ABSTRACT
BACKGROUND: The safety of the living donor in living-donor liver transplantation (LDLT) is always the first priority, meanwhile, the graft-to-recipient weight ratio (GRWR) and the anatomy of the liver allograft must also not be compromised in order to warrant tranplatation success. When it comes to the allograft of the right lobe of the liver without the middle hepatic vein (R-M), the outflow and adequate drainage for the territory of middle hepatic vein (MHV) is one critical concern. Despite publications in some high-volume transplant centers on the positive results of using expanded polytetrafluoroethylene (ePTFE) grafts to substitute those of autologous veins, complications related to the ePTFE graft have not been well discussed. METHODS: From July 2012 to June 2016, 129 adult patients who underwent living donor liver transplantation in Taipei Veterans General Hospital were analyzed. There were 3 cases of adjacent organ erosion with gas bubbles in the lumen of an ePTFE graft, including gastrointestinal (GI) tract penetration in 2 out of the first 15 cases that used the venous graft of ringed expanded polytetrafluoroethylene (rPTFE). The patient survival rate during this period was compared and radiological findings of rPTFE function and clinical signs of erosion with infection were also examined to raise the concerns of safety as well as early detection of complications of rPTFE. RESULTS: The overall 1-year patient survival rate was 90%, of which the right lobe wih MHV (R+M) group was 93.5% and the R-M group was 91.9%. For the mean of GRWR, the R+M group was 1.05 ± 0.19 and R-M group was 1.19 ± 0.27, while those who needed reconstruction with vein grafts was 0.96 ± 0.11. Among the R-M group, 24 out of 88 cases (27.3%) needed reconstruction of MHV tributaries. Of the 24 cases, 15 cases were done with rPTFE and the 1-year patient survival rate of the rPTFE group was 73%, which is significantly worse (P = .008) than the non-rPTFE (89%) and non-reconstructed (97%) groups. The mean GRWR is significantly higher (P = .001) in the non-reconstructed group (1.19 ± 0.27) than in the rPTFE (0.99 ± 0.11) and non-rPTFE (0.94 ± 0.11) groups. The venous grafts patency rate between the different graft types is no different, and there is also significance in warm ischemic time (P = .009) between the non-reconstructed (49 ± 15), rPTFE (81 ± 51), and non-rPTFE (56 ± 18) groups in the mean minutes. CONCLUSION: In cases of fever of unknown cause in patients receiving LDLT with rPTFE graft, a regular computed tomography (CT) scan with contrast and gas bubbles within the graft lumen is the best way for early detection of graft related infection and suspicious GI tract penetration. To decrease the risks of tissue reaction induced by ePTFE graft in LDLT, omentum patches or other inert agents can be introduced as a buffer between the graft and adjacent organs, especially the GI tracts. However, research in material science shall be explored to solve the problem in the future.
Subject(s)
Blood Vessel Prosthesis , Liver Transplantation/adverse effects , Liver Transplantation/methods , Living Donors , Postoperative Complications/etiology , Adult , Blood Vessel Prosthesis/adverse effects , Female , Humans , Male , Middle Aged , Polytetrafluoroethylene , Postoperative Complications/diagnosis , Tomography, X-Ray Computed/methodsABSTRACT
Mutations in endoglin, a TGFß/BMP coreceptor, are causal for hereditary hemorrhagic telangiectasia (HHT). Endoglin-null (Eng-/-) mouse embryos die at embryonic day (E)10.5-11.5 due to defects in angiogenesis. In part, this is due to an absence of vascular smooth muscle cell differentiation and vessel investment. Prior studies from our lab and others have shown the importance of endoglin expression in embryonic development in both endothelial cells and neural crest stem cells. These studies support the hypothesis that endoglin may play cell-autonomous roles in endothelial and vascular smooth muscle cell precursors. However, the requirement for endoglin in vascular cell precursors remains poorly defined. Our objective was to specifically delete endoglin in neural crest- and somite-derived Pax3-positive vascular precursors to understand the impact on somite progenitor cell contribution to embryonic vascular development. Pax3Cre mice were crossed with Eng+/- mice to obtain compound mutant Pax3(Cre/+);Eng+/- mice. These mice were then crossed with homozygous endoglin LoxP-mutated (Eng(LoxP/LoxP)) mice to conditionally delete the endoglin gene in specific lineages that contribute to endothelial and smooth muscle constituents of developing embryonic vessels. Pax3(Cre/+);Eng(LoxP/)(-) mice showed a variety of vascular defects at E10.5, and none of these mice survived past E12.5. Embryos analyzed at E10.5 showed malformations suggestive of misdirection of the intersomitic vessels. The dorsal aorta showed significant dilation with associated vascular smooth muscle cells exhibiting disorganization and enhanced expression of smooth muscle differentiation proteins, including smooth muscle actin. These results demonstrate a requirement for endoglin in descendants of Pax3-expressing vascular cell precursors, and thus provides new insight into the cellular basis underlying adult vascular diseases such as HHT.
Subject(s)
Blood Vessels/embryology , Blood Vessels/metabolism , Intracellular Signaling Peptides and Proteins/metabolism , Neovascularization, Physiologic , Paired Box Transcription Factors/metabolism , Actins/metabolism , Alleles , Animals , Aorta/embryology , Aorta/pathology , Embryo Loss/metabolism , Embryo Loss/pathology , Embryo, Mammalian/abnormalities , Embryo, Mammalian/metabolism , Embryo, Mammalian/pathology , Endoglin , Endothelial Cells/metabolism , Gene Deletion , Integrases/metabolism , Intracellular Signaling Peptides and Proteins/deficiency , Mice , Muscle, Smooth, Vascular/metabolism , Myocytes, Smooth Muscle/metabolism , PAX3 Transcription Factor , Phenotype , Recombination, Genetic/genetics , Somites/blood supply , Staining and LabelingABSTRACT
AIMS: The agarase from Thalassomonas agarivorans BCRC 17492 was cloned and overexpressed in Escherichia coli. The characterization of the novel agarase was performed. METHODS AND RESULTS: The genomic library of T. agarivorans BCRC 17492 was constructed for screening agarase gene. The novel ß-agarase, namely AgaB1, was successfully identified and shared only 57% identity to reported agarase from Alteromonas sp. To characterize the AgaB1 protein, the recombinant AgaB1 can be obtained by heterologous expression in E. coli. The agarase activity of AgaB1 was achieved at 30·25 U per mg at 35°C. According to the analysis of optimal conditions, the highest activity of AgaB1 was attained at 40°C, pH 7·4 and 200 mmol l(-1) NaCl, and half-life of AgaB1 can be maintained for almost 1 h at 40°C. Further determination of substrate hydrolysis indicated that AgaB1 had possession of both endo- and exolytic activity, and neoagarobiose was the major hydrolysis product by TLC and high-performance liquid chromatograph/mass spectrometer (HPLC/MS) analysis. CONCLUSIONS: We have successfully cloned and overexpressed the novel ß-agarase from T. agarivorans BCRC 17492 in E. coli. The high yield and detailed characterization of recombinant AgaB1 was provided. SIGNIFICANT AND IMPACT OF THE STUDY: AgaB1 was the first ß-agarase that was cloned and described from Thalassomonas species. In the light of properties of AgaB1, it has the potential as the biocatalyst for industrial applications.
Subject(s)
Gammaproteobacteria/enzymology , Glycoside Hydrolases/genetics , Amino Acid Sequence , Cloning, Molecular , Disaccharides/metabolism , Escherichia coli/genetics , Gammaproteobacteria/genetics , Glycoside Hydrolases/chemistry , Glycoside Hydrolases/metabolism , Hydrolysis , Recombinant Proteins/metabolismABSTRACT
Based on use of a loop-mediated isothermal amplification (LAMP) identification protocol, this study attempted to detect Lactococcus garvieae in fish by using primer sets designed from an L. garvieae alpha/beta fold family hydrolase gene. Reaction time and temperatures were optimized for 60 min at 60°C with the resulting amplicons visualized by adding SYBR Green I to the reaction tube. The assay specificity was assessed using 45 different bacterial strains. Positive results were observed in all 30 L. garvieae isolates from various aquatic animals. No false-positive results were observed in 15 non-L. garvieae strains. The detection limit of the LAMP assay was 10-fold more sensitive than the conventional polymerase chain reaction (PCR) targeting 16S rDNA when using purified L. garvieae DNA. The detection limit of the LAMP assay was approximately 300 colony-forming units (CFU) using crude bacterial lysates, 100-fold more sensitive than PCR. Furthermore, L. garvieae in spleen, kidney and brain of experimentally challenged tilapia and grey mullet were detected using this optimized LAMP assay. Results of this study demonstrate the effectiveness of LAMP in providing a rapid yet simple test for detecting L. garvieae in fish.
Subject(s)
Fish Diseases/microbiology , Gram-Positive Bacterial Infections/veterinary , Lactococcus/isolation & purification , Polymerase Chain Reaction/veterinary , Animals , Base Sequence , DNA, Bacterial/genetics , Fishes , Gene Expression Regulation, Bacterial/physiology , Gene Expression Regulation, Enzymologic , Gram-Positive Bacterial Infections/diagnosis , Gram-Positive Bacterial Infections/microbiology , Hydrolases/genetics , Hydrolases/metabolism , Lactococcus/pathogenicity , Polymerase Chain Reaction/methods , RNA, Bacterial/genetics , RNA, Ribosomal, 16S/genetics , Sensitivity and Specificity , Species SpecificityABSTRACT
A multiplex polymerase chain reaction (m-PCR) technique was developed as a rapid and accurate diagnostic tool for identifying five major Gram-negative bacilli -Vibrio vulnificus, V. parahaemolyticus, Aeromonas hydrophila, Chryseobacterium meningosepticum and Edwardsiella tarda- that cause major diseases in cultured aquatic animals in Taiwan. The expected amplicons for V. vulnificus, V. parahaemolyticus, A. hydrophila, C. meningosepticum and E. tarda were 410, 368, 685, 180 and 230bp, respectively. The assay was shown to be specific for the target pathogens. The sensitivities of detection were estimated to be 20.5fgâ¼200pg of genomic DNA or 10(2) â¼10(4) colony-forming units (cfu) of bacterial isolates when adopted as PCR templates. The m-PCR was capable of simultaneously amplifying target fragments from bacterial genome DNA mixed with the DNA extracted from viscera and tissues taken from fish without affecting the performance of the method.
Subject(s)
Fish Diseases/diagnosis , Gram-Negative Bacteria/genetics , Gram-Negative Bacterial Infections/veterinary , Multiplex Polymerase Chain Reaction/veterinary , Animals , DNA Primers , Gram-Negative Bacterial Infections/diagnosis , Sensitivity and Specificity , TaiwanABSTRACT
Congenital obstructive nephropathy is a major cause of renal insufficiency in children. Osteopontin (OPN) is a phosphoprotein produced by the kidney that mediates cell adhesion and migration. We investigated the role of OPN in the renal response to unilateral ureteral obstruction (UUO) in neonatal mice. OPN null mutant (-/-) and wild-type (+/+) mice were subjected to sham operation or UUO within the first 2 days of life. At 7 and 21 days of age, fibroblasts (fibroblast-specific protein (FSP)-1), myofibroblasts (alpha-smooth muscle actin (SMA)), and macrophages (F4/80) were identified by immunohistochemical staining. Apoptotic cells were detected by terminal deoxy transferase uridine triphosphate nick end-labeling technique and interstitial collagen by Masson trichrome or picrosirius red stain. Compared to sham-operated or contralateral kidneys, obstructed kidneys showed increases in all parameters by 7 days, with further increases by 21 days. After 21 days UUO, there was an increase in tubular and interstitial apoptosis in OPN -/- mice as compared to +/+ animals (P<0.05). However, FSP-1- and alpha-SMA-positive cells and collagen in the obstructed kidney were decreased in OPN -/- compared to +/+ mice (P<0.05), whereas the interstitial macrophage population did not differ between groups. We conclude that OPN plays a significant role in the recruitment and activation of interstitial fibroblasts to myofibroblasts in the progression of interstitial fibrosis in the developing hydronephrotic kidney. However, OPN also suppresses apoptosis. Future approaches to limit the progression of obstructive nephropathy in the developing kidney will require targeting of specific renal compartments.
Subject(s)
Apoptosis/physiology , Kidney/pathology , Osteopontin/metabolism , Ureteral Obstruction/metabolism , Ureteral Obstruction/pathology , Animals , Animals, Newborn , Collagen/metabolism , Female , Fibroblasts/metabolism , Fibroblasts/pathology , Fibrosis , Gene Expression Regulation , Kidney/metabolism , Kidney Tubules/metabolism , Kidney Tubules/pathology , Macrophages/metabolism , Macrophages/pathology , Male , Mice , Mice, Knockout , Osteopontin/genetics , Ureteral Obstruction/physiopathologyABSTRACT
The use of focused high-intensity light sources for ablative perturbation has been an important technique for cell biological and developmental studies. In targeting subcellular structures many studies have to deal with the inability to target, with certainty, an organelle or large macromolecular complex. Here we demonstrate the ability to selectively target microtubule-based structures with a laser microbeam through the use of enhanced yellow fluorescent protein (EYFP) and enhanced cyan fluorescent protein (ECFP) variants of green fluorescent protein fusions of tubule. Potorous tridactylus (PTK2) cell lines were generated that stably express EYFP and ECFP tagged to the alpha-subunit of tubulin. Using microtubule fluorescence as a guide, cells were irradiated with picosecond laser pulses at discrete microtubule sites in the cytoplasm and the mitotic spindle. Correlative thin-section transmission electron micrographs of cells fixed one second after irradiation demonstrated that the nature of the ultrastructural damage appeared to be different between the EYFP and the ECFP constructs suggesting different photon interaction mechanisms. We conclude that focal disruption of single cytoplasmic and spindle microtubules can be precisely controlled by combining laser microbeam irradiation with different fluorescent fusion constructs. The possible photon interaction mechanisms are discussed in detail.
Subject(s)
Laser Therapy/methods , Microsurgery/methods , Microtubules/radiation effects , Microtubules/ultrastructure , Bacterial Proteins/radiation effects , Dose-Response Relationship, Radiation , Energy Transfer/physiology , Green Fluorescent Proteins/radiation effects , Laser Therapy/instrumentation , Luminescent Proteins/radiation effects , Microsurgery/instrumentation , Microtubules/physiology , Radiation Dosage , Surgery, Computer-Assisted/methodsABSTRACT
The aim of this study was to determine the effects of varying parameters of Er:YAG laser irradiation with and without water spray cooling on root canal dentine in vitro. After horizontally removing tooth crowns from extracted human teeth, roots were axially sectioned into thin slices, exposing the root canal surface. An Er:YAG laser delivered 10-30 J/cm(2) into a 0.4-mm diameter laser spot on the root canal surface. Single pulses of different lengths (80-280 micro s) were applied with and without water spray cooling/irrigation, and sequences of three pulses at a repetition rate of 30 Hz were applied at selected pulse parameters. The irradiated samples were investigated using both confocal laser scanning microscopy (CLSM) and scanning electron microscopy (SEM). At most irradiation conditions, the root canal dentine surface was ablated. Three-dimensional images from CLSM revealed that the cavity walls were not smooth. Depths of the cavities revealed significant differences between the cavities. No debris was observed at the surface of cavities at any irradiation condition. Strong melting and recrystallisation, or unusually flat surfaces with open dentinal tubules were obtained with sequences of three pulses without water cooling. CLSM is an effective tool for investigation of laser effects on root canal dentine. By varying the irradiation conditions, the Er:YAG laser can induce different modifications of root canal surface, which may be very interesting for root canal preparation.
Subject(s)
Dental Pulp Cavity/radiation effects , Dentin/radiation effects , Laser Therapy , Root Canal Preparation , Dental Pulp Cavity/diagnostic imaging , Humans , In Vitro Techniques , Microscopy, Confocal , Microscopy, Electron, Scanning , Root Canal Preparation/methods , Therapeutic Irrigation , Ultrasonography , WaterABSTRACT
Osteopontin is a novel cytokine that is expressed in pulmonary granulomatous disease such as sarcoidosis and tuberculosis. It can regulate macrophage and T cell migration, activation, and cytokine expression, yet its role in granuloma formation and evolution is unknown. We induced hypersensitivity pulmonary granulomas by embolizing Schistosoma mansoni eggs to the lungs of osteopontin-deficient (null mutant) mice and osteopontin-sufficient (wild-type control) mice. Granulomas from osteopontin-null animals were smaller at early time points and contained remarkably few macrophages and macrophage-derived epithelioid cells and giant cells. T cell accumulation was unaffected by osteopontin deficiency. These results demonstrate that osteopontin regulates macrophage accumulation during pulmonary granuloma formation, and may explain the impaired ability of osteopontin-deficient hosts to control mycobacterial disease.
Subject(s)
Cytokines/physiology , Granuloma/pathology , Lung Diseases/pathology , Lung/pathology , Sialoglycoproteins/physiology , Animals , Cell Count , Cytokines/deficiency , Epithelial Cells/pathology , Giant Cells/pathology , Granuloma/etiology , Granuloma/physiopathology , Immunization , Lung/immunology , Lung Diseases/etiology , Lung Diseases/physiopathology , Macrophages/pathology , Mice , Mice, Knockout , Osteopontin , Schistosoma mansoni/immunology , Sialoglycoproteins/deficiency , T-Lymphocytes/pathologyABSTRACT
Osteopontin (OPN) is a chemotactic protein that attracts immune cells, to inflammatory sites. The sensitization phase of allergic cutaneous contact hypersensitivity (CHS) requires the migration of Langerhans cells/dendritic cells (LCs/DCs) from skin to draining lymph nodes. Characterizing OPN function for LC/DC migration we found upregulated OPN expression in hapten sensitized skin and draining lymph nodes. OPN induces chemotactic LC/DC migration, initiates their emigration from the epidermis, and attracts LCs/DCs to draining lymph nodes by interacting with CD44 and alphav integrin. Furthermore, OPN-deficient mice have a significantly reduced CHS response that correlates with an impaired ability of OPN-deficient mice to attract LCs/DCs to draining lymph nodes. In conclusion, OPN is an important factor in the initiation of CHS by guiding LCs/DCs from skin into lymphatic organs.
Subject(s)
Cell Movement/immunology , Dermatitis, Allergic Contact/immunology , Langerhans Cells/immunology , Lymph Nodes/immunology , Sialoglycoproteins/immunology , Animals , Antibodies, Monoclonal/administration & dosage , Antibodies, Monoclonal/immunology , Cell Differentiation , Cells, Cultured , Chemotaxis , Dendritic Cells/cytology , Dendritic Cells/immunology , Disease Models, Animal , Epidermis/immunology , Hyaluronan Receptors/immunology , Injections, Intradermal , Langerhans Cells/cytology , Lymph Nodes/cytology , Mice , Mice, Inbred C57BL , Mice, Knockout , Osteopontin , Receptors, Vitronectin/biosynthesis , Receptors, Vitronectin/immunology , Sialoglycoproteins/administration & dosage , Sialoglycoproteins/genetics , Up-RegulationABSTRACT
OBJECTIVE: To describe the epithelial healing rates observed in freshly cultured rabbit corneas chemically burned with high-concentration hydrochloric acid (HCl) and sodium hydroxide (NaOH) and subsequently treated with phototherapeutic keratectomy (PTK). METHODS: We obtained 126 fresh corneoscleral rims from cadaveric New Zealand white rabbits. Each cornea was exposed to 4-mm cellulose sponges soaked in a solution of topical 0.9% isotonic sodium chloride solution, 2M HCl, or 0.5M NaOH. A transepithelial PTK (6-mm zone; 100-microm ablation depth) was then performed using the excimer laser (150-mJ/cm(2) energy pulse; 20 nanosecond duration; and 10-Hz frequency). Corneas were placed in tissue culture, and 1 cornea from each group was taken out of culture each day after treatment. Re-epithelialization was monitored by means of fluorescein staining, slitlamp photography, and histopathological analysis. RESULTS: Corneas treated with HCl and NaOH exhibited immediate epithelial defects that slowly healed over time. In PTK-treated corneas, the re-epithelialization rate was accelerated compared with that of controls (P =.003 for the HCl group, and P<.001 for the NaOH group). The new epithelial layers were smoother in PTK-treated corneas, as confirmed by results of histopathological analysis. CONCLUSION: Corneal damage caused by HCl and NaOH may be modulated in vitro by PTK in this rabbit model. CLINICAL RELEVANCE: After corneal chemical damage, 193-nm excimer laser PTK accelerates epithelial wound healing.
Subject(s)
Burns, Chemical/metabolism , Epithelial Cells/physiology , Epithelium, Corneal/physiology , Eye Burns/chemically induced , Photorefractive Keratectomy , Wound Healing , Animals , Burns, Chemical/pathology , Cornea/surgery , Corneal Injuries , Fluorophotometry , Hydrochloric Acid , Lasers, Excimer , Organ Culture Techniques , Rabbits , Sodium HydroxideABSTRACT
The Jagged/Notch signaling pathways control cell fate determination and differentiation, and their dysfunction is associated with human pathologies involving cardiovascular abnormalities. To determine the presence of these genes during vascular response to injury, we analyzed expression of Jagged1, Jagged2, and Notch1 through 4 after balloon catheter denudation of the rat carotid artery. Although low levels of Jagged1, Jagged2, and constitutive expression of Notch1 were seen in uninjured endothelium, expression of all was significantly increased in injured vascular cells. High Jagged1 expression was restricted to the regenerating endothelial wound edge, whereas Notch transcripts were abundant in endothelial and smooth muscle cells. To understand the basis for Jagged/Notch control of cellular phenotype, we studied an in vitro model of NIH3T3 cells transfected with a secreted form of the extracellular domain of Jagged1. We report that the soluble Jagged1 protein caused decreased cell-matrix adhesion and cell migration defects. Cadherin-mediated intercellular junctions as well as focal adhesions were modified in soluble Jagged1 transfectants, demonstrating that cell-cell contacts and adhesion plaques may be targets of Jagged/Notch activity. We suggest that Jagged regulation of cell-cell and cell-matrix interactions may contribute to the control of cell migration in situations of tissue remodeling in vivo.
Subject(s)
Carotid Artery Injuries/genetics , Gene Expression , Membrane Proteins/genetics , Multigene Family , Wounds, Nonpenetrating/genetics , 3T3 Cells , Animals , Calcium-Binding Proteins , Carotid Artery Injuries/pathology , Carotid Artery Injuries/physiopathology , Cell Communication/physiology , Cell Movement/physiology , Cell-Matrix Junctions/physiology , In Vitro Techniques , Intercellular Signaling Peptides and Proteins , Jagged-1 Protein , Jagged-2 Protein , Mice , Phenotype , Proteins/physiology , Rats , Receptors, Notch , Serrate-Jagged Proteins , Wounds, Nonpenetrating/pathology , Wounds, Nonpenetrating/physiopathologyABSTRACT
BACKGROUND AND OBJECTIVE: Most of the in vitro work to characterize the effects of clinical laser surgery on corneal tissues has concentrated on the effects on stromal keratocytes and endothelium with little attention being paid to corneal epithelium. Our purpose is to describe the epithelial healing rates observed in freshly cultured rabbit corneas treated with phototherapeutic keratectomy (PTK). STUDY DESIGN/MATERIALS AND METHODS: Corneas were placed in a simple organ culture system, with media change every 2 days. A clinical excimer laser was used to perform a 6 mm diameter, 100 microm depth transepithelial PTK on 24 cultured rabbit corneas, 1 day after culture initiation. For each post-treatment day, one experimental and one control cornea were removed from culture and stained with fluorescein, photographed, and fixed for histology. Epithelial defect area was measured with digital imaging software and analyzed statistically to assess the re-epithelialization rate. RESULTS: Control corneas, maintained in culture for 1-4 days, had no epithelial defects. Those corneas treated with PTK exhibited an immediate epithelial defect that slowly healed over 3 days. This was confirmed on histopathological analysis. A significant linear trend in re-epithelialization across the time points studied was found (F = 80.48, P = 0.0029). The slope of the linear regression model showed an estimate rate of re-epithelialization of -6.70 over the 3 days. CONCLUSION: We have described the development of a simple, whole organ, rabbit cornea culture model for re-epithelialization after PTK. Our rates of epithelial healing resemble those found in the literature in live rabbit models. Therefore, this model may possibly be used to monitor epithelial wound healing in different corneal diseases or injuries.
Subject(s)
Epithelium, Corneal/radiation effects , Models, Biological , Photorefractive Keratectomy/adverse effects , Radiation Injuries, Experimental/etiology , Radiation Injuries, Experimental/pathology , Wound Healing/radiation effects , Animals , Disease Models, Animal , Epithelium, Corneal/injuries , Epithelium, Corneal/pathology , Fluorescein , Lasers, Excimer , Organ Culture Techniques , Rabbits , Time FactorsABSTRACT
Product R (Reticulose) is a peptide-nucleic acid immunomodulator recently shown to enhance the expression of mRNAs encoding pro-inflammatory cytokines. Interleukin 8 (IL-8) and macrophage chemoattractant protein-1 (MCP-1) are pro-inflammatory chemokines involved in immune cell mobilization and stimulation. To determine whether Product R acts by upregulating these chemokines, we assayed its effects on the expression of IL-8 and MCP-1 mRNAs and proteins by human monocytic U937 cells and by adherent peripheral blood mononuclear cells (PBMCs). U937 cells were cultured for 0-21 days in media containing 0-20% Product R or phosphate-buffered saline (PBS). Compared to control cultures, cells cultured in Product R expressed increased amounts of IL-8 and MCP-1 mRNAs, as measured by reverse transcriptase-polymerase chain reaction (RT-PCR). Product R also increased secretion of IL-8 and MCP-1, as measured by enzyme-linked immunosorbent assay (ELISA), and boosted secretion induced by bacterial lipopolysaccharide (LPS), in a time- and dose-dependent manner. In adherent PBMCs, Product R increased IL-8 and MCP-1 secretion, but reduced LPS-induced MCP-1 secretion. While mRNAs encoding the IL-8 receptor, CXCR2, and the MCP-1 receptor, CCR2, were increased in U937 cells cultured in 5-10% Product R, we observed no change in binding of receptor-specific antibodies. These findings suggest that Product R upregulates the expression of IL-8 and MCP-1, which may boost immune system activity in virally-infected patients.
Subject(s)
Adjuvants, Immunologic/pharmacology , Chemokine CCL2/metabolism , Interleukin-8/metabolism , Monocytes/metabolism , Peptide Nucleic Acids/pharmacology , U937 Cells/metabolism , Apoptosis/drug effects , Apoptosis/immunology , Chemokine CCL2/biosynthesis , Humans , Interleukin-8/biosynthesis , Monocytes/drug effects , Monocytes/immunology , Receptors, CCR2 , Receptors, Chemokine/biosynthesis , Receptors, Interleukin-8A/biosynthesis , U937 Cells/drug effects , U937 Cells/immunologyABSTRACT
We have previously demonstrated that the expression of the soluble extracellular domain of the transmembrane ligand for Notch receptors, Jagged 1 (sJ1), in NIH 3T3 cells results in the formation of a matrix-dependent chord-like phenotype, the loss of contact inhibition of growth, and an inhibition of pro-alpha 1(I) collagen expression. In an effort to define the mechanism by which sJ1 induces this phenotype, we report that sJ1 transfectants display biochemical and cytoskeletal alterations consistent with the activation of Src. Indeed, cotransfection of sJ1 transfectants with a dominant-negative mutant of Src resulted in the loss of matrix-dependent chord formation and correlated with the restoration of type I collagen expression and contact inhibition of growth. We also report that the sJ1-mediated induction of Src activity and related phenotypes, including chord formation, may result from the inhibition of endogenous Jagged 1-mediated Notch signaling since it was not possible to detect an sJ1-dependent induction of CSL-dependent transcription in these cells. Interestingly, NIH 3T3 cells transfected with dominant-negative (but not constitutively active) mutants of either Notch 1 or Notch 2 displayed a similar Src-related phenotype as the sJ1 transfectants. These data suggest that the ability of sJ1 to mediate chord formation is Src-dependent and requires the repression of endogenous Jagged 1-mediated Notch signaling, which is tolerant to the destabilization of the actin cytoskeleton, a mediator of cell migration.
Subject(s)
Membrane Proteins/physiology , Oncogene Protein pp60(v-src)/metabolism , Proteins/physiology , 3T3 Cells , Animals , Calcium-Binding Proteins , Cortactin , Intercellular Signaling Peptides and Proteins , Jagged-1 Protein , Membrane Proteins/genetics , Membrane Proteins/metabolism , Mice , Microfilament Proteins/metabolism , Mutagenesis, Site-Directed , Phenotype , Phosphorylation , Proteins/genetics , Proteins/metabolism , Receptors, Notch , Serrate-Jagged Proteins , Spectrometry, Fluorescence , Transfection , Tyrosine/metabolismABSTRACT
An epizootic bacterial infection in the giant freshwater prawn Macrobranchium rosenbergii occurred in Taiwan from May to June 1999. The cumulative mortality was approximately 30 to 75%. The diseased prawns showed opaque and whitish muscles and were approximately 2 mo old with total lengths from 5 to 6 cm. Histopathologically, they showed marked edema and necrotic lesions with inflammation in the muscles and hepatopancreas. Bacteria isolated using brain heart infusion medium or tryptic soy agar were Gram-positive and ovoid. Three isolates from diseased prawns at different farms were tested using the API 20 Strepsystem and conventional tests and identified as Lactococcus garvieae. Experimental infections with these isolates gave gross signs and histopathological changes similar to those seen in the naturally infected prawns. The LD50 value of isolate MR1 was 6.6 x 10(5) colony forming units/prawn. Identification of MR1 was confirmed by a PCR assay for L. garvieae that gave the expected amplicon of 1100 bp. In addition, its 16S rDNA sequence (GenBank accession number AF283499) gave 99% sequence identity to Enterococcus seriolicida (synonym L. garvieae; GenBank accession number AF061005). This is the first report of confirmed L. garvieae infection in prawn aquaculture.
Subject(s)
Decapoda/microbiology , Lactococcus/isolation & purification , RNA, Ribosomal, 16S/analysis , Animals , Aquaculture , Base Sequence , Colony Count, Microbial , Lactococcus/classification , Lactococcus/genetics , Molecular Sequence Data , Muscles/microbiology , Muscles/pathology , Polymerase Chain Reaction/veterinary , TaiwanABSTRACT
PURPOSE: Vascular endothelial growth factor (VEGF) causes widespread retinal vascular dilation, produces breakdown of the blood-retinal barrier, and is implicated in ocular neovascularization (NV). Basic fibroblast growth factor (bFGF) also has been implicated in the production of ocular NV. This study was performed to investigate the ability of simultaneous sustained intravitreal release of both VEGF and bFGF to induce robust retinal NV in the rabbit. METHODS: Intravitreal implantation of sustained-release Hydron polymeric pellets containing both 20 microg of VEGF and 20 microg of bFGF was performed on adult male Dutch belted rabbits. In other animals either 20 microg or 50 microg bFGF-containing pellets was implanted intravitreally; also, either 20 microg VEGF or 50 microg VEGF-containing pellets was implanted. Control rabbits received either blank polymeric pellets or a pellet containing 30 microg bovine serum albumin. Eyes were examined by indirect ophthalmoscopy after surgery at 24 hrs, 48 hrs, 4 days, 7 days, 14 days, 21 days, and 28 days. Findings were documented by color fundus photography and fluorescein angiography (FA). Eyes were enucleated and prepared for histologic analysis at 28 days following intravitreal implantation of the VEGF/bFGF-containing pellets. RESULTS: In all eyes implanted with VEGF/bFGF pellets, dilation and tortuosity of existing blood vessels were observed within 48 hrs after pellet implantation. The progression of retinal vascular changes was rapid and occurred over the entire optic disk and medullary rays between 4 and 7 days. Hemorrhage occurred as early as 14 days after VEGF/bFGF pellet implantation. In eyes with massive hemorrhage, total traction retinal detachment developed after the second week. The presence of abnormal tissues at the vitreo-retinal interface within 28 days was demonstrated by light microscopy while FA showed profuse leakage of dye from anomalous vessels within the first week. Neither bFGF-exposed eyes nor control eyes showed any vascular changes. Eyes that received only VEGF-containing pellets exhibited tortuosity of existing vessels, but neither hemorrhaging nor retinal detachment occurred. CONCLUSIONS: These results demonstrate that retinal vascular changes leading to hemorrhaging is produced rapidly in the rabbit by simultaneous intravitreal release of both VEGF and bFGF. Understanding how these growth factors induce retinal NV may suggest novel therapeutic treatment strategies.
Subject(s)
Endothelial Growth Factors/toxicity , Fibroblast Growth Factor 2/toxicity , Lymphokines/toxicity , Retinal Hemorrhage/chemically induced , Retinal Neovascularization/chemically induced , Retinal Vessels/drug effects , Animals , Delayed-Action Preparations , Drug Combinations , Drug Implants , Fluorescein Angiography , Fundus Oculi , Male , Ophthalmoscopy , Rabbits , Retinal Detachment/chemically induced , Retinal Detachment/pathology , Retinal Hemorrhage/pathology , Retinal Neovascularization/pathology , Retinal Vessels/pathology , Vascular Endothelial Growth Factor A , Vascular Endothelial Growth Factors , Vitreous BodyABSTRACT
Osteopontin (OPN) is a secreted phosphoprotein shown to function in wound healing, inflammation, and tumor progression. Expression of OPN is often co-localized with members of the matrix metalloproteinase (MMP) family. We report that OPN is a novel substrate for two MMPs, MMP-3 (stromelysin-1) and MMP-7 (matrilysin). Three cleavage sites were identified for MMP-3 in human OPN, and two of those sites were also cleaved by MMP-7. These include hydrolysis of the human Gly166-Leu167, Ala201-Tyr202 (MMP-3 only), and Asp210-Leu211 peptide bonds. Only the N-terminal Gly-Leu cleavage site is conserved in rat OPN (Gly151-Leu152). These sites are distinct from previously reported cleavage sites in OPN for the proteases thrombin or enterokinase. We found evidence for the predicted MMP cleavage fragments of OPN in vitro in tumor cell lines, and in vivo in remodeling tissues such as the postpartum uterus, where OPN and MMPs are co-expressed. Furthermore, cleavage of OPN by MMP-3 or MMP-7 potentiated the function of OPN as an adhesive and migratory stimulus in vitro through cell surface integrins. We predict that interaction of MMPs with OPN at tumor and wound healing sites in vivo may be a mechanism of regulation of OPN bioactivity.
Subject(s)
Matrix Metalloproteinase 3/metabolism , Matrix Metalloproteinase 7/metabolism , Sialoglycoproteins/metabolism , Amino Acids/chemistry , Animals , Binding Sites , Blotting, Western , CHO Cells , Cell Adhesion , Cell Movement , Cricetinae , Dose-Response Relationship, Drug , Female , HeLa Cells , Humans , Immunoblotting , In Situ Hybridization , Integrins/metabolism , Matrix Metalloproteinase 3/chemistry , Matrix Metalloproteinase 7/chemistry , Models, Chemical , Osteopontin , Peptides/chemistry , Protein Binding , Rats , Recombinant Proteins/metabolism , Sialoglycoproteins/chemistry , Signal Transduction , Substrate Specificity , Uterus/metabolism , Wound HealingABSTRACT
Osteopontin (OPN), an extracellular matrix protein, is expressed in the myocardium with hypertrophy and failure. We tested the hypothesis that OPN plays a role in left ventricular (LV) remodeling after myocardial infarction (MI). Accordingly, OPN expression and LV structural and functional remodeling were determined in wild-type (WT) and OPN knockout (KO) mice 4 weeks after MI. Northern analysis showed increased OPN expression in the infarcted region, peaking 3 days after MI and gradually decreasing over the next 28 days. In the remote LV, OPN expression was biphasic, with peaks at 3 and 28 days. In situ hybridization and immunohistochemical analyses showed increased OPN mRNA and protein primarily in the interstitium. Infarct size, heart weight, and survival were similar in KO and WT mice after MI (P=NS), whereas the lung wet weight/dry weight ratio was increased in the KO mice (P<0.005 versus sham-operated mice). Peak LV developed pressure was reduced to a similar degree after MI in the KO and WT mice. The number of terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL)-positive myocytes was similar in KO and WT mice after MI. In contrast, post-MI LV chamber dilation was approximately twice as great in KO versus WT mice (P<0.001). Myocyte length increased after MI in WT mice (P<0.001) but not in KO mice. Electron microscopy showed increased collagen content in WT mice after MI but not in KO mice after MI. Type I collagen content was increased approximately 3-fold and approximately 7-fold in remote and infarcted regions, respectively, of WT hearts after MI but not in KO hearts (P<0.01 versus WT hearts). Likewise, Northern analyses showed increased collagen I(alpha(1)) mRNA after MI in remote regions of WT hearts but not in KO hearts. Thus, increased OPN expression plays an important role in regulating post-MI LV remodeling, at least in part, by promoting collagen synthesis and accumulation.