Detection, epidemiology and host spectrum of cowpox and Borna disease virus infections

Several orthopoxviruses (OPV) and Borna disease virus (BDV) are enveloped, zoonotic viruses with a wide geographical distribution. OPV antibodies cross-react, and former smallpox vaccination has therefore protected human populations from another OPV infection, rodent-borne cowpox virus (CPXV). Cowpox in humans and cats usually manifests as a mild, self-limiting dermatitis and constitutional symptoms, but it can be severe and even life-threatening in the immunocompromised. Classical Borna disease is a progressive meningoencephalomyelitis in horses and sheep known in central Europe for centuries. Nowadays the virus or its close relative infects humans and also several other species in central Europe and elsewhere, but the existence of human Borna disease with its suspected neuropsychiatric symptoms is controversial. The epidemiology of BDV is largely unknown, and the present situation is even more intriguing following the recent detection of several-million-year-old, endogenized BDV genes in primate and various other vertebrate genomes. The aims of this study were to elucidate the importance of CPXV and BDV in Finland and in possible host species, and particularly to 1) establish relevant methods for the detection of CPXV and other OPVs as well as BDV in Finland, 2) determine whether CPXV and BDV exist in Finland, 3) discover how common OPV immunity is in different age groups in Finland, 4) characterize possible disease cases and clarify their epidemiological context, 5) establish the hosts and possible reservoir species of these viruses and their geographical distribution in wild rodents, and 6) elucidate the infection kinetics of BDV in the bank vole. An indirect immunofluorescence assay and avidity measurement were established for the detection, timing and verification of OPV or BDV antibodies in thousands of blood samples from humans, horses, ruminants, lynxes, gallinaceous birds, dogs, cats and rodents. The mostly vaccine-derived OPV seroprevalence was found to decrease gradually according to the year of birth of the sampled human subjects from 100% to 10% in those born after 1977. On the other hand, OPV antibodies indicating natural contact with CPXV or other OPVs were commonly found in domestic and wild animals: the horse, cow, lynx, dog, cat and, with a prevalence occasionally even as high as 92%, in wild rodents, including some previously undetected species and new regions. Antibodies to BDV were detected in humans, horses, a dog, cats, and for the first time in wild rodents, such as bank voles (Myodes glareolus). Because of the controversy within the human Borna disease field, extra verification methods were established for BDV antibody findings: recombinant nucleocapsid and phosphoproteins were produced in Escherichia coli and in a baculovirus system, and peptide arrays were additionally applied. With these verification assays, Finnish human, equine, feline and rodent BDV infections were confirmed. Taken together, wide host spectra were evident for both OPV and BDV infections based on the antibody findings, and OPV infections were found to be geographically broadly distributed. PCR amplification methods were utilised for hundreds of blood and tissue samples. The methods included conventional, nested and real-time PCRs with or without the reverse transcription step and detecting four or two genes of OPVs and BDV, respectively. OPV DNA could be amplified from two human patients and three bank voles, whereas no BDV RNA was detected in naturally infected individuals. Based on the phylogenetic analyses, the Finnish OPV sequences were closely related although not identical to a Russian CPXV isolate, and clearly different from other CPXV strains. Moreover, the Finnish sequences only equalled each other, but the short amplicons obtained from German rodents were identical to monkeypox virus, in addition to German CPXV variants. This reflects the close relationship of all OPVs. In summary, RNA of the Finnish BDV variant could not be detected with the available PCR methods, but OPV DNA infrequently could. The OPV species infecting the patients of this study was proven to be CPXV, which is most probably also responsible for the rodent infections. Multiple cell lines and some newborn rodents were utilised in the isolation of CPXV and BDV from patient and wildlife samples. CPXV could be isolated from a child with severe, generalised cowpox. BDV isolation attempts from rodents were unsuccessful in this study. However, in parallel studies, a transient BDV infection of cells inoculated with equine brain material was detected, and BDV antigens discovered in archival animal brains using established immunohistology. Thus, based on several independent methods, both CPXV and BDV (or a closely related agent) were shown to be present in Finland. Bank voles could be productively infected with BDV. This experimental infection did not result in notable pathological findings or symptoms, despite the intense spread of the virus in the central and peripheral nervous system. Infected voles commonly excreted the virus in urine and faeces, which emphasises their possible role as a BDV reservoir. Moreover, BDV RNA was regularly reverse transcribed into DNA in bank voles, which was detected by amplifying DNA by PCR without reverse transcription, and verified with nuclease treatments. This finding indicates that BDV genes could be endogenized during an acute infection. Although further transmission studies are needed, this experimental infection demonstrated that the bank vole can function as a potential BDV reservoir. In summary, multiple methods were established and applied in large panels to detect two zoonoses novel to Finland: cowpox virus and Borna disease virus. Moreover, new information was obtained on their geographical distribution, host spectrum, epidemiology and infection kinetics.

Enhanced photodynamic effect of cobalt(III) dipyridophenazine complex on thyrotropin receptor expressing HEK293 cells

Ternary cobalt(III) complexes CoL(B)] (1-3) of a trianionic tetradentate phenolate-based ligand (L) and phenanthroline bases (B), viz. 1,10-phenanthroline (phen in 1), dipyridoquinoxaline (dpq in 2) and dipyridophenazine (dppz in 3) are synthesized, characterized from X-ray crystallographic, analytical and spectral techniques, and their utility in photodynamic therapy (PDT) of thyroid diseases caused by TSH receptor dysfunction is probed. The complexes display a visible spectral band within the PDT spectral window at similar to 690 nm. Photodynamic potential was estimated through DNA cleavage activity of the dpq and dppz complexes in UV-A light of 365 nm and red light of 676 nm. The reactions proceed via the hydroxyl radical pathway. The complexes retain their DNA photocleavage activity in red light under anaerobic conditions, a situation normally prevails in hypoxic tumor core. Investigation into the photocytotoxic potential of these complexes showed that the dppz complex 3 is approximately 4-fold more active in the HEK293 cells expressing human thyrotropin receptor (HEK293-hTSHR) than in the parental cell line and has an insignificant effect on an unrelated human cervical carcinoma cell line (HeLa). Photoexcitation of complex 3 in HEK293-hTSHR cells leads to damage hTSHR as evidenced from the decrease in cAMP formation both in absence and presence of hTSH and decrease in the TSHR immunofluorescence with a concomitant cytoplasmic translocation of the membrane protein, cadherin. The involvement of hTSHR is evidenced from the ability of complex 3 to bind to the extracellular domain of hTSHR (hTSHR-ECD) with a K-d value of 81 nM and from the photocleavage of hTSHR-ECD.

Behaviour of the germ cell specific lamin through mammalian spermatogenesis as probed with monoclonal antibodies.

We had earlier identified a 60 kDa nuclear lamin protein (lamin(g)) unique to the germ cells of rat testis which was subsequently shown to be antigenically conserved in germ cells of grasshopper, rooster, frog and plants. We have now obtained eight monoclonal antibodies in mouse against this lamin(g) antigen. While all the eight Mabs reacted with lamin(g) antigen in an immunoblot analysis, only three Mabs (A(11)C(7), A(11)D(4), C1F7) showed strong reactivity in the immunofluorescence analysis of the germ cells. The Mabs A(11)C(7) and A(11)D(4) showed a slight cross-reactivity with rat liver lamin B. Indirect immunofluorescence analysis of pre-meiotic, meiotic and post-meiotic germ cells with Mabs have shown that while the lamin(g) is localized in the lamina structures of spermatogonia and round spermatids, it is localized to the phase dense regions of pachytene spermatocytes which is in conformity with our previous observations using rabbit polyclonal antibodies. The localization of the antigen in the germ cells was also confirmed by immunohistochemical staining of the thin sections of seminiferous tubules. By immunostaining the surface spread pachytene spermatocytes, the antigen was further localized to the telomeric ends of the paired homologous chromosomes. Using anti-somatic lamin B antibodies, we have also demonstrated the absence of somatic lamins in meiotic and post-meiotic germ cells. The lamina structure of pre-meiotic spermatogonial nucleus contains both somatic lamin B and lamin(g) as evidenced by immunofluorescence studies with two differently fluorochrome labelled anti-lamin B and anti-lamin(g) antibodies. The selective retention of lamin(g) in the pachytene spermatocytes is probably essential for anchoring the telomeric ends of the paired chromosomes to the inner nuclear membrane.

Inhibition of nuclear protein import by a monoclonal antibody against a novel class of nuclear pore proteins

Nuclear import of proteins is mediated by the nuclear pore complexes in the nuclear envelope and requires the presence of a nuclear localization signal (NLS) on the karyophilic protein. In this paper, we describe studies with a monoclonal antibody, Mab E2, which recognizes a class of nuclear pore proteins of 60-76 kDa with a common phosphorylated epitope on rat nuclear envelopes. The Mab Ea-reactive proteins fractionated with the relatively insoluble pore complex-containing component of the envelope and gave a finely punctate pattern of nuclear staining in immunofluorescence assays. The antibody did not bind to any cytosolic proteins. Mab E2 inhibited the interaction of a simian virus 40 large T antigen NLS peptide with a specific 60-kDa NLS-binding protein from rat nuclear envelopes in photoaffinity labeling experiments. The antibody blocked the nuclear import of NLS-albumin conjugates in an in vitro nuclear transport assay with digitonin-permeabilized cells, but did not affect passive diffusion of a small nonnuclear protein, lysozyme, across the pore. Mab E2 may inhibit protein transport by directly interacting with the 60-kDa NLS-binding protein, thereby blocking signal-mediated nuclear import across the nuclear pore complex. (C) 1994 Academic Press, Inc.

Modulating effect of gelonin gp330 conjugate on Heymann's nephritis induced in rats - A pathomorphological evaluation

Heymann's nephritis (HN) in rats induced by injecting renal proximal tubule brush border protein gp330, is an animal model replicating human autoimmune membranous glomerulonephritis(1). Endogenous IgG gets deposited between the foot processes in the epithelial side of the glomerulus and causes complement-mediated membrane injury, leading to proteinuria and basement membrane thickening. We investigated the effect of a toxin, gelonin conjugated to gp330 and targetted against antigp330-producing cells in ameliorating immune injury and nephrotic state in rats. The groups of animals injected with purified gp330 revealed by immunofluorescence, characteristic granular deposits of IgG along the basement membrane. The rats intravenously injected with gelonin gp330 conjugate, four days after the antigenic challenge with gp330 in two doses, showed amelioration of the nephrotic state and appreciable reduction in glomerular IgG deposits against immune injury. This substantiates our earlier biochemical results and corroborates the possibility of using toxins conjugated to specific antigen in treating antibody-mediated autoimmune diseases.

Japanese encephalitis virus infection of mouse cell lines: ability to prime mice for generation of virus specific cytotoxic T lymphocytes and differences in CTL recognisable viral determinants

Ten different mouse cell lines were examined for Japanese encephalitis virus (JEV) infection in vitro and then tested for their ability to generate virus specific cytotoxic T lymphocytes (CTL). Among all cell lines examined, Neuro La (a neuroblastoma) was readily infected with JEV as examined by immunofluorescence and viral replication. Among other cells, P388D1, RAW 264.7 (Macrophage origin), Sp2/0 (B-cell Hybridoma), YAC-1 (T-cell lymphoma), and L929 (Fibroblast) were semipermissive to JEV infection. The cytopathic effects caused by progressive JEV infection varied from cell line to cell line. In the case of YAC-1 cells long-term viral antigen expression was observed without significant alterations in cell viability. Intermediate degrees of cytopathicity are seen in RAW 264.7 and L929 cells while infection of PS, Neuro 2a, P388D1 and Sp2/0 caused major viability losses. All infected cell lines were able to prime adult BALB/c (H-2(d)) mice for the generation of secondary JEV specific CTL. In contrast to YAC-1, the permissive neuroblastoma cell line Neuro 2a (H-2K(k)D(d)) was found to be least efficient in its ability to stimulate anti-viral CTL generation. Cold target competition studies demonstrated that both Neuro 2a and YAC-1 (H-2K(k)D(d)) cells expressed similar viral determinants that are recognised by CTL, suggesting that the reason for the lower ability of Neuro 2a to stimulate anti-viral CTL was not due to lack of viral CTL determinants. These findings demonstrate that a variety of mouse cell lines can be infected with Japanese encephalitis virus, and that these infected cells could be utilised to generate virus specific CTL in BALB/c mice.

Angelica archengelica extract induced perturbation of rat skin and tight junctional protein (ZO-1) of HaCaT cells

Background and purpose of the study: Herbal enhancers compared to the synthetic ones have shown less toxis effects. Coumarins have been shown at concentrations inhibiting phospoliphase C-Y (Phc-Y) are able to enhance tight junction (TJ) permeability due to hyperpoalation of Zonolous Occludense-1 (ZO-1) proteins. The purpose of this study was to evaluate the influence of ethanolic extract of Angelica archengelica (AA-E) which contain coumarin on permeation of repaglinide across rat epidermis and on the tight junction plaque protein ZO-1 in HaCaT cells. Methods: Transepidermal water loss (TEWL) from the rat skin treated with different concentrations of AA-E was assessed by Tewameter. Scanning and Transmission Electron Microscopy (TEM) on were performed on AA-E treated rat skin portions. The possibility of AA-E influence on the architecture of tight junctions by adverse effect on the cytoplasmic ZO-1 in HaCaT cells was investigated. Finally, the systemic delivery of repaglinide from the optimized transdermal formulation was investigated in rats. Results: The permeation of repaglinide across excised rat epidermis was 7-fold higher in the presence of AA-E (5% w/v) as compared to propylene glycol:ethanol (7:3) mixture. The extract was found to perturb the lipid microconstituents in both excised and viable rat skin, although, the effect was less intense in the later. The enhanced permeation of repaglinide across rat epidermis excised after treatment with AA-E (5% w/v) for different periods was in concordance with the high TEWL values of similarly treated viable rat skin. Further, the observed increase in intercellular space, disordering of lipid structure and corneocyte detachment indicated considerable effect on the ultrastructure of rat epidermis. Treatment of HaCaT cell line with AA-E (0.16% w/v) for 6 hrs influenced ZO-1 as evidenced by reduced immunofluorescence of anti-TJP1 (ZO-1) antibody in Confocal Laser Scanning Microscopy studies (CLSM) studies. The plasma concentration of repaglinide from transdermal formulation was maintained higher and for longer time as compared to oral administration of repaglinide. Major conclusion: Results suggest the overwhelming influence of Angelica archengelica in enhancing the percutaneous permeation of repaglinide to be mediated through perturbation of skin lipids and tight junction protein (ZO-1).

Mutations in WDR62, encoding a centrosomal and nuclear protein, in Indian primary microcephaly families with cortical malformations

Primary microcephaly is an autosomal recessive disorder characterized by smaller than normal brain size and mental retardation. It is genetically heterogeneous with seven loci: MCPH1-MCPH7. We have previously reported genetic analysis of 35 families, including the identification of the MCPH7 gene STIL. Of the 35 families, three families showed linkage to the MCPH2 locus. Recent whole-exome sequencing studies have shown that the WDR62 gene, located in the MCPH2 candidate region, is mutated in patients with severe brain malformations. We therefore sequenced the WDR62 gene in our MCPH2 families and identified two novel homozygous protein truncating mutations in two families. Affected individuals in the two families had pachygyria, microlissencephaly, band heterotopias, gyral thickening, and dysplastic cortex. Using immunofluorescence study, we showed that, as with other MCPH proteins, WDR62 localizes to centrosomes in A549, HepG2, and HaCaT cells. In addition, WDR62 was also localized to nucleoli. Bioinformatics analysis predicted two overlapping nuclear localization signals and multiple WD-40 repeats in WDR62. Two other groups have also recently identified WDR62 mutations in MCPH2 families. Our results therefore add further evidence that WDR62 is the MCPH2 gene. The present findings will be helpful in genetic diagnosis of patients linked to the MCPH2 locus.

An Exported Heat Shock Protein 40 Associates with Pathogenesis-Related Knobs in Plasmodium falciparum Infected Erythrocytes

Cell surface structures termed knobs are one of the most important pathogenesis related protein complexes deployed by the malaria parasite Plasmodium falciparum at the surface of the infected erythrocyte. Despite their relevance to the disease, their structure, mechanisms of traffic and their process of assembly remain poorly understood. In this study, we have explored the possible role of a parasite-encoded Hsp40 class of chaperone, namely PFB0090c/PF3D7_0201800 (KAHsp40) in protein trafficking in the infected erythrocyte. We found the gene coding for PF3D7_0201800 to be located in a chromosomal cluster together with knob components KAHRP and PfEMP3. Like the knob components, KAHsp40 too showed the presence of PEXEL motif required for transport to the erythrocyte compartment. Indeed, sub-cellular fractionation and immunofluorescence analysis (IFA) showed KAHsp40 to be exported in the erythrocyte cytoplasm in a stage dependent manner localizing as punctuate spots in the erythrocyte periphery, distinctly from Maurer's cleft, in structures which could be the reminiscent of knobs. Double IFA analysis revealed co-localization of PF3D7_0201800 with the markers of knobs (KAHRP, PfEMP1 and PfEMP3) and components of the PEXEL translocon (Hsp101, PTEX150). KAHsp40 was also found to be in a complex with KAHRP, PfEMP3 and Hsp101 as confirmed by co-immunoprecipitation assay. Our results suggest potential involvement of a parasite encoded Hsp40 in chaperoning knob assembly in the erythrocyte compartment.

Identification of an exported heat shock protein 70 in Plasmodium falciparum

Host cell remodelling is a hallmark of malaria pathogenesis. It involves protein folding, unfolding and trafficking events and thus participation of chaperones such as Hsp70s and Hsp40s is well speculated. Until recently, only Hsp40s were thought to be the sole representative of the parasite chaperones in the exportome. However, based on the re-annotated Plasmodium falciparum genome sequence, a putative candidate for exported Hsp70 has been reported, which otherwise was known to be a pseudogene. We raised a specific antiserum against a C-terminal peptide uniquely present in PfHsp70-x. Immunoblotting and immunofluorescence-based approaches in combination with sub-cellular fractionation by saponin and streptolysin-O have been taken to determine the expression and localization of PfHsp70-x in infected erythrocyte. The re-annotated sequence of PfHsp70-x reveals it to be a functional protein with an endoplasmic reticulum signal peptide. It gets maximally expressed at the schizont stage of intra-erythrocytic life cycle. Majority of the protein localizes to the parasitophorous vacuole and some of it gets exported to the erythrocyte compartment where it associates with Maurer's clefts. The identification of an exported parasite Hsp70 chaperone presents us with the fact that the parasite has evolved customized chaperones which might be playing crucial roles in aspects of trafficking and host cell remodelling.

Identification of heat shock factor binding protein in Plasmodium falciparum

Background: Heat shock factor binding protein (HSBP) was originally discovered in a yeast two-hybrid screen as an interacting partner of heat shock factor (HSF). It appears to be conserved in all eukaryotes studied so far, with yeast being the only exception. Cell biological analysis of HSBP in mammals suggests its role as a negative regulator of heat shock response as it appears to interact with HSF only during the recovery phase following exposure to heat stress. While the identification of HSF in the malaria parasite is still eluding biologists, this study for the first time, reports the presence of a homologue of HSBP in Plasmodium falciparum. Methods: PfHSBP was cloned and purified as his-tag fusion protein. CD (Circular dichroism) spectroscopy was performed to predict the secondary structure. Immunoblots and immunofluorescence approaches were used to study expression and localization of HSBP in P. falciparum. Cellular fractionation was performed to examine subcellular distribution of PfHSBP. Immunoprecipitation was carried out to identify HSBP interacting partner in P. falciparum. Results: PfHSBP is a conserved protein with a high helical content and has a propensity to form homo-oligomers. PfHSBP was cloned, expressed and purified. The in vivo protein expression profile shows maximal expression in trophozoites. The protein was found to exist in oligomeric form as trimer and hexamer. PfHSBP is predominantly localized in the parasite cytosol, however, upon heat shock, it translocates to the nucleus. This study also reports the interaction of PfHSBP with PfHSP70-1 in the cytoplasm of the parasite. Conclusions: This study emphasizes the structural and biochemical conservation of PfHSBP with its mammalian counterpart and highlights its potential role in regulation of heat shock response in the malaria parasite. Analysis of HSBP may be an important step towards identification of the transcription factor regulating the heat shock response in P. falciparum.

Plasmodium berghei glycine cleavage system T-protein is non-essential for parasite survival in vertebrate and invertebrate hosts

T-protein, an aminomethyltransferase, represents one of the four components of glycine cleavage system (GCS) and catalyzes the transfer of methylene group from H-protein intermediate to tetrahydrofolate (THF) forming N-5, N-10-methylene THF (CH2-THF) with the release of ammonia. The malaria parasite genome encodes T-, H- and L-proteins, but not P-protein which is a glycine decarboxylase generating the aminomethylene group. A putative GCS has been considered to be functional in the parasite mitochondrion despite the absence of a detectable P-protein homologue. In the present study, the mitochondrial localization of T-protein in the malaria parasite was confirmed by immunofluorescence and its essentiality in the entire parasite life cycle was studied by targeting the T-protein locus in Plasmodium berghei (Pb). PbT knock out parasites did not show any growth defect in asexual, sexual and liver stages indicating that the T-protein is dispensable for parasite survival in vertebrate and invertebrate hosts. The absence of P-protein homologue and the non-essentiality of T protein suggest the possible redundancy of GCS activity in the malaria parasite. Nevertheless, the H- and L-proteins of GCS could be essential for malaria parasite because of their involvement in alpha-lcetoacid dehydrogenase reactions. (C) 2014 Elsevier B.V. All rights reserved.

Lobe-specific Expression of Phosphodiesterase 5 in Rat Prostate

OBJECTIVE To investigate the level and location of phosphodiesterase 5 (PDE5) expression in rat prostate. METHODS The ventral, dorsal, and lateral lobes of rat prostate were examined for PDE5 expression by Western blotting. Intact rat urogenital complex, including the urinary bladder and accessory reproductive glands, was examined for PDE5 expression by immunohistochemistry. Individual prostatic lobes were further examined by immunofluorescence for expression of PDE5, alpha-smooth muscle actin, and rat endothelial cell antigen. RESULTS Western blot analysis showed that PDE5 was expressed at a significantly lower level in dorsal lobe (DL) than in ventral lobe (VL) or lateral lobe (LL). Immunohistochemistry and immunofluorescence analyses showed that PDE5 was expressed in both acinar epithelium and periacinar smooth muscle. However, although similar levels of smooth muscle PDE5 expression were observed in all 3 prostatic lobes, significantly lower level of epithelial PDE5 expression was found in DL compared with VL or LL. In prostatic blood vessels, PDE5 expression was clearly visible in the endothelium but not as easily detectable in the smooth muscle. CONCLUSION PDE5 was expressed in the acinar epithelium and periacinar smooth muscle of rat prostate. However, the epithelial PDE5 expression was significantly less in DL than in VL or LL. Regardless, the acinar wall, not the blood vessel wall, is the predominant PDE5 expression site in rat prostate. (C) 2015 Elsevier Inc.

Characterization of Precursor PfHsp60 in Plasmodium falciparum Cytosol during Its Asexual Development in Human Erythrocytes

Mitochondrial heat shock protein 60 (Hsp60) is a nuclear encoded gene product that gets post-translationally translocated into the mitochondria. Using multiple approaches such as immunofluorescence experiments, isoelectric point analysis with two-dimensional gel electrophoresis, and mass spectrometric identification of the signal peptide, we show that Hsp60 from Plasmodium falciparum (PfHsp60) accumulates in the parasite cytoplasm during the ring, trophozoite, and schizont stages of parasite development before being imported into the parasite mitochondria. Using co-immunoprecipitation experiments with antibodies specific to cytoplasmic PfHsp90, PfHsp70-1, and PfHsp60, we show association of precursor PfHsp60 with cytoplasmic chaperone machinery. Metabolic labeling involving pulse and chase indicates translocation of the precursor pool into the parasite mitochondrion during chase. Analysis of results obtained with Geldanamycin treatment confirmed precursor PfHsp60 to be one of the clients for PfHsp90. Cytosolic chaperones bind precursor PfHsp60 prior to its import into the mitochondrion of the parasite. Our data suggests an inefficient co-ordination in the synthesis and translocation of mitochondrial PfHsp60 during asexual growth of malaria parasite in human erythrocytes.

Isolation of mineralizing Nestin+ Nkx6.1+ vascular muscular cells from the adult human spinal cord

Background: The adult central nervous system (CNS) contains different populations of immature cells that could possibly be used to repair brain and spinal cord lesions. The diversity and the properties of these cells in the human adult CNS remain to be fully explored. We previously isolated Nestin(+) Sox2(+) neural multipotential cells from the adult human spinal cord using the neurosphere method (i.e. non adherent conditions and defined medium). -- Results: Here we report the isolation and long term propagation of another population of Nestin(+) cells from this tissue using adherent culture conditions and serum. QPCR and immunofluorescence indicated that these cells had mesenchymal features as evidenced by the expression of Snai2 and Twist1 and lack of expression of neural markers such as Sox2, Olig2 or GFAP. Indeed, these cells expressed markers typical of smooth muscle vascular cells such as Calponin, Caldesmone and Acta2 (Smooth muscle actin). These cells could not differentiate into chondrocytes, adipocytes, neuronal and glial cells, however they readily mineralized when placed in osteogenic conditions. Further characterization allowed us to identify the Nkx6.1 transcription factor as a marker for these cells. Nkx6.1 was expressed in vivo by CNS vascular muscular cells located in the parenchyma and the meninges. -- Conclusion: Smooth muscle cells expressing Nestin and Nkx6.1 is the main cell population derived from culturing human spinal cord cells in adherent conditions with serum. Mineralization of these cells in vitro could represent a valuable model for studying calcifications of CNS vessels which are observed in pathological situations or as part of the normal aging. In addition, long term propagation of these cells will allow the study of their interaction with other CNS cells and their implication in scar formation during spinal cord injury.