Transglutaminase binding fusion protein linked to SLPI reduced corneal inflammation and neovascularization.
ABSTRACT: To study the effect of topical administration of a fusion protein (PF-MC) made up of N-terminal portion of the protease inhibitor Trappin-2 (which is a substrate of transglutaminasa-2) and SLPI (protein with anti-inflammatory, anti-bacterial and anti-viral ability), in an animal model of corneal inflammation and angiogenesis.An alkali injury was produced with a filter paper of 3?mm with 1?N NaOH during 40?seconds on the right cornea of 36 male Sprague Dawley rats, under general anesthesia. Animals were divided into three groups according to treatment. Group 1 was treated with 10 ul of PF-MC (200 ug/ml; n?=?12), Group 2, with 10 ul of SLPI (200 ug/ml; n?=?12) and Group 3 was treated with buffer (10 ul; n?=?12) topically administered four times a day for up to 7?days. Half of the animals were sacrificed at day 3 before making a re-epithelialization time analysis with fluorescein staining at 18 and 24?hours. In the remaining animals corneal opacity was studied and digital photographs were taken at day 7 before doing euthanasia. Eyes were processed for histology and immunofluorescence.Corneal ulcerated area was significantly lower in PF-MC treated animals compared to SLPI and buffer-treated animals at 18?hours and 24?hours postinjury. A clear cornea and fundus red reflex was only found among PF-MC treated animals. Histological analysis revealed a stratified corneal epithelium with at least three layers in all PF-MC animals at day 7. In this group there was a reduced number of PMNs in the corneal stroma at 3 and 7?days of follow-up. Besides, corneal neovascularization was much more extended in SLPI and Buffer animals than in animals treated with PF-MC.The binding of SLPI with Cementoin to transglutaminase seems to be an effective strategy to treat corneal inflammation and angiogenesis.
Project description:Local insulin delivery has been shown to improve osseous healing in diabetic animals. The purpose of this study was to quantify the effects of local intramedullary delivery of saline or Ultralente insulin (UL) on various fracture healing parameters using an in vivo non-diabetic BB Wistar rat model. Quantitation of local insulin levels showed a rapid release of insulin from the fractured femora, demonstrating complete release at 2 days. RT-PCR analysis revealed that the expression of early osteogenic markers (Col1?2, osteopontin) was significantly enhanced with UL treatment when compared with saline controls (p?<?0.05). Significant differences in VEGF?+?cells and vascularity were evident between the treatment and control groups at day 7 (p?<?0.05). At day 21, histomorphometric analysis demonstrated a significant increase in percent mineralized tissue in the UL-treated animals compared with controls (p?<?0.05), particularly within the subperiosteal region of the fracture callus. Mechanical testing at 4 weeks showed significantly greater mechanical strength for UL-treated animals (p?<?0.05), but healing in control animals caught up at 6 weeks post-fracture. These results suggest that the primary osteogenic effect of UL during the early stages of fracture healing (1-3 weeks) is through an increase in osteogenic gene expression, subperiosteal angiogenesis, and mineralized tissue formation.
Project description:Diel and seasonal vertical migrations of zooplankton represent a widespread phenomenon occurring in marine and freshwater environments. Diel migrations are panoceanic, while seasonal migrations usually occur in temperate and polar areas. This paper describes differences in the diel and seasonal vertical migrations in the Drake Passage north and south of the Polar Front (PF). We analyzed material of 85 stations collected in spring of 2008 and 2010 (October-November) and in summer of 2010 and 2011 (January) within the 0-300 m depth range during various time of a day. At each station we sampled the upper mixed (UL), the middle (ML), and the deeper layers (DL) bounded by hydrological gradients. Diel migrations were significantly different south and north of the PF in terms of total abundance, biomass, diversity and individual taxa density. In both seasons, mesoplankton dielly migrated between the ML/DL and the UL north of the PF and between layers below 300 m and the DL and ML south of the PF. Deeper range of diel migrations south of the PF was coupled with a general mesoplankton descent in summer period compared to spring. Conversely, north of the PF, mesoplankton ascended to upper layers in summer, which was mirrored in lesser depths of diel migrations. The differences in the plankton distribution on both sides of the PF are likely associated with variations of vertical distribution of phytoplankton. Some abundant taxa such as Aetideus sp. and Oithona plumifera showed both common (nighttime ascend) and inverted (nighttime descend) vertical migrations depending on season and position related to the PF.
Project description:Unilateral inner ear damage is followed by behavioral recovery due to central vestibular compensation. The dose-dependent therapeutic effect of Ginkgo biloba extract EGb 761 on vestibular compensation was investigated by behavioral testing and serial cerebral [18F]-Fluoro-desoxyglucose ([18F]-FDG)-?PET in a rat model of unilateral labyrinthectomy (UL). Five groups of 8 animals each were treated with EGb 761-supplemented food at doses of 75, 37.5 or 18.75 mg/kg body weight 6 weeks prior and 15 days post UL (groups A,B,C), control food prior and EGb 761-supplemented food (75 mg/kg) for 15 days post UL (group D), or control food throughout (group E). Plasma levels of EGb 761 components bilobalide, ginkgolide A and B were analyzed prior and 15 days post UL. Behavioral testing included clinical scoring of nystagmus, postural asymmetry, head roll tilt, body rotation during sensory perturbation and instrumental registration of mobility in an open field before and 1, 2, 3, 5, 7, 15 days after UL. Whole-brain [18F]-FDG-?PET was recorded before and 1, 3, 7, 15 days after UL. The EGb 761 group A (75 mg/kg prior/post UL) showed a significant reduction of nystagmus scores (day 3 post UL), of postural asymmetry (1, 3, 7 days post UL), and an increased mobility in the open field (day 7 post UL) as compared to controls (group E). Application of EGb 761 at doses of 37.5 and 18.75 mg/kg prior/post UL (groups B,C) resulted in faster recovery of postural asymmetry, but did not influence mobility relative to controls. Locomotor velocity increased with higher plasma levels of ginkgolide A and B. [18F]-FDG-?PET revealed a significant decrease of the regional cerebral glucose metabolism (rCGM) in the vestibular nuclei and cerebellum and an increase in the hippocampal formation with higher plasma levels of ginkgolides and bilobalide 1 and 3 days post UL. Decrease of rCGM in the vestibular nucleus area and increase in the hippocampal formation with higher plasma levels persisted until day 15 post UL. In conclusion, Ginkgo biloba extract EGb 761 improves vestibulo-ocular motor, vestibulo-spinal compensation, and mobility after UL. This rat study supports the translational approach to investigate EGb 761 at higher dosages for acceleration of vestibular compensation in acute vestibular loss.
Project description:The development of the cornea, a highly specialized transparent tissue located at the anterior of the eye, is coordinated by a variety of molecules and cells. Here, we report that mast cells (MCs), recently found to be involved in morphogenesis, played a potentially important role in corneal development in mice. We show that two different waves of MC migration occurred during corneal development. In the first wave, MCs migrated to the corneal stroma and became distributed throughout the cornea. This wave occurred by embryonic day 12.5, with MCs disappearing from the cornea at the time of eyelid opening. In the second wave, MCs migrated to the corneal limbus and became distributed around limbal blood vessels. The number of MCs in this region gradually increased after birth and peaked at the time of eyelid opening in mice, remaining stable after postnatal day 21. We also show that integrin ?4?7 and CXCR2 were important for the migration of MC precursors to the corneal limbus and that c-Kit-dependent MCs appeared to be involved in the formation of limbal blood vessels and corneal nerve fibers. These data clearly revealed that MCs participate in the development of the murine cornea.
Project description:The severity of bloodstream infections (BSI) depends on pathogen, source, and host factors. Secretory leukocyte protease inhibitor (SLPI) counteracts tissue damage, balances inflammation, and is increased in pneumonia and sepsis. We aimed to evaluate whether SLPI production differs depending on etiology, disease severity, and sex in BSI and to correlate SLPI with markers of inflammation and immunosuppression. Of the adult patients with BSI, 109 were included and sampled repeatedly, from hospital admission through day 28. Controls (blood donors) were sampled twice. SLPI in plasma was measured with enzyme-linked immunosorbent assay (ELISA) technique. Streptococcus pneumoniae and Staphylococcus aureus etiology were associated with higher SLPI than Escherichia coli on days 1-2 and 3. On day 1-2, subjects with sepsis had higher SLPI concentrations than those with non-septic BSI. Pneumonia was associated with higher SLPI than a non-pulmonary source of infection. SLPI co-varied with inflammatory markers. SLPI concentrations did not differ with regard to sex in the full cohort, but men with pneumonia had higher SLPI than women on day 1-2. S. pneumoniae and S. aureus BSI were associated with higher SLPI, when compared to E. coli. Severity and pneumonia, as well as male sex in the pneumonia sub-cohort, were factors independently associated with higher SLPI.
Project description:Regulation of immune-like cell properties of periodontal ligament (PDL) cells is not understood. We investigate the importance of secretory leukocyte protease inhibitor (SLPI) for production of pro-inflammatory cytokines in human PDL cells.PDL cells were isolated from teeth extracted for orthodontic reasons. Cellular location of SLPI was investigated by immunocytochemistry. Cytokine transcript and protein expression were assessed by quantitative real-time RT-PCR and Western blotting. SLPI gene activity was knocked-down by siRNA. NF-κB signaling was assessed by measuring IκBα, and phosphorylated p65 and p105 protein expression.PDL cells showed cytoplasmic expression of SLPI. Cellular expression level of SLPI negatively correlated to LPS-induced stimulation of IL-6 and MCP-1. Both SLPI gene activity and protein were reduced by about 70% in PDL cells treated with SLPI siRNA compared to cells treated with non-coding construct. Treatment with SLPI siRNA was associated with up-regulation of both basal and LPS-stimulated IL-6, MCP-1 and TLRs mRNA expression. The up-regulation of MCP-1 transcript in SLPI siRNA-treated cells was confirmed on protein level. SLPI siRNA-treatment enhanced the phosphorylated NF-κB p105 protein expression.SLPI regulates PDL cell pro-inflammatory cytokine expression and modulates NF-κB signaling, suggesting that SLPI governs the immune cell-like properties of PDL cells.
Project description:Sexually mature (mid-May) adult female goldfish received a single injection of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP; 50 ug/g body weight at 5 uL/g body weight) or 0.6% saline on day 0 and a single injection of alpha-methyl-p-tyrosine (aMPT; 240 ug/g body weight at 5 uL/g body weight) or 0.6% saline on Day 5 with the goal of severely deplete catecholamine (dopamine and norepinephrine) levels. Hypothalamus tissue was extracted on Day 6. Telencephalon tissue was extracted on Day 6. The objective of this study was to examine gene expression changes in the neuroendocrine brain in response to catecholamine depletion. Overall design: A common reference design was used for both the hypothalamus and telencephalon groups: Hypothalamus: 3 independent groups of hypothalami from sexually mature female goldfish treated with 50 ug/g MPTP + 240 ug/g aMPT hybridized against a common vehicle control. An additional group was used for a dye-swap. Slides 4, 17, 21, 52. Telencephalon: 3 independent groups of telencephali from sexually mature female goldfish treated with 50 ug/g MPTP + 240 ug/g aMPT hybridized against a common vehicle control. An additional group was used for a dye-swap. Slides 28, 34, 43, 48.
Project description:Bitter melon (Momordica charantia, MC) has been used in traditional Korean medicine in treating diabetes. In addition, some reports were emerged, showing the antifertility activities of MC in mammals. We investigated the effects of ethanolic MC extract on the reproductive activity of golden hamsters whose spermatogenetic capacity is controlled by their photoperiods. The animals were divided into 4 groups: long photoperiod (LP) control, short photoperiod (SP) control, and LP animals treated with MC. The animals were orally ingested with low (0.03 g/kg) or high (0.15 g/kg) concentrations of the ethanolic extracts for 8 weeks on the daily basis. The control animals received the vehicle. The animals were then mated with age-matched females, experienced pregnancy. As results, the LP control animals showed active large testes but SP control animals displayed remarkably reduced testes. The animals treated with both concentrations of MC extracts demonstrated large testes, indicating fertile activity as animals in LP. LP control animals had litters as expected, but SP controls had no litters at all. MC extract showed the same results as LP animals in generating offsprings. These results suggest that the MC extract does not change the photoperiodic influence on reproductive activity of male golden hamsters.
Project description:Many frequently prescribed drugs are non-genotoxic carcinogens (NGC) in rodent liver. Their mode of action and health risks for humans remain to be elucidated. Here, we investigated the impact of two model NGC, the anti-epileptic drug phenobarbital (PB) and the contraceptive cyproterone acetate (CPA), on intrahepatic epithelial-mesenchymal crosstalk and on growth of first stages of hepatocarcinogenesis. Unaltered hepatocytes (HC) and preneoplastic HC (HCPREN) were isolated from rat liver for primary culture. DNA replication of HC and HCPREN was increased by in vitro treatment with 10 µM CPA, but not 1 mM PB. Next, mesenchymal cells (MC) obtained from liver of rats treated with either PB (50 mg/kg bw/day) or CPA (100 mg/kg bw/day), were cultured. Supernatants from both types of MC raised DNA synthesis of HC and HCPREN. This indicates that PB induces replication of HC and HCPREN only indirectly, via growth factors secreted by MC. CPA, however, acts on HC and HCPREN directly as well as indirectly via mesenchymal factors. Transcriptomics and bio-informatics revealed that PB and CPA induce extensive changes in the expression profile of MC affecting many growth factors and pathways. MC from PB-treated rats produced and secreted enhanced levels of HBEGF and GDF15, factors found to suppress apoptosis and/or induce DNA synthesis in cultured HC and HCPREN. MC from CPA-treated animals showed enhanced expression and secretion of HGF, which strongly raised DNA replication of HC and HCPREN. In conclusion, our findings reveal profound effects of two prototypical NGC on the hepatic mesenchyme. The resulting release of factors, which suppress apoptosis and/or enhance cell replication preferentially in cancer prestages, appears to be crucial for tumor promotion by NGC in the liver.
Project description:Sexually mature (mid-May) adult female goldfish received a single injection of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP; 50 ug/g body weight at 5 uL/g body weight) or 0.6% saline on day 0 and a single injection of alpha-methyl-p-tyrosine (aMPT; 240 ug/g body weight at 5 uL/g body weight) or 0.6% saline on Day 5 with the goal of severely deplete catecholamine (dopamine and norepinephrine) levels. Hypothalamus tissue was extracted on Day 6. Telencephalon tissue was extracted on Day 6. The objective of this study was to examine gene expression changes in the neuroendocrine brain in response to catecholamine depletion. A common reference design was used for both the hypothalamus and telencephalon groups: Hypothalamus: 3 independent groups of hypothalami from sexually mature female goldfish treated with 50 ug/g MPTP + 240 ug/g aMPT hybridized against a common vehicle control. An additional group was used for a dye-swap. Slides 4, 17, 21, 52. Telencephalon: 3 independent groups of telencephali from sexually mature female goldfish treated with 50 ug/g MPTP + 240 ug/g aMPT hybridized against a common vehicle control. An additional group was used for a dye-swap. Slides 28, 34, 43, 48.