Journal: International Journal of Molecular Sciences

Article Title: Expression of Carbonic Anhydrase I in Motor Neurons and Alterations in ALS

doi: 10.3390/ijms17111820

Figure Lengend Snippet: CA1 is expressed in human spinal cord motor neurons. Images of the normal human spinal cord immune-stained with CA1 or CA2 antibody using the DAB method (brown color) counter-stained with hematoxylin (blue color). The GαCA1 (1:500) and HRP-RbαCA2 (1:500) antibodies were used for this experiment. ( A ) A low magnification image of the ventral horn of spinal cord stained with the CA1 antibody. Two representative motor neurons are indicated by arrows. The white scale bar indicates 0.25 mm; ( B , C ) Higher magnification of spinal cord images stained with the CA1 antibody; ( D , E ) Higher magnification of spinal cord images stained with the CA2 antibody; The black scale bar indicates 50 μm for ( B – E ).

Article Snippet: Both recombinant human CA1 (#2180-CA) and CA2 (#2184-CA) were purchased from R&D Systems (Minneapolis, MN, USA).

Techniques: Staining

Journal: International Journal of Molecular Sciences

Article Title: Expression of Carbonic Anhydrase I in Motor Neurons and Alterations in ALS

doi: 10.3390/ijms17111820

Figure Lengend Snippet: The punctate CA1-immunoreactivity co-localizes with the ER marker in control spinal cord motor neurons. Images are motor neurons double-labeled with fluorescent antibodies against CA1 ( red , GαCA1, 1:100) and PDI ( green ) or neurofilaments (SM31, SM32, green ) and the overlapped signals are shown in the far right panels. Images were from spinal cord sections of three control subjects Two representative motor neurons of each double-labeling set are presented: CA1 and PDI ( A – C , A’ – C’ ); CA1 & SM31 ( D – F , D’ – F’ ); and CA1 and SM32 ( G – I , G’ – I’ ). The white scale bar indicates 20 μm.

Article Snippet: Both recombinant human CA1 (#2180-CA) and CA2 (#2184-CA) were purchased from R&D Systems (Minneapolis, MN, USA).

Techniques: Marker, Control, Labeling

Journal: International Journal of Molecular Sciences

Article Title: Expression of Carbonic Anhydrase I in Motor Neurons and Alterations in ALS

doi: 10.3390/ijms17111820

Figure Lengend Snippet: CA1 is differentially regulated from CA2 in ALS spinal cord. ( A ) Western blot analysis of the proteins from either the cytosolic (cyto) or microsomal (mv) fractionation extracted from the spinal cords of the control or ALS subjects probed with CA1 (HRP-GαCA1, 1:5000), CA2 (HRP-RbαCA2, 1:5000), SOD1, and PDI antibodies. An equal amount of proteins were used for each lane for either “cyto” or “mv” fraction; ( B ) Quantitative analyses of the differences in the intensities of immune-reactive signals for each protein between the control and ALS groups. All data points were indicated for each group. The dotted horizontal and solid vertical lines in each group represent “Mean ± SD” of the group value. p values are indicated for each graph, and * indicates p < 0.05.

Article Snippet: Both recombinant human CA1 (#2180-CA) and CA2 (#2184-CA) were purchased from R&D Systems (Minneapolis, MN, USA).

Techniques: Western Blot, Fractionation, Control

Journal: International Journal of Molecular Sciences

Article Title: Expression of Carbonic Anhydrase I in Motor Neurons and Alterations in ALS

doi: 10.3390/ijms17111820

Figure Lengend Snippet: Altered CA1-immunoreactive patterns in ALS pathology. Spinal cord sections were immunohistochemically stained for CA1-immunoreactivity (brown, GαCA1, 1:500) and counter-stained with hematoxylin ( blue ). Three randomly selected images (indicated by 1–3) were shown for each sample. Samples labeled in black ( A – C ) are from control subjects; and those labeled in red ( D – E ) are from ALS patients. Neurons with the normal punctate CA1-immunoreactive distribution are indicated by black arrows and those with altered CA1-immunoreactive pattern were indicated by red arrows. The black scale bar indicates 100 μm.

Article Snippet: Both recombinant human CA1 (#2180-CA) and CA2 (#2184-CA) were purchased from R&D Systems (Minneapolis, MN, USA).

Techniques: Staining, Labeling, Control

Journal: International Journal of Molecular Sciences

Article Title: Expression of Carbonic Anhydrase I in Motor Neurons and Alterations in ALS

doi: 10.3390/ijms17111820

Figure Lengend Snippet: Long-term expression of CA1 decreases HEK293 cell survival. The expression of either GFP or CA1 was induced by 0.25 μg/mL doxycycline (DOX) in HEK293 cells at different times. The expression of GFP ( A ) and CA1 ( B ) in absence (−) and presence (+) of DOX was examined at 96 h by Western blot analysis; CA1 antibody (ab1088367, 1:5000) was used for ( B ); The rate of cell survival was measured by the WST8 assay at the indicated time (48, 96 and 144 h, respectively) in absence (−) and presence (+) of DOX for GFP ( C ) and CA1 ( D ). The data are the average of three independent experiments and expressed as “Mean ± SEM”. * indicates p value < 0.05. The p values (not indicated by *) for GFP at 48, 96, 144 h, and CA1 at 48 h are 0.68, 0.80, 0.20, and 0.15, respectively.

Article Snippet: Both recombinant human CA1 (#2180-CA) and CA2 (#2184-CA) were purchased from R&D Systems (Minneapolis, MN, USA).

Techniques: Expressing, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: Expression of Carbonic Anhydrase I in Motor Neurons and Alterations in ALS

doi: 10.3390/ijms17111820

Figure Lengend Snippet: Expression of CA1 in HEK293 cells induces activation of apoptosis indicated by the cleavage of both PARP-1 and Caspase-3. CA1 expression was induced by 0.25 μg/mL doxycycline (DOX) for 96 h and HEK293 cells were analyzed via FACS using PARP-1 and Caspase-3 antibodies which recognized only cleaved protein fragments, in absence (−) or presence (+) of DOX. The expression of GFP was used as the control. ( A , C ) Representative FACS profiles of measuring cleaved PARP-1 ( A ) and cleaved Caspase-3 ( C ) in HEK293 cells; The histogram is plotted with the cleaved Caspase-3 or cleaved PARP-1 fluorescent intensity ( x -axis) against cell counts ( y -axis). M1 and M2 gates mark the cell population used to observe the fluorescence shift across the x -axis. The percentage of the cells in M1 and M2 was indicated directly above the gated line; ( B , D ) Bar graphs representing the degrees of PARP-1 ( B ) and Caspase-3 ( D ) cleavage as indicated by the percentage of M2 gated cells. The data are the average of three independent experiments and expressed as “Mean ± SEM”. * indicates p value < 0.05.

Article Snippet: Both recombinant human CA1 (#2180-CA) and CA2 (#2184-CA) were purchased from R&D Systems (Minneapolis, MN, USA).

Techniques: Expressing, Activation Assay, Control, Fluorescence

Journal: Journal of visualized experiments : JoVE

Article Title: Using microarrays to interrogate microenvironmental impact on cellular phenotypes in cancer.

doi: 10.3791/58957

Figure Lengend Snippet: The full list of ECM proteins and conditions that are used in the MEMA experiments. The uniprot ID, stock concentrations, and final working concentrations are provided. In some instances, the printed condition represents a protein complex or a combination of multiple proteins, which is indicated in the notes column.

Article Snippet: Cadherin-20 (CDH20) , R & D Systems , 5604-CA , ECM.

Techniques: Concentration Assay

Journal: Journal of visualized experiments : JoVE

Article Title: Using microarrays to interrogate microenvironmental impact on cellular phenotypes in cancer.

doi: 10.3791/58957

Figure Lengend Snippet: Materials

Article Snippet: Cadherin-20 (CDH20) , R & D Systems , 5604-CA , ECM.

Techniques: Imaging, Inverted Microscopy, Microarray, Electron Microscopy

Journal: Retrovirology

Article Title: Increased expression of OX40 is associated with progressive disease in patients with HTLV-1-associated myelopathy/tropical spastic paraparesis

doi: 10.1186/1742-4690-10-51

Figure Lengend Snippet: Tax-dependent constitutive expression of OX40 in HTLV-1-infected T-cell lines and Tax-inducible JPX-9 cell line. A. Representative histograms of OX40 expression in 6 HTLV-1 infected T-cell lines (HUT-102, MT-1, MT-2, MT-4, C5/MJ, SLB-1) and two HTLV-1-uninfected T-cell lines (CEM-OX40L and CEM-OX40). Shaded histograms represent the isotype control. Tax+ or Tax- means whether these cells express Tax (Tax+) or not (Tax-). B. Representative histograms of OX40L expression in 6 HTLV-1 infected T-cell lines (HUT-102, MT-1, MT-2, MT-4, C5/MJ, SLB-1) and two HTLV-1-uninfected T-cell lines (CEM-OX40L and CEM-OX40). Shaded histograms are isotype controls. C. Flow cytometric analysis of expression of OX40 after induction of Tax in JPX-9 cells. D. Flow cytometric analysis of expression of OX40L after induction of Tax in JPX-9 cells. E. Soluble OX40 and OX40L levels in cell culture supernatant and cell lysate from 6 HTLV-1 infected T-cell lines (HUT-102, MT-1, MT-2, MT-4, C5/MJ, SLB-1) and three HTLV-1-uninfected T-cell lines (CEM-mock, CEM-OX40L and CEM-OX40). F. Soluble OX40 and OX40L levels in cell culture supernatant and cell lysate from JPX-9 cell line treated with CdCl 2 along with the induction of viral transactivator Tax.

Article Snippet: Recombinant human OX40 ligand/TNFSF4 and recombinant human OX40/TNFRSF4/Fc Chimera were purchased from R&D Systems (Minneapolis, MN) and used for the standard curve in sOX40L and sOX40 ELISA, respectively.

Techniques: Expressing, Infection, Cell Culture

Journal: Retrovirology

Article Title: Increased expression of OX40 is associated with progressive disease in patients with HTLV-1-associated myelopathy/tropical spastic paraparesis

doi: 10.1186/1742-4690-10-51

Figure Lengend Snippet: OX40 is specifically expressed on the surface of T cells naturally infected with HTLV-1 that have the potential to produce pro-inflammatory cytokines. A. OX40 was detected on CD4 + T cells of HAM/TSP patient with anti-OX40 mAb (clones B-7B5) after 16 hours in vitro cultivation in the absence of any growth factors or mitogen. B. OX40L was not detected on CD4 + T cells of HAM/TSP patient with anti-OX40L mAb (clones 5A8) after 16 hours in vitro cultivation in the absence of any growth factors or mitogen. C. Tax protein was detected in CD4 + T cells of HAM/TSP patient after 16 hours in vitro cultivation. D. OX40 was expressed almost exclusively in naturally infected CD4 + T cells that also expressed Tax in HAM/TSP patient. E. Both HTLV-1 tax and OX40 mRNA expression in CD4 + T cells was increased after 16 hours in vitro cultivation. F. The frequency of pro-inflammatory cytokine positive cells within the OX40 + CD4 + and Tax + CD4 + populations from HTLV-1 infected individuals are significantly higher than OX40 - CD4 + and Tax - CD4 + T cells, respectively (p<0.001, Student’s t- test). One representative experiment of HAM/TSP patient (HAM/TSP1) is shown.

Article Snippet: Recombinant human OX40 ligand/TNFSF4 and recombinant human OX40/TNFRSF4/Fc Chimera were purchased from R&D Systems (Minneapolis, MN) and used for the standard curve in sOX40L and sOX40 ELISA, respectively.

Techniques: Infection, Clone Assay, In Vitro, Expressing

Journal: Retrovirology

Article Title: Increased expression of OX40 is associated with progressive disease in patients with HTLV-1-associated myelopathy/tropical spastic paraparesis

doi: 10.1186/1742-4690-10-51

Figure Lengend Snippet: Increased expression of OX40 in vivo in rapidly progressive HAM/TSP patients. A. The plasma levels of soluble OX40 (sOX40) measured by ELISA. The plasma levels of sOX40 in typical HAM/TSP patients (chronic HAM: n=20), asymptomatic carriers (ACs: n=9) and normal uninfected healthy controls (NCs: n=13). B. No correlation between the plasma levels of sOX40 and HTLV-1 proviral load (tax copies/10,000PBMCs) from 29 HTLV-1 infected individuals (20 chronic HAM/TSP patients and 9 ACs). Data were analyzed by Spearman rank correlation. C. The cerebrospinal fluid (CSF) levels of sOX40 in rapidly progressive HAM/TSP patients (n=3), chronic HAM/TSP patients (n=22) and other neurological diseases including multiple sclerosis (MS) (n=12), aseptic meningitis (n=8), systemic lupus erythematosus (SLE) with neurological manifestations (n=5), chronic inflammatory demyelinating polyneuropathy (CIDP) (n=9), Guillain-Barré syndrome (GBS) (n=6), and amyotrophic lateral sclerosis (ALS) (n=9). Chronic HAM/TSP means typical cases fulfilling diagnostic criteria and rapidly progressive HAM/TSP is defined by patients’ incapacity to walk unaided within three months after symptoms’ onset. D. The levels of sOX40 in the CSF from HTLV-1 infected other inflammatory neurological diseases (HTLV-1+ OINDs), i.e. any inflammatory neurological disorders except for HAM/TSP which occurred in HTLV-1 infected individuals, was not significantly different from that of chronic HAM/TSP, whereas the levels of sOX40 from HTLV-1+ OINDs was significantly increased than that of non-infected OINDs (HTLV-1- OINDs). HTLV-1+ OINDs: 1 multiple sclerosis (MS), 1 SLE with neurological manifestations, 4 aseptic meningitis. HTLV-1- OINDs: 9 MS, 5 SLE with neurological manifestations, 7 CIDP, 5 GBS.

Article Snippet: Recombinant human OX40 ligand/TNFSF4 and recombinant human OX40/TNFRSF4/Fc Chimera were purchased from R&D Systems (Minneapolis, MN) and used for the standard curve in sOX40L and sOX40 ELISA, respectively.

Techniques: Expressing, In Vivo, Enzyme-linked Immunosorbent Assay, Infection, Diagnostic Assay

Journal: Retrovirology

Article Title: Increased expression of OX40 is associated with progressive disease in patients with HTLV-1-associated myelopathy/tropical spastic paraparesis

doi: 10.1186/1742-4690-10-51

Figure Lengend Snippet: The expression of pro-inflammatory cytokines in peripheral blood mononuclear cells of HTLV-1 infected individuals

Article Snippet: Recombinant human OX40 ligand/TNFSF4 and recombinant human OX40/TNFRSF4/Fc Chimera were purchased from R&D Systems (Minneapolis, MN) and used for the standard curve in sOX40L and sOX40 ELISA, respectively.

Techniques: Expressing, Infection

Journal: Retrovirology

Article Title: Increased expression of OX40 is associated with progressive disease in patients with HTLV-1-associated myelopathy/tropical spastic paraparesis

doi: 10.1186/1742-4690-10-51

Figure Lengend Snippet: Clinical and laboratory findings of HAM/TSP patients for whom paired CSF and plasma samples were tested for soluble OX40 (sOX40)

Article Snippet: Recombinant human OX40 ligand/TNFSF4 and recombinant human OX40/TNFRSF4/Fc Chimera were purchased from R&D Systems (Minneapolis, MN) and used for the standard curve in sOX40L and sOX40 ELISA, respectively.

Techniques:

Journal: Retrovirology

Article Title: Increased expression of OX40 is associated with progressive disease in patients with HTLV-1-associated myelopathy/tropical spastic paraparesis

doi: 10.1186/1742-4690-10-51

Figure Lengend Snippet: Expression of OX40 in inflammatory mononuclear cells in spinal cord lesions of HAM/TSP patient with short disease duration and progressive symptoms. We studied autopsy specimens from 9 HAM/TSP patients by immunohistochemical staining. A. No OX40 positive cells are detected in the spinal cord lesion without active inflammation of a HAM/TSP patient with a long duration of illness. Magnification: ×40. B. Many infiltrating mononuclear cells are positively stained by anti-OX40 mAb in the spinal cord lesion with active inflammation of HAM/TSP patient with 2.5 years of illness. Magnification: ×40. C. There was reduced or no OX40L protein expression in spinal cord tissues of HAM/TSP patients. OX40L showed only low background staining and there was no OX40L positive staining on inflammatory mononuclear cells in the spinal cord lesions. Magnification: ×20. D. Positive control staining for OX40L positive CEM-OX40L cells. Magnification: ×20. Bar: 50 μm.

Article Snippet: Recombinant human OX40 ligand/TNFSF4 and recombinant human OX40/TNFRSF4/Fc Chimera were purchased from R&D Systems (Minneapolis, MN) and used for the standard curve in sOX40L and sOX40 ELISA, respectively.

Techniques: Expressing, Immunohistochemical staining, Staining, Positive Control

Journal: Retrovirology

Article Title: Increased expression of OX40 is associated with progressive disease in patients with HTLV-1-associated myelopathy/tropical spastic paraparesis

doi: 10.1186/1742-4690-10-51

Figure Lengend Snippet: Anti-OX40 monoclonal antibody specifically eliminated naturally infected CD4 + T cells via antibody-dependent cell-mediated cytotoxicity (ADCC) in cultured PBMCs. Anti-OX40 mAb (clone B-7B5) reduces the percentage of Tax-positive cells, whereas the isotype control mAb (clone 2C2: anti-HIV-1 gp21, mouse IgG1) has no effect on Tax expression (1st, 2nd, and 3rd panels from left). Culture of PBMCs with anti-CD16/CD32 (Fc receptor) antibody to block Fc receptors abolishes Tax suppression by anti-OX40 mAb (4th panels from left). F(ab’) 2 fragment do not suppress Tax exppression (right panels).

Article Snippet: Recombinant human OX40 ligand/TNFSF4 and recombinant human OX40/TNFRSF4/Fc Chimera were purchased from R&D Systems (Minneapolis, MN) and used for the standard curve in sOX40L and sOX40 ELISA, respectively.

Techniques: Infection, Cell Culture, Expressing, Blocking Assay