Journal: Human Reproduction (Oxford, England)

Article Title: Chronic hyperandrogenemia in the presence and absence of a western-style diet impairs ovarian and uterine structure/function in young adult rhesus monkeys

doi: 10.1093/humrep/dex338

Figure Lengend Snippet: Photomicrographs illustrating immunohistochemical staining for estrogen receptor 1 (ESR1), progesterone receptor (PGR) MMP26, TIMP3, Ki-67 and androgen receptor (AR) in the endometrial functionalis zone of the macaque uterus from representative females in each treatment group (C, T, WSD, T+WSD). Brown staining denotes positive expression of proteins. Sections are counterstained with hematoxylin (blue) staining. ESR1, PGR, Ki-67 and AR staining is nuclear, while MMP26 and TIMP3 show cytoplasmic localization. Inset shows a negative control with an irrelevant antibody (Anti-Br(d)U). TIMP3 staining was localized to the predecidual cells around the spiral arteries.

Article Snippet: Antibodies used were against estrogen receptor 1 (ESR1,ER-ID5; Cat#: MS-354-P, Thermo Fisher Scientific), progesterone receptor (PGR, Cat#: Ms-298-P, 1 μg, Lab Vision/NeoMarkers, Fremont, CA, USA), androgen receptor (AR-F39 (Cat#: MU256, 1/50, BioGenex, Fremont, CA, USA), Ki67 (Cat#: MU370-UC, 1/200, BioGenex), MMP26 (Cat# ab57636; Abcam, Cambridge, MA, USA) and TIMP3 (Cat#:MAB973, R&D Systems, Inc. Minneapolis, MN, USA).

Techniques: Immunohistochemical staining, Staining, Expressing, Negative Control

Journal: Human Reproduction (Oxford, England)

Article Title: Chronic hyperandrogenemia in the presence and absence of a western-style diet impairs ovarian and uterine structure/function in young adult rhesus monkeys

doi: 10.1093/humrep/dex338

Figure Lengend Snippet: Expression of ESR1, PGR, MMP26 and TIMP3 mRNAs as detected by qRT-PCR in endometrial biopsies collected from macaques in the mid-luteal phase of the cycle. Significant (P < 0.05) differences between individual treatment groups are denoted by different uppercase letters above columns.

Article Snippet: Antibodies used were against estrogen receptor 1 (ESR1,ER-ID5; Cat#: MS-354-P, Thermo Fisher Scientific), progesterone receptor (PGR, Cat#: Ms-298-P, 1 μg, Lab Vision/NeoMarkers, Fremont, CA, USA), androgen receptor (AR-F39 (Cat#: MU256, 1/50, BioGenex, Fremont, CA, USA), Ki67 (Cat#: MU370-UC, 1/200, BioGenex), MMP26 (Cat# ab57636; Abcam, Cambridge, MA, USA) and TIMP3 (Cat#:MAB973, R&D Systems, Inc. Minneapolis, MN, USA).

Techniques: Expressing, Quantitative RT-PCR

Journal: Transplantation

Article Title: Urinary TIMP-2 Predicts the Presence and Duration of Delayed Graft Function in Donation After Circulatory Death Kidney Transplant Recipients

doi: 10.1097/tp.0000000000002472

Figure Lengend Snippet: FIGURE 2. TIMP-2 and IGFBP7 values were significantly different for patients with and without fDGF. Median ± IQR of both TIMP-2 and IGFBP7 are plotted for patients with and without fDGF. TIMP-2 values were significantly higher for patients with fDGF as compared to patients without fDGF (A). This also applied for the IGFBP7 values (B). Interestingly, TIMP-2 values of all patients decreased in the first 10 days after transplantation, where as IGFBP7 values increased. fDGF, functionally defined delayed graft function; IGFBP7, insulin-like growth factor-binding protein 7; IQR, interquartile range; TIMP-2, tissue inhibitor of metalloproteinases-2.

Article Snippet: Urinary TIMP-2 and IGFBP7 were quantified using sandwich immunoassays (ELISA, DTM200; R&D Systems, Minneapolis, MN for TIMP-2 and EK0991, Boster Biological Technology, Pleasanton, CA for IGFBP7, respectively) according to the manufacturer’s instructions.

Techniques: Transplantation Assay, Binding Assay

Journal: Transplantation

Article Title: Urinary TIMP-2 Predicts the Presence and Duration of Delayed Graft Function in Donation After Circulatory Death Kidney Transplant Recipients

doi: 10.1097/tp.0000000000002472

Figure Lengend Snippet: FIGURE 3. ROC curves of TIMP-2 and IGFBP7 in the prediction of fDGF and prolonged fDGF The absolute concentration of TIMP-2 on day 1 could adequately identify patients with fDGF, and this further improved in case TIMP-2 was corrected for osmolality (TIMP-2/ mOsm) (A). The concentration of IGFBP7 on day 1, on the other hand, showed only limited predictive value also when corrected for osmolality (IGFBP7/mOsm) (B). On day 10, both TIMP-2 and TIMP-2/mOsm could also adequately predict prolonged fDGF duration ≥21 days (C), whereas IGFBP7 and IGFBP7/mOsm could not (D). fDGF, functionally defined delayed graft function; IGFBP7, insulin-like growth factor-binding protein 7; ROC, receiver operating characteristic; TIMP-2, tissue inhibitor of metalloproteinases-2.

Article Snippet: Urinary TIMP-2 and IGFBP7 were quantified using sandwich immunoassays (ELISA, DTM200; R&D Systems, Minneapolis, MN for TIMP-2 and EK0991, Boster Biological Technology, Pleasanton, CA for IGFBP7, respectively) according to the manufacturer’s instructions.

Techniques: Concentration Assay, Binding Assay

Journal: Transplantation

Article Title: Urinary TIMP-2 Predicts the Presence and Duration of Delayed Graft Function in Donation After Circulatory Death Kidney Transplant Recipients

doi: 10.1097/tp.0000000000002472

Figure Lengend Snippet: FIGURE 5. TIMP-2/mOsm and IGFBP7/mOsm in tertiles. Patients were divided into tertiles based on TIMP-2/mOsm and IGFBP7/ mOsm concentrations, and the percentage of patients with fDGF and prolonged fDGF in each tertile was calculated. On day 1, patients without fDGF predominantly grouped together in the tertile with the lowest values for TIMP-2/mOsm, (77%), whereas patients with fDGF (92%) were in the middle or highest tertile (A). For IGFBP7/mOsm, a comparable figure was seen, with slightly lower percentages (B). On day 10, patients with prolonged fDGF predominantly grouped together in the tertile with the highest TIMP-2/mOsm values (83.3%), and none had TIMP-2/mOsm values in the lowest tertile (C). In contrast, patients with prolonged fDGF did not group together according to their IGFBP7/mOsm values (D). fDGF, functionally defined delayed graft function; IGFBP7, insulin-like growth factor-binding protein 7; TIMP-2, tissue inhibitor of metalloproteinases-2.

Article Snippet: Urinary TIMP-2 and IGFBP7 were quantified using sandwich immunoassays (ELISA, DTM200; R&D Systems, Minneapolis, MN for TIMP-2 and EK0991, Boster Biological Technology, Pleasanton, CA for IGFBP7, respectively) according to the manufacturer’s instructions.

Techniques: Binding Assay

Journal: Cancer Research

Article Title: TIMP1 Triggers Neutrophil Extracellular Trap Formation in Pancreatic Cancer

doi: 10.1158/0008-5472.can-20-4125

Figure Lengend Snippet: Figure 1. Pathologic levels of TIMP1 induce NET forma- tion. A, GSEA of published proteome data derived from human PDAC tissue. Illustrated are biological processes that significantly correlate with TIMP1 protein levels (creation parameters: FDR q-value ≤0.05; normalized enrichment score > 2.0; overlap size > 100). B, Representative confocal microscopy images of primary human neutrophils stimulated with 500 ng/mL TIMP1 or left untreated. Neutrophils were stained for DNA (blue), MPO (green), or NE (magenta). Scale bars in overview images, 50 mm. Scale bars in detail images, 20 mm. C, Quantification of DNA-occupied areas as read- out for NET formation after stimulation of primary human neutrophils with increasing concentrations of TIMP1 (c ¼ 0 ng/mL, n ¼ 42; c ¼ 50 ng/mL, n ¼ 12; 250 ng/mL, n ¼ 12; 500 ng/mL, n ¼ 21; or 2,000 ng/mL, n ¼ 12). D, Quantification of DNA-occupied areas as read- out for NET-formation in the presence of healthy donor-matched plasma with (n ¼ 15), or without (n ¼15) supplementation of 500 ng/ mL recombinant human (rh) TIMP1. Results are represented as the mean SEM. For statistical analyses, a two-sided paired t test in case of normal distribution or a nonparametric Mann–Whitney test in the absence of normal distribution was employed. , P ≤0.01; , P ≤0.001; n.s., nonsignificant.

Article Snippet: Plasma TIMP1 levels were determined in healthy human donors or patients with PDAC using the respective DuoSet ELISA kit (R&D Systems) according to manufacturer’s instructions.

Techniques: Derivative Assay, Confocal Microscopy, Staining, Clinical Proteomics, Recombinant, MANN-WHITNEY

Journal: Cancer Research

Article Title: TIMP1 Triggers Neutrophil Extracellular Trap Formation in Pancreatic Cancer

doi: 10.1158/0008-5472.can-20-4125

Figure Lengend Snippet: Figure 2. TIMP1 mediates NET formation via interaction with CD63. A, C, and D, Quantification of DNA-occupied areas after stimulation of primary human neutrophils. A, Neutrophils were treated with 500 ng/mL recombinant human TIMP1 (n ¼ 18), equimolar levels of recombinant N-TIMP1 (n ¼ 18), or were left untreated (n ¼ 24). B, Quantification of TIMP1 surface binding to different neutrophil populations. Neutrophils were treated with or without fluorescently labeled TIMP1 and analyzed for TIMP1 binding and CD63 surface expression using flow cytometry. Geometric means of TIMP1 Alexa Fluor 488 binding are represented as the mean SEM of three different donors. Statistical differences between groups were analyzed employing the Kruskal–Wallis test due to absence of equal variances (, P ≤0.001). C, Neutrophils were treated with TIMP1 (n ¼ 15) or were left untreated (n ¼ 12). To block the TIMP1/CD63 interaction, neutrophils were preincubated with an antibody against CD63 before stimulation with TIMP1 (n ¼ 6) or were only incubated with anti-CD63 antibodies (n ¼ 6). D, Neutrophils were treated with TIMP1 (n ¼ 24), were left untreated (n ¼ 21), or were stimulated with TIMP1, which was preincubated with a peptide resembling amino acids 152 – 201 of the large extracellular loop of CD63 (n ¼ 18), or were incubated with the peptide only (n ¼ 18). Results are represented as the means SEM. E and F, Neutrophils were primed as indicated and analyzed for CD63 expression by FACS analysis (nonprimed, n ¼ 4; TNFa-primed, n ¼ 4; GMCSF-primed, n ¼ 3; IFNg-primed, n ¼ 3; E), or were subsequently stimulated with TIMP1 (F). Cell-free DNA released by nonprimed neutrophils (n ¼ 18), TNFa-primed (n ¼ 21), GMCSF-primed (n ¼ 6), or IFNg-primed (n ¼ 6) neutrophils was normalized to nonprimed neutrophils. For statistical analyses, a two-sided paired t test in case of normal distribution or nonparametric Mann–Whitney test in the absence of normal distribution was employed. , P ≤0.05; , P ≤0.01; , P ≤0.001; n.s., nonsignificant.

Article Snippet: Plasma TIMP1 levels were determined in healthy human donors or patients with PDAC using the respective DuoSet ELISA kit (R&D Systems) according to manufacturer’s instructions.

Techniques: Recombinant, Binding Assay, Labeling, Expressing, Cytometry, Blocking Assay, Incubation, MANN-WHITNEY

Journal: Cancer Research

Article Title: TIMP1 Triggers Neutrophil Extracellular Trap Formation in Pancreatic Cancer

doi: 10.1158/0008-5472.can-20-4125

Figure Lengend Snippet: Figure 3. TIMP1/CD63-triggered NET formation involves ERK activation. A–C, Densitometric analysis of Western blots of cell lysates from neutrophils treated as indicated. A, Neutrophils were treated with 500 ng/mL recombinant wild-type TIMP1 (n ¼ 3), equimolar levels of recombinant N-TIMP1 (n ¼ 3), or were left untreated (n ¼ 3). B, Neutrophils were treated with 500 ng/mL TIMP1 (n ¼ 5), were preincubated with an antibody against CD63 before stimulation with TIMP1 (n ¼ 3), or were left untreated (n ¼ 5). C, Neutrophilswere stimulated with 500 ng/mL TIMP1 (n ¼ 4), were treated with TIMP1,whichwas preincubated with LEL-CD63 (n ¼ 3), or were left untreated (n ¼ 4). Bar graphs indicate the mean SEM of ERK phosphorylation normalized to GAPDH and related to the untreated control. For statistical analyses, a one-sample t test was employed. , P ≤0.05. D, Quantification of DNA-occupied areas after stimulation of primary human neutrophils. Neutrophils were stimulated with 500 ng/mL TIMP1 (n ¼ 15) or were left untreated (n ¼ 14) after pretreatment with an inhibitor of ERK activation (U0126; n ¼ 15) or DMSO. Results are represented as the means SEM. For statistical analyses, a nonparametric Mann–Whitney test due to absence of normal distribution was employed. , P ≤0.05; n.s., nonsignificant.

Article Snippet: Plasma TIMP1 levels were determined in healthy human donors or patients with PDAC using the respective DuoSet ELISA kit (R&D Systems) according to manufacturer’s instructions.

Techniques: Activation Assay, Western Blot, Recombinant, Phospho-proteomics, Control, MANN-WHITNEY

Journal: Cancer Research

Article Title: TIMP1 Triggers Neutrophil Extracellular Trap Formation in Pancreatic Cancer

doi: 10.1158/0008-5472.can-20-4125

Figure Lengend Snippet: Figure 4. NET formation in PDAC is TIMP1–dependent and determines cancer survival. A, Isolation of murine tumors and NET staining. Immunofluorescence microscopy of pancreatic tissue from KPC mice with visualization of DNA (blue), MPO (green), and citH3 (magenta). Arrowheads, NET-forming neutrophils. Scale bars, 50 mm. B, Correlation between plasma levels of TIMP1 and NET-formation in murine pancreatic tumors (n ¼ 9). C, Quantification of NET-forming neutrophils in TIMP1high

Article Snippet: Plasma TIMP1 levels were determined in healthy human donors or patients with PDAC using the respective DuoSet ELISA kit (R&D Systems) according to manufacturer’s instructions.

Techniques: Isolation, Staining, Microscopy, Clinical Proteomics

Journal: Cancer Research

Article Title: TIMP1 Triggers Neutrophil Extracellular Trap Formation in Pancreatic Cancer

doi: 10.1158/0008-5472.can-20-4125

Figure Lengend Snippet: Figure 5. TIMP1 correlates with local and systemic NET formation in patients with PDAC. A, Representative confocal microscopy images of immunofluorescence staining of pancreatic tissue from a human PDAC patient with stainings for DNA (blue), MPO (green), citH3 (magenta), and TIMP1 (red). NETs were defined as colocalization of MPO, citH3, and DNA, and surface of NETs (yellow) was displayed employing Imaris software. Areas expressing high levels of TIMP1 are indicated in red and surrounded by dashed lines. Total surface of NETs was determined employing Imaris software and compared between areas expressing high levels of TIMP1 and areas expressing low levels of TIMP1. Results are represented as means, normalized to areas with low TIMP1 levels. Whiskers indicate min to max values of three different pictures. For statistical analyses, a one-sample t test was employed. , P ≤0.05. Scale bars, 20 mm. B, Plasma TIMP1 levels from individual PDAC patientswith low NETs [<0.1225 plasma NETs (corrected absorbance at 450 nm), n ¼ 17] and patients with PDAC with high NETs [≥0.1225 plasma NETs (corrected absorbance at 450 nm), n ¼ 51] are shown as dots; medians of both groups are indicated as crossbars; whiskers indicate min to max values. For statistical analyses, a nonparametric Mann– Whitney test due to absence of normal distribution was employed. , P ≤0.05. C, Correlation of relative plasma NET levels with plasma TIMP1 levels from patients with PDAC (n ¼ 68). For statistical analyses, a Spearman rank correlation due to absence of normal distribution was employed. Spearman rank correlation coefficient (R) and a linear trend line are indicated.

Article Snippet: Plasma TIMP1 levels were determined in healthy human donors or patients with PDAC using the respective DuoSet ELISA kit (R&D Systems) according to manufacturer’s instructions.

Techniques: Confocal Microscopy, Staining, Software, Expressing, Clinical Proteomics, MANN-WHITNEY

Journal: Cancer Research

Article Title: TIMP1 Triggers Neutrophil Extracellular Trap Formation in Pancreatic Cancer

doi: 10.1158/0008-5472.can-20-4125

Figure Lengend Snippet: Figure 6. Plasma TIMP1/NET levels predict survival of patients with PDAC. A–E, Kaplan–Meier survival curves of patients with PDAC grouped according to their plasma TIMP1 levels (A, C, and E), plasma NET levels (B, C, and E), or serum CA19–9 levels (D and E). Log-rank tests (Mantel–Cox test) were used for statistical analyses and Cox regression analysis was employed to calculate HRs, including the corresponding 95.0% confidence intervals, between indicated groups. Cut-off values, as determined by maximally selected log-rank statistics: plasma TIMP1, 174.42 ng/mL; plasma NETs, 0.1225 corrected absorbance at 450 nm; serum CA19–9, 1088 U/ml. One-year overall survival rates of distinct patient groups: TIMP1low (n ¼ 29 patients), 86.1%; TIMP1high (n ¼ 39 patients), 45.2%; NETlow (n ¼ 17 patients), 83.9%; NEThigh (n ¼ 51 patients), 53.9%; TIMP1lowNETlow (TINElow, n ¼ 9 patients), 100.0%; TIMP1lowNEThigh/TIMP1highNETlow (TINEint, n ¼ 26 patients), 69.3%; TIMP1highNEThigh (TINEhigh, n ¼ 32 patients), 43.1%; CA19–9low (n ¼ 54 patients), 63.8%; CA19–9high (n ¼ 8 patients), not defined, patients of this group died within one year; TINECA 0 (no parameter above respective cut-off value, n ¼ 10 patients): 16.1%; TINECA 1 (one parameter above respective cut-off value, n ¼ 20 patients), 32.3%; TINECA 2 (two parameters above respective cut-off value, n ¼ 25 patients), 40,3%; TINECA 3 (three parameters above respective cut-off value, n ¼ 7 patients), 11.3%.

Article Snippet: Plasma TIMP1 levels were determined in healthy human donors or patients with PDAC using the respective DuoSet ELISA kit (R&D Systems) according to manufacturer’s instructions.

Techniques: Clinical Proteomics