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411.
RS Redman GS Kerr JB Payne TR Mikuls J Huang HR Sayles 《Biotechnic & histochemistry》2016,91(2):77-85
Serum procalcitonin (ProCT) is elevated in response to bacterial infections, whereas high sensitivity C-reactive protein (hsCRP) is a nonspecific inflammatory marker that is increased by excess adipose tissue. We examined the efficacy of ProCT and hsCRP as biomarkers of periodontitis in the saliva and serum of patients with arthritis, which is characterized by variable levels of systemic inflammation that potentially can confound the interpretation of inflammatory biomarkers. Blood and unstimulated whole saliva were collected from 33 patients with rheumatoid arthritis (RA) and 50 with osteoarthritis (OA). Periodontal status was assessed by full mouth examination and patients were categorized as having no/mild, moderate or severe periodontitis by standard parameters. Salivary and serum ProCT and hsCRP concentrations were compared. BMI, diabetes, anti-inflammatory medications and smoking status were ascertained from the patient records. Differences between OA and RA in proportionate numbers of patients were compared for race, gender, diabetes, adiposity and smoking status. Serum ProCT was significantly higher in arthritis patients with moderate to severe and severe periodontitis compared with no/mild periodontitis patients. There were no significant differences in salivary ProCT or salivary or serum hsCRP in RA patients related to periodontitis category. Most of the OA and RA patients were middle aged or older, 28.9% were diabetic, 78.3% were overweight or obese, and slightly more than half were either current or past smokers. The OA and RA groups differed by race, but not gender; blacks and males were predominant in both groups. The OA and RA groups did not differ in terms of controlled or uncontrolled diabetes, smoking status or BMI. The RA patients had been prescribed more anti-inflammatory medication than the OA patients. Our results demonstrate that circulating ProCT is a more discriminative biomarker for periodontitis than serum hsCRP in patients with underlying arthritis. Any elevation in salivary and serum hsCRP due to periodontitis apparently was overshadowed by differences among these patients in factors that influence CRP, such as the extent of inflammation between RA and OA, the extent of adipose tissue, the use of anti- inflammatory medications and smoking status. Although our study showed no differences in salivary ProCT related to severity of periodontitis, this biomarker also may be useful with further refinement. 相似文献
412.
mRNA-mediated gene delivery into human progenitor cells promotes highly efficient protein expression 总被引:1,自引:0,他引:1
Wiehe JM Ponsaerts P Rojewski MT Homann JM Greiner J Kronawitter D Schrezenmeier H Hombach V Wiesneth M Zimmermann O Torzewski J 《Journal of cellular and molecular medicine》2007,11(3):521-530
Gene transfer into human CD34+ haematopoietic progenitor cells (HPC) and multi-potent mesenchymal stromal cells (MSC) is an essential tool for numerous in vitro and in vivo applications including therapeutic strategies, such as tissue engineering and gene therapy. Virus based methods may be efficient, but bear risks like tumorigenesis and activation of immune responses. A safer alternative is non-viral gene transfer, which is considered to be less efficient and accomplished with high cell toxicity. The truncated low affinity nerve growth factor receptor (ALNGFR) is a marker gene approved for human in vivo application. Human CD34+ HPC and human MSC were transfected with in vitro-transcribed mRNA for DeltaLNGFR using the method of nucleofection. Transfection efficiency and cell viability were compared to plasmid-based nucleofection. Protein expression was assessed using flow cytometry over a time period of 10 days. Nucleofection of CD34+ HPC and MSC with mRNA resulted in significantly higher transfection efficiencies compared to plasmid transfection. Cell differentiation assays were performed after selecting DeltaLNGFR positive cells using a fluorescent activating cell sorter. Neither cell differentiation of MSC into chondrocytes, adipocytes and osteoblasts, nor differentiation of HPC into burst forming unit erythroid (BFU-E) colony forming unit-granulocyte, erythrocyte, macrophage and megakaryocyte (CFU-GEMM), and CFU-granulocyte-macrophage (GM) was reduced. mRNA based nucleofection is a powerful, highly efficient and non-toxic approach for transient labelling of human progenitor cells or, via transfection of selective proteins, for transient manipulation of stem cell function. It may be useful to transiently manipulate stem cell characteristics and thus combine principles of gene therapy and tissue engineering. 相似文献
413.
Sara S Roscioni Loes EM Kistemaker Mark H Menzen Carolina RS Elzinga Reinoud Gosens Andrew J Halayko Herman Meurs Martina Schmidt 《Respiratory research》2009,10(1):1-17
Background
Platelet-derived growth factor A (PDGF-A) signals solely through PDGF-Rα, and is required for fibroblast proliferation and transdifferentiation (fibroblast to myofibroblast conversion) during alveolar development, because pdgfa-null mice lack both myofibroblasts and alveoli. However, these PDGF-A-mediated mechanisms remain incompletely defined. At postnatal days 4 and 12 (P4 and P12), using mouse lung fibroblasts, we examined (a) how PDGF-Rα correlates with ki67 (proliferation marker) or alpha-smooth muscle actin (αSMA, myofibroblast marker) expression, and (b) whether PDGF-A directly affects αSMA or modifies stimulation by transforming growth factor beta (TGFβ).Methods
Using flow cytometry we examined PDGF-Rα, αSMA and Ki67 in mice which express green fluorescent protein (GFP) as a marker for PDGF-Rα expression. Using real-time RT-PCR we quantified αSMA mRNA in cultured Mlg neonatal mouse lung fibroblasts after treatment with PDGF-A, and/or TGFβ.Results
The intensity of GFP-fluorescence enabled us to distinguish three groups of fibroblasts which exhibited absent, lower, or higher levels of PDGF-Rα. At P4, more of the higher than lower PDGF-Rα + fibroblasts contained Ki67 (Ki67+), and Ki67+ fibroblasts predominated in the αSMA + but not the αSMA- population. By P12, Ki67+ fibroblasts comprised a minority in both the PDGF-Rα + and αSMA+ populations. At P4, most Ki67+ fibroblasts were PDGF-Rα + and αSMA- whereas at P12, most Ki67+ fibroblasts were PDGF-Rα- and αSMA-. More of the PDGF-Rα + than - fibroblasts contained αSMA at both P4 and P12. In the lung, proximate αSMA was more abundant around nuclei in cells expressing high than low levels of PDGF-Rα at both P4 and P12. Nuclear SMAD 2/3 declined from P4 to P12 in PDGF-Rα-, but not in PDGF-Rα + cells. In Mlg fibroblasts, αSMA mRNA increased after exposure to TGFβ, but declined after treatment with PDGF-A.Conclusion
During both septal eruption (P4) and elongation (P12), alveolar PDGF-Rα may enhance the propensity of fibroblasts to transdifferentiate rather than directly stimulate αSMA, which preferentially localizes to non-proliferating fibroblasts. In accordance, PDGF-Rα more dominantly influences fibroblast proliferation at P4 than at P12. In the lung, TGFβ may overshadow the antagonistic effects of PDGF-A/PDGF-Rα signaling, enhancing αSMA-abundance in PDGF-Rα-expressing fibroblasts. 相似文献414.
Inositol polyphosphatases (IPPases), particularly those that can hydrolyze myo-inositol hexakisphosphate (Ins P6), are of biotechnological interest for their ability to reduce the metabolically unavailable organic phosphate content of
feedstuffs and to produce lower inositol polyphosphates (IPPs) for research and pharmaceutical applications. Here, the gene
coding for a new protein tyrosine phosphatase (PTP)-like IPPase was cloned from Megasphaera elsdenii (phyAme), and the biochemical properties of the recombinant protein were determined. The deduced amino acid sequence of PhyAme is
similar to known PTP-like IPPases (29–44% identity), and the recombinant enzyme displayed strict specificity for IPP substrates.
Optimal IPPase activity was displayed at an ionic strength of 250 mM, a pH of 5.0, and a temperature of 60°C. In order to
elucidate its stereospecificity of Ins P6 dephosphorylation, a combination of high-performance ion-pair chromatography and kinetic studies was conducted. PhyAme displayed
a stereospecificity that is unique among enzymes belonging to this class in that it preferentially cleaved Ins P6 at one of two phosphate positions, 1D-3 or 1D-4. PhyAme followed two distinct and specific routes of hydrolysis, predominantly
degrading Ins P6 to Ins(2)P via: (a) 1D-Ins(1,2,4,5,6)P5, 1D-Ins(1,2,5,6)P4, 1D-Ins(1,2,6)P3, and 1D-Ins(1,2)P2 (60%) and (b) 1D-Ins(1,2,3,5,6)P5, 1D-Ins(1,2,3,6)P4, Ins(1,2,3)P3, and d/l-Ins(1,2)P2 (35%). 相似文献
415.
Wanfen Xiong Rebecca Knispel Jason MacTaggart Timothy C. Greiner Stephen J. Weiss B. Timothy Baxter 《The Journal of biological chemistry》2009,284(3):1765-1771
During arterial aneurysm formation, levels of the membrane-anchored matrix
metalloproteinase, MT1-MMP, are elevated dramatically. Although MT1-MMP is
expressed predominately by infiltrating macrophages, the roles played by the
proteinase in abdominal aortic aneurysm (AAA) formation in vivo
remain undefined. Using a newly developed chimeric mouse model of AAA, we now
demonstrate that macrophage-derived MT1-MMP plays a dominant role in disease
progression. In wild-type mice transplanted with MT1-MMP-null marrow, aneurysm
formation induced by the application of CaCl2 to the aortic surface
was almost completely ablated. Macrophage infiltration into the aortic media
was unaffected by MT1-MMP deletion, and AAA formation could be reconstituted
when MT1-MMP+/+ macrophages, but not MT1-MMP+/+
lymphocytes, were infused into MT1-MMP-null marrow recipients. In
vitro studies using macrophages isolated from either
WT/MT1-MMP-/- chimeric mice, MMP-2-null mice, or MMP-9-null mice
demonstrate that MT1-MMP alone plays a dominant role in macrophage-mediated
elastolysis. These studies demonstrate that destruction of the elastin fiber
network during AAA formation is dependent on macrophage-derived MT1-MMP, which
unexpectedly serves as a direct-acting regulator of macrophage proteolytic
activity.Development and progression of abdominal aortic aneurysm
(AAA)2 is a complex
process that, untreated, can lead to tissue failure, hemorrhage, and death
(1). Destruction of the orderly
elastin lamellae of the vessel wall is considered the sine qui non of arterial
aneurysm formation (2) as adult
tissues cannot regenerate normal elastin fibers
(3). Moreover, the elastin
degradation products are chemotactic for inflammatory cells and serve to
amplify the local injury (4).
Although several types of elastolytic proteases are elevated in AAA tissue
(5-9),
studies using murine models of AAA and targeted protease deletion suggest that
matrix metalloproteinases (MMPs), particularly the secreted proteases, MMP-2
and MMP-9, play key roles in the pathologic remodeling of the elastin lamellae
that lead to AAA (7,
8).Membrane-type 1 MMP (MT1-MMP) is the prototypical member of a family of
membrane-tethered MMPs (10).
Recent studies indicate that MT1-MMP expression is elevated in human AAA
tissues and that infiltrating macrophages are the primary source of the
proteinase in aortic lesions
(11-13).
Although indirect evidence suggests that MT1-MMP may participate in the
control of monocyte/macrophage motile responses as well as interactions with
the vessel wall during transmigration
(14,
15), the role(s) played by
MT1-MMP in regulating macrophage proteolytic activity or AAA formation in
vivo remains undefined.Using a murine model of AAA and mice with a targeted deletion of MT1-MMP in
myelogenous cell populations, we now demonstrate that macrophage-derived
MT1-MMP is required for elastin degradation and aneurysm formation.
Importantly, macrophages are not dependent on MT1-MMP for infiltrating aortic
tissues but instead use the protease to directly regulate their elastolytic
potential in an MMP-2- and MMP-9-independent fashion. These studies define a
new and unexpected role for MT1-MMP in controlling macrophage elastolytic
activity in the in vitro and in vivo settings. 相似文献
416.
Smita Jaiswal Todd Pearson Heather Friberg Leonard D. Shultz Dale L. Greiner Alan L. Rothman Anuja Mathew 《PloS one》2009,4(10)
Background
The lack of a suitable animal model to study viral and immunological mechanisms of human dengue disease has been a deterrent to dengue research.Methodology/Principal Findings
We sought to establish an animal model for dengue virus (DENV) infection and immunity using non-obese diabetic/severe combined immunodeficiency interleukin-2 receptor γ-chain knockout (NOD-scid IL2rγnull) mice engrafted with human hematopoietic stem cells. Human CD45+ cells in the bone marrow of engrafted mice were susceptible to in vitro infection using low passage clinical and established strains of DENV. Engrafted mice were infected with DENV type 2 by different routes and at multiple time points post infection, we detected DENV antigen and RNA in the sera, bone marrow, spleen and liver of infected engrafted mice. Anti-dengue IgM antibodies directed against the envelope protein of DENV peaked in the sera of mice at 1 week post infection. Human T cells that developed following engraftment of HLA-A2 transgenic NOD-scid IL2rγnull mice with HLA-A2+ human cord blood hematopoietic stem cells, were able to secrete IFN-γ, IL-2 and TNF-α in response to stimulation with three previously identified A2 restricted dengue peptides NS4b 2353(111–119), NS4b 2423(181–189), and NS4a 2148(56–64).Conclusions/Significance
This is the first study to demonstrate infection of human cells and functional DENV-specific T cell responses in DENV-infected humanized mice. Overall, these mice should be a valuable tool to study the role of prior immunity on subsequent DENV infections. 相似文献417.
418.
Repeated subcutaneous (s.c.) injections of recombinant granulocyte-macrophage colony-stimulating factor (recGM-CSF) for 4-5 days can enrich an immunization site with antigen-presenting cells (APC), which has been correlated with improved immune responses in experimental and clinical studies. A recombinant vaccinia virus encoding the GM-CSF gene (rV-GM-CSF) has been developed and can generate specific antitumour immunity in a whole tumour cell vaccine. In the present study, we examined whether rV-GM-CSF could produce and release GM-CSF locally which, in turn, might enrich a site of immunization for APC as previously shown for recGM-CSF. S.c. injection of rV-GM-CSF significantly (P<0.05) enhanced the percentage and overall number of APC, measured by class II expression levels, in the regional lymph nodes that drain the injection site. Dose- and temporal-dependent studies showed class II expression levels in the draining lymph nodes were maximally enhanced 5-7 days after a single injection of 10(7)plaque-forming units (pfu) of rV-GM-CSF. Flow cytometry revealed that the increase in class II expression resulted from (i) a higher class II expression level on CD19(+)B cells and (ii) an increase in the number of CD11c(+)/class II(+)professional APC within the draining lymph nodes. Moreover, isolation of lymph nodes from rV-GM-CSF-treated mice revealed their capacity to support higher levels of antigen-specific T cell proliferation and allospecific cytotoxic responses. A comparison between a single injection of rV-GM-CSF and a 4-day course of recGM-CSF revealed comparable changes in class II expression and functional T cell assays. GM-CSF can be delivered in a recombinant poxvirus, and the local production of the cytokine results in cellular and phenotypic changes that are similar to those of recGM-CSF. The ability to utilize rV-GM-CSF as a single inoculum may be more compatible with traditional immunization strategies. 相似文献
419.
E S Medlock I Goldschneider D L Greiner L Shultz 《Journal of immunology (Baltimore, Md. : 1950)》1987,138(11):3590-3597
We have presented evidence in a previous paper that the development of prothymocytes, pre-B cells, and TdT+ lymphoid precursor cells in the bone marrow of motheaten (me/me) and viable motheaten (mev/mev) mice is defective. In the present study, we have used a selective culture system that supports the generation of rat- and mouse-origin TdT+ bone marrow lymphoid cells in vitro to further investigate the early stages of lymphopoiesis in me/me and mev/mev mice. The results demonstrate that bone marrow stromal cell feeder layers derived from me/me and mev/mev mice do not support the growth of rat TdT+ cells in vitro, whereas stromal cell feeder layers from heterozygous (+/-) littermates and wild type (+/+) control mice do. Moreover, composite feeder layers formed by mixing as few as one part me/me and mev/mev bone marrow cells with 7 to 9 parts +/- littermate bone marrow cells also fail to effectively support the generation of TdT+ cells in vitro. In contrast to me/me and mev/mev mice, other mutant mouse models of autoimmune (NZB, NZB/W), immunodeficient (nu/nu), and hemopoietic (W/Wv, Sl/Sld) disorders form feeder layers that support normal to elevated levels of TdT+ cell growth in vitro. Thus, to date, only the me/me and mev/mev mutant mice have been found to lack the appropriate microenvironment for the generation of TdT+ bone marrow cells. Histologic analysis of the stromal cell feeder layers that are formed in our culture system shows that multilayered cellular patches, which normally are the most active sites of TdT+ cell development in vitro, are absent in feeder layers of me/me and mev/mev cells. Moreover, feeder layers from mev/mev mice contain a population of MAC 1+, basophilic, nonvacuolated, macrophage-like cells; whereas feeder layers from control mice contain MAC 1+, eosinophilic, vacuolated macrophage-like cells. Stromal cell feeder layers formed by mixtures of me/me or mev/mev and control mouse bone marrow cells contain numerous multilayered cellular patches and vacuolated mononuclear cells, but also contain large numbers of basophilic mononuclear cells. These composite feeder layers have a disproportionately reduced capacity to support the generation of TdT+ cells in vitro. Although the stromal microenvironment of me/me and mev/mev bone marrow does not support the growth of TdT+ cells in vivo or in vitro, the bone marrow from these mutant mice contains detectable numbers of pre-TdT+ cells. Thus, when cultured on normal mouse feeder layers, mutant mouse bone marrow rapidly generates TdT+ cells in vitro, albeit at significantly reduced levels as compared to +/- littermate controls.(ABSTRACT TRUNCATED AT 400 WORDS) 相似文献
420.
It has recently been demonstrated that aPY, a peptide which has significant homology with neuropeptide Y (NPY) is present in extracts of anglerfish islets. The purpose of this study was to determine whether cells or nerves which contain NPY-like immunoreactivity could be identified in anglerfish islet tissue and whether aPY is synthesized by this tissue. Antisera against bovine pancreatic polypeptide (BPP), NPY and the 200 kd neurofilament polypeptide were used for immunohistochemical analysis of islets. Identical cells were stained by both the NPY and BPP antisera. The NPY and 200 kd neurofilament antisera also labeled nerve fibers in the tissue which were not stained with the BPP antiserum. The nature of the NPY-like peptide synthesized in islet cells was determined by subjecting differentially radioactively labeled Mr 2,500-8,000 peptides from islet extracts to reverse phase HPLC. Labeled aPY was unequivocally identified in the extracts and was labeled appropriately (as predicted from its sequence) with 13 different radioactive amino acids. These results demonstrate that one form of NPY-like peptide synthesized in anglerfish islets is aPY. The form of NPY-like peptide which was immunolocalized in nerves remains to be determined. 相似文献