Journal: PLoS ONE

Article Title: Autoreactivity to Glucose Regulated Protein 78 Links Emphysema and Osteoporosis in Smokers

doi: 10.1371/journal.pone.0105066

Figure Lengend Snippet: A .) Anti-GRP78 autoantibody prevalence was not significantly associated with COPD severity (GOLD stages) . Numbers within columns denote subject n. B .) Anti-GRP78 autoantibodies are significantly associated with emphysema prevalence (%) in the smoking subjects. OR = odds ratio; CI = confidence interval. C .) Emphysema scores per CT were also significantly greater among those subjects with anti-GRP78 autoantibodies.

Article Snippet: Membranes were incubated with 1∶1000 dilutions of mouse anti-human GRP78 mAb (R&D Systems) at 4°C, followed by 1∶4000 dilutions of chicken anti-mouse IgG-HRP (Santa Cruz Biotechnology, Santa Cruz, CA).

Techniques:

Journal: PLoS ONE

Article Title: Autoreactivity to Glucose Regulated Protein 78 Links Emphysema and Osteoporosis in Smokers

doi: 10.1371/journal.pone.0105066

Figure Lengend Snippet: A .) T scores (left panel) and osteoporosis prevalence (at either/both hip or spine) (right panel) among the smoking cohort. B .) Serum levels of bone turnover metabolite collagen type 1 cross-linked C-telopeptide (CTX) were greatest among smokers with anti-GRP78 autoantibodies. The lowest, second lowest, middle, second highest, and highest lines represent 10 th , 25 th , median, 75 th , and 90 th percentiles, respectively. Means are denoted by solid squares. C .) Serum levels of bone turnover metabolite type 1 (N-terminal) procollagen (P1NP) were greatest among smokers with anti-GRP78 autoantibodies. D .) The relationship between GRP78 autoantibody positivity and the concurrent co-existences of low BMD and emphysema in smokers is significant in both genders, but greatest in males.

Article Snippet: Membranes were incubated with 1∶1000 dilutions of mouse anti-human GRP78 mAb (R&D Systems) at 4°C, followed by 1∶4000 dilutions of chicken anti-mouse IgG-HRP (Santa Cruz Biotechnology, Santa Cruz, CA).

Techniques:

Journal: PLoS ONE

Article Title: Autoreactivity to Glucose Regulated Protein 78 Links Emphysema and Osteoporosis in Smokers

doi: 10.1371/journal.pone.0105066

Figure Lengend Snippet: A .) Compared to normal lung (Left panel), emphysematous lungs (middle and right panels) demonstrated increased immunostaining in macrophages (yellow arrows) and alveolar epithelial cells (blue arrows). Magnification x100 left and middle panels, and magnification x400 in the right panel, n = 3. B .) GRP78 was also greater in bronchoalveolar lavage fluid (BALF) from emphysematous lungs compared to normal preparations. Lanes 1 and 8 are rGRP78 standards. Lanes 2–7 are BALF from individual emphysematous lung explants; lanes 9–14 are BALF from normal lung explants. All specimen lanes were loaded with equal amounts of BALF proteins. C .) Indirect immunofluorescent assays showed anti-GRP78 IgG isolated from patients bind to alveolar macrophages from normal lung explants (panel a), and osteoclasts derived from bone marrow (panel b). Normal human IgG control is illustrated in panel c.

Article Snippet: Membranes were incubated with 1∶1000 dilutions of mouse anti-human GRP78 mAb (R&D Systems) at 4°C, followed by 1∶4000 dilutions of chicken anti-mouse IgG-HRP (Santa Cruz Biotechnology, Santa Cruz, CA).

Techniques: Immunostaining, Isolation, Derivative Assay, Control

Journal: PLoS ONE

Article Title: Autoreactivity to Glucose Regulated Protein 78 Links Emphysema and Osteoporosis in Smokers

doi: 10.1371/journal.pone.0105066

Figure Lengend Snippet: A .) Mean fluorescence intensity (MFI) for phosphorylated NFkB among paired, concurrent autologous CD14 + derived macrophages was increased in all 10 normal specimens after incubation with patient-derived autoantibodies to GRP78 (α-GRP78), relative to control cells treated with normal human IgG. Patient derived anti-GRP78 autoantibodies also increased macrophage production of IL-8 ( B .), CCL-2 ( C .) and MMP9 ( C .). Population means are denoted with a horizontal line.

Article Snippet: Membranes were incubated with 1∶1000 dilutions of mouse anti-human GRP78 mAb (R&D Systems) at 4°C, followed by 1∶4000 dilutions of chicken anti-mouse IgG-HRP (Santa Cruz Biotechnology, Santa Cruz, CA).

Techniques: Fluorescence, Derivative Assay, Incubation, Control

Journal: PLoS ONE

Article Title: Autoreactivity to Glucose Regulated Protein 78 Links Emphysema and Osteoporosis in Smokers

doi: 10.1371/journal.pone.0105066

Figure Lengend Snippet: A .) Addition of GRP78 to PBMNC cultures increased CD4 T-cell proliferation (BrdU uptake) relative to media controls (no added protein) (n = 47), unlike addition of elastin split products (ESP). P c denotes alpha level corrected for multiple comparisons. B .) GRP78-induced CD4 T-cell proliferation was greatest among cultures from smokers with emphysema (n = 34). Specific indices (SI) of proliferation were calculated as %CD4 T-cells incorporating BrdU in GRP78-supplemented cultures minus incorporation in concurrent media controls. C .) GRP78 also increased percentages of CD4 T-cells that produced IFN-gamma again, unlike effects of ESP. D .) IFN-gamma production was also greatest in the CD4 T-cells from the emphysematous smokers. Specific indices (SI) were calculated as %CD4 T-cells producing IFN-gamma production in GRP78-supplemented cultures minus that of concurrent controls.

Article Snippet: Membranes were incubated with 1∶1000 dilutions of mouse anti-human GRP78 mAb (R&D Systems) at 4°C, followed by 1∶4000 dilutions of chicken anti-mouse IgG-HRP (Santa Cruz Biotechnology, Santa Cruz, CA).

Techniques: Produced

Journal: Science advances

Article Title: Aging compromises human islet beta cell function and identity by decreasing transcription factor activity and inducing ER stress.

doi: 10.1126/sciadv.abo3932

Figure Lengend Snippet: Fig. 2. Beta cell TFs, ER stress, and autophagy markers in aging beta cells. Immunohistochemistry and confocal microscopy of human pancreas formalin-fixed paraffin- embedded samples from adults (22 to 35 years old) and old adults (61 to 79 years old). Human islets were stained with (A) insulin, PDX1, NKX6-1, and/or NKX2-2; (B) insulin and HSPA5 or XBP1; and (C) insulin, LC3A/B, and LAMP1. (D) Quantification of LC3-LAMP1 colocalization, area of beta cell cytosol covered by LAMP1+ granules, and LAMP1+ granule size. Each dot represents data from a single region of ~140 m2 of human islets. Here, 1 region per islet, 10 regions per donor were quantified. In (A) and (B), the islet region is demarcated by the dotted yellow line. Scale bars, 50 m (A and B), 20 m (C), and 5 m (inset of C). CI, confidence interval.

Article Snippet: After blocking, the tissue sections were incubated overnight at room temperature with primary antibodies raised against insulin [guinea pig, Dako (catalog no. IR002, lot no. 10149939) at 1:4], 53BP1 [rabbit, Bethyl Laboratories (catalog no. IHC- 0001) at 1:100], 8HG [rabbit, Novus Biologicals (catalog no. NB110-96878) at 1:100], NKX6.1 [rabbit, Cell Signaling Technology (catalog no. D8O4R) at 1:100 dilution], NKX2.2 [mouse, Developmental Studies Hybridoma Bank (catalog no. 74.5A5) at 1:40 dilution], PDX1 [goat at 1:10,000; (75)], NCL [rabbit, Cell Signaling Technology (catalog no. D4C7O) at 1:100], XBP1 [rabbit, Thermo Fisher Scientific (PA5-27650) at 1:100], HSPA5 [rabbit, Novus Biologicals (NBP1-06274) at 1:100], LC3A/B [rabbit, Cell Signaling Technology (catalog no. 12741) at 1:100], and LAMP1 [Developmental Studies Hybridoma Bank (catalog no. 1D4B) at 1:50].

Techniques: Immunohistochemistry, Confocal Microscopy, Formalin-fixed Paraffin-Embedded, Staining