Systemic Inflammatory Response in Spontaneous Subarachnoid Hemorrhage from Aneurysmal Rupture versus Subarachnoid Hemorrhage of Unknown Origin.

Objective: It is well known that spontaneous non-aneurysmal subarachnoid hemorrhage (SAH), also known as sine materia SAH (smSAH), has usually a better course and prognosis than its aneurysmal counterpart (aSAH). This might depend on different inflammatory mechanisms initiated by bleeding events of different origins. The aim of the present study was to explore the systemic inflammatory response in spontaneous SAH, comparing aSAH and smSAH. Methods: We performed a prospective observational study over a consecutive series of patients with SAH. For these patients, we collected all clinical data and, furthermore, performed venous blood sampling over six time points to analyze blood cells. We further performed the analysis of lymphocytes and monocytes by means of flow cytometry to quantify common subtypes. Statistical analysis included a t-student test, Chi-square test, multivariate logistic regression, and ROC analysis. Results: 48 patients were included: six (12.5%) with a diagnosis of spontaneous smSAH, and forty-two patients (87.5%) with aSAH. Significant differences on Day 0 were found for neutrophils and a systemic neuro-inflammatory index, namely, systemic inflammatory response index (SIRI). At the ROC analysis, neutrophil-to-lymphocyte ratio (NLR), lymphocyte-to-monocyte ratio (LMR), and SIRI exhibited satisfactory predictive power on day 0. At the multivariable logistic regression analysis, the combined index (NLR, LMR, SIRI at day 0) yielded an OR of 0.59 (95% CI 0.29-1.21]). LMR at day 0 yielded an OR of 1.25 ([95% CI 0.94-1.68]), NLR at day 0 exhibited an OR of 0.68 ([95% CI 0.42-1.09]), and SIRI at day 0 displayed an OR of 0.31 ([95% CI 0.06-1.49]). Conclusion: This preliminary study indicated a possible role of some inflammatory indices that point out the importance of innate and adaptive immunity in the etiopathogenetic mechanisms. Drugs modulating these responses could eventually counteract or, at least, reduce secondary damage associated with SAH.

An Early Increase of Blood Leukocyte Subsets in Aneurysmal Subarachnoid Hemorrhage Is Predictive of Vasospasm.

Objective: Vasospasm is a severe complication in patients with aneurysmal subarachnoid hemorrhage (aSAH) and cannot be reliably predicted. Its pathophysiology remains elusive with the current body of evidence suggesting inflammation as one of the main driving forces. We here aimed to analyze circulating immune cell subsets over time in patients with aSAH with or without vasospasm. Methods: We performed a prospective observational study recruiting patients with spontaneous aSAH. Peripheral blood withdrawn at pre-specified time-points after aSAH, day 0, days 3-4, 6-8, 10-11, 13-15, and 18-21. Flow cytometry analysis, cell blood counts, and laboratory and diagnostic parameters were performed. Patients were monitored by transcranial Doppler for vasospasm as well as by advanced imaging and divided into a group with (VS) and without vasospasm VS (NVS). Results: We included 42 patients for study analysis, 21 VS and 21 NVS. An early significant increase at day 0 in platelet, leukocyte, neutrophil, lymphocyte, NK lymphocyte, monocyte, and CD 14++ CD16- DR+ monocyte counts was found in patients with later ensuing vasospasm. The early differences in platelets, leukocytes, lymphocytes, and NK lymphocytes remained significant on multivariate analysis. Conclusions: An early increase of immune cellular subsets in aSAH may contribute to predict VS.

Teriflunomide treatment reduces B cells in patients with MS.

OBJECTIVE: To study the immunomodulatory effect of teriflunomide on innate and adaptive immune cell populations through a pilot, open-label, observational study in a cohort of patients with relapsing-remitting MS. METHODS: Blood lymphocytes were isolated from 10 patients with MS before and after 3 or 12 months of treatment. Adaptive and innate immune cell subsets were analyzed by flow cytometry as follows: B cells (memory, regulatory, and mature subsets), T cells (effector and regulatory subsets), and natural killer (NK) cells (CD56dim and CD56bright subsets). RESULTS: Our results show that teriflunomide significantly reduces absolute counts of total CD19+ B cells and mature and regulatory B-cell subsets. T cells were affected to a lesser extent, with a trend in reduction of absolute counts for both T effector CD4+ cells (Th1, Th17 and Th1/17) and T regulatory CD8+ and CD4+ cells. Teriflunomide had no detectable effect on NK-cell numbers. CONCLUSIONS: In our small cohort, teriflunomide treatment affects mainly and significantly on B-cell numbers, while having a milder effect on T-cell numbers. Larger cohorts are necessary to confirm these findings and understand the effect of teriflunomide on the functionality of these cells.

Human mesenchymal stem cells target adhesion molecules and receptors involved in T cell extravasation.

INTRODUCTION: Systemic delivery of bone marrow-derived mesenchymal stem cells (MSC) seems to be of benefit in the treatment of multiple sclerosis (MS), an autoimmune disease of the central nervous system (CNS) sustained by migration of T cells across the brain blood barrier (BBB) and subsequent induction of inflammatory lesions into CNS. MSC have been found to modulate several effector functions of T cells. In this study, we investigated the effects of MSC on adhesion molecules and receptors on T cell surface that sustain their transendothelial migration. METHODS: We used different co-culture methods combined with real-time PCR and flow cytometry to evaluate the expression both at the mRNA and at the plasma-membrane level of α4 integrin, ß2 integrin, ICAM-1 and CXCR3. In parallel, we assessed if MSC are able to modulate expression of adhesion molecules on the endothelial cells that interact with T cells during their transendothelial migration. RESULTS: Our in vitro analyses revealed that MSC: (i) inhibit proliferation and activation of both peripheral blood mononuclear cells (PBMC) and CD3(+)-selected lymphocytes through the release of soluble factors; (ii) exert suppressive effects on those surface molecules highly expressed by activated lymphocytes and involved in transendothelial migration; (iii) inhibit CXCL10-driven chemotaxis of CD3(+) cells; (iv) down-regulated expression of adhesion molecules on endothelial cells. CONCLUSIONS: Taken together, these data demonstrate that the immunosuppressive effect of MSC does not exclusively depends on their anti-proliferative activity on T cells, but also on the impairment of leukocyte migratory potential through the inhibition of the adhesion molecules and receptors that are responsible for T cell trafficking across BBB. This could suggest a new mechanism through which MSC modulate T cell responses.

Neuroprotective features of mesenchymal stem cells.

Bone marrow (BM) derived mesenchymal stem cells (MSC) differentiate into cells of the mesodermal lineage but also, under certain experimental circumstances, into cells of the neuronal and glial lineage. Their therapeutic translation has been significantly boosted by the demonstration that MSC display significant also anti-proliferative, anti-inflammatory and anti-apoptotic features. These properties have been exploited in the effective treatment of experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis where the inhibition of the autoimmune response resulted in a significant neuroprotection. A significant rescue of neural cells has been achieved also when MSC were administered in experimental brain ischemia and in animals undergoing brain or spinal cord injury. In these experimental conditions BM-MSC therapeutic effects are likely to depend on paracrine mechanisms mediated by the release of growth factors, anti-apoptotic molecules and anti-inflammatory cytokines creating a favorable environment for the regeneration of neurons, remyelination and improvement of cerebral flow. For potential clinical application BM-MSC offer significant practical advantages over other types of stem cells since they can be obtained from the adult BM and can be easily cultured and expanded in vitro under GMP conditions displaying a very low risk of malignant transformation. This review discusses the targets and mechanisms of BM-MSC mediated neuroprotection.

Regulation of human mesenchymal stem cell functions by an autocrine loop involving NAD+ release and P2Y11-mediated signaling.

In several cell types, a regulated efflux of NAD(+) across Connexin 43 hemichannels (Cx43 HC) can occur, and extracellular NAD(+) (NAD(+)(e)) affects cell-specific functions. We studied the capability of bone marrow-derived human mesenchymal stem cells (MSC) to release intracellular NAD(+) through Cx43 HC. NAD(+) efflux, quantified by a sensitive enzymatic cycling assay, was significantly upregulated by low extracellular Ca(2+) (5-6-fold), by shear stress (13-fold), and by inflammatory conditions (3.1- and 2.5-fold in cells incubated with lipopolysaccharide (LPS) or at 39°C, respectively), as compared with untreated cells, whereas it was downregulated in Cx43-siRNA-transfected MSC (by 53%) and by cell-to-cell contact (by 45%). Further, we show that NAD(+)(e) activates the purinergic receptor P2Y(11) and a cyclic adenosin monophosphate (cAMP)/cyclic ADP-ribose/[Ca(2+)](i) signaling cascade, involving the opening, unique to MSC, of L-type Ca(2+) channels. Extracellular NAD(+) enhanced nuclear translocation of cAMP/Ca(2+)-dependent transcription factors. Moreover, NAD(+), either extracellularly added or autocrinally released, resulted in stimulation of MSC functions, including proliferation, migration, release of prostaglandin E(2) and cytokines, and downregulation of T lymphocyte proliferation compared with controls. No detectable modifications of MSC markers and of adipocyte or osteocyte differentiation were induced by NAD(+)(e). Controls included Cx43-siRNA transfected and/or NAD(+)-glycohydrolase-treated MSC (autocrine effects), and NAD(+)-untreated or P2Y(11)-siRNA-transfected MSC (exogenous NAD(+)). These findings suggest a potential beneficial role of NAD(+)(e) in modulating MSC functions relevant to MSC-based cell therapies.

Fibroblast growth factor 2 and platelet-derived growth factor, but not platelet lysate, induce proliferation-dependent, functional class II major histocompatibility complex antigen in human mesenchymal stem cells.

OBJECTIVE: To document the specificity and the mechanism of induction of a novel class II major histocompatibility complex (MHC) antigen by mitogenic growth factors in human mesenchymal stem cells (MSCs) expanded in vitro for translational applications. METHODS: Expression of class II MHC molecules was measured in human MSCs and differentiated cells expanded in the presence of fibroblast growth factor 2 (FGF-2), platelet-derived growth factor BB (PDGF-BB), human platelet lysate, or interferon-γ (IFNγ). The roles of cell proliferation and growth factor-induced signaling pathways were investigated as well as the class II MHC assembly machinery and functional capacity. RESULTS: FGF-2 and, to a lesser extent, PDGF-BB induced in adult human MSCs the expression of HLA-DR (normally induced by inflammatory cytokines), which was able to stimulate CD4+ T cells via superantigen binding. In contrast to IFNγ, FGF induced HLA-DR expression only in human MSCs proliferating under its mitogenic effect and not in mouse MSCs or in differentiated human cells. Although it induced cell proliferation, human platelet lysate did not cause HLA-DR expression in human MSCs. HLA-DR expression occurred following FGF-specific binding to its receptor(s), mainly FGF receptor 1, without inducing IFNγ or tumor necrosis factor α expression. Both MAPK/ERK-1/2 and phosphatidylinositol 3-kinase/Akt controlled cell proliferation and HLA-DR expression, but only MAPK/ERK-1/2 controlled the induction of the class II MHC transcription activator protein CIITA, the major determinant of HLA-DR transcription. CONCLUSION: The induction of functional HLA-DR in proliferating progenitor MSCs is a property of human MSCs that have been expanded with mitogenic growth factors. This has potential biologic significance in the regulation and/or protection of progenitor cell subpopulations under sustained mitogenic proliferation and needs to be taken into account when expanding MSCs for use in in vivo applications.

Simultaneous determination of triazines and their main transformation products in surface and urban wastewater by ultra-high-pressure liquid chromatography-tandem mass spectrometry.

This work describes the optimization, validation and application of an ultra-high-pressure liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method for the quantification and confirmation of 11 compounds (atrazine, simazine, terbuthylazine, terbumeton, terbutryn and their main transformation products) in surface and wastewater samples. Several of these analytes are included in the list of priority substances in the framework on European Water Policy. The application of this method to water samples reveals that the most relevant transformation products (TPs) should be incorporated into current analytical methods to obtain a more realistic knowledge of water quality regarding pesticide contamination. TPs are generally more polar and mobile than parents and can be transported to the aquatic environment more easily than their precursors. This can explain their concentrations found in water, which in many cases are much higher than intact triazines. To efficiently combine UHPLC with MS/MS, a fast-acquisition triple quadrupole mass analyser was used. Working in selected reaction monitoring mode, up to three simultaneous transitions per compound were acquired, allowing a reliable quantification and confirmation at nanogram per litre levels. The method developed includes a pre-concentration step based on solid-phase extraction (OASIS HLB cartridges). Satisfactory recoveries (70-120%) and relative standard deviations (<20%) were obtained for all compounds in different water sample types spiked at two concentrations (0.025 and 0.1 microg/L in surface water; 0.25 and 1.0 microg/L in effluent wastewater; 0.5 and 2.0 microg/L in influent wastewater). The optimized method was found to have excellent sensitivity with instrumental detection limits as low as 0.03 pg. In addition, the influence of the matrix constituents on the ionization efficiency and the extraction recovery was studied in different types of Italian and Spanish surface and urban wastewater. Signal suppressions were observed for all compounds, especially for influent wastewater. The use of isotope-labelled internal standards was found to be the best approach to assure an accurate quantification in all matrix samples.

Catastrophic NAD+ depletion in activated T lymphocytes through Nampt inhibition reduces demyelination and disability in EAE.

Nicotinamide phosphoribosyltransferase (Nampt) inhibitors such as FK866 are potent inhibitors of NAD(+) synthesis that show promise for the treatment of different forms of cancer. Based on Nampt upregulation in activated T lymphocytes and on preliminary reports of lymphopenia in FK866 treated patients, we have investigated FK866 for its capacity to interfere with T lymphocyte function and survival. Intracellular pyridine nucleotides, ATP, mitochondrial function, viability, proliferation, activation markers and cytokine secretion were assessed in resting and in activated human T lymphocytes. In addition, we used experimental autoimmune encephalomyelitis (EAE) as a model of T-cell mediated autoimmune disease to assess FK866 efficacy in vivo. We show that activated, but not resting, T lymphocytes undergo massive NAD(+) depletion upon FK866-mediated Nampt inhibition. As a consequence, impaired proliferation, reduced IFN-gamma and TNF-alpha production, and finally autophagic cell demise result. We demonstrate that upregulation of the NAD(+)-degrading enzyme poly-(ADP-ribose)-polymerase (PARP) by activated T cells enhances their susceptibility to NAD(+) depletion. In addition, we relate defective IFN-gamma and TNF-alpha production in response to FK866 to impaired Sirt6 activity. Finally, we show that FK866 strikingly reduces the neurological damage and the clinical manifestations of EAE. In conclusion, Nampt inhibitors (and possibly Sirt6 inhibitors) could be used to modulate T cell-mediated immune responses and thereby be beneficial in immune-mediated disorders.

The plant hormone abscisic acid stimulates the proliferation of human hemopoietic progenitors through the second messenger cyclic ADP-ribose.

Abscisic acid (ABA) is a hormone involved in pivotal physiological functions in higher plants, such as response to abiotic stress and control of seed dormancy and germination. Recently, ABA was demonstrated to be autocrinally produced by human granulocytes, beta pancreatic cells, and mesenchymal stem cells (MSC) and to stimulate cell-specific functions through a signaling pathway involving the second messenger cyclic ADP-ribose (cADPR). Here we show that ABA expands human uncommitted hemopoietic progenitors (HP) in vitro, through a cADPR-mediated increase of the intracellular calcium concentration ([Ca(2+)](i)). Incubation of CD34(+) cells with micromolar ABA also induces transcriptional effects, which include NF-kappaB nuclear translocation and transcription of genes encoding for several cytokines. Human MSC stimulated with a lymphocyte-conditioned medium produce and release ABA at concentrations sufficient to exert growth-stimulatory effects on co-cultured CD34(+) cells, as demonstrated by the inhibition of colony growth in the presence of an anti-ABA monoclonal antibody. These results provide a remarkable example of conservation of a stress hormone and of its second messenger from plants to humans and identify ABA as a new hemopoietic growth factor involved in the cross-talk between HP and MSC.

P2X7-mediated increased intracellular calcium causes functional derangement in Schwann cells from rats with CMT1A neuropathy.

Charcot-Marie-Tooth (CMT) is the most frequent inherited neuromuscular disorder, affecting 1 person in 2500. CMT1A, the most common form of CMT, is usually caused by a duplication of chromosome 17p11.2, containing the PMP22 (peripheral myelin protein-22) gene; overexpression of PMP22 in Schwann cells (SC) is believed to cause demyelination, although the underlying pathogenetic mechanisms remain unclear. Here we report an abnormally high basal concentration of intracellular calcium ([Ca(2+)](i)) in SC from CMT1A rats. By the use of specific pharmacological inhibitors and through down-regulation of expression by small interfering RNA, we demonstrate that the high [Ca(2+)](i) is caused by a PMP22-related overexpression of the P2X7 purinoceptor/channel leading to influx of extracellular Ca(2+) into CMT1A SC. Correction of the altered [Ca(2+)](i) in CMT1A SC by small interfering RNA or with pharmacological inhibitors of P2X7 restores functional parameters of SC (migration and release of ciliary neurotrophic factor), which are typically defective in CMT1A SC. More significantly, stable down-regulation of the expression of P2X7 restores myelination in co-cultures of CMT1A SC with dorsal root ganglion sensory neurons. These results establish a pathogenetic link between high [Ca(2+)](i) and impaired SC function in CMT1A and identify overexpression of P2X7 as the molecular mechanism underlying both abnormalities. The development of P2X7 inhibitors is expected to provide a new therapeutic strategy for treatment of CMT1A neuropathy.

C-C chemokine receptor 6-regulated entry of TH-17 cells into the CNS through the choroid plexus is required for the initiation of EAE.

Interleukin 17-producing T helper cells (T(H)-17 cells) are important in experimental autoimmune encephalomyelitis, but their route of entry into the central nervous system (CNS) and their contribution relative to that of other effector T cells remain to be determined. Here we found that mice lacking CCR6, a chemokine receptor characteristic of T(H)-17 cells, developed T(H)-17 responses but were highly resistant to the induction of experimental autoimmune encephalomyelitis. Disease susceptibility was reconstituted by transfer of wild-type T cells that entered into the CNS before disease onset and triggered massive CCR6-independent recruitment of effector T cells across activated parenchymal vessels. The CCR6 ligand CCL20 was constitutively expressed in epithelial cells of choroid plexus in mice and humans. Our results identify distinct molecular requirements and ports of lymphocyte entry into uninflamed versus inflamed CNS and suggest that the CCR6-CCL20 axis in the choroid plexus controls immune surveillance of the CNS.

Ascorbic acid pre-treated quartz stimulates TNF-alpha release in RAW 264.7 murine macrophages through ROS production and membrane lipid peroxidation.

BACKGROUND: Inhalation of crystalline silica induces a pulmonary fibrotic degeneration called silicosis caused by the inability of alveolar macrophages to dissolve the crystalline structure of phagocytosed quartz particles. Ascorbic acid is capable of partially dissolving quartz crystals, leading to an increase of soluble silica concentration and to the generation of new radical sites on the quartz surface. The reaction is specific for the crystalline forms of silica. It has been already demonstrated an increased cytotoxicity and stronger induction of pro-inflammatory cyclooxygenase-2 (COX-2) by ascorbic acid pre-treated quartz (QA) compared to untreated quartz (Q) in the murine macrophage cell line RAW 264.7. METHODS: Taking advantage of the enhanced macrophage response to QA as compared to Q particles, we investigated the first steps of cell activation and the contribution of early signals generated directly from the plasma membrane to the production of TNF-alpha, a cytokine that activates both inflammatory and fibrogenic pathways. RESULTS: Here we demonstrate that TNF-alpha mRNA synthesis and protein secretion are significantly increased in RAW 264.7 macrophages challenged with QA as compared to Q particles, and that the enhanced response is due to an increase of intracellular ROS. Plasma membrane-particle contact, in the absence of phagocytosis, is sufficient to trigger TNF-alpha production through a mechanism involving membrane lipid peroxidation and this appears to be even more detrimental to macrophage survival than particle phagocytosis itself. CONCLUSION: Taken together these data suggest that an impairment of pulmonary macrophage phagocytosis, i.e. in the case of alcoholic subjects, could potentiate lung disease in silica-exposed individuals.

Reciprocal interactions between human mesenchymal stem cells and gammadelta T cells or invariant natural killer T cells.

The immunomodulatory activities of human mesenchymal stem cells (MSCs) provide a rational basis for their application in the treatment of immune-mediated diseases, such as graft versus host disease and multiple sclerosis. The effects of MSCs on invariant natural killer T (iNKT) and gammadelta T cells, both involved in the pathogenesis of autoimmune diseases, are unknown. Here, we investigated the effects of MSCs on in vitro expansion of these unconventional T-cell populations. MSCs inhibited iNKT (Valpha24(+)Vbeta11(+)) and gammadelta T (Vdelta2(+)) cell expansion from peripheral blood mononuclear cells in both cell-to-cell contact and transwell systems. Such inhibition was partially counteracted by indomethacin, a prostaglandin E(2) inhibitor. Block of indoleamine 2,3-deoxygenase and transforming growth factor beta1 did not affect Valpha24(+)Vbeta11(+) and Vdelta2(+) cell expansion. MSCs inhibited interferon-gamma production by activated Valpha24(+)Vbeta11(+) and impaired CD3-mediated proliferation of activated Valpha24(+)Vbeta11(+) and Vdelta2(+) T cells, without affecting their cytotoxic potential. MSCs did not inhibit antigen processing/presentation by activated Vdelta2(+) T cells to CD4(+) T cells. In contrast, MSCs were lysed by activated Vdelta2(+) T cells through a T-cell receptor-dependent mechanism. These results are translationally relevant in view of the increasing interest in MSC-based therapy of autoimmune diseases.

Different cellular and molecular mechanisms for early and late-onset myelin protein zero mutations.

Mutations in the gene MPZ, encoding myelin protein zero (MPZ), cause inherited neuropathies collectively called Charcot-Marie-Tooth type 1B (CMT1B). Based on the age of onset, clinical and pathological features, most MPZ mutations are separable into two groups: one causing a severe, early-onset, demyelinating neuropathy and a second, causing a late-onset neuropathy with prominent axonal loss. To investigate potential pathomechanisms underlying the two phenotypes, we transiently transfected HeLa cells with two late-onset (T95M, H10P) and two early-onset (H52R, S22_W28 deletion) mutations and analyzed their effects on intracellular protein trafficking, glycosylation, cell viability and intercellular adhesion. We found that the two late-onset mutations were both transported to the cell membrane and moderately reduced MPZ-mediated intercellular adhesion. The two early-onset mutations caused two distinct abnormalities. H52R was correctly glycosylated and trafficked to the plasma membrane, but strongly affected intercellular adhesion. When co-expressed with wild-type MPZ (wtMPZ), a functional dominant negative effect was observed. Alternatively, S22_W28 deletion was retained within the cytoplasm and reduced both adhesion caused by wtMPZ and cellular viability. Since the same trafficking patterns were observed in transfected murine Schwann cells, they are not an artifact of heterologous cell expression. Our results suggest that at least some late-onset mutations cause a partial loss of function in the transfected cells, whereas multiple abnormal gain of function pathways can result in early-onset neuropathy. Further characterization of these pathways will lead to a better understanding of the pathogenesis of CMT1B and a rational basis for treating these debilitating inherited neuropathies.

Multipotent mesenchymal stromal cells from amniotic fluid: solid perspectives for clinical application.

BACKGROUND: Mesenchymal stromal cells are multipotent cells considered to be of great promise for use in regenerative medicine. However, the cell dose may be a critical factor in many clinical conditions and the yield resulting from the ex vivo expansion of mesenchymal stromal cells derived from bone marrow may be insufficient. Thus, alternative sources of mesenchymal stromal cells need to be explored. In this study, mesenchymal stromal cells were successfully isolated from second trimester amniotic fluid and analyzed for chromosomal stability to validate their safety for potential utilization as a cell therapy product. DESIGN AND METHODS: Mesenchymal stromal cells were expanded up to the sixth passage starting from amniotic fluid using different culture conditions to optimize large-scale production. RESULTS: The highest number of mesenchymal stromal cells derived from amniotic fluid was reached at a low plating density; in these conditions the expansion of mesenchymal stromal cells from amniotic fluid was significantly greater than that of adult bone marrow-derived mesenchymal stromal cells. Mesenchymal stromal cells from amniotic fluid represent a relatively homogeneous population of immature cells with immunosuppressive properties and extensive proliferative potential. Despite their high proliferative capacity in culture, we did not observe any karyotypic abnormalities or transformation potential in vitro nor any tumorigenic effect in vivo. CONCLUSIONS: Fetal mesenchymal stromal cells can be extensively expanded from amniotic fluid, showing no karyotypic abnormalities or transformation potential in vitro and no tumorigenic effect in vivo. They represent a relatively homogeneous population of immature mesenchymal stromal cells with long telomeres, immunosuppressive properties and extensive proliferative potential. Our results indicate that amniotic fluid represents a rich source of mesenchymal stromal cells suitable for banking to be used when large amounts of cells are required.

The molecular signature of therapeutic mesenchymal stem cells exposes the architecture of the hematopoietic stem cell niche synapse.

BACKGROUND: The hematopoietic stem cells (HSCs) niche of the bone marrow is comprised of HSCs, osteoblasts, endothelial cells and a stromal component of non-hematopoietic multipotent cells of mesenchymal origin named "mesenchymal stem cells" (MSCs). RESULTS: Here we studied the global transcriptional profile of murine MSCs with immuno-therapeutic potential and compared it with that of 486 publicly available microarray datasets from 12 other mouse tissues or cell types. Principal component analysis and hierarchical clustering identified a unique pattern of gene expression capable of distinctively classifying MSCs from other tissues and cells. We then performed an analysis aimed to identify absolute and relative abundance of transcripts in all cell types. We found that the set of transcripts uniquely expressed by MSCs is enriched in transcription factors and components of the Wnt signaling pathway. The analysis of differentially expressed genes also identified a set of genes specifically involved in the HSC niche and is complemented by functional studies that confirm the findings. Interestingly, some of these genes play a role in the maintenance of HSCs in a quiescent state supporting their survival and preventing them from proliferating and differentiating. We also show that MSCs modulate T cell functions in vitro and, upon in vivo administration, ameliorate experimental autoimmune encephalomyelitis (EAE). CONCLUSION: Altogether, these findings provide novel and important insights on the mechanisms of T cell function regulation by MSCs and help to cement the rationale for their application in the treatment of autoimmune diseases.

Human mesenchymal stem cells promote survival of T cells in a quiescent state.

Mesenchymal stem cells (MSC) are part of the bone marrow that provides signals supporting survival and growth of bystander hematopoietic stem cells (HSC). MSC modulate also the immune response, as they inhibit proliferation of lymphocytes. In order to investigate whether MSC can support survival of T cells, we investigated MSC capacity of rescuing T lymphocytes from cell death induced by different mechanisms. We observed that MSC prolong survival of unstimulated T cells and apoptosis-prone thymocytes cultured under starving conditions. MSC rescued T cells from activation induced cell death (AICD) by downregulation of Fas receptor and Fas ligand on T cell surface and inhibition of endogenous proteases involved in cell death. MSC dampened also Fas receptor mediated apoptosis of CD95 expressing Jurkat leukemic T cells. In contrast, rescue from AICD was not associated with a significant change of Bcl-2, an inhibitor of apoptosis induced by cell stress. Accordingly, MSC exhibited a minimal capacity of rescuing Jurkat cells from chemically induced apoptosis, a process disrupting the mitochondrial membrane potential regulated by Bcl-2. These results suggest that MSC interfere with the Fas receptor regulated process of programmed cell death. Overall, MSC can inhibit proliferation of activated T cells while supporting their survival in a quiescent state, providing a model of their activity inside the HSC niche. Disclosure of potential conflicts of interest is found at the end of this article.

Extracellular NAD+ is an agonist of the human P2Y11 purinergic receptor in human granulocytes.

Micromolar concentrations of extracellular beta-NAD+ (NAD(e)+) activate human granulocytes (superoxide and NO generation and chemotaxis) by triggering: (i) overproduction of cAMP, (ii) activation of protein kinase A, (iii) stimulation of ADP-ribosyl cyclase and overproduction of cyclic ADP-ribose (cADPR), a universal Ca2+ mobilizer, and (iv) influx of extracellular Ca2+. Here we demonstrate that exposure of granulocytes to millimolar rather than to micromolar NAD(e)+ generates both inositol 1,4,5-trisphosphate (IP3) and cAMP, with a two-step elevation of intracellular calcium levels ([Ca2+]i): a rapid, IP3-mediated Ca2+ release, followed by a sustained influx of extracellular Ca2+ mediated by cADPR. Suramin, an inhibitor of P2Y receptors, abrogated NAD(e)+-induced intracellular increases of IP3, cAMP, cADPR, and [Ca2+]i, suggesting a role for a P2Y receptor coupled to both phospholipase C and adenylyl cyclase. The P2Y(11) receptor is the only known member of the P2Y receptor subfamily coupled to both phospholipase C and adenylyl cyclase. Therefore, we performed experiments on hP2Y(11)-transfected 1321N1 astrocytoma cells: micromolar NAD(e)+ promoted a two-step elevation of the [Ca2+]i due to the enhanced intracellular production of IP3, cAMP, and cADPR in 1321N1-hP2Y(11) but not in untransfected 1321N1 cells. In human granulocytes NF157, a selective and potent inhibitor of P2Y(11), and the down-regulation of P2Y(11) expression by short interference RNA prevented NAD(e)+-induced intracellular increases of [Ca2+]i and chemotaxis. These results demonstrate that beta-NAD(e)+ is an agonist of the P2Y(11) purinoceptor and that P2Y(11) is the endogenous receptor in granulocytes mediating the sustained [Ca2+]i increase responsible for their functional activation.

Mechanisms of the adaptive immune response inside the central nervous system during inflammatory and autoimmune diseases.

In this review we will discuss the unique features that make the central nervous system (CNS) a specialized microenvironment where immune responses are tightly regulated in order to properly face pathogens without damaging the neural cells. We will show how every paradigm of this theoretical model has been addressed by the scientific literature over the past decades providing new insights on the immune response within the CNS. In particular, new light has been shed on the trafficking of the immune cells inside and outside the CNS. Dendritic cells (DCs) have been described in the context of structures in direct contact with the cerebrospinal fluid (CSF) and their migration, upon antigen encounter, outside the CNS into deep cervical lymph nodes (DCLNs) has been further clarified. T-cells, B-cells, and antibody-secreting cells (ASCs) have been found in the CSF and CNS parenchymal lesions of inflammatory disorders and their phenotype depicted. Moreover, in chronically inflamed CNS, ectopic lymphoid structures have been observed and a germinal center reaction similar to the one found in peripheral lymph nodes has been described. These structures may play a role in the maintenance and expansion of the local autoimmune response. Although the complex interactions between immune and neural cells still remain far to be elucidated, the data discussed here suggest that the physiopathology of the adaptive immune response inside the CNS mimics, although in a mitigated fashion, what occurs in other organs and tissues.