Direct presentation of a melanocyte-associated antigen in peripheral lymph nodes induces cytotoxic CD8+ T cells.

Encounter of self-antigens in the periphery by mature T cells induces tolerance in the steady-state. Hence, it is not understood why the same peripheral antigens are also promiscuously expressed in the thymus to mediate central tolerance. Here, we analyzed CD8(+) T-cell tolerance to such an antigen constituted by ovalbumin under the control of the tyrosinase promoter. As expected, endogenous CD8(+) T-cell responses were altered in the periphery of transgenic mice, resulting from promiscuous expression of the self-antigen in mature medullary epithelial cells and deletion of high-affinity T cells in the thymus. In adoptive T-cell transfer experiments, we observed constitutive presentation of the self-antigen in peripheral lymph nodes. Notably, this self-antigen presentation induced persisting cytotoxic cells from high-affinity CD8(+) T-cell precursors. Lymph node resident melanoblasts expressing tyrosinase directly presented the self-antigen to CD8(+) T cells, independently of bone marrow-derived antigen-presenting cells. This peripheral priming was independent of the subcellular localization of the self-antigen, indicating that this mechanism may apply to other melanocyte-associated antigens. Hence, central tolerance by promiscuous expression of peripheral antigens is a mandatory, rather than a superfluous, mechanism to counteract the peripheral priming, at least for self-antigens that can be directly presented in lymph nodes. The peripheral priming by lymph node melanoblasts identified here may constitute an advantage for immunotherapies based on adoptive T-cell transfer.

Recanalization and Collaterals Predict Outcome in Proximal MCA Occlusion, but neither Penumbra nor Core of Stroke

Data on new predictors of outcome include penumbra core or collaterals.Objective: To test the predictive value of recanalization, collaterals, penumbra and core of ischemia for functional outcome in a large group of patients with MCA occlusion. Method: Consecutive events included prospectively in the Acute Stroke Registry and Analysis of Lausanne from April 2002 to April 2009 with an acute stroke due to proximal MCA occlusion (M1) were considered for analysis. Acute CTA were reviewed to grade the collaterals (dichotomized in poor __50% or good _50% compared to the normal side) and localization of M1 occlusion (proximal or mid-distal). Acute CTP were reviewed and reconstructed to determine penumbra, core and stroke index (penumbra/penumbra_core) of brain ischemia. Good outcome was defined by mRS 0-2 at 3 months.Results: Among 242 events (115 male, mean NIHSS 18.1, SD 5.8, mean age 66, SD 15), 42% were treated with intravenous thrombolysis, and 3% with intraarterial thrombolysis. Collateral status was rated as poor in 53% of events and proximal M1 occlusion was present in 64%. Recanalization determined at 24 hours with CTA was complete in 26% events and partial/absent in 54%.CTP was available for 212 events. Mean penumbra was 88.6 cm3 (median 84.4, SD 53.8), mean core was 54.1 cm3 (median 46.2, SD 45.7) and stroke index was 64% (median 68%, SD 25%). Good outcome was observed in 87 events (36%) and was associated in multivariate logistic regression with thrombolysis (p_0.02, OR_2.5, 95% CI 1.2-5.4), recanalization (p_0.001, OR_4.1, 95% CI 1.9-8.9), lower NIHSS (p_0.001, OR_0.84, 95% CI 0.78-0.91), male gender (p_0.01, OR_2.8, 95% CI 1.3-5.9), mRS prior to stroke (p_0.02, OR_0.5, 95% CI 0.28-0.9) and good collateral status (p_0.005, OR_3, 95% CI 1.4-6.4). Nor penumbra, nor core, nor stroke index were significant in the multivariate model, even if an association was present in the univariate model between good functional outcome and penumbra (p_0.004, OR_1.008, 95% CI 1.003-1.01), core (p_0.001, OR_0.98, 95% CI 0.976-0.99) and strokeindex (p_0.001, OR_16.7, 95% CI 4.6 59.9).Conclusion: MCA recanalization is the best predictor for good functional outcome, followed by collateral status. CTP data did not predict the functional outcome in our large group of M1 occlusion. Author Disclosures: C. Odier: None. P. Michel: Research Grant; Significant; Paion, Lundbeck. Speakers; Modest; Boehringer-Ingelheim. Consultant/Advisory Board; Modest; Boehringer- Ingelheim. Consultant/Advisory Board; Significant; Servier, Lundbeck.

A Missense Mutation in PRPF6 Causes Impairment of pre-mRNA Splicing and Autosomal-Dominant Retinitis Pigmentosa.

Retinitis pigmentosa (RP) is an inherited form of retinal degeneration that leads to progressive visual-field constriction and blindness. Although the disease manifests only in the retina, mutations in ubiquitously expressed genes associated with the tri-snRNP complex of the spliceosome have been identified in patients with dominantly inherited RP. We screened for mutations in PRPF6 (NM_012469.3), a gene on chromosome 20q13.33 encoding an essential protein for tri-snRNP assembly and stability, in 188 unrelated patients with autosomal-dominant RP and identified a missense mutation, c.2185C>T (p.Arg729Trp). This change affected a residue that is conserved from humans to yeast and cosegregated with the disease in the family in which it was identified. Lymphoblasts derived from patients with this mutation showed abnormal localization of endogenous PRPF6 within the nucleus. Specifically, this protein accumulated in the Cajal bodies, indicating a possible impairment in the tri-snRNP assembly or recycling. Expression of GFP-tagged PRPF6 in HeLa cells showed that this phenomenon depended exclusively on the mutated form of the protein. Furthermore, analysis of endogenous transcripts in cells from patients revealed intron retention for pre-mRNA bearing specific splicing signals, according to the same pattern displayed by lymphoblasts with mutations in other PRPF genes. Our results identify PRPF6 as the sixth gene involved in pre-mRNA splicing and dominant RP, corroborating the hypothesis that deficiencies in the spliceosome play an important role in the molecular pathology of this disease.

Overcoming the heterologous bias: an in vivo functional analysis of multidrug efflux transporter, CgCdr1p in matched pair clinical isolates of Candida glabrata.

We have taken advantage of the natural milieu of matched pair of azole sensitive (AS) and azole resistant (AR) clinical isolates of Candida glabrata for expressing its major ABC multidrug transporter, CgCdr1p for structure and functional analysis. This was accomplished by tagging a green fluorescent protein (GFP) downstream of ORF of CgCDR1 and integrating the resultant fusion protein at its native chromosomal locus in AS and AR backgrounds. The characterization confirmed that in comparison to AS isolate, CgCdr1p-GFP was over-expressed in AR isolates due to its hyperactive native promoter and the GFP tag did not affect its functionality in either construct. We observed that in addition to Rhodamine 6 G (R6G) and Fluconazole (FLC), a recently identified fluorescent substrate of multidrug transporters Nile Red (NR) could also be expelled by CgCdr1p. Competition assays with these substrates revealed the presence of overlapping multiple drug binding sites in CgCdr1p. Point mutations employing site directed mutagenesis confirmed that the role played by unique amino acid residues critical to ATP catalysis and localization of ABC drug transporter proteins are well conserved in C. glabrata as in other yeasts. This study demonstrates a first in vivo novel system where over-expression of GFP tagged MDR transporter protein can be driven by its own hyperactive promoter of AR isolates. Taken together, this in vivo system can be exploited for the structure and functional analysis of CgCdr1p and similar proteins wherein the artefactual concerns encountered in using heterologous systems are totally excluded.

Transient prenatal expression of NPY-Y1 receptor in trigeminal axons innervating the mystacial vibrissae.

Using immunohistochemistry in combination with confocal laser scanning microscopy, we studied the ontogeny of neuropeptide Y-Y1 receptor (Y1-R) expression in the trigeminal system of the rat. The study was limited to the nerve fibers innervating the mystacial pad and the trigeminal ganglia. In the trigeminal ganglia, Y1-R-immunoreactive (IR) neurons were first observed at E16.5. At this same stage some nerve fibers in the trigeminal ganglia also exhibited Y1-R-like immunoreactivity (LI). Strongly Y1-R-IR nerve fibers innervating the follicles of the mystacial vibrissae were first observed at E18. After double labeling, the Y1-R-LI was found to be colocalized with the neuronal marker protein gene product 9.5. At P1 only weak labeling for the Y1-R was found around the vibrissae follicles, whereas the neurons in the trigeminal ganglia were intensely labeled. The same was true for the adult rat, but at this stage no Y1-R labeling at all was observed in nerve fibers around the vibrissal follicles. These results strongly support an axonal localization of the Y1-R at this developmental stage. The transient expression of the Y1-R during prenatal mystacial pad development suggests a role for the Y1-R in the functional development of the vibrissae.

Bortezomib reverses the proliferative and antiapoptotic effect of neuropeptides on prostate cancer cells.

Objectives:  Neuropeptides are important signal initiators in advanced prostate cancer, partially acting through activation of nuclear factor kappa B. Central to nuclear factor kappa B regulation is the ubiquitin-proteasome system, pharmacological inhibition of which has been proposed as an anticancer strategy. We investigated the putative role of the proteasome inhibitor bortezomib in neuropeptides signaling effects on prostate cancer cells. Methods:  Human prostate cancer cell lines, LNCaP and PC-3, were used to examine cell proliferation, levels of proapoptotic (caspase-3, Bad) and cell cycle regulatory proteins (p53, p27, p21), as well as total and phosphorylated Akt and p44/42 mitogen-activated protein kinase proteins. Furthermore, 20S proteasome activity, subcellular localization of nuclear factor kappa B and transcription of nuclear factor kappa B target genes, interleukin-8 and vascular endothelial growth factor, were assessed. Results:  Neuropeptides (endothelin-1, bombesin) increased cell proliferation, whereas bortezomib decreased proliferation and induced apoptosis, an effect maintained after cotreatment with neuropeptides. Bad, p53, p21 and p27 were downregulated by neuropeptides in PC-3, and these effects were reversed with the addition of bortezomib. Neuropeptides increased proteasomal activity and nuclear factor kappa B levels in PC-3, and these effects were prevented by bortezomib. Interleukin-8 and vascular endothelial growth factor transcripts were induced after neuropeptides treatment, but downregulated by bortezomib. These results coincided with the ability of bortezomib to reduce mitogen-activated protein kinase signaling in both cell lines. Conclusions:  These findings are consistent with bortezomib-mediated abrogation of neuropeptides-induced proliferative and antiapoptotic signaling. Thus, the effect of the drug on the neuropeptides axis needs to be further investigated, as neuropeptide action in prostate cancer might entail involvement of the proteasome.

Dynamic regulation of notch 1 and notch 2 surface expression during T cell development and activation revealed by novel monoclonal antibodies.

It is well established that Notch signaling plays a critical role at multiple stages of T cell development and activation. However, detailed analysis of the cellular and molecular events associated with Notch signaling in T cells is hampered by the lack of reagents that can unambiguously measure cell surface Notch receptor expression. Using novel rat mAbs directed against the extracellular domains of Notch1 and Notch2, we find that Notch1 is already highly expressed on common lymphoid precursors in the bone marrow and remains at high levels during intrathymic maturation of CD4(-)CD8(-) thymocytes. Notch1 is progressively down-regulated at the CD4(+)CD8(+) and mature CD4(+) or CD8(+) thymic stages and is expressed at low levels on peripheral T cells. Immunofluorescence staining of thymus cryosections further revealed a localization of Notch1(+)CD25(-) cells adjacent to the thymus capsule. Notch1 was up-regulated on peripheral T cells following activation in vitro with anti-CD3 mAbs or infection in vivo with lymphocytic chorio-meningitis virus or Leishmania major. In contrast to Notch1, Notch2 was expressed at intermediate levels on common lymphoid precursors and CD117(+) early intrathymic subsets, but disappeared completely at subsequent stages of T cell development. However, transient up-regulation of Notch2 was also observed on peripheral T cells following anti-CD3 stimulation. Collectively our novel mAbs reveal a dynamic regulation of Notch1 and Notch2 surface expression during T cell development and activation. Furthermore they provide an important resource for future analysis of Notch receptors in various tissues including the hematopoietic system.

Lateralization of olfactory processing: Differential impact of right and left temporal lobe epilepsies.

Olfactory processes were reported to be lateralized. The purpose of this study was to further explore this phenomenon and investigate the effect of the hemispheric localization of epileptogenic foci on olfactory deficits in patients with temporal lobe epilepsy (TLE). Olfactory functioning was assessed in 61 patients and 60 healthy control (HC) subjects. The patients and HC subjects were asked to rate the intensity, pleasantness, familiarity, and edibility of 12 common odorants and then identify them. Stimulations were delivered monorhinally in the nostril ipsilateral to the epileptogenic focus in TLE and arbitrarily in either the left or the right nostril in the HC subjects. The results demonstrated that regardless of the side of stimulation, patients with TLE had reduced performance in all olfactory tasks compared with the HC subjects. With regard to the side of the epileptogenic focus, patients with left TLE judged odors as less pleasant and had more difficulty with identification than patients with right TLE, underlining a privileged role of the left hemisphere in the emotional and semantic processing of odors. Finally, irrespective of group, a tendency towards a right-nostril advantage for judging odor familiarity was found in agreement with a prominent role of the right hemisphere in odor memory processing.

In vivo visualization of F-actin structures during the development of the moss Physcomitrella patens.

* The 'in planta' visualization of F-actin in all cells and in all developmental stages of a plant is a challenging problem. By using the soybean heat inducible Gmhsp17.3B promoter instead of a constitutive promoter, we have been able to label all cells in various developmental stages of the moss Physcomitrella patens, through a precise temperature tuning of the expression of green fluorescent protein (GFP)-talin. * A short moderate heat treatment was sufficient to induce proper labeling of the actin cytoskeleton and to allow the visualization of time-dependent organization of F-actin structures without impairment of cell viability. * In growing moss cells, dense converging arrays of F-actin structures were present at the growing tips of protonema cell, and at the localization of branching. Protonema and leaf cells contained a network of thick actin cables; during de-differentiation of leaf cells into new protonema filaments, the thick bundled actin network disappeared, and a new highly polarized F-actin network formed. * The controlled expression of GFP-talin through an inducible promoter improves significantly the 'in planta' imaging of actin.

Fusion of the human gene for the polyubiquitination co-effector UEV-1 with Kua, a newly identified gene

UEV proteins are enzymatically inactive variants of the E2 ubiquitin-conjugating enzymes that regulate noncanonical elongation of ubiquitin chains. In Saccharomyces cerevisiae, UEV is part of the RAD6-mediated error-free DNA repair pathway. In mammalian cells, UEV proteins can modulate c-FOS transcription and the G2-M transition of the cell cycle. Here we show that the UEV genes from phylogenetically distant organisms present a remarkable conservation in their exon–intron structure. We also show that the human UEV1 gene is fused with the previously unknown gene Kua. In Caenorhabditis elegans and Drosophila melanogaster, Kua and UEV are in separated loci, and are expressed as independent transcripts and proteins. In humans, Kua and UEV1 are adjacent genes, expressed either as separate transcripts encoding independent Kua and UEV1 proteins, or as a hybrid Kua–UEV transcript, encoding a two-domain protein. Kua proteins represent a novel class of conserved proteins with juxtamembrane histidine-rich motifs. Experiments with epitope-tagged proteins show that UEV1A is a nuclear protein, whereas both Kua and Kua–UEV localize to cytoplasmic structures, indicating that the Kua domain determines the cytoplasmic localization of Kua–UEV. Therefore, the addition of a Kua domain to UEV in the fused Kua–UEV protein confers new biological properties to this regulator of variant polyubiquitination.[Kua cDNAs isolated by RT-PCR and described in this paper have been deposited in the GenBank data library under accession nos. AF1155120 (H. sapiens) and AF152361 (D. melanogaster). Genomic clones containing UEV genes: S. cerevisiae, YGL087c (accession no. Z72609); S. pombe, c338 (accession no. AL023781); P. falciparum, MAL3P2 (accession no. AL034558); A. thaliana, F26F24 (accession no. AC005292); C. elegans, F39B2 (accession no. Z92834); D. melanogaster, AC014908; and H. sapiens, 1185N5 (accession no. AL034423). Accession numbers for Kua cDNAs in GenBank dbEST: M. musculus, AA7853; T. cruzi, AI612534. Other Kua-containing sequences: A. thaliana genomic clones F10M23 (accession no. AL035440), F19K23 (accession no. AC000375), and T20K9 (accession no. AC004786).

Coat color dilution in mice because of inactivation of the melanoma antigen MART-1.

Melanoma antigen recognized by T cells 1 (MART-1) is a melanoma-specific antigen, which has been thoroughly studied in the context of immunotherapy against malignant melanoma and which is found only in the pigment cell lineage. However, its exact function and involvement in pigmentation is not clearly understood. Melanoma antigen recognized by T cells 1 has been shown to interact with the melanosomal proteins Pmel17 and OA1. To understand the function of MART-1 in pigmentation, we developed a new knockout mouse model. Mice deficient in MART-1 are viable, but loss of MART-1 leads to a coat color phenotype, with a reduction in total melanin content of the skin and hair. Lack of MART-1 did not affect localization of melanocyte-specific proteins nor maturation of Pmel17. Melanosomes of hair follicle melanocytes in MART-1 knockout mice displayed morphological abnormalities, which were exclusive to stage III and IV melanosomes. In conclusion, our results suggest that MART-1 is a pigmentation gene that is required for melanosome biogenesis and/or maintenance.

The TPTE gene family: cellular expression, subcellular localization and alternative splicing.

The human TPTE (Transmembrane Phosphatase with TEnsin homology) gene family encodes a PTEN-related tyrosine phosphatase with four potential transmembrane domains. Chromosomal mapping revealed multiple copies of the TPTE gene on chromosomes 13, 15, 21, 22 and Y. Human chromosomes 13 and 21 copies encode two functional proteins, TPIP (TPTE and PTEN homologous Inositol lipid Phosphatase) and TPTE, respectively, whereas only one copy of the gene exists in the mouse genome. In the present study, we show that TPTE and TPIP proteins are expressed in secondary spermatocytes and/or prespermatids. In addition, we report the existence of several novel alternatively spliced isoforms of these two proteins with variable number of transmembrane domains. The latter has no influence on the subcellular localization of these different peptides as shown by co-immunofluorescence experiments. Finally, we identify another expressed TPTE copy, mapping to human chromosome 22, whose transcription appears to be under the control of the LTR of human endogenous retrovirus RTVL-H3.

Chitosan-based nanogels for selective delivery of photosensitizers to macrophages and improved retention in and therapy of articular joints.

Macrophages play key roles in inflammatory disorders. Therefore, they are targets of treatments aiming at their local destruction in inflammation sites. However, injection of low molecular mass therapeutics, including photosensitizers, in inflamed joints results in their rapid efflux out of the joints, and poor therapeutic index. To improve selective uptake and increase retention of therapeutics in inflamed tissues, hydrophilic nanogels based on chitosan, of which surface was decorated with hyaluronate and which were loaded with one of three different anionic photosensitizers were developed. Optimal uptake of these functionalized nanogels by murine RAW 264.7 or human THP-1 macrophages as models was achieved after <4h incubation, whereas only negligible uptake by murine fibroblasts used as control cells was observed. The uptake by cells and the intracellular localization of the photosensitizers, of the fluorescein-tagged chitosan and of the rhodamine-tagged hyaluronate were confirmed by fluorescence microscopy. Photodynamic experiments revealed good cell photocytotoxicity of the photosensitizers entrapped in the nanogels. In a mouse model of rheumatoid arthritis, injection of free photosensitizers resulted in their rapid clearance from the joints, while nanogel-encapsulated photosensitizers were retained in the inflamed joints over a longer period of time. The photodynamic treatment of the inflamed joints resulted in a reduction of inflammation comparable to a standard corticoid treatment. Thus, hyaluronate-chitosan nanogels encapsulating therapeutic agents are promising materials for the targeted delivery to macrophages and long-term retention of therapeutics in leaky inflamed articular joints.

Cellular distribution of glucose and monocarboxylate transporters in human brain white matter and multiple sclerosis lesions.

To ensure efficient energy supply to the high demanding brain, nutrients are transported into brain cells via specific glucose (GLUT) and monocarboxylate transporters (MCT). Mitochondrial dysfunction and altered glucose metabolism are thought to play an important role in the progression of neurodegenerative diseases, including multiple sclerosis (MS). Here, we investigated the cellular localization of key GLUT and MCT proteins in human brain tissue of non-neurological controls and MS patients. We show that in control brain tissue GLUT and MCT proteins were abundantly expressed in a variety of central nervous system cells, particularly in microglia and endothelial cells. In active MS lesions, GLUTs and MCTs were highly expressed in infiltrating leukocytes and reactive astrocytes. Astrocytes manifest increased MCT1 staining and maintain GLUT expression in inactive lesions, whereas demyelinated axons exhibit significantly reduced GLUT3 and MCT2 immunoreactivity in inactive lesions. Finally, we demonstrated that the co-transcription factor peroxisome proliferator-activated receptor gamma co-activator 1-alpha (PGC-1α), an important protein involved in energy metabolism, is highly expressed in reactive astrocytes in active MS lesions. Overexpression of PGC-1α in astrocyte-like cells resulted in increased production of several GLUT and MCT proteins. In conclusion, we provide for the first time a comprehensive overview of key nutrient transporters in white matter brain samples. Moreover, our data demonstrate an altered expression of these nutrient transporters in MS brain tissue, including a marked reduction of axonal GLUT3 and MCT2 expression in chronic lesions, which may impede efficient nutrient supply to the hypoxic demyelinated axons thereby contributing to the ongoing neurodegeneration in MS. GLIA 2014;62:1125-1141.

Multiparametric brainstem segmentation using a modified multivariate mixture of Gaussians

The human brainstem is a densely packed, complex but highly organised structure. It not only serves as a conduit for long projecting axons conveying motor and sensory information, but also is the location of multiple primary nuclei that control or modulate a vast array of functions, including homeostasis, consciousness, locomotion, and reflexive and emotive behaviours. Despite its importance, both in understanding normal brain function as well as neurodegenerative processes, it remains a sparsely studied structure in the neuroimaging literature. In part, this is due to the difficulties in imaging the internal architecture of the brainstem in vivo in a reliable and repeatable fashion. A modified multivariate mixture of Gaussians (mmMoG) was applied to the problem of multichannel tissue segmentation. By using quantitative magnetisation transfer and proton density maps acquired at 3 T with 0.8 mm isotropic resolution, tissue probability maps for four distinct tissue classes within the human brainstem were created. These were compared against an ex vivo fixated human brain, imaged at 0.5 mm, with excellent anatomical correspondence. These probability maps were used within SPM8 to create accurate individual subject segmentations, which were then used for further quantitative analysis. As an example, brainstem asymmetries were assessed across 34 right-handed individuals using voxel based morphometry (VBM) and tensor based morphometry (TBM), demonstrating highly significant differences within localised regions that corresponded to motor and vocalisation networks. This method may have important implications for future research into MRI biomarkers of pre-clinical neurodegenerative diseases such as Parkinson's disease.