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Background: Mesenchymal stromal cells may represent an ideal candidate to deliver anti-cancer drugs. In a previous study, we demonstrated that exposure of mouse bone marrow derived stromal cells to Doxorubicin led them to acquire anti-proliferative potential towards co-cultured haematopoietic stem cells (HSCs). We thus hypothesized whether freshly isolated human bone marrow Mesenchymal stem cells (hMSCs) and mature murine stromal cells (SR4987 line) primed in vitro with anti-cancer drugs and then localized near cancer cells, could inhibit proliferation., Methods and Principal Findings: Paclitaxel (PTX) was used to prime culture of hMSCs and SR4987. Incorporation of PTX into hMSCs was studied by using FICT-labelled-PTX and analyzed by FACS and confocal microscopy. Release of PTX in culture medium by PTX primed hMSCs (hMSCsPTX) was investigated by HPLC. Culture of Endothelial cells (ECs) and aorta ring assay were used to test the anti-angiogenic activity of hMSCsPTX and PTX primed SR4987(SR4987PTX), while anti-tumor activity was tested in vitro on the proliferation of different tumor cell lines and in vivo by co-transplanting hMSCsPTX and SR4987PTX with cancer cells in mice. Nevertheless, despite a loss of cells due to chemo-induced apoptosis, both hMSCs and SR4987 were able to rapidly incorporate PTX and could slowly release PTX in the culture medium in a time dependent manner. PTX primed cells acquired a potent anti-tumor and anti-angiogenic activity in vitro that was dose dependent, and demonstrable by using their conditioned medium or by co-culture assay. Finally, hMSCsPTX and SR4987PTX co-injected with human cancer cells (DU145 and U87MG) and mouse melanoma cells (B16) in immunodeficient and in syngenic mice significantly delayed tumor takes and reduced tumor growth., Conclusions: These data demonstrate, for the first time, that without any genetic manipulation, mesenchymal stromal cells can uptake and subsequently slowly release PTX. This may lead to potential new tools to increase efficacy of cancer therapy.

Background: A fundamental problem in cancer research is identifying the cell type that is capable of sustaining neoplastic growth and its origin from normal tissue cells. Recent investigations of a variety of tumor types have shown that phenotypically identifiable and isolable subfractions of cells possess the tumor-forming ability. In the present paper, using two lineage-related human melanoma cell lines, primary melanoma line IGR39 and its metastatic derivative line IGR37, two main observations are reported. The first one is the first phenotypic evidence to support the origin of melanoma cancer stem cells (CSCs) from mutated tissue-specific stem cells; and the second one is the identification of a more aggressive subpopulation of CSCs in melanoma that are CXCR6+., Methods/findings: We defined CXCR6 as a new biomarker for tissue-specific stem cell asymmetric self-renewal. Thus, the relationship between melanoma formation and ABCG2 and CXCR6 expression was investigated. Consistent with their non-metastatic character, unsorted IGR39 cells formed significantly smaller tumors than unsorted IGR37 cells. In addition, ABCG2+ cells produced tumors that had a 2-fold greater mass than tumors produced by unsorted cells or ABCG2- cells. CXCR6+ cells produced more aggressive tumors. CXCR6 identifies a more discrete subpopulation of cultured human melanoma cells with a more aggressive MCSC phenotype than cells selected on the basis of the ABCG2+ phenotype alone., Conclusions/significance: The association of a more aggressive tumor phenotype with asymmetric self-renewal phenotype reveals a previously unrecognized aspect of tumor cell physiology. Namely, the retention of some tissue-specific stem cell attributes, like the ability to asymmetrically self-renew, impacts the natural history of human tumor development. Knowledge of this new aspect of tumor development and progression may provide new targets for cancer prevention and treatment.

UCB (human umbilical cord blood) contains cells able to differentiate into non-haematopoietic cell lineages. It also contains cells similar to primitive ESCs (embryonic stem cells) that can differentiate into pancreatic-like cells. However, few data have been reported regarding the possibility of expanding these cells or the differential gene expression occurring in vitro. In this study, we expanded formerly frozen UCB cells by treatment with SCF (stem cell factor) and GM-CSF (granulocyte-macrophage colony stimulating factor) in the presence of VPA (valproic acid). Gene expression profiles for beta cell differentiation and pluripotency (embryo stem cell phenotype) were analysed by RT-PCR and immunocytochemistry. The results show a dramatic expansion (>150-fold) of haematopoietic progenitors (CD45+/CD133+) which also expressed embryo markers of pluripotency (nanog, kfl-4, sox-2, oct-3/4 and c-myc), nestin, and pancreatic markers such as pax-4, ngn-3, pdx-1 and syt-1 (that is regulated by pdx-1 and provides the cells with a Ca++ regulation mechanism essential for insulin exocytosis). Our results show that UCB cells can be expanded to produce large numbers of cells of haematopoietic lineage that naturally (without the need of retroviral vectors or transposons) express a gene pattern compatible with endocrine pancreatic precursors and markers of pluripotency. Further investigations are necessary to clarify, first, whether in this context, the embryogenes expressed are functional or not, and secondly, since these cells are safer than cells transfected with retroviral vectors or transposons, whether they would represent a potential tool for clinical application.

The study of Lesch-Nyhan-diseased (LND) human brain is crucial for understanding how mutant hypoxanthine-phosphoribosyltransferase (HPRT) might lead to neuronal dysfunction. Since LND is a rare, inherited disorder caused by a deficiency of the enzyme HPRT, human neural stem cells (hNSCs) that carry this mutation are a precious source for delineating the consequences of HPRT deficiency and for developing new treatments. In our study we have examined the effect of HPRT deficiency on the differentiation of neurons in hNSCs isolated from human LND fetal brain. We have examined the expression of a number of transcription factors essential for neuronal differentiation and marker genes involved in dopamine (DA) biosynthetic pathway. LND hNSCs demonstrate aberrant expression of several transcription factors and DA markers. HPRT-deficient dopaminergic neurons also demonstrate a striking deficit in neurite outgrowth. These results represent direct experimental evidence for aberrant neurogenesis in LND hNSCs and suggest developmental roles for other housekeeping genes in neurodevelopmental disease. Moreover, exposure of the LND hNSCs to retinoic acid medium elicited the generation of dopaminergic neurons. The lack of precise understanding of the neurological dysfunction in LND has precluded development of useful therapies. These results evidence aberrant neurogenesis in LND hNSCs and suggest a role for HPRT gene in neurodevelopment. These cells combine the peculiarity of a neurodevelopmental model and a human, neural origin to provide an important tool to investigate the pathophysiology of HPRT deficiency and more broadly demonstrate the utility of human neural stem cells for studying the disease and identifying potential therapeutics.

Background: Identification of neoplastic cells in cerebrospinal fluid (CSF) by cytological analysis is the key diagnostic feature of leptomeningeal metastasis (LM). Because of the lack of sensitivity of this test, considerable efforts have been made to identify alternative diagnostic markers. Data from the literature suggest that measurement of tumor markers (TM) in CSF may be helpful for improving the diagnosis., Methods: We analyzed the concentrations of the TM carcinoembryonic antigen (CEA), CA15.3, CA125 and CA19.9 in both CSF and serum from 18 patients with neoplastic meningitis diagnosed by CSF cytology. We also performed these same measurements in 50 patients affected by other neurological diseases (OND) in order to evaluate putative intrathecal synthesis. In addition, CSF and serum concentrations of the proangiogenic factor VEGF (vascular endothelial growth factor) were evaluated., Results: All LM patients showed intrathecal synthesis for at least one TM. In one patient, a negative CSF cytology after treatment paralleled normalization of tumor marker synthesis. None of the OND patients displayed intrathecal TM synthesis. The VEGF Index (CSF/serum VEGF relative to CSF/serum albumin ratios) was significantly higher in LM patients compared with the control group. However, significant overlap between LM patients and values seen in those with OND was observed., Conclusions: Evaluation of intrathecal TM synthesis is a specific, sensitive, reliable, and reproducible diagnostic tool, and is useful to support diagnosis of carcinomatous meningitis.

A role for CD4(+)CD25(+) regulatory T cells (Tregs) in the control of allergic diseases has been postulated. We developed a mouse model in which anaphylaxis is induced in SJL mice by immunization and challenge with the fragment of self myelin proteolipid protein (PLP)(139-151), that is not expressed in the thymus, but not with fragment 178-191 of the same protein, that is expressed in the thymus. In this study, we show that resistance to anaphylaxis is associated with naturally occurring CD4(+)CD25(+) Tregs specific for the self peptide expressed in the thymus. These cells increase Foxp3 expression upon Ag stimulation and suppress peptide-induced proliferation of CD4(+)CD25(-) effector T cells. Depletion of Tregs with anti-CD25 in vivo significantly diminished resistance to anaphylaxis to PLP(178-191), suggesting an important role for CD4(+)CD25(+) Tregs in preventing the development of allergic responses to this thymus-expressed peptide. These data indicate that naturally occurring CD4(+)CD25(+) Tregs specific for a peptide expressed under physiological conditions in the thymus are able to suppress the development of a systemic allergic reaction to self.

Valproic acid (VPA) has been recently investigated for its anticancer properties in different tumors, including malignant gliomas. The aim of the present work was to evaluate the effects of VPA, alone or in combination with other chemotherapeutic drugs, on in vitro growth of human glioma cell lines. A172, U373, U138, U87, and SW1783 were treated with VPA alone or in combination with mitoxantrone, etoposide, or 1,3-bis(2-chloroethyl)-l-nitrosourea (BCNU). The effects of treatments on cell growth were assessed with crystal violet staining and analyzed using the combination index (CI). The percentage of apoptotic cells and the DNA content for cell cycle phases detection were also investigated by flow cytometry. Despite a certain variability, glioma cell lines were rather resistant to the drugs tested. Addition of VPA decreased the IC50 of the chemotherapeutic agents in all cell lines tested. This effect was more evident with BCNU. The synergic effect of the association of VPA and BCNU was related to an increased block of cell cycle with accumulation in S-G2/M phases of cell cycle rather than an increased programmed cell death. In our experimental model, VPA showed anticancer properties per se on human glioma cell lines and our data support the hypothesis that, if used in association with conventional chemotherapy, it might improve the effects of single chemotherapeutic agents.

We investigated in vitro the properties of selected populations of cancer stem-like cells defined as tumorospheres that were obtained from human glioblastoma. We also assessed their potential and capability of differentiating into mature cells of the central nervous system. In vivo, their tumorigenicity was confirmed after transplantation into the brain of non-obese diabetic/severe combined immunodeficient (NOD-SCID) mice. The angiogenic potential of tumorospheres and glioblastoma-derived cells grown as adherent cells was revealed by evaluating the release of angiogenic factors such as vascular endothelial growth factor and CXCL12 by ELISA, as well as by rat aortic ring assay. The proliferative response of tumorospheres in the presence of CXCL12 was observed for the first time. Multidrug resistance-associated proteins 1 and 3 as well as other molecules conferring multidrug resistance were higher when compared with primary adherent cells derived from the same tumor. Finally, we obtained cells from the tumor developing after grafting that clearly expressed the putative neural stem cell marker CD133 as shown by FACS analysis and also nestin and CXCR4. The cells' positivity for glial fibrillary acidic protein was very low. Moreover these cells preserved their angiogenic potential. We conclude that human glioblastoma could contain tumor cell subsets with angiogenic and chemoresistance properties and that this chemoresistance potential is highly preserved by immature cells whereas the angiogenic potential is, to a higher extent, a property of mature cells. A better understanding of the features of these cell subsets may favor the development of more specifically targeted therapies., ((c) 2006 Wiley-Liss, Inc.)

Since the intrinsic resistance of prostate carcinoma likely reflects a low susceptibility to drug-induced apoptosis, in this study we explored the possibility of sensitizing prostate carcinoma cells to apoptosis by combination of TRAIL with camptothecins. Indeed, these agents are known to activate different pathways of apoptosis. Topotecan- and gimatecan induced moderate up-regulation of TRAIL-R1 and -R2 which resulted in a different cell response to the combination in androgen-independent cells (DU-145 and PC-3). In DU-145 cells apoptosis was increased by lower TRAIL concentrations and was earlier than in PC-3 cells, as shown using Annexin V-binding assay. The relative resistance of PC-3 cells to drug-induced apoptosis was associated with constitutive Akt activation, higher levels of cFLIP-L and Bcl-2, and lower levels of Bax. The different expression/activation of apoptosis-related factors appears to influence the sensitization of prostate carcinoma cells by TRAIL. Potentiation of camptothecin-induced apoptosis by TRAIL appears dependent on cooperation between extrinsic and intrinsic pathways, as documented by loss of the sensitization to apoptosis following reduction of caspase 8 after small interfering RNA transfection. The efficacy of the approach may be critically dependent on the intrinsic susceptibility to apoptosis of different tumors. These observations support that the activation of multiple signals could enhance apoptotic response and suggest the therapeutic interest of the TRAIL/camptothecin combination.

Twitcher (GALC(twi/twi)) is the murine model of globoid cell leukodystrophy (GLD or Krabbe disease), a disease caused by mutations of the lysosomal enzyme galactocerebrosidase (GALC). To verify the therapeutic potential on twitcher of neural stem/progenitor cells (NSPC), we transduced them with a GALC lentiviral vector. Brain injection of NSPC-GALC increased survival of GALC(twi/twi) from 36.1 +/- 4.1 to 52.2 +/- 5.6 days (P < 0.0001). Detection of GALC activity and flow cytometry showed that NSPC-GALC and NSPC expressing the green fluorescent protein were attracted to the posterior area of twitcher brain, where demyelination occurs first. GALC(twi/twi) microglia, also more abundant in posterior regions of the brain, released significant amounts of the cytotoxic cytokine TNF-alpha when matched with NSPC-GALC. Thus, in murine GLD, and possibly in other demyelinating diseases, NSPC are attracted to regions of active demyelination but have limited survival and therapeutic potential if attacked by activated macrophages/microglia.

Malignant gliomas are associated with risk of thromboembolism, but the molecular link between tumor and peripheral pro-coagulant status has not been elucidated. Vascular Endothelial Growth Factor (VEGF), tissue-type Plasminogen Activator (tPA), Plasminogen Activator Inhibitor-1 (PAI-1) and lipoprotein (lp) (a) influence the pro-coagulant status. To assess whether the presence of the tumor influenced the peripheral levels of VEGF, tPA, PAI-1 and lp(a), we studied the expression and secretion of VEGF, tPA, PAI-1 and lp(a) in glioma specimens, in peripheral blood and in primary glioma-derived cultures. We also measured lp(a), VEGF, tPA and PAI-1 in the peripheral circulation of patients, before and after surgery for glioma. VEGF, tPA and PAI-1 were expressed in glioma specimens. Glioma cells were indeed a major source of tPA and PAI-1; these molecules were significantly more expressed in glioma than in patient's blood cells. Lp(a) was rarely expressed in glioma specimens and not expressed in blood cells. In glioma, VEGF, tPA and PAI-1 were localized mainly in tumor cells; tPA was localized also in the extracellular matrix and PAI-1 in tumor vascular lumen. Glioma cells were indeed able to produce and release VEGF, tPA and PAI-1. After surgery, peripheral levels of VEGF and PAI-1 were increased, while tPA and lp(a) were unchanged. The great amount of VEGF, tPA and PAI-1 produced by glioma could influence peripheral levels of these molecules. The partial resection of the tumor by surgery was not able to decrease plasma levels of these molecules.

Heparins represent the first choice for prevention and treatment of venous thromboembolism. In particular, low molecular weight heparins (LMWHs) provide pharmacokinetic advantages compared to unfractionated heparin (UFH): longer half-life, better bioavailability, and lower binding to plasma proteins. In the last years results of preclinical and clinical studies have suggested that LMWH may be able to inhibit cell growth, cell invasion, and angiogenesis, which are key mechanisms involved in tumor progression, possibly influencing favorable clinical outcome in at least a proportion of cancer patients. In this work we investigated the effect of LMWH (enoxaparin) on cell growth and cell invasion in primary cell cultures obtained from high-grade glioma specimens: 5 anaplastic astrocytoma (AA) and 13 glioblastoma multiforme (GBM). Apoptosis and expression of the thrombin receptor PAR1 were also assessed. A significant decrease in tumor cell growth was observed after treatment with 10 U/ml (-21%; p = 0.001) and 100 U/ml (-26%; p < 0.001); tumor cells from AA (grade III; WHO) were more affected by LMWH treatment compared to cell lines from GBM (grade IV; WHO). The antiproliferative effect was more pronounced in cell cultures displaying higher expression of PAR1. Glioma cell cultures were able to invade a model of basement membrane (Matrigel matrix) in standard culture conditions, but migration was not modulated significantly by LMWH treatment at any of the concentrations tested (1, 10, 100 U/ml). In conclusion, our results confirm the antineoplastic effect of LMWH, suggesting a potential direct role on tumor cell growth in high grade gliomas.

Background and Purpose: The gene-encoding plasminogen activator inhibitor type 1 (PAI-1) has a common 4G/5G 'functional' polymorphism, and people homozygous for the 4G allele have higher PAI-1 plasma concentrations. The 4G/4G genotype is associated with increased risk of myocardial infarction but paradoxically protects against stroke. We hypothesized that this paradox may be explained via an effect of the PAI-1 polymorphism on plasma lipids., Methods: We studied 71 consecutive Italian patients referred to our Institute for first stroke or vascular cognitive impairment. PAI-1 gene 4G/5G polymorphism, total plasma cholesterol, plasma triglycerides, sex, age, smoking, oral contraceptive use, statin therapy, hypertension, diabetes, and history of myocardial infarction were examined., Results: The 4G/4G genotype was significantly associated with high cholesterol (p = 0.003) but not with triglycerides (p = 0.39). Adjusted odds ratios were: 5.8 for 4G/4G vs. 4G/5G (95% CI, 3.1-23.0), and 15.9 for 4G/4G vs. 5G/5G (95% CI, 2.4-105.0)., Conclusions: This finding may explain the involvement of the PAI-1 polymorphism in the clustering of atherothrombotic risk factors, and why people with the 4G/4G genotype are at increased risk for myocardial infarction. Stroke is not so clearly related to hypercholesterolemia and other effects of the 4G/4G genotype (perhaps increased PAI-1 expression) may protect against stroke., (Copyright 2006 S. Karger AG, Basel.)

The survival of patients with malignant gliomas is still unsatisfactory despite multimodality treatment, therefore new therapeutic strategies are required. Tumor necrosis factor apoptosis related ligand (TRAIL/Apo2L), a member of the tumor necrosis factor superfamily, may induce apoptotic cell death in several tumors, but not in normal cells, upon binding with specific receptors. In the present study, the expression and function of TRAIL receptors (TRAIL-R1/DR4 and TRAIL-R2/DR5) has been investigated in five human glioma cell lines (U87, U138, U373, A172, SW1783) in ex vivo tumors and in primary cultures obtained from the tumors. Our data show that gliomas preferentially express TRAIL R2 and that treatment with topotecan, a topoisomerase I inhibitor, significantly up-regulates its expression as detected by flow cytometry and western blotting. Moreover, in most cases, treatment with topotecan resulted in an increased sensitivity to TRAIL-dependent apoptosis, although cyclohexymide had to be added to induce apoptosis. On glioma cell lines, the effects of irradiation on TRAIL receptors were also analysed. In our experimental conditions, irradiation with 2 Gy had a modest additive effect on TRAIL-dependent apoptosis and was not able to modulate TRAIL receptor expression.

The chiral antidepressant venlafaxine (VEN) is both a serotonin and a norepinephrine uptake inhibitor. CYP2D6 and CYP3A4 contribute to its metabolism, which has been shown to be stereoselective. Ten CYP2D6 genotyped and depressive (F32x and F33x, ICD-10) patients participated in an open study on the pharmacokinetic and pharmacodynamic consequences of a carbamazepine augmentation in VEN non-responders. After an initial 4-week treatment with VEN (195 +/- 52 mg/day), the only poor metabolizer out of 10 depressive patients had the highest plasma concentrations of S-VEN and R-VEN, respectively, whereas those of R-O-demethyl-VEN were lowest. Five non-responders completed the second 4-week study period, during which they were submitted to a combined VEN-carbamazepine treatment. In the only non-responder to this combined treatment, there was a dramatic decrease of both enantiomers of VEN, O-demethylvenlafaxine, N-desmethylvenlafaxine and N, O-didesmethylvenlafaxine in plasma, which suggests non-compliance, although metabolic induction by carbamazepine cannot entirely be excluded. The administration of carbamazepine [mean +/- SD, range: 360 +/- 89 (200-400) mg/day] over 4 weeks did not result in a significant modification of the plasma concentrations of the enantiomers of VEN and its O- and N-demethylated metabolites in the other patients. In conclusion, these preliminary observations suggest that the combination of VEN and carbamazepine represents an interesting augmentation strategy by its efficacy, tolerance and absence of pharmacokinetic modifications. However, these findings should be verified in a more comprehensive study.

CXCL12 (stromal cell-derived factor-1/CXCL12) regulates leukocyte, endothelial and hematopoietic precursor migration, bone-marrow myelopoiesis and angiogenesis. CXCL12 and its receptor CXCR4 are over-expressed in malignant gliomas, which are highly vascularized tumors with a poor prognosis. We studied the expression of CXCL12 and CXCR4 in glioma cell lines, endothelial cells, tissue sections and endocavitary fluids from patients with gliomas. We then analyzed the proliferative and the apoptotic effect of CXCL12 in endothelial cells and glioma primary cultures. We observed the release of CXCL12 in supernatants of human brain microvascular endothelial cells and at variable levels, in post-surgical endocavitary fluids. CXCL12 was expressed in both glioma and endothelial cells as assessed by immunostaining of surgical brain sections. CXCR4 was found in cells lines and primary cultures from malignant gliomas as well as in endothelial cells and was increased by vascular endothelial growth factor and basic fibroblast growth factor (bFGF). CXCL12 inhibited bFGF-induced proliferation of endothelial cells and increased the survival of endothelial cells. The survival of primary cells obtained from glioma specimens was also enhanced in the presence of CXCL12. We point out the presence and the release of CXCL12 in tumor microenvironment and we observed a modulating effect of CXCL12 on proliferation and survival of both endothelial and tumoral cells. Our data support in vivo studies suggesting a role in angiogenesis played by CXCL12, which could represent a possible prognostic factor.

Purpose: Deep venous thrombosis/pulmonary embolism (DVT/PE) is a frequent complication in the course of cancer, particularly in brain tumors. We investigated genetic and plasma factors possibly associated with risk of DVT/PE in patients with high-grade glioma., Experimental Design: In a case-control study, we studied polymorphisms of the genes coding for factor II (G20210A), factor V (G1691A), methylenetetrahydrofolate-reductase (C677T), tissue-type plasminogen activator (tPA; insertion/deletion), plasminogen activator inhibitor-1 (PAI-1; 4G/5G), and vascular endothelial growth factor (VEGF; C936T). We also measured plasma levels of D-dimer, lipoprotein (lp) (a), homocysteine, VEGF, tPA, and PAI-1, comparing healthy control patients with patients with glioma or with patients with neurological nonneoplastic disease (multiple sclerosis)., Results: Genotype frequencies of polymorphisms analyzed were similar in patients with glioma and in healthy matched population. D-dimer, lp (a), homocysteine, VEGF, tPA, and PAI-1 plasma levels were significantly higher in patients with glioma than in healthy controls, whereas patients having neurological nonneoplastic disease had plasma values of these molecules not significantly different from healthy controls. VEGF, tPA, and PAI-1 were also found at high-plasma levels in patients carrying genotypes that, in healthy controls, were associated with "low-producing" phenotypes., Conclusions: Genetic risk factors alone did not explain the high incidence of DVT/PE observed in patients with glioma. Higher plasma levels of molecules influencing the coagulation pathways indicate that the tumor itself might confer an increased risk of DVT/PE; thus, D-dimer, homocysteine, lp (a), VEGF, tPA, and PAI-1 look like good candidates to be evaluated as DVT/PE prognostic factors.

Despite progresses in surgery and treatments of malignant gliomas, prognosis of these tumors remains poor, with a median life expectancy of 12 months in glioblastomas. Chemotherapy (mostly with nitrosoureas) has been demonstrated to prolong overall survival, but the entity of this improvement is slight and disease recurrence/progression is the rule, stressing the need for multimodality treatment. In this work we investigated the effect of association of temozolomide (TMZ), an orally bioavailable alkylating agent, with three chemotherapeutic drugs, liposomal doxorubicin (DOXO), cis-platinum (CDDP). and topotecan (TP), on cell growth of A 172, U373, U138, U87, and SW1783 (all human glioma cell lines). Results indicate a synergistic effect (CI < 1) of TMZ in association with liposomal DOXO and CDDP on cell growth inhibition in most of the studied cell lines (A172, U373, U138, U87). Synergistic effect also has been obtained after treatment of A 172 and U373 with TMZ and TP in association. In conclusion, our results confirm the potential effect of association of chemotherapic drugs with different mechanisms of action in the treatment of gliomas.

Multiple sclerosis (MS) is a demyelinating disease of the central nervous system (CNS) mediated by blood-derived immune cells invading the CNS. This invasion could be determined by chemokines, and their role within the MS-affected brain is still poorly defined. We investigated the expression by RT-PCR and protein release by ELISA of the interferon-gamma (IFN-gamma)-inducible chemokines in human brain microvascular endothelial cells (HBMECs) and astrocytes. The monokine induced by IFN-gamma (Mig) behaves as a homing chemokine constitutively expressed in HBMECs and astrocytes, whereas the IFN-gamma-inducible 10-kDa protein (IP-10) and IFN-inducible T cell alpha-chemoattractant (I-TAC) are induced only after inflammatory stimuli. The biologic activity of IFN-gamma-inducible chemokines from an endothelial source was analyzed, and the transendothelial migration of activated lymphocytes was partly antagonized by specific antibodies, especially anti-Mig antibody. Our data highlight the capability of cells of the CNS to activate the chemoattractant machinery in a proinflammatory environment and in MS.

The expression of the death receptor Fas/CD95 is cell type-specific and can be modulated by different cytotoxic treatments. In spite of a frequent expression of Fas/CD95 in high-grade gliomas, these tumours are typically refractory to conventional therapy. Using a human glioblastoma cell line (GBM), we explored the possibility of modulating susceptibility to Fas/CD95-mediated apoptosis following cytotoxic treatment. GBM cells were sensitive to the antiproliferative effects of topoisomerase I inhibitors (topotecan and a novel lipophilic analog CPT83) and taxol, less sensitive to cisplatin and, in any case, rather resistant to drug-induced apoptosis. This pattern of cellular response was consistent with p53 mutation. GBM cells expressed low levels of Fas/CD95, which was associated with low susceptibility to antibody-stimulated Fas/CD95-mediated apoptosis. A significant up-regulation of Fas/CD95 expression was detected after exposure to topotecan and CPT83, whereas cisplatin induced a low increase and taxol did not modify Fas/CD95 expression. In addition, after treatment with topoisomerase I inhibitors, the up-regulation of Fas/CD95 resulted in an increased susceptibility of GBM cells to antibody-stimulated Fas/CD95-mediated apoptosis, while no synergistic effects were detected after treatment with cisplatin or taxol. Our data suggest that Fas/CD95 up-regulation can be a common response to DNA damage, whereas sensitisation to Fas/CD95-mediated apoptosis appears to be dependent on the type of DNA damage and on the pathway of cellular response. The observed effects might have important therapeutic implications for the design of novel therapeutic strategies in the treatment of malignant gliomas.

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., (Copyright 2001 Academic Press.)

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.

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.

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.

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., (Copyright 2000 Wiley-Liss, Inc.)

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.

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.

The association of primary sclerosing cholangitis (PSC) to HLA class II genes was studied by comparing patients from five different European populations. Deduced HLA-DRB1, DQA1, DQB1 haplotypes of 256 PSC patients from England, Italy, Norway, Spain and Sweden were compared to those observed in 764 ethnically-matched controls. Increased frequencies of the DRB1*03, DQA1*0501, DQB1*02 (RR=3.0, P<0.00001) and the DRB1*13, DQA1*0103, DQB1*0603 haplotypes (RR=2.4, P<0.0001) were observed in all five patient groups. A total of 16% of the PSC patients were homozygous for the DRB1*03, DQA1*0501, DQB1*02 haplotype compared to 1% of the controls (RR=20, P<0.0001). The DRB1*04, DQA1*03, DQB1*0302 haplotype was significantly reduced in frequency(RR=0.4, P<0.00001). Among Norwegian, Swedish and British patients that did not carry neither the DRB1*03, DQA1*0501, DQB1*02 nor the DRB1*13, DQA1*0103, DQB1*0603 haplotype, an increased frequency of the DRB1*15, DQA1*0102, DQB1*0602 haplotype was observed (RR=2.0, P<0.0001). Thus, PSC was found to be positively associated to three different HLA class II haplotypes (i.e. the DRB1*03, DQA1*0501, DQB1*02, the DRB1*15, DQA1*0102, DQB1*0602 and the DRB1*13, DQA1*0103, DQB1*0603 haplotypes) and negatively associated to one HLA class II haplotype (i.e. the DRB1*04, DQB1*0302 haplotype).

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.

Migraine, particularly migraine with aura (MA), may be a risk factor for ischemic stroke (IS). The reasons for this association are unknown. We investigated the presence of genetic abnormalities of the protein C system in 83 MA patients, 31 IS patients, and 124 healthy controls, all aged under 45 years. We found an increased frequency of activated protein C resistance due to Arg506Gln factor V mutation, and of protein S deficiency in both disorders, with figures higher than those reported in the general population and significantly different from those found in controls. These prothrombotic genetic abnormalities may be shared risk factors in IS and MA, and may play a role in increasing the risk of cerebrovascular disease in migraineurs.

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.

In order to investigate whether brain endothelial cells activation and/or damage could be selectively monitorized, soluble vascular cell adhesion molecule 1 (sVCAM-1) and thrombomodulin (TM) levels were studied in serum and cerebrospinal fluid (CSF) of 39 multiple sclerosis (MS) patients in various phases of the disease, 19 patients with other non-inflammatory neurological diseases (OND) and 15 patients with inflammatory neurological diseases (IND). No differences in sVCAM-1 CSF levels were detected, except for lower levels in IND compared to OND. Serum TM levels were lower in IND compared to progressive MS patients. Moreover, a significant decrease both in VCAM index and in TM index was detected in IND compared to all other groups. TM index was higher in MS patients in progression as compared to OND. The combined analysis of sVCAM-1 and TM might be a useful tool in monitoring brain endothelium activation or damage in different phases of MS.

The in vitro effects of dexamethasone (Dx) and low and high-dose 6-methylprednisolone (MP) on the expression of adhesion molecules ICAM-1,VCAM-1 and class II antigen HLA-DR on human brain endothelial cells (HBECs) was studied. HBECs were obtained from the surgical specimen of a multiple sclerosis patient undergoing brain surgery for vascular aneurysm. HBECs obtained from apparently normal brain capillaries of surgical specimens of two patients undergoing brain surgery for a meningioma and a low-grade glioma, respectively, were used as controls. The effect of steroids was studied both in the basal condition and after stimulation with proinflammatory cytokines (gamma-IFN and TNF-alpha). In order to detect possible endothelium local tissue specific differences, the experiment was repeated using human umbilical vein endothelial cells (HUVECs). Only high-dose MP was able to down-regulate TNF-alpha-induced VCAM-1 expression on endothelial cells.

CSF and serum levels of soluble Fas were studied in MS patients, in patients with various neurological diseases and in healthy controls. We did not detect differences in serum sFas levels between MS patients and controls. In CSF, despite sFas levels being similar in all patients studied, a statistically significant correlation between sFas CSF/sFas serum ratio and BBB damage (expressed as albumin CSF/albumin serum ratio) was detected in non-MS neurological disease, but not in MS patients. The normalized ratio (sFas CSF/sFas serum)/(Alb CSF/Alb serum) was significantly increased in MS patients compared to patients with non-inflammatory neurological disease suggesting an intrathecal synthesis of soluble Fas in MS. The percentage of apoptotic mononuclear cells was higher in CSF as compared to peripheral blood; moreover a lower proportion of apoptotic cells was found in CSF of MS patients. The findings lend support to the involvement of sFas in MS pathogenesis and suggest that a lower apoptosis in CSF may be a feature of the disease.

Methylprednisolone (MP) is a synthetic steroid commonly used in the treatment of multiple sclerosis (MS) relapses. It has a wide spectrum of activities on immune cells: it might also act by preventing mononuclear cell/endothelium adhesion. We studied adhesion phenomena between cultured human umbilical vein endothelial cells (HUVECs) and PBMNCs (CD45+, CD14+) from 6 MS patients treated in vivo with MP. We also studied fluctuations in CD11a and CD18 levels on lymphocytes and monocytes, as well as changes in serum sICAM-1 and sVCAM-1 concentrations. After MP treatment, PBMNCs adhesion to endothelium decreased at 3 h, while it went back to baseline levels at 24 h. A tendency to increase in both CD11a and CD18 on the surface of lymphocytes was detected, while an increase in serum sVCAM-1 was seen at 3 h.

The in vivo effects on the expression of adhesion molecules and on the adhesion between mononuclear cells and multiple sclerosis human brain endothelial cells (MS-HBECs) were investigated at the beginning of beta-IFN-1b treatment of MS patients. MS-HBECs were isolated from a surgical specimen obtained from an MS patient undergoing brain surgery for vascular aneurysm. 48 h after the first single administration of beta-IFN-1b, PBMNCs of 10 MS patients were analyzed for HLA-DR, CD11a, CD18 and VLA-4 expression and the adhesion between PBMNCs and both stimulated and unstimulated MS-HBECs evaluated. sICAM-1 and sVCAM-1 dosage in the serum of the patients was checked as well. The experiments were repeated using HUVECs in order to detect possible endothelial organ-specific differences. The experiments were also performed after six months of beta-INF-1b treatment on HUVECs. No significant effects on mononuclear cells/endothelium adhesion were detected at 48 h, but adhesion of PBMNCs to HUVECs decreased at six months. An increase in HLA-DR and VLA-4 and a decrease of CD18 was detected in monocytes. The serum level of sVCAM-1 increased at T2 and was still higher than at T0 at six months. The effect of the beta-IFN-1b treatment on both MS-HBECs and HUVECs, was selectively studied in vitro by testing the expression of cytokine-induced adhesion molecules HLA-DR, ICAM-1 and VCAM-1. The in vitro experiments confirmed that beta-IFN-1b is able to antagonize gamma-IFN-induced HLA-DR expression on MS human brain endothelial cells without relevant effects on VCAM-1 and ICAM-1.

A non-radioactive technique for the typing of tumour necrosis factor (TNF) microsatellite alleles has been applied in the study of an Italian population. The frequency of the TNFa 10 allele was markedly increased compared with other European populations.

The ability of human brain endothelial cells to produce mRNA for interleukin-10, and release IL-10 in culture supernatants after in vitro stimulation with LPS, TNF-alpha and gamma-IFN was assessed and compared to that of astrocytes, peripheral blood mononuclear cells and human umbilical vein endothelial cells. IL-1 beta and beta 2-microglobulin release were also analysed. IL-10 and TNF-alpha mRNA presence was investigated in normal brain as well as in three plaques from two multiple sclerosis patients. While increased IL-1 beta and beta 2-microglobulin release in the supernatants of stimulated cells could be detected in all the studied cell lineages, IL-10 mRNA and protein release was only seen in LPS-stimulated PBMNCs. Similarly, mRNA for IL-10 was not detected in CNS tissues, while TNF-alpha was present in all plaques. The lack of production of significant amounts of IL-10 by astrocytes and human brain endothelial cells suggests that these cells may not be the primary source of in vivo IL-10-mediated down-regulation of immune reactions within the central nervous system.

Eight patients affected by central nervous system tumours disseminating via cerebrospinal fluid received rIL-2 immunotherapy according to three different protocols involving intrathecal administration followed or not by systemic infusion. Immunological monitoring included serial evaluation of CSF leukocytes, CSF and peripheral blood CD3-CD56+ cells, and NK activity. The most marked rise in CSF leukocyte levels was induced by daily intrathecal rIL2 administration, which also induced increased PB NK activity. Systemic rIL2 infusion following intrathecal treatment maintained a high percentage of CSF CD3-CD56+ cells, but not CSF leukocytes at high levels. Clinical conditions improved after treatment in two patients, worsened in one and remained substantially unchanged in the remaining five. The side effects of intrathecal rIL2 treatment included fever, confusion, and seizures, and there were marked interindividual variations in the immunological response.

We have analyzed the distribution of tumor necrosis factor (TNF) a and -b microsatellite alleles in HLA-DQ and -DR typed Swedish patients with multiple sclerosis (MS) (n = 122) and ethnically matched control subjects (n = 178). We found significant differences in the frequencies of TNFa and TNFb alleles between patients and controls. TNFaII was significantly associated with MS. This was also the case for the combination of TNFaII with TNFb4. However, TNFaII (alone or in combination with TNFb4) did not show any disease association independent of DQA1*0102/ DQB1*0602/DR2, whereas the previously reported strong association with HLA-DQA1*0102/DQB1*0602/DR2 in Scandinavian populations was confirmed. Therefore the association of TNFaII (and TNFb4) is most likely secondary to the increase of DQA1*0102/DQB1*0602/DR2 in MS patients. The proportion of TNFa6 positive individuals was lower among DR2-negative MS patients than among DR2-negative controls (P = 0.08). Since the presence of the TNFa6 allele correlates with low TNF alpha production in response to lipopolysaccharide, it could be speculated that DR2-negative MS patients have an increased risk of being high TNF alpha producers in response to exogenous stimuli.

We studied the allelic constitution at the HLA class II DQA1, DQB1, DRB1 and DPB1 in 94 Italian multiple sclerosis (MS) patients and 98 controls. No significant increase in the frequency of DR2 alleles was detected among MS patients, as previously observed both in European and some Italian studies. A slight increase was found for the DQA1*0301 and DQB1*0602 alleles in the MS patients. No significant association was found with the glutamine residue at position 34 of the DQ alpha chain, which was noted previously in MS patients from northern Europe.

The immunological therapy of cancer has been proposed in a number of neoplasms (Borden, Sondel, 1989; Foon, 1989; Rosenberg, 1992) and has recently been adopted in the treatment of Central Nervous System (CNS) tumors in combination with conventional surgical and radiotherapeutical approach. In this context, loco-regional administration of immunomodulating agents (for instance in post-surgical cavity) allows to achieve much higher in situ concentrations than by systemic route. Since these treatments have potential adverse effects, careful assessment of clinical and immunological parameters in phase I trials is needed. CNS tumors disseminating via Cerebrospinal Fluid (CSF) pathways offer a stimulating opportunity for intrathecal immunotherapy. In this context, alpha-IFN and IL2 (alone or in combination with LAK cells) have been employed either loco-regionally or intrathecally (Merchant, Mc Vicar, Merchant & Young, 1992; Schiller, Hank, Storer, Borchert, Moore, Albertini, Bechhofer, Wesley, Brown, Bastin & Sondel, 1993). The rationale for the use of both these substances includes the known anti-tumor action of alpha-IFN (Mahaley, Urso, Whaley, Blue, Williams, Guaspari & Selker, 1985; Nagai, 1988) and the ability of r-IL2 to generate activated cells effective in lysing tumor cell targets (Hayes, Moore, Pierz, Chen, Da Rosso, Nirenberg & Allen, 1993). We treated 3 patients (2 affected by disseminating cerebellar medulloblastoma, 1 by disseminating thalamic glioblastoma) by intrathecal r-IL2 via recervoir. In the first 2 patients, this treatment was preceded by alpha-IFN (also intrathecally). Monitoring of immunological effects of the treatment schedule involved kinetics of CSF and serum TNF-alpha, IL2s and IL2R during the first day of r-IL2 treatment, as well as on day +2 and +4 of both r-IL2 cycles, and assessment of CSF cells, protein and CSF and PB NK cell activity and CD3-CD56+ cells during the course of all treatment cycles. We also assessed clinical and neuroradiological effects of immunotherapy.