capacity, both in vitro and in vivo, once primed with Paclitaxel (PTX). In this report we investigate whether adipose tissue-derived MSCs (AT-MSCs) behave similarly to BM-MSCs in their uptake and release of PTX in sufficient amounts to inhibit tumor proliferation in vitro. According to a standardized procedure, PTX primed AT-MSCs (AT-MSCsPTX) were washed and then subcultured to harvest their conditioned medium, which was then tested to evaluate its in vitro anti-tumor potential. We observed that AT-MSCsPTX were able to uptake PTX and release it in a time-dependent manner and that the released drug was active in vitro against proliferation of leukemia, anaplastic osteosarcoma, prostatic carcinoma and neuroblastoma cell lines. These data confirm that AT-MSCs, as well as BM-MSCs, can be loaded in vitro with anti-cancer drugs. While the harvesting of BM-MSCs requires invasive procedures, AT-MSCs can be prepared from fat samples taken with little patient discomfort. For this reason, this source of stromal cells represents an important alternative to BM-MSCs in developing new tools for carrying and delivering anti-cancer drugs into tumor microenvironments.

Immunophenotype-related microRNA expression in canine chronic lymphocytic leukemia.

MicroRNAs (miRNAs) are posttranscriptional regulatory noncoding RNAs used to profile human hematopoietic tumors. In this study, some mature miRNAs was quantitated in peripheral blood from dogs with chronic lymphocytic leukemia (CLL). Relative expression data were normalised against four endogenous controls (let-7a, miR-17-5p, miR-26b, and miR-223) selected by geNorm analysis. The results revealed distinct miRNA patterns in CLL depending on the immunophenotype. Also in dogs, the different miRNAs expression could reflect developmental lineage and tumor differentiation. The similar genetics, physiology and exposure to environment in dogs and humans make the miRNA expression study in canine CLL attractive for comparative oncology.

ZAP-70 and Syk expression in canine lymphoid cells and preliminary results on leukaemia cases.

Zeta-chain-associated protein (ZAP-70) and spleen tyrosine kinase (Syk) are structurally and functionally homologous tyrosine kinases playing a role in the T- and B-cell signal transduction. Their activation can lead to lymphokine production, cytolitic activity, antibody secretion, cell proliferation, differentiation, survival and phagocytosis. Anomalous ZAP-70 and Syk expression is reported to be related to tumor formation and progression, and ZAP-70 immunoreactivity is a good prognostic marker of disease progression in human chronic lymphocytic leukaemia (CLL). Until now, to our knowledge, there are no reports about canine ZAP-70 and Syk expression profiles. In the present study, a RT-PCR procedure for the quali-quantitative evaluation of canine ZAP-70 and Syk transcripts was designed. The expression patterns of canine ZAP-70 and Syk mRNAs were evaluated in canine leukocyte subpopulations and in peripheral whole blood samples from healthy dogs and from dogs with different types of leukaemia. Similarly to humans, normal canine CD4+ and CD8+ T cells showed high expression of ZAP-70, whereas Syk was abundantly expressed in normal CD21+ B cells. The expression profile of ZAP-70 and Syk was markedly different in canine normal and leukaemic blood. Decreased Syk expression was detected in dogs with T-cell CLL, whereas decreased ZAP-70 expression was detected in dogs with B-cell CLL and B-cell acute lymphocytic leukaemia (ALL). The comparison of ZAP-70 and Syk mRNA levels between normal and leukaemic peripheral whole blood showed that the expression ratio ZAP-70/Syk is subjected to modification depending on the leukaemia status of patients. The results of the present work open an interesting topic for leukaemogenesis investigation and are the basis for further studies for a proper evaluation of the potential utility of these parameters for the diagnosis and prognosis of canine T- and B-cell leukaemia.

Pravastatin in vivo reduces mononuclear cell migration through endothelial monolayers.

The objective was to evaluate pravastatin modulation on peripheral blood mononuclear cell (PBMC) migration across endothelial monolayers. Eleven hypercholesterolaemic patients were treated with pravastatin 20 mg/day. At baseline (T0), after 40 days (T40) and after 6 months (T 6 months) of treatment total serum cholesterol, low-density lipoprotein (LDL), high-density lipoprotein, triglycerides, C-reactive protein, as well as tumour necrosis factor-alpha (TNF-alpha) and metalloproteinases-9 plasma levels were evaluated. At the same time points the effect of pravastatin on migration of PBMCs through a monolayer of murine brain endothelial cells was studied both in basal conditions and after endothelial stimulation with recombinant mouse TNF-alpha 10 ng/ml for 24 h. Seven volunteers were used as healthy controls. Significant decreases in total cholesterol, LDL and triglycerides as well as inhibition of transmigration were observed. PBMCs transmigration in patients prior to pravastatin therapy was higher than in healthy controls. These results suggest that pravastatin could be of benefit in a spectrum of diseases characterised by extravasation of PBMCs into the central nervous system.

Expression of drug resistance proteins Pgp, MRP1, MRP3, MRP5 and GST-pi in human glioma.

Chemotherapy in glioma is poorly effective: the blood-brain barrier and intrinsic and/or acquired drug resistance of tumor cells could partly explain this lack of major effect. We investigated expression of P-glycoprotein (Pgp), multidrug resistance protein (MRP) 1, MRP3, MRP5 and glutathione-S-transferase pi (GST-pi) in malignant glioma patients. Cytofluorimetric analysis of 48 glioma specimens and 21 primary cultures showed high levels of MRP1, moderate levels of MRP5 and low levels of Pgp, GST-pi and MRP3. Immunohistochemistry (25 glioma specimens) showed expression of GST-pi (66.7% of cases), MRP1 (51.3%), MRP5 (45.8%), Pgp (34.8%) and MRP3 (29.9%) in tumor cells. Moreover, analysis of tumor samples by real time quantitative PCR showed mRNA expression of all investigated genes. Tumor vasculature, analyzed in glioma specimens and in tumor derived endothelial cells, showed expression of all investigated proteins. Non-tumor brain samples (from a patient with arteriovenous malformation and from one with epilepsy), normal human astrocytes and cultured endothelial cells were also analyzed: astrocytes and endothelial cells expressed the highest levels of the investigated proteins, mainly MRP1 and MRP5. No significant differences in proteins expression were detected between primary or recurrent gliomas, suggesting that glioma chemoresistance is mostly intrinsic. Therefore, we detected, for the first time, the presence of MRP3 and MRP5 on glioma specimens--both in tumor and endothelial cells--and we delineated an expression profile of chemoresistance proteins in glioma. The possible association of inhibitors of drug efflux pumps with chemotherapy could be investigated to improve drugs delivery into the tumor and their cytotoxic effects.

Atypical transverse myelitis due to cytomegalovirus in an immunocompetent patient.

Cytomegalovirus (CMV)-associated transverse myelitis is rare in immunocompetent patients. We report a 73-year-old man with no evidence of immune compromise, who developed acute transverse myelitis. Cerebrospinal fluid pleocytosis indicated central nervous system inflammation, and spinal MRI showed weak signal hypointensity in T1, hyperintensity in T2 and DP between C7 and T2, but no contrast enhancement. High CSF anti- CMV IgG index with normal CSF IgG index indicated intact blood-brain barrier, and supported the diagnosis of CMV-induced myelitis in an immunocompetent patient.

Fotemustine combined with procarbazine in recurrent malignant gliomas: a phase I study with evaluation of lymphocyte 06-alkylguanine-DNA alkyltransferase activity.

The aims of this phase I study in patients with recurrent malignant gliomas were to determine the maximum tolerated dose (MTD) and toxicity profile of fotemustine when combined with a fixed dose of procarbazine (PCZ), and to evaluate the extent of O6-alkylguanine-DNA alkyltransferase (ATase) depletion in circulating lymphocytes during treatment. Sixteen patients received an induction cycle consisting of 100 mg/day oral PCZ for 12 consecutive days and a 1-h intravenous infusion of fotemustine given 4 h after PCZ on days 5 and 12 at escalated doses (50, 75, 100 and 125 mg/m2/day). After a 6-week rest period, a maximum of 4 maintenance cycles (PCZ 300 mg/day, 4 days; fotemustine, day 4) was given every 4 weeks. ATase activity was measured on days 1, 5 and 12 over 4 h after PCZ intake. Fifteen patients had previously received at least one nitrosourea-based chemotherapy, associated with PCZ in 12 cases. The MTD of fotemustine was 125 mg/m2 (days 5 and 12) with myelosuppression as the dose limiting toxicity (DLT). At this dose level, half of patients experienced grade 3 anemia, neutropenia or thrombopenia. No extra-hematological DLT was observed. No significant depletion of ATase activity by PCZ was evidenced. One partial response and 7 stable diseases were obtained leading to a disease control rate of 50%. The median times to progression and survival were 2.6 and 9.7 months, respectively. This combined regimen of PCZ and fotemustine was well tolerated with a good disease control rate in heavily pretreated glioma patients and merits further investigation in phase II studies.

Tumour necrosis factor microsatellites and HLA-DRB1*, HLA-DQA1*, and HLA-DQB1* alleles in Peruvian patients with rheumatoid arthritis.

OBJECTIVE: To study the association between rheumatoid arthritis (RA) and HLA and tumour necrosis factor (TNF) polymorphism in Peruvian mestizo patients in comparison with ethnically similar controls. METHODS: Seventy nine patients with RA and 65 ethnically matched healthy controls were genotyped for HLA-DRB1, HLA-DQA1, HLA-DQB1, and TNFalpha and TNFbeta alleles using PCR amplification. Clinical severity was assessed as mild, moderate, or severe in 35 of the patients. RESULTS: TNFalpha6 showed the strongest association with disease susceptibility. The TNFalpha6 allele was more common in patients than in controls (p<0.0076) and the proportion of patients with at least one copy of this allele was greater (p<0.015, relative risk 2.35). Among the HLA-DRB1* alleles with the shared epitope sequence, only the DRB1*1402 allele was significantly increased in patients compared with controls (p<0.0311), as was the proportion of patients with at least one copy of this allele (p<0.0232, relative risk 2.74). In contrast, the overall frequency of alleles with the shared epitope was not different in patients and controls. The haplotype HLA-DRB1*1402-DQB1*0301-DQA1*0401 was significantly more common in patients. TNFalpha6 was more common in patients whether or not they had this haplotype. None of the 11 patients lacking the TNFalpha6 allele had severe disease. CONCLUSIONS: This study shows for the first time that TNF gene polymorphism is associated with susceptibility to RA in a non-white population. TNFalpha6 and HLA-DRB1*1402 independently conferred significantly increased risk in Peruvian mestizo patients.

Modulation of experimental allergic encephalomyelitis in Lewis rats by administration of a peptide of Fas ligand.

The effects of modulation of apoptosis in experimental allergic encephalomyelitis (EAE) in Lewis rats have been investigated using a peptide of the Fas-Ligand protein (FasL-p). The peptide was administered both subcutaneously and intra-cerebro-ventricularly (i.c.v.) after EAE induction. Rats treated subcutaneously with FasL-p showed a worse clinical score as compared to saline treated animals, while i.c.v. treatment with FasL-p did not modify significantly the severity of EAE. Apoptotic lymphomonocytes (identified by TUNEL) infiltrating the brain and the spinal cord were decreased in rats treated i.c.v. with FasL-p. The data suggest that the Fas/Fas-ligand pathway may be modulated by treatments with peptides of Fas-Ligand and that it may be at work within the central nervous system in EAE.

Dying neural cells activate glia through the release of a protease product.

The close relationship between neurodegeneration and gliosis could play a relevant role in propagating the degenerative event in the brain. Although there is evidence of the neurotoxicity of activated glia, the ability of damaged neurons to modulate glial response remains unexplored. Exposure of primary glial cells to damaged or dead hippocampal neurons was followed by glial release of tumor necrosis factor-alpha (TNF-alpha). This release was reduced by a partial prevention of neural death. By contrast, no TNF-alpha was released when glial cells were exposed to damaged murine fibroblasts. Exposure of glial cells to the cerebrospinal fluid (CSF) of patients with Alzheimer's disease was also followed by TNF-alpha release, while the CSF of subjects with nondegenerative brain disorders evoked no response. These data suggest that damaged neurons both in vitro and in vivo release factor(s) that activate glial response. Heat treatment of sonicated neurons or use of a mixture of protease inhibitors, among them the caspase inhibitors Z-DEVD-FMK and Z-YVAD-FMK, prevented TNF-alpha release from glial cells. We conclude that a primary neurodegenerative event may induce glial response by releasing a neurospecific protein factor via activation of a caspase.

Modulation of fas-ligand (Fas-L) on human microglial cells: an in vitro study.

The expression of Fas-Ligand (Fas-L) on microglia could be relevant in multiple sclerosis immunopathology. The present study was performed to evaluate in vitro the expression of Fas-L in human microglial cells both unstimulated and after stimulation with IFN-gamma, beta-IFN-1b and beta-IFN-1b+IFN-gamma. Cells were stimulated for 6,12, 24 and 48 h. Surface Fas-L was evaluated by flow cytometry, total Fas-L by Western blot, whereas mRNA for Fas-L was measured by RT-PCR. We also evaluated the capacity of microglial cells to induce, in vitro, apoptosis on Fas-positive T leukemia Jurkat cells. Our results showed a constitutive expression of Fas-L on microglia. IFN-gamma downregulated the expression of the molecule, while beta-IFN-1b and beta-IFN-1b+IFN- gamma did not. The amount of surface Fas-L was related to the ability of microglial cells to induce apoptosis in Fas-positive target cells, which was partly inhibited by blockade of the Fas-Fas-L pathway.

Corticosteroids protect oligodendrocytes from cytokine-induced cell death.

The present data show that the simultaneous exposure to tumor necrosis factor-alpha (TNFalpha) and interferon-gamma (IFNgamma) induces cell death with characteristics of apoptosis in cultured rat oligodendrocytes; TNFalpha alone was ineffective. We have also demonstrated that different corticosteroids (aldosterone, deoxycorticosterone, dexamethasone and corticosterone) protect rat oligodendrocytes in culture from apoptosis induced by TNFalpha plus IFNgamma. This effect seems to be exerted via the interaction with both type I and type II corticosteroid receptors since all steroids considered are effective. Since oligodendrocyte apoptosis represents an important event in multiple sclerosis and in several demyelinating diseases, the present observations might be considered an interesting background for further researches directed to the possibility of controlling in vivo the death of these cells.

Tumor necrosis factor microsatellite polymorphisms in Italian glioblastoma patients.

Tumor necrosis factor (TNF) genes map on the short arm of chromosome 6 and have been described to contain several polymorphic regions, the most informative of which are TNFa (13 alleles) and TNFb (8 alleles) microsatellites. We analyze TNFa and TNFb microsatellite polymorphisms in 58 Italian patients with glioblastoma (GBL) and 95 unrelated healthy controls. At the TNFb locus, we detected a statistically significant decrease in the TNFb4 allele in GBL patients compared with the controls (P = 0.002; Pcorr = 0.015). Among the patients, 8 were homozygous TNFb4 (+/+), 23 were TNFb4 heterozygous (+/-), and 27 were negative for TNFb4 (-/-). In a comparison with the controls, we detected a statistically significant difference (P = 0.017). In fact, although no difference was detected in +/-, statistically significant differences were detected both for an increase in -/- and for a decrease in +/+ in the patient groups (P = 0.006 and P = 0.047, respectively). These data suggest that TNFb4-negative individuals might preferentially develop a Th2-type immune response that could lead to a reduction in antitumor activity.

Circulating intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1) and plasma thrombomodulin levels in glioblastoma patients.

Cellular adhesion molecules have been implicated in tumor progression and metastasis. Serum samples from 22 patients with glioblastoma (GBM), before surgery, and 19 sex and age matched healthy controls were analyzed for circulating levels of ICAM-1 and VCAM-1. At the same time also soluble plasma thrombomodulin, a marker of endothelial cell damage and activation, was detected. Soluble ICAM-1 and VCAM-1 levels were comparable in glioblastoma patients and healthy controls, while plasma thrombomodulin (TM) was significantly increased in cancer patients. There was no correlation between thrombomodulin levels and the presence of an intratumoral hemorrhage detected by CT scan, while entity of post-contrast enhacemement at CT correlated with higher TM levels. Further study with serial sampling of GBM patients and correlation with enhancement at CT will allow to ascertain the value of serum TM as a marker of disease recurrence or angiogenesis in those tumors.

Immunocompetence of human microvascular brain endothelial cells: cytokine regulation of IL-1beta, MCP-1, IL-10, sICAM-1 and sVCAM-1.

Endothelia from the brains of four patients undergoing neurosurgery, including one multiple sclerosis (MS) patient, were studied in vitro to determine cytokine and chemokine production; the release of soluble adhesion molecules was also investigated. The same procedure was repeated on human umbilical vein endothelial cells (HUVECs) in order to detect possible district-specific differences. After isolation, the endothelium was cultured and stimulated with gamma-interferon (IFN), tumour necrosis factor alpha (TNF-alpha) and LPS. The results showed that brain endothelium, in our experimental conditions, does not produce interleukin (IL)-10 and produces lower amounts of IL-1beta and soluble intercellular adhesion molecule-(sICAM-1) than HUVECs do; no differences were detected in soluble vascular cell adhesion molecule-(sVCAM-1) production. MCP-1 mRNA was detected both without and after stimulation with TNF-alpha and gamma-IFN in HUVECs and MS human brain endothelial cells (HBECs), while in non-MS-HBECs it was found only after gamma-IFN stimulation.