Characterization of E14 spinal cord-derived progenitor cells.

<p>Progenitor cells obtained from E14 fetal spinal cord formed sphere in the growth medium containing EGF/FGF2 (A: phase contrast image) and they were nestin (B: identical field to A) and BrdU immunoreactive (C). After dissociation, these cells were seeded on PEI-coated coverslips in no EGF/FGF2 and 1%FBS containing culture medium (D: phase contrast image). At 1div, over 90% of the dissociated progenitor cells were still nestin immunoreactive (E: identical field to D) and there were also trkB (F) and/or CNTFRα immunoreactive (G). These cells slowed or stopped dividing and during differentiation about 30% of the cells were labeled with BrdU at 7 div (H). At 7 div, these cells were triple-stained with anti-TuJ1 for neuron (I), GFAP for astrocyte (J) and O4 for oligodendrocyte (K). Approximately 50% of these cells differentiated into astrocytes.</p

Neurotrophic factor gene profile of E14 spinal cord derived-progenitor cells during differentiation <i>in vitro.</i>

<p>After 2–4 passages, cells were seeded on the PEI-coated culture flasks at a density of 2×10⁵ cells/ml in the differentiation medium (no EGF/FGF2, 1% FBS). Culture cells were harvested at 1, 3 and 7 days in vitro (div). As a control cells were harvested immediately after dissociation (0 div). Total RNA (5 µg) was loaded at each lane for RNase protection assay. Before differentiation (0 div), there were two prominent gene expression of BDNF and CNTF. While BDNF gene expression decreased significantly at 3 and 7 div, CNTF gene expression increased significantly at 3 and 7 div. Each value represented mean ± SEM of % GAPDH obtained from three independent experiments. GAPDH was used as an internal control to normalize for loading differences. * Significant difference compared to 0 div (p<0.05, Tukey <i>post hoc</i> test).</p

Neutralization of endogenous CNTF inhibited the differentiation of spinal progenitor cells into astrocytes.

<p>After 2–4 passages, cells were seeded on PEI-coated coverslips at a density of 2×10⁵ cells/ml in the differentiation medium with different concentrations of anti-CNTF antibody (0–500 µg/ml). At 7div the culture cells were triple-stained with TuJ1 (A), GFAP (C) and O4 (E). Neutralization of endogenous CNTF by polyclonal antibody (100 µg/ml and more) significantly decreased the number of astrocytes (D), but did not affect the numbers of neurons (B) or oligodendrocytes (F). * Significant difference compared to the control (p<0.05, Tukey <i>post hoc</i> test).</p

Effect of neutralization of endogenous CNTF on the survival and proliferation of spinal progenitor cells.

<p>Comparisons of total cell number (A) and the percentage of BrdU (B) and nestin (C) positive cells to total cell number between control and neutralization groups (anti-CNTF antibody, 100 µg/ml). Neutralization of endogenous CNTF did not affect the survival or proliferation of spinal progenitor cells, but increased the number of undifferentiated progenitor cells expressing nestin (C). * Significant difference between two groups (p<0.05, Mann-Whitney U-test).</p

Effects of additional application of exogenous neurotrophic factors on the differentiation of spinal progenitor cells.

<p>Treatments of cultures were as follows in each bar graph: (1) control (normal rat IgG 100 µg/ml); (2) BDNF (20 ng/ml); (3) NT-3 (20 ng/ml); (4) anti-CNTF (100 µg/ml); (5) anti-CNTF +BDNF; (6) anti-CNTF + NT-3. At 7 div after each treatment, cells were triple-stained with TuJ1 for neuron, GFAP for astrocyte and O4 for oligodendrocyte. Combined treatments of anti-CNTF plus exogenous BDNF (B, E, H) or NT-3 (C, F, I) not only inhibited the astrocytic differentiation (lanes 5 and 6 in D, E, F), but also promoted the differentiation of spinal progenitor cells into neurons (lane 5 in A, B) or oligodendrocytes (lane 6 in G, I), respectively. The application of exogenous BDNF alone (lane 2 in A) or NT-3 alone (lane 3 in G) also increased the numbers of neuron or oligodendrocytes, respectively. However, the differentiation of progenitor cells into astrocytes was not inhibited by these treatments (lanes 2 and 3 in D). Compared to the cultures treated by NT-3 alone (lane 3 in G), there was a significant increase in the number of oligodendrocytes in the cultures treated by anti-CNTF plus NT-3 (lane 6 in G). * Significant difference compared to the control (lane 1 in A, D, G) (p<0.05, Tukey <i>post hoc</i> test). # Significant difference compared to NT-3 alone (lane 3 in G) (p<0.05, Tukey <i>post hoc</i> test).</p

Neurotrophic factor gene profile of spinal cord during development.

<p>Spinal cord tissues were harvested from embryos or neonates at each developmental age. Total RNA (10 µg) was loaded at each lane for RNase protection assay (A). There were three different temporal patterns of neurotrophic factor gene expression. NT-3 gene expression peaked at E14 and then declined sharply (D). BDNF gene expression (C), followed by NGF (B) and GDNF (F), increased between E19 and P10. Initial moderate expression of CNTF increased significantly at P10 and P17 (G). Each value represented mean ± SEM of % GAPDH obtained from four independent experiments. GAPDH was used as an internal control to normalize for loading differences. * Significant difference compared to the other developmental ages except (*), # Significant difference compared to the other developmental ages including (*) (p<0.05, Tukey <i>post hoc</i> test).</p

Demographic, clinical and therapeutic data at the time of an enrollment in IORRA.

<p>*Maximum value of RF measured in the cohort project during 2000–2010 for each individual was used.</p>†<p>Cut-off  = 4.5 IU/ml.</p><p>IORRA, Institute of Rheumatology Rheumatoid Arthritis cohort study; BMI, body mass index; DAS28, disease activity score in 28 joints; J-HAQ, the Japanese version of Health Assessment Questionnaire; RF, rheumatoid factor; ACPA, anti-citrullinated peptide antibody; TKR, total knee replacement; DMARDs, disease modifying antirheumatic drugs.</p

Multivariate Cox proportional hazards model of each SNP associated with the occurrence of hip fracture.

<p>All analyses were adjusted for independent non-genetic factors: age, body mass index, Japanese version of Health Assessment Questionnaire disability score, and history of total knee replacement. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0104587#pone.0104587-Furuya1" target="_blank">[7]</a>.</p><p>*Alleles are listed as major allele/minor allele.</p><p>SNP, single nucleotide polymorphism; MAF, minor allele frequency; HR, hazard ratio; CI, confidence interval; <i>OPG</i>, osteoprotegerin; <i>ZBTB40</i>, zinc finger and BTB domain containing 40; <i>MHC</i>, major histocompatibility complex; <i>RANK</i>, receptor activator of the nuclear factor-κB; <i>SPTBN1</i>, spectrin β nonerythrocytic 1; <i>LRP4</i>, low-density lipoprotein receptor-related protein 4.</p

The systemic overexpression of TACE causes no overt defects.

<p>(A) A schematic of the transgene construct. pA, polyadenylation signal sequence. (B) Gross morphology of the 8-week-old control (Ctrl) and <i>Tace</i>-Tg (Tg) mice. (C) Quantitative RT-PCR analysis of <i>Tace</i> expression in the liver, lung, skin, spleen, bone marrow cells (BM), and thymus from 8-week-old control (Ctrl) and <i>Tace</i>-Tg (Tg) mice. The expression level of <i>Tace</i> in each organ of the control mice is set to 1. Bars, S.D. *p<0.05. **p<0.005. (D) Western blot analysis using anti-HA, anti-TACE, and anti-β-Actin antibodies. (E) Hematoxylin and eosin-stained sections of the spleen, liver, and tibia from 8-week-old control, <i>Tace</i>-Tg, and <i>Tace</i>-tg/<i>Tace^−/−</i> (Tg/<i>Tace^−/−</i>) mice.</p

Stepwise multiple regression analysis on risk factors for radiographic progression (n = 830).

<p>Multiple R squared value = 0.055.</p><p>95% CI, 95% confidence interval; ACPA, anti-citrullinated peptide antibody; SE, shared epitope; <i>PADI4</i>, peptidyl arginine deiminase type IV.</p