Transplanted neural stem/precursor cells instruct phagocytes and reduce secondary tissue damage in the injured spinal cord.

Transplanted neural stem/precursor cells possess peculiar therapeutic plasticity and can simultaneously instruct several therapeutic mechanisms in addition to cell replacement. Here, we interrogated the therapeutic plasticity of neural stem/precursor cells after their focal implantation in the severely contused spinal cord. We injected syngeneic neural stem/precursor cells at the proximal and distal ends of the contused mouse spinal cord and analysed locomotor functions and relevant secondary pathological events in the mice, cell fate of transplanted neural stem/precursor cells, and gene expression and inflammatory cell infiltration at the injured site. We used two different doses of neural stem/precursor cells and two treatment schedules, either subacute (7 days) or early chronic (21 days) neural stem/precursor cell transplantation after the induction of experimental thoracic severe spinal cord injury. Only the subacute transplant of neural stem/precursor cells enhanced the recovery of locomotor functions of mice with spinal cord injury. Transplanted neural stem/precursor cells survived undifferentiated at the level of the peri-lesion environment and established contacts with endogenous phagocytes via cellular-junctional coupling. This was associated with significant modulation of the expression levels of important inflammatory cell transcripts in vivo. Transplanted neural stem/precursor cells skewed the inflammatory cell infiltrate at the injured site by reducing the proportion of 'classically-activated' (M1-like) macrophages, while promoting the healing of the injured cord. We here identify a precise window of opportunity for the treatment of complex spinal cord injuries with therapeutically plastic somatic stem cells, and suggest that neural stem/precursor cells have the ability to re-programme the local inflammatory cell microenvironment from a 'hostile' to an 'instructive' role, thus facilitating the healing or regeneration past the lesion.

Post-acute delivery of erythropoietin induces stroke recovery by promoting perilesional tissue remodelling and contralesional pyramidal tract plasticity.

The promotion of post-ischaemic motor recovery remains a major challenge in clinical neurology. Recently, plasticity-promoting effects have been described for the growth factor erythropoietin in animal models of neurodegenerative diseases. To elucidate erythropoietin's effects in the post-acute ischaemic brain, we examined how this growth factor influences functional neurological recovery, perilesional tissue remodelling and axonal sprouting of the corticorubral and corticobulbar tracts, when administered intra-cerebroventricularly starting 3 days after 30 min of middle cerebral artery occlusion. Erythropoietin administered at 10 IU/day (but not at 1 IU/day), increased grip strength of the contralesional paretic forelimb and improved motor coordination without influencing spontaneous locomotor activity and exploration behaviour. Neurological recovery by erythropoietin was associated with structural remodelling of ischaemic brain tissue, reflected by enhanced neuronal survival, increased angiogenesis and decreased reactive astrogliosis that resulted in reduced scar formation. Enhanced axonal sprouting from the ipsilesional pyramidal tract into the brainstem was observed in vehicle-treated ischaemic compared with non-ischaemic animals, as shown by injection of dextran amines into both motor cortices. Despite successful remodelling of the perilesional tissue, erythropoietin enhanced axonal sprouting of the contralesional, but not ipsilesional pyramidal tract at the level of the red and facial nuclei. Moreover, molecular biological and histochemical studies revealed broad anti-inflammatory effects of erythropoietin in both hemispheres together with expression changes of plasticity-related molecules that facilitated contralesional axonal growth. Our study establishes a plasticity-promoting effect of erythropoietin after stroke, indicating that erythropoietin acts via recruitment of contralesional rather than of ipsilesional pyramidal tract projections.

Neurosphere-derived multipotent precursors promote neuroprotection by an immunomodulatory mechanism.

In degenerative disorders of the central nervous system (CNS), transplantation of neural multipotent (stem) precursor cells (NPCs) is aimed at replacing damaged neural cells. Here we show that in CNS inflammation, NPCs are able to promote neuroprotection by maintaining undifferentiated features and exerting unexpected immune-like functions. In a mouse model of chronic CNS inflammation, systemically injected adult syngeneic NPCs use constitutively activated integrins and functional chemokine receptors to selectively enter the inflamed CNS. These undifferentiated cells survive repeated episodes of CNS inflammation by accumulating within perivascular areas where reactive astrocytes, inflamed endothelial cells and encephalitogenic T cells produce neurogenic and gliogenic regulators. In perivascular CNS areas, surviving adult NPCs induce apoptosis of blood-borne CNS-infiltrating encephalitogenic T cells, thus protecting against chronic neural tissue loss as well as disease-related disability. These results indicate that undifferentiated adult NPCs have relevant therapeutic potential in chronic inflammatory CNS disorders because they display immune-like functions that promote long-lasting neuroprotection.

Delayed post-ischaemic neuroprotection following systemic neural stem cell transplantation involves multiple mechanisms.

Recent evidence suggests that neural stem/precursor cells (NPCs) promote recovery in animal models with delayed neuronal death via a number of indirect bystander effects. A comprehensive knowledge of how transplanted NPCs exert their therapeutic effects is still lacking. Here, we investigated the effects of a delayed transplantation of adult syngenic NPCs--injected intravenously 72 h after transient middle cerebral artery occlusion--on neurological recovery, histopathology and gene expression. NPC-transplanted mice showed a significantly improved recovery from 18 days post-transplantation (dpt) onwards, which persisted throughout the study. A small percentage of injected NPCs accumulated in the brain, integrating mainly in the infarct boundary zone, where most of the NPCs remained undifferentiated up to 30 dpt. Histopathological analysis revealed a hitherto unreported very delayed neuroprotective effect of NPCs, becoming evident at 10 and 30 dpt. Tissue survival was associated with downregulation of markers of inflammation, glial scar formation and neuronal apoptotic death at both mRNA and protein levels. Our data highlight the relevance of very delayed degenerative processes in the stroke brain that are intimately associated with inflammatory and glial responses. These processes may efficaciously be antagonized by (stem) cell-based strategies at time-points far beyond established therapeutic windows for pharmacological neuroprotection.

Persistent inflammation alters the function of the endogenous brain stem cell compartment.

Endogenous neural stem/precursor cells (NPCs) are considered a functional reservoir for promoting tissue homeostasis and repair after injury, therefore regenerative strategies that mobilize these cells have recently been proposed. Despite evidence of increased neurogenesis upon acute inflammatory insults (e.g. ischaemic stroke), the plasticity of the endogenous brain stem cell compartment in chronic CNS inflammatory disorders remains poorly characterized. Here we show that persistent brain inflammation, induced by immune cells targeting myelin, extensively alters the proliferative and migratory properties of subventricular zone (SVZ)-resident NPCs in vivo leading to significant accumulation of non-migratory neuroblasts within the SVZ germinal niche. In parallel, we demonstrate a quantitative reduction of the putative brain stem cells proliferation in the SVZ during persistent brain inflammation, which is completely reversed after in vitro culture of the isolated NPCs. Together, these data indicate that the inflamed brain microenvironment sustains a non cell-autonomous dysfunction of the endogenous CNS stem cell compartment and challenge the potential efficacy of proposed therapies aimed at mobilizing endogenous precursors in chronic inflammatory brain disorders.

A new promoter polymorphism in the alpha-1-antichymotrypsin gene is a disease modifier of Alzheimer's disease.

Increased levels of alpha-1-antichymotrypsin (ACT), a protease inhibitor and an acute phase protein, have been found in the brain and peripheral blood of patients with Alzheimer's disease (AD). Patients from northern Italy with a clinical diagnosis of probable AD, and patients with early onset AD (EOAD) from UK with AD neuropathological diagnosis were genotyped for a new polymorphism in the promoter region of the ACT gene which has been shown to affect ACT expression. A subset of patients with clinical AD from northern Italy was also followed up for 2 years and monitored for cognitive decline. The ACT TT promoter genotype was associated with an increased risk of EOAD independently from the presence of the apolipoprotein E (APOE) epsilon 4 allele. After manifestation of the disease the ACT TT genotype was also associated with faster cognitive decline in patients with the APOE allele epsilon 4. The ACT gene appears to influence the early clinical development of the disease, and the interaction of the ACT and APOE genes affects clinical progression of AD.

Genetic polymorphisms and cerebrospinal fluid levels of tissue inhibitor of metalloproteinases 1 in sporadic Alzheimer's disease.

Tissue inhibitor of metalloproteinases 1 (TIMP-1) inhibits several proteinases including a disintegrin and metalloproteinase 10 (ADAM10), a major alpha-secretase that cleaves the beta-amyloid precursor protein within its amyloidogenic Abeta domain. The gene encoding TIMP-1 (TIMP 1) maps to the short arm of the X chromosome, in a region previously suggested as conferring genetic susceptibility for Alzheimer's disease (AD). To determine whether genetic variability of TIMP 1 contributes to the pathogenesis of AD, we analysed one single nucleotide polymorphism within TIMP 1 and one single nucleotide polymorphism in the 5'-untranslated region of TIMP 1 in patients with AD and control subjects from two independent and ethnically different populations. We did not observe any association between TIMP 1 genotypes and the diagnosis of AD in men or women. We also measured TIMP-1 protein levels in the cerebrospinal fluid of patients with AD, healthy control subjects, and patients with other neurological disorders. TIMP-1 levels were similar in all groups. In addition, no significant differences were observed after stratification for TIMP 1 genotypes. Our data show that neither genetic variability nor protein levels of TIMP-1 are associated with AD.

Absence of linkage between the interleukin-6 gene (-174 G/C) polymorphism and migraine.

To assess the role of interleukin-6 (IL-6) in migraine, we analyzed the -174 G/C IL-6 gene polymorphism in 268 patients with migraine and 305 controls. No significant difference in the distribution of IL-6 genotypes (chi(2)=0.601, P=0.74) and allelic frequencies (chi(2)=0.024, P=0.876) was found. When patients were subdivided into subgroups (migraine with aura, migraine without aura and mixed headaches), IL-6 alleles were similarly distributed. Comparison of the clinical features of the disease with the -174 G/C IL-6 genotypes showed no significant difference. In conclusion, we found no significant association between the -174 G/C IL-6 polymorphism and the occurrence or the clinical features of migraine.

Apolipoprotein E gene polymorphisms in patients with migraine.

To investigate the role of apolipoprotein E (APOE) polymorphisms in migraine, we analyzed the APOE genotypes of 241 migraine patients and 587 controls. The results of a Chi-square analysis indicated that APOE alleles were similarly distributed (chi(2)=2.89, P=0.24) between cases and controls. However, we found a significant (P<0.001) increase of the varepsilon2-varepsilon4 genotype in the group of migraine patients. Patients were divided into three subgroups: migraine with aura; migraine without aura; and mixed headaches (migraine associated with tension-type headache). Subgroup analysis showed that the varepsilon2-varepsilon4 genotype was significantly increased only in patients with mixed headaches. The stratification of patients by APOE status did not reveal significant associations with the clinical features of the disease. In conclusion, we observed no significant association between APOE polymorphisms and migraine. The association between the APOE varepsilon2-varepsilon4 genotype and the tension-type headache deserves further evaluation.

Detection of TNF and TNF receptor mRNA in cells and tissues.

We describe a semiquantitative reverse transcriptase polymerase chain reaction (RT-PCR) technique for the quantification of the messenger RNA of human and murine tumor necrosis factor alpha (TNFalpha) and related receptors. This protocol can be adapted for blood, peripheral blood lymphocytes, or other tissues. We propose a dot-blot technique which, if properly set up, is fast and quantitatively reliable. We describe two different detection protocols employing either radioactive or, alternatively for laboratories that cannot or do not want to use radioactivity, fluorescent-labeled probes. We also describe our calculations for relative quantification, based on the use of a positive control sample that becomes the reference value used to compare experimental samples. These protocols have as their aim to provide a flexible tool that can be employed in several different human and murine experimental settings by laboratories with different equipment.

Immune regulatory neural stem/precursor cells protect from central nervous system autoimmunity by restraining dendritic cell function.

BACKGROUND: The systemic injection of neural stem/precursor cells (NPCs) provides remarkable amelioration of the clinico-pathological features of experimental autoimmune encephalomyelitis (EAE). This is dependent on the capacity of transplanted NPCs to engage concurrent mechanisms of action within specific microenvironments in vivo. Among a wide range of therapeutic actions alternative to cell replacement, neuroprotective and immune modulatory capacities of transplanted NPCs have been described. However, lacking is a detailed understanding of the mechanisms by which NPCs exert their therapeutic plasticity. This study was designed to identify the first candidate that exemplifies and sustains the immune modulatory capacity of transplanted NPCs. METHODOLOGY/PRINCIPAL FINDINGS: To achieve the exclusive targeting of the peripheral immune system, SJL mice with PLP-induced EAE were injected subcutaneously with NPCs and the treatment commenced prior to disease onset. NPC-injected EAE mice showed significant clinical improvement, as compared to controls. Exogenous NPCs lacking the expression of major neural antigens were reliably (and for long-term) found at the level of draining lymph nodes, while establishing sophisticated anatomical interactions with lymph node cells. Importantly, injected NPCs were never found in organs other than lymph nodes, including the brain and the spinal cord. Draining lymph nodes from transplanted mice showed focal up-regulation of major developmental stem cell regulators, such as BMP-4, Noggin and Sonic hedgehog. In lymph nodes, injected NPCs hampered the activation of myeloid dendritic cells (DCs) and steadily restrained the expansion of antigen-specific encephalitogenic T cells. Both ex vivo and in vitro experiments identified a novel highly NPC-specific-BMP-4-dependent-mechanism hindering the DC maturation. CONCLUSION/SIGNIFICANCE: The study described herein, identifies the first member of the TGF beta/BMP family of stem cell regulators as a novel tolerogenic factor released by NPCs. Full exploitation of this pathway as an efficient tool for vaccination therapy in autoimmune inflammatory conditions is underway.

A polymorphism in the interleukin-1alpha gene influences the clinical features of migraine.

OBJECTIVE: To evaluate whether a particular genotype of the interleukin-1alpha (IL1A) gene affects the clinical features of migraine. BACKGROUND: Proinflammatory mediators have been reported to play a role in the pathophysiology of migraine. Recent studies suggest that polymorphisms in the interleukin-1 genes influence the age at onset and subsequent course of several chronic inflammatory diseases. METHODS: In a group of 269 patients with migraine, we tested the association of the -889 C/T biallelic polymorphism of the IL1A gene with several clinical features of the disease. RESULTS: Patients with migraine carrying the T/T genotype show an age at onset of the disease that is significantly (P <.01) lower than IL1A C/C or C/T carriers. In addition, the same genotype was significantly (P <.05) more frequent in patients with migraine with aura than in patients with migraine without aura. CONCLUSIONS: The results of our study suggest a role for the IL1A gene in modifying the clinical features of migraine.

Injection of adult neurospheres induces recovery in a chronic model of multiple sclerosis.

Widespread demyelination and axonal loss are the pathological hallmarks of multiple sclerosis. The multifocal nature of this chronic inflammatory disease of the central nervous system complicates cellular therapy and puts emphasis on both the donor cell origin and the route of cell transplantation. We established syngenic adult neural stem cell cultures and injected them into an animal model of multiple sclerosis--experimental autoimmune encephalomyelitis (EAE) in the mouse--either intravenously or intracerebroventricularly. In both cases, significant numbers of donor cells entered into demyelinating areas of the central nervous system and differentiated into mature brain cells. Within these areas, oligodendrocyte progenitors markedly increased, with many of them being of donor origin and actively remyelinating axons. Furthermore, a significant reduction of astrogliosis and a marked decrease in the extent of demyelination and axonal loss were observed in transplanted animals. The functional impairment caused by EAE was almost abolished in transplanted mice, both clinically and neurophysiologically. Thus, adult neural precursor cells promote multifocal remyelination and functional recovery after intravenous or intrathecal injection in a chronic model of multiple sclerosis.