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1.
Neuropathol Appl Neurobiol ; 32(3): 351-6, 2006 Jun.
Article in English | MEDLINE | ID: mdl-16640654

ABSTRACT

Immunohistochemistry for alpha-synuclein has become the histological technique of choice for the diagnosis for Parkinson's disease, Dementia with Lewy bodies and Multiple System Atrophy (http://www.ICDNS.org). Nevertheless, no standardised protocol has been proposed. We have reviewed 242 of the 270 studies published until June 2005 that mentioned immunohistochemistry for anti-alpha synuclein on human tissue and we found that only 75 (31%) used commercial antibodies. We also noted that protocols, particularly dilution and antigen unmasking, varied between studies, even when the same antibody was employed. In order to establish a standardised protocol for alpha-synuclein immunohistochemistry, which can be applied in diagnostic neuropathology we tested seven commercial monoclonal antibodies in brains of subjects with Parkinson's disease, dementia with Lewy bodies, multiple system atrophy, multiple sclerosis with incidental Lewy bodies and aged-matched normal brain and determined for each antibody the best suited protocol for antigen unmasking. We evaluated the intensity of immunolabelling in Lewy bodies, neuropil threads, dendrites, pre-synaptic terminals, granular cytoplasmic positivity, peri-axonal positivity, glial inclusions and non-specific immunolabelling. Although our results showed that all the antibodies detected alpha-synuclein inclusions, differences were noted between antibodies, particularly with regard to the detection of glial inclusions. From our study, the best antibodies of the seven tested appeared to be those directed against amino acids 116-131 and 15-123 and we suggest them to be used in routine diagnostic practice for alpha-synucleinopathies.


Subject(s)
Antibodies, Monoclonal , Immunohistochemistry/standards , alpha-Synuclein/metabolism , Brain/pathology , Humans , alpha-Synuclein/immunology
2.
Neuromuscul Disord ; 12(2): 183-6, 2002 Feb.
Article in English | MEDLINE | ID: mdl-11738361

ABSTRACT

We have previously shown that galectin-1 is a factor capable of converting mouse dermal fibroblasts to the myogenic lineage [Cell Transplant 2000;9:519]. Here, we report that human dermal fibroblasts are also capable of expressing the myogenic marker, desmin, when grown in muscle-cell-conditioned media. Furthermore, the human foetal skin cells also express this marker when grown in the presence of galectin-1. These results highlight the importance of galectin-1 in the conversion of both human and murine skin cells to a myogenic lineage. Thus galectin-1 could be an important tool for use in autologous cell therapies for the treatment of human muscular dystrophies.


Subject(s)
Adjuvants, Immunologic/pharmacology , Desmin/metabolism , Fibroblasts/metabolism , Hemagglutinins/pharmacology , Skin/metabolism , Animals , Biomarkers , Cells, Cultured , Culture Media, Conditioned , Desmin/analysis , Fetus , Fibroblasts/cytology , Fibroblasts/drug effects , Galectin 1 , Gestational Age , Humans , Mice , Skin/drug effects
3.
Cell Transplant ; 9(4): 519-29, 2000.
Article in English | MEDLINE | ID: mdl-11038068

ABSTRACT

Using the mdx mouse model for human Duchenne muscular dystrophy we have shown that a cell population residing in the dermis of C57B1/10ScSn mouse skin is capable of converting to a myogenic lineage when implanted into the mdx muscle environment. It was important to determine the characteristics of the converting cell. A previous in vitro study indicated that 10% of cells underwent conversion but only when the cells were grown in medium previously harvested from a myogenic culture. In the present study we cloned cells derived from the dermis to identify the converting cells. Clones grown in normal growth medium showed no conversion, but when grown in medium conditioned by muscle cells around 40% conversion was achieved in several individual clones. We investigated whether the protein beta-galactoside binding protein (betaGBP), which is secreted by myoblasts and acts as a cell growth regulator of fibroblasts. could be a candidate factor responsible for conversion. Medium harvested from COS-1 cells infected with a construct containing betaGBP has been used for this investigation. Growth of dermal fibroblasts in medium enriched with this factor showed a high rate of conversion to cells expressing muscle-specific factors.


Subject(s)
Cell Differentiation , Dermis/cytology , Desmin/metabolism , Fibroblasts/cytology , Hemagglutinins/pharmacology , Muscles/cytology , Animals , COS Cells , Cells, Cultured , Clone Cells , Culture Media, Conditioned , Electrophoresis, Agar Gel , Electrophoresis, Polyacrylamide Gel , Fibroblasts/metabolism , Galectins , Genes, Reporter , Hemagglutinins/genetics , Humans , Mice , Mice, Inbred mdx , Muscles/metabolism , Muscular Dystrophy, Animal , Muscular Dystrophy, Duchenne , Plasmids , Transfection
4.
Eur Respir J ; 13(3): 660-7, 1999 Mar.
Article in English | MEDLINE | ID: mdl-10232443

ABSTRACT

The effects of the mucolytic agent, dithioerythritol (DTE), and the temperature at which sputum processing is conducted on cellular and biochemical markers in induced sputum was assessed. Samples from healthy and atopic asthmatic subjects were treated with either DTE or phosphate-buffered saline (PBS) at 22 or 37 degrees C and compared for cell counts and concentrations of histamine, tryptase, eosinophil cationic protein (ECP), free interleukin (IL)-8, immunoglobulin (Ig)A, IL-8/IgA complexes and secretory component (SC). In addition, the influence of DTE on in vitro mediator release from blood eosinophils, basophils and bronchoalveolar lavage (BAL) mast cells was studied. Processing with DTE improved cytospin quality and increased the cell yield and measurable ECP, tryptase, IgA and SC, but reduced levels of histamine in PBS-treated samples and had no effect on IL-8. Cell counts or mediator levels were similar when sputum was processed at 22 or 37 degrees C, even though DTE induced blood basophils and BAL mast cells to release histamine at 37 degrees C. In spiking experiments, recovery of added ECP, tryptase, total IL-8 and histamine from sputum was similar in DTE- and PBS-processed sputum, but reduced for free IL-8 in PBS-treated samples. In conclusion, dithioerythritol improves cell and mediator recovery without causing cell activation when sputum processing is conducted at room temperature. The extent of recovery depends on the mediator studied.


Subject(s)
Asthma/diagnosis , Dithioerythritol , Ribonucleases , Sputum/chemistry , Sputum/cytology , Basophils/chemistry , Biomarkers/analysis , Blood Proteins/analysis , Bronchial Provocation Tests , Bronchoalveolar Lavage Fluid/chemistry , Bronchoalveolar Lavage Fluid/cytology , Cell Count , Electrolytes/analysis , Eosinophil Granule Proteins , Eosinophils/chemistry , Eosinophils/cytology , Female , Histamine/analysis , Humans , Inflammation Mediators/analysis , Interleukins/analysis , Male , Mast Cells/chemistry , Osmolar Concentration , Prognosis , Reference Values , Sensitivity and Specificity , Severity of Illness Index , Temperature
6.
Am J Respir Cell Mol Biol ; 14(1): 95-103, 1996 Jan.
Article in English | MEDLINE | ID: mdl-8534491

ABSTRACT

The interaction of cells with the extracellular matrix can alter cell responses and is regulated by integrins on the cell surface. We used monoclonal antibodies to the VLA-4 integrins CD29 and CD49d followed by an F(ab')2 fragment of rabbit anti-mouse immunoglobulin G1 to crosslink integrins on the surface of human lung mast cells and basophils. Crosslinking either CD29 or CD49d caused a significant histamine release (HR) from the basophils of most asthmatic donors (10 of 14 for CD49d and 7 of 10 for CD29) (HR = 21 +/- 5%, n = 10, P < 0.005 for CD29 and HR = 19 +/- 4%, n = 14, P < 0.01 for CD49d) yet failed to initiate HR from the basophils of non-atopic and atopic donors (HR was 1 +/- 0.5% for CD29 and 1 +/- 0.5% for CD49d, n = 10, P = NS). Crosslinking either CD29 or CD49d also failed to initiate histamine release from human lung mast cells (HR was 1 +/- 1% for CD29 and 2 +/- 1% for CD49d). The basophils of asthmatic donors responded to 100 and 30 micrograms/ml tissue fibronectin (HR = 12 +/- 2% and 10 +/- 3% for 100 and 30 micrograms/ml fibronectin, respectively, n = 18, P < 0.05), whereas basophils of nonasthmatic patients again failed to degranulate (HR was 0 +/- 0.4% and 1 +/- 0.6%, respectively, n = 11, P = NS). In contrast to the basophil, crosslinking of either CD29 or CD49d failed to initiate histamine release in human lung mast cells (HR = 1 +/- 1% for CD29 and 2 +/- 1%, n = 15). Human lung mast cells were also unresponsive to tissue fibronectin (100 and 30 micrograms/ml) (HR = 1 +/- 1%, n = 5). The tyrosine kinase inhibitor, genistein, significantly reduced CD29- and CD49d-induced HR (inhibition = 83 +/- 7% for CD29 and 77 +/- 6% for CD49d, n > or = 5, P < 0.05). A second tyrosine kinase inhibitor, piceatannol, also significantly reduced both CD29- and CD49d-induced HR (inhibition was 62 +/- 19% for CD29 and 56 +/- 14% for CD49d, n = 7, P < or = 0.05). Integrin crosslinking also affected the response to a second, immunoglobulin E (IgE)-dependent stimulus. Both CD29 and CD49d clustering significantly inhibited anti-IgE-induced histamine release from the human basophil. Inhibition was 30 +/- 5%, n = 18, P < or = 0.001 for CD29 versus 40 +/- 6% for CD49d. In summary, we have shown that crosslinking the beta 1 integrins using either monoclonal antibodies or tissue fibronectin can initiate mediator release from the basophils of asthmatic patients by a mechanism which appears to be tyrosine kinase-mediated. In addition, clustering of integrins modulates the response to a second IgE-dependent signal.


Subject(s)
Basophils/physiology , Integrins/chemistry , Integrins/physiology , Lung/cytology , Mast Cells/physiology , Antibodies, Monoclonal , Antigens, CD/chemistry , Antigens, CD/immunology , Antigens, CD/physiology , Cell Degranulation/drug effects , Cross-Linking Reagents , Enzyme Inhibitors/pharmacology , Fibronectins/pharmacology , Genistein , Histamine Release/drug effects , Humans , Immunoglobulin E/pharmacology , Integrin alpha4 , Integrin beta1/chemistry , Integrin beta1/immunology , Integrin beta1/physiology , Isoflavones/pharmacology , Protein-Tyrosine Kinases/antagonists & inhibitors , Stilbenes/pharmacology
7.
Int Arch Allergy Immunol ; 107(1-3): 151-3, 1995.
Article in English | MEDLINE | ID: mdl-7542062

ABSTRACT

The interaction of cells with surfaces or components of the extracellular matrix alters cell responses and is regulated by integrins on the cell surface. We have used monoclonal antibodies to CD29 and CD49d followed by an F(ab)2 fragment of rabbit anti-mouse IgG1 to cross-link the integrins on the surface of human lung mast cells and basophils. We found that cross-linking either CD29 or CD49d failed to initiate mediator release from the basophils of non-atopic and atopic donors [histamine release (HR) = 1 +/- 0.5% for CD29 and 1 +/- 0.5% for CD49d, n = 10, NS]. In contrast we found that clustering CD29 caused significant HR from the basophils of asthmatic donors (HR = 21 +/- 5%, n = 10, p < 0.005). Clustering of CD49d also caused significant degranulation in the same donors (HR = 9 +/- 3%, n = 10, p < 0.11). Incubating the basophils of these asthmatic donors with a synthetic RGD peptide significantly reduced CD29- and CD49d-induced histamine release. CS-1 peptide was also found to inhibit CD29-induced histamine release but had no significant effect on CD49d-induced histamine release. The tyrosine kinase inhibitors, genistein and piceatannol, completely ablated CD29- and CD49d-induced degranulation. In summary, we have shown that cross-linking integrins can initiate mediator release from the basophils of asthmatic patients and that this appears to involve recognition of RGD and activation of tyrosine kinase.


Subject(s)
Antigens, CD/physiology , Basophils/metabolism , Integrins/physiology , Lung/cytology , Mast Cells/metabolism , Antibodies, Monoclonal/immunology , Antibodies, Monoclonal/pharmacology , Antigens, CD/immunology , Asthma/immunology , Asthma/pathology , Carrier Proteins/pharmacology , Fibronectins/metabolism , Genistein , Histamine Release/drug effects , Histamine Release/physiology , Humans , Integrin alpha4 , Integrin beta1 , Integrins/immunology , Isoflavones/pharmacology , Oligopeptides/pharmacology , Protein-Tyrosine Kinases/antagonists & inhibitors , Receptor Aggregation/drug effects , Receptor Aggregation/physiology , Stilbenes/pharmacology
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