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  • Nicotine accelerates diabetes-induced retinal changes. 24911405

    To investigate the effects of nicotine on retinal alterations in early-stage diabetes in an established rodent model.Sprague-Dawley rats were examined using a combination of confocal scanning laser ophthalmoscopy and spectral domain optical coherence tomography to determine changes in retinal structure in response to nicotine exposure, diabetes and the combined effects of nicotine and diabetes. Diabetes was induced by a single injection of 65 mg/kg streptozotocin and nicotine injections were administered subcutaneously daily. Retinal thickness in the superior, inferior, nasal and temporal quadrants were determined based on the spectral domain optical coherence tomography (SD-OCT) volume scans (20° × 20°) centered on the optic disc. Segmentation of discrete retinal layers was performed on a subset of SD-OCT cross-sections to further examine changes in each treatment group. Survival of neurons within the ganglion cell layer (GCL) was assessed by confocal morphometric imaging.The control group did not experience any significant change throughout the study. The nicotine treatment group experienced an average decrease in total retinal thickness (TRT) of 9.4 µm with the majority of the loss localized within the outer nuclear layer (ONL) as determined by segmentation analysis (p less than  0.05). The diabetic group exhibited a trend toward decreased TRT while segmentation analysis of the diabetic retinopathy (DR) group revealed significant thinning within the ONL (p less than  0.05). The combination of nicotine and diabetes revealed a significant increase of 8.9 µm in the TRT (p less than  0.05) accompanied by a decrease in the number of GCL neurons.We demonstrated significant temporal changes in retinal morphology in response to nicotine exposure, diabetes and with the combined effects of nicotine and diabetes. These findings may have implications in determining treatment strategies for diabetic patients using products containing nicotine, such as cigarettes, smokeless tobacco, electronic cigarettes or smoking cessation products.
    Document Type:
    Reference
    Product Catalog Number:
    MAB377
    Product Catalog Name:
    Anti-NeuN Antibody, clone A60

  • AHI1 is required for photoreceptor outer segment development and is a modifier for retinal degeneration in nephronophthisis. 20081859

    Degeneration of photoreceptors is a common feature of ciliopathies, owing to the importance of the specialized ciliary structure of these cells. Mutations in AHI1, which encodes a cilium-localized protein, have been shown to cause a form of Joubert syndrome that is highly penetrant for retinal degeneration. We show that Ahi1-null mice fail to form retinal outer segments and have abnormal distribution of opsin throughout their photoreceptors. Apoptotic cell death of photoreceptors occurs rapidly between 2 and 4 weeks of age in these mice and is significantly (P = 0.00175 and 0.00613) delayed by a reduced dosage of opsin. This phenotype also shows dosage-sensitive genetic interactions with Nphp1, another ciliopathy-related gene. Although it is not a primary cause of retinal blindness in humans, we show that an allele of AHI1 is associated with a more than sevenfold increase in relative risk of retinal degeneration within a cohort of individuals with the hereditary kidney disease nephronophthisis. Our data support context-specific roles for AHI1 as a contributor to retinopathy and show that AHI1 may explain a proportion of the variability in retinal phenotypes observed in nephronophthisis.
    Document Type:
    Reference
    Product Catalog Number:
    MAB5316

  • Identification of early response genes and pathway activated by androgens in the initial segment and caput regions of the regressed rat epididymis. 20660069

    To identify the initial response to androgens and estrogens in the orchidectomized, regressed epididymis, we determined the gene expression changes triggered by the administration of either of two metabolites of testosterone, 5alpha-dihydrotestosterone (DHT) or 17beta-estradiol (E2), in the regressed rat epididymis. Adult rats were orchidectomized and 8 d later implanted with either empty implants (control), DHT-filled-, or E2-filled-polydioxanone implants. Rats were euthanized 12 h, 1 d, and 7 d later, and RNA was extracted and probed on Rat230-2.0 Affymetrix arrays. Probe sets that respond to DHT or E2 were identified at early time points; although the expression of some was repressed, the expression of many others was either transiently or chronically elevated. Nerve growth factor receptor (Ngfr) and S100 calcium binding protein G (S100g) were two E2 up-regulated genes detected at 12 h. Among the genes that showed a dramatic early response to DHT were endothelin 1 (Edn1), bone morphogenetic protein 4 (Bmp4), and IGF binding protein 3 (Igfbp3), which were suppressed, and IGF-I (Igf1), which was induced. Genes that were up- or down-regulated by DHT were classified based on biological function. Using PathwayStudio 4.0, we identified genes that were linked and directly influenced either the expression or regulation of one another. Epidermal growth factor and IGF-I play an important role in the pathway due to their function in regulation and expression of many other genes. These results provide novel insights into the impact of androgen action on the expression of genes that are important for epididymal function.
    Document Type:
    Reference
    Product Catalog Number:
    07-313

  • Remyelination of cytokine- or antibody-demyelinated CNS aggregate cultures is inhibited by macrophage supplementation. 14730701

    Remyelination in CNS aggregate cultures is determined both by macrophage enrichment and the mode of demyelination. Despite the same degree of myelin loss, accumulation of MBP in anti-MOG antibody-demyelinated aggregates overtakes that of controls, while recovery is significantly delayed following IFN-gamma-induced demyelination. In antibody-treated cultures, remyelination was associated with a significant increase in culture supernatant levels of TGF-beta1, FGF-2, and PDGF-AA as well as an induction of TNF-alpha immediately following removal of the demyelinating insult. The impaired recovery in IFN-gamma-treated cultures, denoted by a significant reduction in TGF-beta1, was reversed by treatment with hrTGF-beta1. Macrophage supplementation of the cultures prior to the addition of either demyelinating agent induced a greater degree of myelin loss followed by incomplete remyelination in both cases. This failure to remyelinate was associated in both groups with a several-fold elevation in TNF-alpha and with modest increases in PDGF-AA and FGF-2 in the antibody-treated cultures. In contrast, macrophage supplementation to mature cultures in the absence of any demyelinating treatment resulted in enhanced accumulation of MBP associated with a promyelinative growth factor and TNF-alpha profile similar to that in aggregates enriched with macrophages at the outset of the culture period. Hence, effector elements of the adaptive immune response appear to override promyelinogenic in favor of proinflammatory macrophage factors in mature CNS aggregates, counteracting the potential for myelin repair.
    Document Type:
    Reference
    Product Catalog Number:
    AB980

  • Expression of apoptosis-related genes during human preimplantation embryo development: potential roles for the Harakiri gene product and Caspase-3 in blastomere fragmenta ... 12606589

    In order to resolve the mechanisms and reasons of cellular fragmentation it is crucial to understand what genes may be responsible for regulation of this process. We report herein that human oocytes and preimplantation embryos possess abundant levels of transcripts encoding cell death suppressors, Mcl-1, Bcl-x and Bag-1, and the cell death inducer genes, Bax and Caspase-2. Lower but detectable levels of mRNA expression for the Bfl-1/a1, Bcl-w, Harakiri (Hrk) and Caspase-3 genes were also detected during all developmental stages. We also performed analysis of gene expression in single human embryos exhibiting various degrees of fragmentation at the 2-, 4- and 8-cell stages. At the 4-cell stage, embryos displaying 30-50% fragmentation showed a significant increase in Hrk mRNA levels (P = 0.016). Immunostaining with anti-Hrk antibody confirmed increased staining in some, but not all, fragmented embryos. While Caspase-3 transcripts were elevated in both 4- and 8-cell embryos exhibiting a severe degree of fragmentation, this difference did not reach statistical significance. However, accumulation of Caspase-3 mRNA in fragmented embryos was paralleled by an induction of Caspase-3-like activity. These findings suggest that cellular fragmentation in a subset of human preimplantation embryos could be regulated by certain components of a genetic programme of cell death.
    Document Type:
    Reference
    Product Catalog Number:
    MAB1273
    Product Catalog Name:
    Anti-Mitochondria Antibody, surface of intact mitochondria, clone 113-1

  • Matrix metalloproteinases in the restorative proctocolectomy pouch of pediatric ulcerative colitis. 22912554

    To investigate matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs) in pouch mucosa of pediatric onset ulcerative colitis (UC).In this cross-sectional study, 28 patients with pediatric onset UC underwent ileal pouch biopsy 13 years (median) after proctocolectomy. Expression of MMPs-3, -7, -8, -9, -12 and -26 and TIMPs-1, -2 and -3 in samples was examined using immunohistochemichal methods, and another biopsy was used to evaluate the grade of histological inflammation. Two investigators independently graded the immunohistochemical specimens in a semiquantitative fashion, using a scale marking staining intensity as follows: 0 = less than 20 positive cells; 1 = 20-50 positive cells; 2 = 50-200 positive cells; 3 = over 20 positive cells. Fecal calprotectin and blood inflammatory markers [serum C-reactive protein (CRP) and erythrocyte sedimentation rate] were determined during a follow-up visit to examine correlations between these markers and the expression of MMPs and TIMPs.Of the 28 patients with pediatric onset UC, nine had not experienced pouchitis, whereas thirteen reported a single episode, and six had recurrent pouchitis (≥ 4 episodes). At the time of the study, six patients required metronidazole. In all of the others, the most recent episode of pouchitis had occurred over one month earlier, and none were on antibiotics. Only four samples depicted no sign of inflammation, and these were all from patients who had not had pouchitis. Two samples were too small to determine the grade of inflammation, but both had suffered pouchitis, the other recurrent. No sample depicted signs of colonic metaplasia. Most pouch samples showed expression of epithelial (e) and stromal (s) MMP-3 (e, n = 22; s, n = 20), MMP-7 (e, n = 28; s, n = 27), MMP-12 (e, n = 20; s, n =24), TIMP-2 (e, n = 23; s, n = 23) and MMP-3 (e, n = 23; s, n = 28) but MMP-8 (e, n = 0; s, n = 1), MMP-9 (e, n = 0; s, n = 9) and MMP-26 (e, n = 0; s, n = 3) and TIMP-1 (n = 0, both) were lacking. In samples with low grade of inflammatory activity, the epithelial MMP-3 and MMP-7 expression was increased (r = -0.614 and r = -0.472, respectively, P less than 0.05 in both). MMPs and TIMPs did not correlate with the markers of inflammation, fecal calprotectin, erythrocyte sedimentation rate, or CRP, with the exception of patients with low fecal calprotectin (less than 100 μg/g) in whom a higher expression of epithelial MMP-7 was found no differences in MMP- or TIMP-profiles were seen in patients with a history of pouchitis compared to ones with no such episodes. Anastomosis with either straight ileoanal anastomosis or ileoanal anastomosis with J-pouch did depict differences in MMP- or TIMP-expression.The expression of MMPs pediatric UC pouch in the long-term shares characteristics with inflammatory bowel disease, but inflammation cannot be classified as a reactivation of the disease.
    Document Type:
    Reference
    Product Catalog Number:
    MAB3315
    Product Catalog Name:
    Anti-MMP-7 Antibody, clone 141-7B2

  • Refractive lenticule re-implantation after myopic ReLEx: a feasibility study of stromal restoration after refractive surgery in a rabbit model. 22743323

    To investigate the potential of refractive lenticule (RL) storage and re-implantation in vivo as a method for reversing RL extraction (ReLEx) and restoring corneal stromal volume.ReLEx [-6.00 diopter (D) correction] was performed on six New Zealand White rabbits in one eye. Each extracted RL was tagged and orientated before storage at -80°C for 28 days. Each RL was then re-implanted autologously in the correct orientation after flap relifting. All animals were monitored for 28 days before being euthanized for immunohistochemical analysis. Unoperated fellow eyes were used as controls. All animals had regular pre- and postoperative slit lamp photography, in vivo confocal microscopy, anterior segment optical coherence tomography (AS-OCT), keratometry, and topography.No intra-operative complications occurred and RL re-implantation was performed without complication. A mild intrastromal haziness was noted on day 3 after re-implantation (corneal haze grade: 2.20 ± 0.45), but corneas were clear on day 28 (0.20 ± 0.27). RL re-implantation restored central corneal thickness, and keratometric and topographic indices to near pre-operative values. Wound healing processes, marked by fibronectin and tenascin, and a few inflammatory cells were present along the re-implanted lenticular interfaces. No myofibroblasts formation, and Ki67- and TUNEL-positive cells were observed in the corneal stroma on postoperative day 28.RL storage and re-implantation is a feasible technique for restoring stromal volume after myopic ReLEx, and may provide a method for restoring tissue in ectatic corneas, or provide an opportunity for further refractive surgery and presbyopic treatment.
    Document Type:
    Reference
    Product Catalog Number:
    MAB1940
    Product Catalog Name:
    Anti-Fibronectin Antibody, cellular, clone DH1

  • Augmentation of radiation response by motesanib, a multikinase inhibitor that targets vascular endothelial growth factor receptors. 20507929

    Motesanib is a potent inhibitor of vascular endothelial growth factor receptors (VEGFR) 1, 2, and 3, platelet-derived growth factor receptor, and Kit receptors. In this report we examine the interaction between motesanib and radiation in vitro and in head and neck squamous cell carcinoma (HNSCC) xenograft models.In vitro assays were done to assess the impact of motesanib on VEGFR2 signaling pathways in human umbilical vein endothelial cells (HUVEC). HNSCC lines grown as tumor xenografts in athymic nude mice were utilized to assess the in vivo activity of motesanib alone and in combination with radiation.Motesanib inhibited VEGF-stimulated HUVEC proliferation in vitro, as well as VEGFR2 kinase activity. Additionally, motesanib and fractionated radiation showed additive inhibitory effects on HUVEC proliferation. In vivo combination therapy with motesanib and radiation showed increased response compared with drug or radiation alone in UM-SCC1 (P less than 0.002) and SCC-1483 xenografts (P = 0.001); however, the combination was not significantly more efficacious than radiation alone in UM-SCC6 xenografts. Xenografts treated with motesanib showed a reduction of vessel penetration into tumor parenchyma, compared with control tumors. Furthermore, triple immunohistochemical staining for vasculature, proliferation, and hypoxia showed well-defined spatial relationships among these parameters in HNSCC xenografts. Motesanib significantly enhanced intratumoral hypoxia in the presence and absence of fractionated radiation.These studies identify a favorable interaction when combining radiation and motesanib in HNSCC models. The data presented suggest that motesanib reduces blood vessel penetration into tumors and thereby increases intratumoral hypoxia. These findings suggest that clinical investigations examining combinations of radiation and motesanib are warranted in HNSCC.
    Document Type:
    Reference
    Product Catalog Number:
    05-321
    Product Catalog Name:
    Anti-Phosphotyrosine Antibody, clone 4G10®

  • Serum analysis of tryptophan catabolism pathway: correlation with Crohn's disease activity. 21823214

    Indoleamine 2,3 dioxygenase-1 (IDO1) is a tryptophan catabolizing enzyme with immunotolerance-promoting functions. We sought to determine if increased gut expression of IDO1 in Crohn's disease (CD) would result in detectable changes in serum levels of tryptophan and the initial IDO1 pathway catabolite, kynurenine.Individuals were prospectively enrolled through the Washington University Digestive Diseases Research Center. The Montreal Classification was used for disease phenotyping. Disease severity was categorized by the Physician's Global Assessment. Serum tryptophan and kynurenine were measured by high-pressure liquid chromatography. IDO1 immunohistochemical staining was performed on formalin-fixed tissue blocks.In all, 25 CD patients and 11 controls were enrolled. Eight CD patients had serum collected at two different timepoints and levels of disease activity compared. Strong IDO1 expression exists in both the lamina propria and epithelium during active CD compared to controls. Suppressed serum tryptophan levels and an elevated kynurenine/tryptophan (K/T) ratio were found in individuals with active CD as compared to those in remission or the control population. K/T ratios correlated positively with disease activity as well as with C-reactive protein and erythrocyte sedimentation rate. In the subgroup of CD patients with two serum measurements, tryptophan levels were elevated while kynurenine levels and the K/T ratio lowered as the disease activity lessened.IDO1 expression in CD is associated with lower serum tryptophan and an elevated K/T ratio. These levels may serve as a reasonable objective marker of gut mucosal immune activation and as a surrogate for CD activity.
    Document Type:
    Reference
    Product Catalog Number:
    Multiple
    Product Catalog Name:
    Multiple

  • DNA methylation affects nuclear organization, histone modifications, and linker histone binding but not chromatin compaction. 17485486

    DNA methylation has been implicated in chromatin condensation and nuclear organization, especially at sites of constitutive heterochromatin. How this is mediated has not been clear. In this study, using mutant mouse embryonic stem cells completely lacking in DNA methylation, we show that DNA methylation affects nuclear organization and nucleosome structure but not chromatin compaction. In the absence of DNA methylation, there is increased nuclear clustering of pericentric heterochromatin and extensive changes in primary chromatin structure. Global levels of histone H3 methylation and acetylation are altered, and there is a decrease in the mobility of linker histones. However, the compaction of both bulk chromatin and heterochromatin, as assayed by nuclease digestion and sucrose gradient sedimentation, is unaltered by the loss of DNA methylation. This study shows how the complete loss of a major epigenetic mark can have an impact on unexpected levels of chromatin structure and nuclear organization and provides evidence for a novel link between DNA methylation and linker histones in the regulation of chromatin structure.
    Document Type:
    Reference
    Product Catalog Number:
    MAB3446
    Product Catalog Name:
    Anti-Heterochromatin Protein-1 α Antibody, clone 2HP-2G9