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1.
J Intern Med ; 280(2): 177-202, 2016 Aug.
Article in English | MEDLINE | ID: mdl-27165517

ABSTRACT

There are more than 30 human proteins whose aggregation appears to cause degenerative maladies referred to as amyloid diseases or amyloidoses. These disorders are named after the characteristic cross-ß-sheet amyloid fibrils that accumulate systemically or are localized to specific organs. In most cases, current treatment is limited to symptomatic approaches and thus disease-modifying therapies are needed. Alzheimer's disease is a neurodegenerative disorder with extracellular amyloid ß-peptide (Aß) fibrils and intracellular tau neurofibrillary tangles as pathological hallmarks. Numerous clinical trials have been conducted with passive and active immunotherapy, and small molecules to inhibit Aß formation and aggregation or to enhance Aß clearance; so far such clinical trials have been unsuccessful. Novel strategies are therefore required and here we will discuss the possibility of utilizing the chaperone BRICHOS to prevent Aß aggregation and toxicity. Type 2 diabetes mellitus is symptomatically treated with insulin. However, the underlying pathology is linked to the aggregation and progressive accumulation of islet amyloid polypeptide as fibrils and oligomers, which are cytotoxic. Several compounds have been shown to inhibit islet amyloid aggregation and cytotoxicity in vitro. Future animal studies and clinical trials have to be conducted to determine their efficacy in vivo. The transthyretin (TTR) amyloidoses are a group of systemic degenerative diseases compromising multiple organ systems, caused by TTR aggregation. Liver transplantation decreases the generation of misfolded TTR and improves the quality of life for a subgroup of this patient population. Compounds that stabilize the natively folded, nonamyloidogenic, tetrameric conformation of TTR have been developed and the drug tafamidis is available as a promising treatment.


Subject(s)
Amyloidosis/therapy , Alzheimer Disease/therapy , Amyloid/physiology , Amyloidosis/physiopathology , Animals , Diabetes Mellitus, Type 2/therapy , Humans , Liver Transplantation , Prealbumin/physiology
2.
Oncogene ; 27(18): 2525-31, 2008 Apr 17.
Article in English | MEDLINE | ID: mdl-17982484

ABSTRACT

Deregulated Her2/ErbB2 receptor tyrosine kinase drives tumorigenesis and tumor progression in a variety of human tissues. Her2 transmits oncogenic signals through phosphorylation of its cytosolic domain. To study innate cellular mechanisms for containing Her2 oncogenic phosphorylation, a siRNA phosphatase library was screened for cellular phosphatase(s) that enhance phosphorylation in the signaling motif of Her2 after knockdown. We found that silencing protein tyrosine phosphatase PTPN13 significantly augmented growth factor-induced phosphorylation of the Her2 signaling domain and promoted the invasiveness of Her2-deregulated cancer cells. In addition, we discovered that growth factor-induced phosphorylation of PTPN13 was essential for the dephosphorylation of Her2 suggesting a negative feedback mechanism induced by growth factor to inhibit cellular Her2 activity through PTPN13. Importantly, we showed that PTPN13 mutations previously reported in human tumors significantly reduced the phosphatase activity of PTPN13, and consequently elevated the oncogenic potential of Her2 and the invasiveness of Her2-overexpressing human cancer cells. Taken together, these results suggest that cellular PTPN13 inhibits Her2 activity by dephosphorylating the signal domain of Her2 and plays a role in attenuating invasiveness and metastasis of Her2 overactive tumors.


Subject(s)
Cell Transformation, Neoplastic/metabolism , Protein Tyrosine Phosphatase, Non-Receptor Type 13/metabolism , Receptor, ErbB-2/metabolism , Signal Transduction , Cell Transformation, Neoplastic/genetics , Cell Transformation, Neoplastic/pathology , HeLa Cells , Humans , Mutation , Neoplasm Invasiveness , Neoplasm Metastasis , Phosphorylation , Protein Structure, Tertiary/genetics , Protein Tyrosine Phosphatase, Non-Receptor Type 13/antagonists & inhibitors , Protein Tyrosine Phosphatase, Non-Receptor Type 13/genetics , RNA, Small Interfering/genetics , Receptor, ErbB-2/genetics , Signal Transduction/genetics
3.
Am J Pathol ; 157(6): 1849-62, 2000 Dec.
Article in English | MEDLINE | ID: mdl-11106558

ABSTRACT

Functional inhibition of tissue factor (TF) has been shown to improve coronary blood flow after myocardial ischemia/reperfusion (I/R) injury. TF initiates the coagulation protease cascade, resulting in the generation of the serine protease thrombin and fibrin deposition. Thrombin can also contribute to an inflammatory response by activating various cell types, including vascular endothelial cells. We used a rabbit coronary ligation model to investigate the role of TF in acute myocardial I/R injury. At-risk areas of myocardium showed increased TF expression in the sarcolemma of cardiomyocytes, which was associated with a low level of extravascular fibrin deposition. Functional inhibition of TF activity with an anti-rabbit TF monoclonal antibody administered either 15 minutes before or 30 minutes after coronary ligation reduced infarct size by 61% (P = 0.004) and 44% (P = 0.014), respectively. Similarly, we found that inhibition of thrombin with hirudin reduced infarct size by 59% (P = 0.014). In contrast, defibrinogenating the rabbits with ancrod had no effect on infarct size, suggesting that fibrin deposition does not significantly contribute to infarct size. Functional inhibition of thrombin reduced chemokine expression and inhibition of either TF or thrombin reduced leukocyte infiltration. We propose that cardiomyocyte TF initiates extravascular thrombin generation, which enhances inflammation and injury during myocardial I/R.


Subject(s)
Myocardial Infarction/etiology , Myocardial Infarction/pathology , Myocardial Ischemia/complications , Myocardial Reperfusion Injury/complications , Myocarditis/etiology , Thrombin/physiology , Thromboplastin/physiology , Animals , Antibodies, Monoclonal/pharmacology , Antithrombins/pharmacology , Cell Movement/physiology , Chemokines/antagonists & inhibitors , Fibrin/metabolism , Fibrinogen/metabolism , Hirudins/pharmacology , Microscopy, Electron , Myocardial Ischemia/metabolism , Myocardial Ischemia/pathology , Myocardial Reperfusion Injury/metabolism , Myocardial Reperfusion Injury/pathology , Myocarditis/prevention & control , Myocardium/pathology , Neutrophils/physiology , Rabbits , Thrombin/antagonists & inhibitors , Thromboplastin/antagonists & inhibitors , Thromboplastin/immunology
4.
Mol Cell Biol ; 20(13): 4543-52, 2000 Jul.
Article in English | MEDLINE | ID: mdl-10848581

ABSTRACT

The p38 group of kinases belongs to the mitogen-activated protein (MAP) kinase superfamily with structural and functional characteristics distinguishable from those of the ERK, JNK (SAPK), and BMK (ERK5) kinases. Although there is a high degree of similarity among members of the p38 group in terms of structure and activation, each member appears to have a unique function. Here we show that activation of p38gamma (also known as ERK6 or SAPK3), but not the other p38 isoforms, is required for gamma-irradiation-induced G(2) arrest. Activation of the MKK6-p38gamma cascade is sufficient to induce G(2) arrest in cells, and expression of dominant negative alleles of MKK6 or p38gamma allows cells to escape the DNA damage-induce G(2) delay. Activation of p38gamma is dependent on ATM and leads to activation of Cds1 (also known as Chk2). These data suggest a model in which activation of ATM by gamma irradiation leads to the activation of MKK6, p38gamma, and Cds1 and that activation of both MKK6 and p38gamma is essential for the proper regulation of the G(2) checkpoint in mammalian cells.


Subject(s)
Calcium-Calmodulin-Dependent Protein Kinases/metabolism , Calcium-Calmodulin-Dependent Protein Kinases/radiation effects , Cell Cycle/radiation effects , Mitogen-Activated Protein Kinases/metabolism , Ataxia Telangiectasia Mutated Proteins , CDC2 Protein Kinase/drug effects , CDC2 Protein Kinase/metabolism , Calcium-Calmodulin-Dependent Protein Kinases/genetics , Cell Cycle Proteins/metabolism , Cell Cycle Proteins/radiation effects , Cell Death/radiation effects , Checkpoint Kinase 2 , DNA Damage/radiation effects , DNA-Binding Proteins , Enzyme Inhibitors/pharmacology , Fibroblasts/cytology , Fibroblasts/radiation effects , G2 Phase/radiation effects , Gamma Rays , HeLa Cells/radiation effects , Humans , Imidazoles/pharmacology , Isoenzymes , MAP Kinase Kinase 1 , MAP Kinase Kinase 5 , MAP Kinase Kinase 6 , MAP Kinase Kinase 7 , Mitogen-Activated Protein Kinase 7 , Mitogen-Activated Protein Kinase Kinases/genetics , Mitogen-Activated Protein Kinase Kinases/metabolism , Mitogen-Activated Protein Kinase Kinases/radiation effects , Mitogen-Activated Protein Kinases/genetics , Mitogen-Activated Protein Kinases/radiation effects , Phosphorylation , Protein Kinases/metabolism , Protein Serine-Threonine Kinases/genetics , Protein Serine-Threonine Kinases/metabolism , Protein Serine-Threonine Kinases/radiation effects , Pyridines/pharmacology , Signal Transduction , Tumor Suppressor Proteins , cdc25 Phosphatases/metabolism , cdc25 Phosphatases/radiation effects , p38 Mitogen-Activated Protein Kinases
5.
J Leukoc Biol ; 67(5): 705-11, 2000 May.
Article in English | MEDLINE | ID: mdl-10811012

ABSTRACT

The p38 family of mitogen-activated protein kinases is believed to mediate a variety of leukocyte responses to pro-inflammatory stimuli. There are four members of the p38 family, and although activation of the different members has been studied in transiently transfected cells much less is known about activation of the endogenous p38s, particularly in myeloid lineage cells. To investigate activation of endogenous p38s, we have made monoclonal antibodies specific for each p38 and have used these antibodies to study p38 activation by pro-inflammatory stimuli in several human monocytic cell lines. Without stimulation endogenous p38alpha kinase activity was readily detectable, whereas that of p38beta, gamma, and delta was barely measurable. In response to inflammatory stimuli, we observed a time- and dose-dependent activation of all four p38s. The kinetics of activation of each of the p38s were similar for each stimulus used, suggesting a common upstream activation pathway. Simultaneous activation of the p38s suggests that all four may be important in inflammation.


Subject(s)
Mitogen-Activated Protein Kinases/metabolism , Antibodies, Monoclonal/pharmacology , Astrocytoma , Cell Line , Enzyme Activation , Escherichia coli , Humans , Inflammation , Interferon-gamma/pharmacology , Kinetics , Lipopolysaccharides/pharmacology , Mitogen-Activated Protein Kinase 12 , Recombinant Proteins/metabolism , Tetradecanoylphorbol Acetate/pharmacology , Transfection , Tumor Cells, Cultured , U937 Cells , p38 Mitogen-Activated Protein Kinases
6.
Proc Natl Acad Sci U S A ; 96(14): 8138-43, 1999 Jul 06.
Article in English | MEDLINE | ID: mdl-10393961

ABSTRACT

We employed a novel mouse line that expresses low levels of human tissue factor (TF) in the absence of murine TF to analyze the role of TF in gestation. Low-TF female mice had a 14-18% incidence of fatal postpartum uterine hemorrhage, suggesting that TF plays an important role in uterine hemostasis. Low-TF female mice mated with low-TF male mice had a 42% incidence of fatal midgestational hemorrhage (n = 41), whereas no fatal midgestational hemorrhages were observed in low-TF female mice mated with wild-type male mice (n = 43). Placentas of low-TF embryos from both low-TF and normal (+/-) TF females were abnormal and contained numerous maternal blood pools in the labyrinth. Placentas of TF null embryos surviving beyond embryonic day 10.5 exhibited similar defects. The mouse maternal-embryonic placental barrier consists of four cellular layers (layers I, II, and III and endothelial cells), where layer I lines the maternal lacunae. Comparison of TF-deficient placentas with control placentas by immunohistochemical and ultrastructural analyses revealed thinning of layer I and a reduction in the number of cellular contacts of layer I trophoblasts spanning the maternal blood space between adjacent trabeculae. These structural changes in low-TF and TF null placentas result in enlarged maternal lacunae, as determined by morphometric analysis, and placental hemorrhage, which leads to midgestational death of low-TF female mice. This study demonstrated that TF is required for uterine hemostasis and revealed an unexpected role of TF in the maintenance of the placental labyrinth.


Subject(s)
Placenta/physiology , Thromboplastin/physiology , Uterine Hemorrhage/physiopathology , Uterus/physiology , Animals , Crosses, Genetic , Female , Hemostasis , Humans , Male , Mice , Mice, Knockout , Placenta/abnormalities , Placenta/blood supply , Pregnancy , Puerperal Disorders/genetics , Puerperal Disorders/pathology , Puerperal Disorders/physiopathology , Thromboplastin/deficiency , Thromboplastin/genetics , Uterine Hemorrhage/genetics , Uterine Hemorrhage/pathology , Uterus/abnormalities , Uterus/blood supply
7.
Infect Immun ; 67(5): 2540-6, 1999 May.
Article in English | MEDLINE | ID: mdl-10225918

ABSTRACT

Tissue factor (TF) is the major activator of the coagulation protease cascade and contributes to lethality in sepsis. Despite several studies analyzing TF expression in animal models of endotoxemia, there remains debate about the cell types that are induced to express TF in different tissues. In this study, we performed a detailed analysis of the induction of TF mRNA and protein expression in two rabbit models of endotoxemia to better understand the cell types that may contribute to local fibrin deposition and disseminated intravascular coagulation. Northern blot analysis demonstrated that lipopolysaccharide (LPS) increased TF expression in the brain, lung, and kidney. In situ hybridization showed that TF mRNA expression was increased in cells identified morphologically as epithelial cells in the lung and as astrocytes in the brain. In the kidney, in situ hybridization experiments and immunohistochemical analysis showed that TF mRNA and protein expression was increased in renal glomeruli and induced in tubular epithelium. Dual staining for TF and vWF failed to demonstrate TF expression in endothelial cells in LPS-treated animals. These results demonstrate that TF expression is induced in many different cell types in LPS-treated rabbits, which may contribute to local fibrin deposition and tissue injury during endotoxemia.


Subject(s)
Lipopolysaccharides/toxicity , Thromboplastin/biosynthesis , Thromboplastin/genetics , Animals , Brain/metabolism , Disease Models, Animal , Endotoxemia/etiology , Endotoxemia/genetics , Endotoxemia/metabolism , Gene Expression/drug effects , Immunohistochemistry , In Situ Hybridization , Kidney/metabolism , Lung/metabolism , RNA, Messenger/genetics , RNA, Messenger/metabolism , Rabbits , Tissue Distribution
8.
J Immunol ; 162(8): 4762-72, 1999 Apr 15.
Article in English | MEDLINE | ID: mdl-10202018

ABSTRACT

Murine experimental meningitis models induced by either Escherichia coli LPS, live Streptococcus pneumoniae, or Listeria monocytogenes were used to study the origin and potential function of soluble CD14 (sCD14) in the brain during bacterial meningitis. Whereas intracerebral infection caused only a minor and/or transient increase of sCD14 levels in the serum, dramatically elevated concentrations of sCD14 were detected in the cerebrospinal fluid. Reverse-transcriptase PCR and FACS analysis of the leukocytes invading the subarachnoid compartment revealed an active amplification of CD14 transcription and concomitant surface expression. These findings were confirmed by in situ hybridization and immunohistochemical analysis. In contrast, parenchymal astrocytes and microglial cells were shown not to significantly contribute to the elevated levels of sCD14. Simultaneous intracerebral inoculation of rsCD14 and S. pneumoniae resulted in a markedly increased local cytokine response. Taken together, these data provide the first evidence that sCD14 can act as an inflammatory co-ligand in vivo. Thus, during bacterial meningitis, sCD14 is massively released by intrathecal leukocytes, and the sCD14 found in the cerebrospinal fluid can play an important role in the pathogenesis of this disease.


Subject(s)
Lipopolysaccharide Receptors/physiology , Meningitis, Bacterial/immunology , Animals , Astrocytes/metabolism , Brain/immunology , Brain/metabolism , Child , Disease Models, Animal , Escherichia coli Infections/blood , Escherichia coli Infections/cerebrospinal fluid , Escherichia coli Infections/immunology , Female , Humans , Injections, Subcutaneous , Leukocytes/immunology , Leukocytes/metabolism , Lipopolysaccharide Receptors/blood , Lipopolysaccharide Receptors/cerebrospinal fluid , Lipopolysaccharide Receptors/genetics , Listeriosis/blood , Listeriosis/cerebrospinal fluid , Listeriosis/immunology , Meningitis, Bacterial/blood , Meningitis, Bacterial/cerebrospinal fluid , Mice , Mice, Inbred C57BL , Microglia/metabolism , Pneumococcal Infections/blood , Pneumococcal Infections/cerebrospinal fluid , Pneumococcal Infections/immunology , RNA, Messenger/biosynthesis , Recombinant Proteins/administration & dosage , Solubility
9.
Blood ; 92(8): 2791-801, 1998 Oct 15.
Article in English | MEDLINE | ID: mdl-9763564

ABSTRACT

von Willebrand factor (vWF) is frequently used as a biochemical marker for endothelial cells (ECs). Despite this, little is known about the relative level of expression and regulation of this hemostatic factor in ECs in different vascular beds in vivo. In the present study, we used quantitative reverse transcription polymerase chain reaction and in situ hybridization analysis to study vWF gene expression in murine tissues. Large differences in the level of vWF mRNA were observed when comparing highly vascularized tissues, with the lung and brain containing 5 to 50 times higher concentrations of vWF mRNA than the kidney and liver. In this regard, ECs of small vessels and some microvessels in the lung and brain expressed abundant vWF mRNA, whereas ECs of similar sized vessels in the liver and kidney expressed relatively low levels. In general, significantly higher levels of vWF mRNA and antigen were demonstrated in ECs of larger vessels compared with microvessels and in venous ECs compared with arterial ECs. Although intraperitoneal administration of endotoxin (or tumor necrosis factor-alpha) increased plasma vWF levels, it had variable effects on the steady-state level of vWF mRNA in murine tissues (ie, it decreased vWF mRNA in many tissues, increased it in others, and had little effect on still others). These results indicate that vWF is differentially expressed and regulated in ECs present in different tissues and within the same vascular bed.


Subject(s)
Endothelium, Vascular/metabolism , Gene Expression Regulation , von Willebrand Factor/genetics , Animals , Brain/blood supply , Endotoxemia/metabolism , Hemostasis , In Situ Hybridization , Kidney/blood supply , Lipopolysaccharides/pharmacology , Liver/blood supply , Lung/blood supply , Lymphoid Tissue/blood supply , Male , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Organ Specificity , RNA, Messenger/biosynthesis , Reverse Transcriptase Polymerase Chain Reaction , Tumor Necrosis Factor-alpha/pharmacology , Tumor Necrosis Factor-alpha/physiology , von Willebrand Factor/biosynthesis
10.
Shock ; 9(3): 157-63, 1998 Mar.
Article in English | MEDLINE | ID: mdl-9525321

ABSTRACT

Recombinant murine interleukin-1beta (IL-1beta) induced a transient increase in plasma levels of CD14 with a peak at 8 h, and this increase in plasma CD14 antigen was accompanied by increased levels of CD14 messenger ribonucleic acid (mRNA) in all organs examined. In most organs, maximal levels of induction were obtained after administration of 125 ng of IL-1beta. Moreover, in situ hybridization studies revealed that CD14 mRNA was induced in both myeloid cells and epithelial cells. Pretreatment of mice with anti-lL-1beta antibodies reduced the subsequent induction of plasma levels of CD14 by lipopolysaccharide (LPS) and significantly reduced the level of induction of CD14 mRNA in kidney and liver. The antibodies did not block LPS mediated induction in lung. Pretreatment with a combination of anti-lL-1beta and anti-tumor necrosis factor (TNF) antibodies was more effective in reducing LPS mediated induction of plasma CD14 and CD14 mRNA in liver than pretreatment with either antibody alone. The combination of anti-lL-1beta and anti-TNF antibodies had no additional effect in kidney and lung over that observed with anti-TNF alone. These studies demonstrate that regulation of CD14 gene expression by LPS in vivo involves multiple signals but is mediated, in part, by the cytokines IL-1beta and TNF-alpha.


Subject(s)
Interleukin-1/pharmacology , Lipopolysaccharide Receptors/drug effects , Lipopolysaccharide Receptors/genetics , Animals , Antibodies/pharmacology , Dose-Response Relationship, Drug , Epithelium/immunology , Female , Gene Expression Regulation/drug effects , Immune Sera/pharmacology , Interleukin-1/immunology , Lipopolysaccharide Receptors/metabolism , Lipopolysaccharides/pharmacology , Mice , Mice, Inbred Strains , RNA, Messenger , Recombinant Proteins/pharmacology , Tumor Necrosis Factor-alpha/immunology
11.
Infect Immun ; 65(11): 4822-31, 1997 Nov.
Article in English | MEDLINE | ID: mdl-9353071

ABSTRACT

We previously demonstrated CD14 gene expression in myeloid and epithelial cells of the mouse and showed that expression of the CD14 gene in both is modulated by lipopolysaccharide (LPS). Here we test the hypothesis that the induction of CD14 in these cells is an indirect effect of LPS, one mediated by tumor necrosis factor alpha (TNF-alpha). TNF-alpha induced a transient increase in levels of CD14 in plasma with a peak at 6 to 8 h, and this increase in levels of CD14 antigen in plasma was accompanied by increased levels of CD14 mRNA in lung, liver, and kidney. Moreover, in situ hybridization studies revealed that CD14 mRNA was induced in both myeloid cells and epithelial cells, the same cells that respond to LPS. Pretreatment of mice with anti-TNF antiserum reduced the LPS-mediated increase in levels of CD14 in plasma and significantly reduced the level of induction of CD14 mRNA in selected epithelial cells in the kidney and liver. The antiserum did not appear to block LPS-mediated induction in myeloid cells in the tissues examined. In C3H/HeJ mice, the epithelial response to LPS was markedly attenuated whereas the response to TNF-alpha was normal. Thus, regulation of CD14 gene expression by LPS differs in epithelial and myeloid cells, with the epithelial responses in kidney and liver being mediated, in part, by TNF-alpha.


Subject(s)
Lipopolysaccharide Receptors/genetics , Lipopolysaccharides/pharmacology , Tumor Necrosis Factor-alpha/physiology , Animals , Female , Gene Expression/drug effects , Immune Sera/immunology , Lipopolysaccharide Receptors/blood , Mice , Mice, Inbred BALB C , Mice, Inbred C3H , Mice, Inbred C57BL , RNA, Messenger/analysis
12.
Am J Pathol ; 150(2): 579-90, 1997 Feb.
Article in English | MEDLINE | ID: mdl-9033272

ABSTRACT

We previously demonstrated that lipopolysaccharide (LPS) induces plasminogen activator inhibitor 1 (PAI-1) gene expression primarily in endothelial cells in most organs of the mouse, with maximal induction by 3 hours. Here we show that induction in the liver occurs in a distinctly different pattern. For example, the increase in PAI-1 mRNA in liver was biphasic with an initial peak at 1 to 2 hours and a second peak at 6 to 8 hours. Moreover, in situ hybridization experiments revealed that PAI-1 mRNA was induced in both endothelial cells and hepatocytes. The endothelial cell response was monophasic and maximal between 1 and 4 hours, whereas the hepatocyte response was biphasic, peaking at 2 hours and again at 6 to 8 hours. To determine possible mechanisms involved in the induction of PAI-1 by LPS, we analyzed the tissues for changes in tumor necrosis factor (TNF)-alpha LPS caused a rapid induction of TNF-alpha mRNA in Kupffer cells, detectable within 15 minutes. Pretreatment of mice with anti-TNF antiserum before challenge with LPS reduced the subsequent increase in plasma levels of PAI-1 by 50 to 70% and significantly reduced the level of induction of PAI-1 mRNA in the liver at both early and late times. Pretreatment appeared to inhibit induction primarily within hepatocytes. These results suggest that LPS may induce PAI-1 in endothelial cells and hepatocytes by different mechanisms.


Subject(s)
Gene Expression Regulation , Lipopolysaccharides/pharmacology , Liver/drug effects , Liver/physiology , Plasminogen Activator Inhibitor 1/genetics , Tumor Necrosis Factor-alpha/physiology , Animals , Female , Liver/cytology , Mice , Mice, Inbred Strains , RNA, Messenger/metabolism , Tissue Distribution
13.
J Exp Med ; 181(3): 857-66, 1995 Mar 01.
Article in English | MEDLINE | ID: mdl-7532683

ABSTRACT

A murine model system was used to study the distribution and regulation of CD14 gene expression in vivo. Western blot analysis failed to detect CD14 in plasma from untreated CB6 (BALB/c x C57Bl6) mice, but showed markedly increased levels of CD14 in plasma from mice treated with lipopolysaccharide (LPS). Plasma levels of CD14 increased in a time- and dose-dependent manner, reaching a maximum between 8 and 16 h. Northern blot analysis of total RNA extracted from mouse tissues revealed low, but significant, levels of CD14 mRNA in many tissues of untreated animals with the highest levels in uterus, adipose tissue, and lung. After intraperitoneal injection of LPS, induction of CD14 gene expression was detected in all organs examined with the extent of induction varying between organs. Induction of CD14 mRNA was both time and dose dependent. Maximum induction in the heart and lung was observed 2-4 h after injection of LPS, while liver and kidney showed maximal induction between 8 and 16 h. In situ hybridization showed that CD14 mRNA was expressed in myeloid cells in many tissues, and that expression in these cells was upregulated by LPS. Unexpectedly, CD14 mRNA was also detected in other cells within tissues, including epithelial cells, and expression in these cell types also was upregulated by LPS. Immunochemical analysis revealed that CD14 antigen colocalized to the cytoplasm of cells expressing CD14 mRNA. These studies demonstrate that CD14 gene expression is not restricted to myeloid cells, and that the level of expression of CD14 is influenced by exposure to LPS.


Subject(s)
Antigens, CD/genetics , Antigens, Differentiation, Myelomonocytic/genetics , Gene Expression Regulation/drug effects , Lipopolysaccharides/pharmacology , Animals , Antigens, CD/biosynthesis , Antigens, CD/blood , Antigens, Differentiation, Myelomonocytic/biosynthesis , Antigens, Differentiation, Myelomonocytic/blood , Lipopolysaccharide Receptors , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , RNA, Messenger/analysis
14.
Br J Cancer ; 65(4): 487-90, 1992 Apr.
Article in English | MEDLINE | ID: mdl-1562456

ABSTRACT

When human melanoma cells are injected into nude mice they usually give rise to tumours that grow progressively and do not elicit a prominent host response. We have recently developed a melanoma cell line, UCT-Mel 7, that did not show these characteristics. In the first place UCT-Mel 7 showed a consistently unusual, phasic growth pattern. After a short initial period of limited growth (phase 1), the tumour ceased growing and remained static for 2-3 months (phase 2). The tumour then regressed (phase 3) to enter a second period of quiescence (phase 4) which was eventually broken by the emergence of a rapidly growing lethal tumour (phase 5). Of particular interest was the fact that the rate at which the tumours grew correlated closely with their collagen content. During the prolonged, phase 2 plateau, the tumours were intensely desmoplastic; rapidly growing phase 5 tumours, that had escaped from dormancy, contained very little collagen and virtually no reticulin. This cell line helps to fill an important need for an experimental system for the study of desmoplasia, dormancy and progression.


Subject(s)
Melanoma/pathology , Animals , Collagen/metabolism , Histocytochemistry , Humans , Mice , Mice, Nude , Neoplasm Transplantation , Reticulin/metabolism , Tumor Cells, Cultured
15.
Int J Cancer ; 50(4): 621-7, 1992 Feb 20.
Article in English | MEDLINE | ID: mdl-1537627

ABSTRACT

When cells from the human malignant melanoma cell line, UCT-Mel 7, were injected into athymic nude mice, tumours developed that were intensely infiltrated with fibrous tissue. In an attempt to reproduce this desmoplastic response in vitro, we co-cultured fibroblasts with UCT-Mel-7 cells, and observed a 2-fold increase in the rate of fibroblast collagen synthesis. This was associated with an increase in the amount of collagen mRNA present in co-cultured fibroblasts. The stimulation was both dose- and time-dependent, with maximal stimulation at a melanoma cell:fibroblast ratio of 1:1. Analysis of the kinetics of proline incorporation into collagen showed that co-culture affected the maximal rate of proline incorporation; no effect was observed on the concentration of proline required for 50% maximal collagen synthesis. The induction of fibroblast collagen synthesis showed an absolute requirement for close proximity between the fibroblasts and the melanoma cells. No soluble fibrogenic factor released by melanoma was detected.


Subject(s)
Collagen/biosynthesis , Fibrosis/metabolism , Melanoma/pathology , Animals , Blotting, Northern , Fibroblasts/metabolism , Fibrosis/pathology , Gene Expression , Humans , In Vitro Techniques , Mannosephosphates/pharmacology , Mice , Mice, Nude , Neoplasm Transplantation , Protease Inhibitors/pharmacology , RNA, Messenger/genetics , Tumor Cells, Cultured
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