Effect of canine mesenchymal stromal cells loaded with paclitaxel on growth of canine glioma and human glioblastoma cell lines.

This study investigated whether canine mesenchymal stromal cells (cMSCs) are able to take up and release paclitaxel (PTX) in active form, and therefore whether they have potential as a tool for therapeutic delivery of this drug. cMSCs from bone marrow and adipose tissue were isolated, expanded and characterised phenotypically. cMSCs were loaded with PTX (cMSCs-PTX) and their capacity for release of PTX was determined by their effect on proliferation of cancer cells. cMSCs-PTX derived from bone marrow and adipose tissue were able to take up and then release active PTX. cMSCs-PTC inhibited proliferation of the canine glioma cell line J3T, and the human glioblastoma cell lines T98G and U87MG. The potential of canine cMSCs-PTX for treatment of canine gliomas should be investigated further.

Genome-wide DNA methylation profiling of recurrent and non-recurrent chordomas.

Chordomas are an aggressive rare type of malignant bone tumors arising from the remnant of the notochord. Chordomas occur mainly in vertebral bones and account for 1-4% of malignant bone tumors. Management and treatment of chordomas are difficult as they are resistant to conventional chemotherapy; therefore, they are mainly treated with surgery and radiation therapy. In this study, we performed DNA methylation profiling of 26 chordomas and normal nucleus pulposus samples plus UCH-1 chordoma cell line using the Illumina Infinium HumanMethylation450 BeadChips. Combined bisulfite restriction analysis and bisulfite sequencing was used to confirm the methylation data. Gene expression was analyzed using RT-PCR before and after 5-aza-2'-deoxycytidine (5-azaDC) treatment of chordoma cell lines. Analysis of the HumanMethylation450 BeadChip data led to the identification of 8,819 loci (2.9%) that were significantly differentially methylated (>0.2 average ß-value difference) between chordomas and nucleus pulposus samples (adjusted P < 0.05). Among these, 5,868 probes (66.5%) were hypomethylated, compared to 2,951 (33.5%) loci that were hypermethylated in chordomas compared to controls. From the 2,951 differentially hypermethylated probes, 33.3% were localized in the promoter region (982 probes) and, among these, 104 probes showed cancer-specific hypermethylation. Ingenuity Pathway Analysis indicates that the cancer-specific differentially methylated loci are involved in various networks including cancer disease, nervous system development and function, cell death and survival, cellular growth, cellular development, and proliferation. Furthermore, we identified a subset of probes that were differentially methylated between recurrent and non-recurrent chordomas. BeadChip methylation data was confirmed for these genes and gene expression was shown to be upregulated in methylated chordoma cell lines after treatment with 5-azaDC. Understanding epigenetic changes in chordomas may provide insights into chordoma tumorigenesis and development of epigenetic biomarkers.

Stemness and osteogenic and adipogenic potential are differently impaired in subcutaneous and visceral adipose derived stem cells (ASCs) isolated from obese donors.

Today adipose tissue is not just considered as the primary energy storage organ, but it is also recognized as an important endocrine tissue and an abundant source of mesenchymal stem cells (adipose-derived stem cells, ASCs). During the last decade, several studies have provided preclinical data on the safety and efficacy of ASCs, supporting their use in cell-based therapy for regenerative medicine purposes. Little is known about the effect of obesity on ASCs properties. Since ASCs differentiation and proliferation are determined by their niche, the differences in body fat distribution and the obesity-related co-morbidities may have several consequences. In this study we compared ASCs of subcutaneous adipose tissue from obese (obS-ASCs) and non-obese (nS-ASCs) donors in order to compare their immunophenotype and osteogenic and adipogenic potential. Moreover, in order to evaluate the possible difference between subcutaneous and visceral fat, obS-ASCs were also compared to ASCs derived from visceral adipose tissue of the same obese donors (obV-ASCs). Our results show that subcutaneous and visceral ASCs derived from obese donors have an impaired cell proliferation, clonogenic ability and immunophenotype. Nevertheless, obS-ASCs are able to differentiate toward osteogenic and adipogenic lineages, although to a small extent with respect to non-obese donors, whereas obV-ASCs lose most of their stem cell characteristics, including multi-differentiation potential. Taken together our findings confirm that not all ASCs present the same behavior, most likely due to their biological microenvironment in vivo. The specific stimuli which can play a key role in ASCs impairment, including the effects of the obesity-related inflammation, should be further investigated to have a complete picture of the phenomenon.

Two bone substitutes analyzed in vitro by porcine and human adipose-derived stromal cells.

Nowadays, the repair of large bone defects is an important goal in orthopaedic and dental fields. Tissue engineering, applied to increase the bone regeneration process, combines suitable scaffolds with either terminally differentiated cells or Mesenchymal Stromal Cells. In vitro studies with Adipose-derived Stromal Cells (ASCs) may identify new bioactive supports, to be tested in preclinical model. In this study, we evaluated the biocompatibility and the osteoinductive properties of two bone substitutes, RegenOSS (RO-1) and a new generation scaffold (RO-2), on both porcine and human ASCs. Porcine ASCs need a prolonged initial phase to adapt to both substitutes; indeed, their growth was initially reduced respect to cells cultured in their absence. In contrast, human ASCs were not negatively affected. However, no toxicity of RO-1 and -2 was observed on both ASC populations which are able to stick to both biomaterials. RO-1 and -2 supported osteogenic differentiation of porcine and human ASCs in a different manner: the presence of RO-1 up-regulated both alkaline phosphatase (ALP) activity and collagen production of human ASCs, whereas in porcine ASCs, RO-2 seemed to up-regulate ALP activity, while the production of collagen is mainly stimulated by the presence of RO-1. We suggest to use not just human ASCs, but also animal ones to select suitable scaffolds to generate bio-constructs in vitro, which then need to be tested in animal model before reaching the market.

Reversine increases multipotent human mesenchymal cells differentiation potential.

Among different human stem cell sources, adult mesenchymal stem cells from bone marrow (BMSCs), and more recently from adipose tissues (ASCs), have shown their capability to differentiate into a variety of different cell types, including osteoblasts, adipocytes, and muscle cells. However, mesenchymal stem cell differentiation toward certain cell types (including skeletal and cardiac muscle), while shown to be achievable, still suffers of low yields and needs to be greatly improved before any therapeutic application could be foreseen. A possible way of achieving this goal is by using a chemical-pharmacological approach to increase stem cell plasticity. Along this line, we envisioned the possibility of pre-treating BMSCs and ASCs with reversine, a synthetic purine that has been shown to induce adult cells de-differentiation. In the current study we tested reversine effects on both BMSCs and ASCs to increase their differentiation toward osteoblasts, smooth and skeletal muscle cells. Reversine pre-treatment, at very low concentration (50 nM), caused a marked increase in the differentiation yields of both BMSCs and ASCs.

Enhanced biological performance of human adipose-derived stem cells cultured on titanium-based biomaterials and silicon carbide sheets for orthopaedic applications.

It is well known that the surface properties of biomaterials may affect bone-healing processes by modulating both cell viability and osteogenic differentiation. In this study we evaluated proliferation and osteogenic differentiation of human adipose-derived stem cells (hASCs) cultured on three prototypes of titanium disks and on thin layers of silicon carbide (SiC-PECVD), a material characterized by a high hardness and wear resistance. Our data indicated that all the tested surfaces supported cell growth, in particular, hASCs seeded on both titanium treated by a double-step etching process (TIT) and titanium modified by two Anodic Spark Deposition processes (TAA) grew better respect to the ones cultured on titanium obtained by KOH alkali etching process on TAA (TAAK). Furthermore, hASCs well colonized SiC-PECVD surface, showing a quite similar viability to cells cultured on plastic (PA). TIT and TAA better supported osteogenic differentiation of hASCs compared to PA, as shown by a marked increase of both alkaline phosphatase activity and calcified extracellular matrix deposition; in contrast TAAK did not positively affect hASCs differentiation. SiC-PECVD did not alter osteogenic differentiation of hASC cells: indeed, ALP and calcium deposition levels were comparable to those of cells cultured on plastic. Furthermore, we observed similar results testing hASCs either pre-differentiated for 14 days in osteogenic medium or directly differentiated on biomaterials. Our study suggests that modifications of titanium surface may improve osteo-integration of implant devices and that SiC-PECVD may represent a valid alternative for the coating of prosthetic devices to reduce wear and metallosis events.

Role of autologous rabbit adipose-derived stem cells in the early phases of the repairing process of critical bone defects.

Adipose-derived stem cells (ASCs) may represent a novel and efficient tool to promote bone regeneration. In this study, rabbit ASCs were expanded in culture and used for the regeneration of full-thickness bone defects in the proximal epiphysis of tibia of 12 New Zealand rabbits. Defects were implanted with graft material as follows: untreated (control), empty hydroxyapatite (HA) disk, ASCs alone, and HA disk seeded with ASCs. Each isolated ASCs population was tested in vitro: they all showed a high proliferation rate, a marked clonogenic ability, and osteogenic differentiation potential. Eight weeks after implantation, macroscopic analyses of all the samples showed satisfactory filling of the lesions without any significant differences in term of stiffness between groups treated with or without cells (p > 0.05). In both the scaffold-treated groups, a good osteointegration was radiographically observed. Even if HA was not completely reabsorbed, ASCs-loaded HA displayed a higher scaffold resorption than the unloaded ones. Histological analyses showed that the osteogenic abilities of the scaffold-treated defects was greater than those of scaffold-free samples, and in particular new formed bone was more mature and more similar to native bone in presence of ASCs. These results demonstrated that autologous ASCs-HA constructs is a potential treatment for the regeneration of bone defects.

Human adipose-derived stem cells as future tools in tissue regeneration: osteogenic differentiation and cell-scaffold interaction.

Tissue engineering is now contributing to new developments in several clinical fields, and mesenchymal stem cells derived from adipose tissue (hASCs) may provide a novel opportunity to replace, repair and promote the regeneration of diseased or damaged musculoskeletal tissue. Our interest was to characterize and differentiate hASCs isolated from twenty-three donors. Proliferation, CFU-F, cytofluorimetric and histochemistry analyses were performed. HASCs differentiate into osteogenic, chondrogenic, and adipogenic lineages, as assessed by tissue-specific markers such as alkaline phosphatase, osteopontin expression and deposition of calcium matrix, lipid-vacuoles formation and Glycosaminoglycans production. We also compared osteo-differentiated hASCs cultured on monolayer and loaded on biomaterials routinely used in the clinic, such as hydroxyapatite, cancellous human bone fragments, deproteinized bovine bone granules, and titanium. Scaffolds loaded with pre-differentiated hASCs do not affect cell proliferation and no cellular toxicity was observed. HASCs tightly adhere to scaffolds and differentiated-hASCs on human bone fragments and bovine bone granules produced, respectively, 3.4- and 2.1-fold more calcified matrix than osteo-differentiated hASCs on monolayer. Moreover, both human and deproteinized bovine bone is able to induce osteogenic differentiation of CTRL-hASCs. Although our in vitro results need to be confirmed in in vivo bone regeneration models, our data suggest that hASCs may be considered suitable biological tools for the screening of innovative scaffolds that would be useful in tissue engineering.

Nefopam, an analogue of orphenadrine, protects against both NMDA receptor-dependent and independent veratridine-induced neurotoxicity.

Nefopam hyghochloride is a potent analgesic compound commercialized in most Western Europe for 20 years, which possesses a profile distinct from that of opioids or anti-inflammatory drugs. Previous evidence suggested a central action of nefopam but the detailed mechanisms remain unclear. While, nefopam structure resembles that of orphenadrine, an uncompetitive NMDA receptor antagonist, here we report that differently from orphenadrine, nefopam (100 microM) failed to protect cultured cerebellar neurons from excitotoxicity following direct exposure of neurons to glutamate. Moreover, nefopam failed to displace MK-801 binding to hippocampal membranes. Nefopam effectively prevented NMDA receptor-mediated early appearance (30 min) of toxicity signs induced by the voltage sensitive sodium channel (VSSC) activator veratridine. The later phase (24 h) of neurotoxicity by veratridine occurring independently from NMDA receptor activation, was also prevented by nefopam. Nefopam effect was not mimicked by the GABA receptor agonist muscimol.

Novel effect of nefopam preventing cGMP increase, oxygen radical formation and neuronal death induced by veratridine.

Nefopam hydrochloride is a potent analgesic compound that possesses a profile distinct from that of opiods or anti-inflammatory drugs. Previous evidence suggested a central action of nefopam but the detailed mechanisms remain unclear. Here we have used cultured cerebellar neurons to test the hypothesis that nefopam may modulate voltage sensitive sodium channel (VSSC) activity. Nefopam (100 microM) effectively prevented NMDA receptor-mediated early appearance (30 min) of toxicity signs induced by the VSSC activator veratridine. Delayed neurotoxicity by veratridine occurring independently from NMDA receptor activation, was also prevented by nefopam. In contrast, excitotoxicity following direct exposure of neurons to glutamate was not affected. Neuroprotection by nefopam was dose-dependent. 50% protection was obtained at 57 microM while full neuroprotection was achieved at 75 microM nefopam. Veratridine-induced sodium influx was completely abolished in nefopam-treated neurons. Intracellular cGMP and oxygen radical formation following VSSC stimulation by veratridine were also effectively prevented by nefopam. Our data are consistent with an inhibitory action of nefopam on VSSC and suggest that nefopam may modulate the release of endogenous glutamate following activation of these channels. This novel action of nefopam may be of great interest for the treatment of neurodegenerative disorders involving excessive glutamate release and neurotransmission.

Alpha-melanocyte-stimulating hormone peptides inhibit HIV-1 expression in chronically infected promonocytic U1 cells and in acutely infected monocytes.

The purpose of the present research was to determine if alpha-melanocyte-stimulating hormone (alpha-MSH) and its C-terminal tripeptide [alpha-MSH (11-13), KPV] alter HIV expression in infected cells. The results indicate that chronically HIV-1-infected promonocytic U1 cells produce alpha-MSH and that immunoneutralization of the endogenous peptide enhances HIV expression. Because U1 cells express the alpha-MSH receptor 1 (MC1R), an autocrine-inhibitory circuit based on the peptide and its receptor likely occurs in these cells. To determine effects of pharmacological concentrations of alpha-MSH peptides on HIV expression, we measured p24 antigen release by TNF-alpha-stimulated U1 cells exposed to a wide range of concentrations of synthetic alpha-MSH and KPV. Viral expression was reduced by both peptides. KPV also effectively reduced HIV replication in acutely infected monocyte-derived macrophages (MDM). The basis of the peptide influence on viral replication is at the transcriptional level; KPV inhibited activation of NF-kappaB that is known to enhance viral expression. Endogenous alpha-MSH likely contributes to natural defense against HIV. However, greater concentrations of synthetic peptide are much more effective in reducing HIV expression in infected cells.

Allergy-associated FcRbeta is a molecular amplifier of IgE- and IgG-mediated in vivo responses.

A role for the Fc receptor beta chain (FcRbeta) in the pathogenesis of allergy has been suggested by genetic studies. FcRbeta is a subunit common to the high-affinity IgE (FcepsilonRI) and low-affinity IgG (FcgammaRIII) receptors, both of which contribute to the initiation of allergic reactions. Current in vitro data suggest that FcRbeta can function as either a positive or negative regulator, leaving a mechanistic explanation for its association with the development of atopy unclear. To address this controversy, we have generated novel mouse models relevant to human Fc receptor function. Analysis of FcepsilonRI- and FcgammaRIII-dependent responses in these mice provides unequivocal genetic evidence that FcRbeta functions as an amplifier of early and late mast cell responses and, remarkably, in vivo anaphylactic responses.

Anaphylaxis mediated through a humanized high affinity IgE receptor.

Mast cells and basophils, which are activated by IgE and allergens through the high affinity IgE receptor (Fc epsilon RI), play a prominent role in anaphylaxis in the mouse. Mice deficient in this receptor become resistant to passive anaphylaxis. As a first step in developing an in vivo model that more closely mimics the IgE-mediated responses in man, we used a combination of transgenic and embryonic stem cell technology to generate a mouse line in which the murine Fc epsilon RI alpha-chain has been replaced with its human homologue. We demonstrate here that these mice express a tetrameric high affinity IgE receptor, in which the human alpha-chain associates with the murine beta- and gamma-chains, and that upon triggering with relevant Ag, this receptor mediates the initiation of the expected intracellular events. In addition, we show that the human alpha-chain restores an anaphylactic response to the nonresponsive alpha-deficient parental mouse line. This "humanized" mouse represents a potentially important model system, not only for studying the role of IgE in human immune responses, but also for testing potential therapeutic reagents that can interfere with responses mediated through the human Fc epsilon RI receptor.

Disruption by interferon-alpha of an autocrine interleukin-6 growth loop in IL-6-dependent U266 myeloma cells by homologous and heterologous down-regulation of the IL-6 receptor alpha- and beta-chains.

IL-6 is an autocrine growth factor for U266 myeloma cells and their growth is inhibited by IFN-alpha or IL-6 mAb. We asked, therefore, whether IFN-alpha-induced growth inhibition involved IL-6. IFN-alpha and mAb against IL-6, the IL-6R alpha-(gp80) or beta-chain (gp130) potently inhibited U266 cells. Remarkably, this effect occurred despite IFN-alpha-augmented secretion of endogenous IL-6. However, examining the IL-6R revealed that IFN-alpha drastically curtailed expression of the IL-6R alpha- and beta-chain. This effect occurred on two different levels (protein and mRNA) and by two different mechanisms (directly and indirectly through IL-6). First, IFN-alpha, but not IL-6, greatly decreased gp80 and, to a lesser extent, gp130 mRNA levels which resulted in a loss of IL-6 binding sites. Second, IFN-alpha-induced IL-6 predominantly down-regulated membrane-bound gp130. IFN-alpha-mediated decrease of gp80 levels was not detected on IL-6-independent myeloma (RPMI 8226) or myeloid cells (U937). We conclude that IFN-alpha inhibited IL-6-dependent myeloma cell growth by depriving U266 cells of an essential component of their autocrine growth loop, a functional IL-6R.

Tumor necrosis factor alpha differentially regulates beta-endorphin concentrations and proopiomelanocortin RNA in the anterior and neurointermediate pituitary in vivo.

We analyzed the effect of tumor necrosis factor alpha (TNF-alpha) on beta-endorphin concentrations and proopiomelanocortin mRNA in the rat anterior and neurointermediate pituitaries. The intraperitoneal injection of 5 micrograms/kg TNF-alpha decreases beta-endorphin in neurointermediate pituicytes 4, 8 and 24 h after the treatment without affecting proopiomelanocortin (POMC) RNA. In contrast, in the anterior pituitary 4 h after the injection of the cytokine, POMC RNA was decreased while the peptide content was increased. These effects can be relevant to the modulation of the pituitary-adrenal axis and immune responses in conditions, such as infections, in which TNF levels are increased.

Involvement of Alu sequences in the cell-specific regulation of transcription of the gamma chain of Fc and T cell receptors.

The Fc epsilon RI-gamma chains are expressed in a variety of hematopoietic cells where they play a critical role in signal transduction. They are part of the high affinity IgE receptor in mast cells, basophils, Langerhans cells, and possibly other cells; a component of the low affinity receptor for IgG (Fc gamma RIIIA or CD16) in natural killer cells and macrophages; and part of the T cell antigen receptor in subsets of T cells. Here we have investigated the transcriptional regulation of the gamma chain gene by analyzing the 2.5-kilobase sequence upstream of the transcription start site. This sequence contains a promoter specific to cells of hematopoietic lineage. However, the tissue specificity of this promoter is only partial because it is active in all of the hematopoietic cells tested here, regardless of whether they constitutively express Fc epsilon RI- gamma chain transcripts. We have identified two adjacent cis-acting regulatory elements, both of which are part of an Alu repeat. The first (-445/-366) is a positive element active in both basophils and T cells. The second (-365/-264) binds to nuclear factors, which appear to be different in basophils and T cells, and acts as a negative element in basophils and as a positive one in T cells. Thus, this Alu repeat (90% identical to Alu consensus sequences) has evolved to become both a positive and negative regulator.

The gene and cDNA for the human high affinity immunoglobulin E receptor beta chain and expression of the complete human receptor.

The high affinity IgE receptor (Fc epsilon RI) is a tetrameric hetero-oligomer composed of an alpha chain, a beta chain, and two disulfide-linked gamma chains. The beta chain contains four transmembrane (TM) segments and long cytoplasmic domains that are thought to play an important role in intracellular signaling. We now report the structural characterization and the sequence of the complete human beta gene and cDNA. The gene spans approximately 10 kilobases and contains seven exons. There is a single transcription initiation site preceded by a TATA box. The first exon codes for the 5'-untranslated region and a portion of the N-terminal cytoplasmic tail. TM-1 is encoded in exons 2 and 3, TM-2 in exons 3 and 4, TM-3 in exon 5, and TM-4 in exon 6. The seventh and final exon encodes the end of the C-terminal cytoplasmic tail and the 3'-untranslated sequence. The human beta gene appears to be a single copy gene. Two corresponding transcripts, detected as a doublet around 3.9 kilobases, are present in cells of mast cell and basophil lineage from different individuals, but not in the other hematopoietic cells tested here. The human beta protein is homologous to rodent beta. The consensus amino acid sequences of human, mouse, and rat beta show 69% identical residues. Analysis of the surface expression of transfected receptors indicates that human alpha gamma and alpha beta gamma complexes are expressed with comparable efficiency. Human beta interacts with human alpha more efficiently than does rat beta, and both rat and mouse beta interact with their corresponding alpha more efficiently than does human beta, demonstrating a species specificity of the alpha/beta interaction.

Characterization of the family of dimers associated with Fc receptors (Fc epsilon RI and Fc gamma RIII).

The receptor for IgE (Fc epsilon RI) is a multimeric complex containing one alpha chain, one beta chain with four transmembrane domains and one homodimer of disulfide-linked gamma-chains. The Fc epsilon RI gamma-chains form additional disulfide-linked dimers with the homologous zeta- and eta-chains, as part of the TCR complex. The low affinity receptor for IgG (Fc gamma RIII)2 on NK cells is also associated with zeta-chains. Here we show that the gamma-chain is expressed in NK cells both as a group of heterogenous gamma gamma homodimers and also as a heterodimer bound to zeta. Fc gamma RIIIA is associated with three types of dimers zeta zeta, gamma zeta, and notably gamma gamma as well. In fact, gamma gamma appears to be the predominant species associating with Fc gamma RIIIA. The surface expressed Fc epsilon RI also associates with the same group of heterogenous gamma gamma homodimers. We also show that there is no C-terminal posttranslational cleavage of gamma occurring before its insertion into the plasma membrane as previously suggested. Thus, like the TCR, Fc gamma RIIIA may form a variety of receptor isoforms, though at present we do not understand the functional implications of these structures.

Cyclosporin A inhibits induction of IL-2 receptor alpha chain expression by affecting activation of NF-kB-like factor(s) in cultured human T lymphocytes.

We have examined the effect and potential mechanism of Cyclosporin A (CsA) on the Interleukin-2-receptor alpha chain (IL-2R alpha) expression in human T-lymphocytes. CsA pretreatment of PHA-activated T-cells led to 30-50% decrease in Tac antigen surface expression and a concomitant decrease in the steady state IL-2R alpha mRNA levels. Transacting factors which recognize a kB-like sequence present in the IL-2R alpha chain regulatory region have been suggested to participate in the transcriptional regulation of the IL-2R alpha gene. Using oligonucleotides corresponding to the 5' regulatory region of the IL-2R alpha gene (i.e. 245 to 291 bp upstream of the start codon) and nuclear extract from resting T lymphocytes, we detected two specific bands by gel mobility shift assay. One of these bands is specifically increased after stimulation with phytohemagglutinin (PHA) and it is inhibited by CsA pretreatment. The same pattern of binding activity has been observed with the tandem repeat of NF-kB binding site present in the enhancer element of the human immunodeficiency virus long terminal repeat (HIV-1 LTR). These data suggest that CsA affects IL-2R receptor alpha chain expression by inhibiting the interaction of transacting factors to kB-like sequences after PHA activation. These findings may be of some relevance for the understanding of the immunosuppressive effects of CsA in normal human T lymphocytes.