PubMed Health⌕ Search

Biomedical subjects

B F Flanagan

Publications and source records attributed to B F Flanagan.

34 records · Page 2Linked to original sources
PubMed1997

TNF-alpha and IL-8 are upregulated in the epidermis of normal human skin after UVB exposure: correlation with neutrophil accumulation and E-selectin expression.

The in vivo response to ultraviolet B (UVB) radiation in skin is characterized by the accumulation of both mononuclear and polymorphonuclear cells within the dermis and an induction of vascular endothelial adhesion molecules. Epidermal production of cytokines (IL-8 and TNF-alpha) has been strongly implicated in the development of UVB-induced inflammation. In the current study, we examined the time course of IL-8 and TNF-alpha mRNA and protein expression in the epidermis over a 24-h period after in vivo UVB irradiation. Also, the induction of adhesion molecule expression and the accumulation of neutrophils within the dermis were followed. We found constitutive expression of both cytokines (mRNA and protein) in the epidermis of unirradiated skin. IL-8 was rapidly upregulated after irradiation and mRNA and protein increased at 4 h, reaching a maximum between 8 and 24 h. TNF-alpha mRNA and protein was minimally increased by 8 h after UVB irradiation and reached a maximum by 24 h. No significant alteration in ICAM-1 or VCAM-1 expression was observed. E-selectin expression, which was absent from control samples, was increased from 4 h onward and also reached a maximum at 24 h, coinciding with peak neutrophil accumulation. A strong correlation (r = 0.96) was found between number of E-selectin-positive vessels and numbers of infiltrating neutrophils at this time. Moreover, because E-selectin expression was increased before any apparent increase in TNF-alpha protein (4 h), TNF-alpha does not appear to be involved in the early induction of the adhesion molecule, but cytokines such as TNF-alpha and IL-8 may act subsequently to augment the inflammatory response.

Adult↗
PubMed1996

Effect of biomaterial surface charge on the inflammatory response: evaluation of cellular infiltration and TNF alpha production.

A rat model was used to investigate the effect of net surface charge on polymer biocompatibility and its potential to modify and stimulate the inflammatory response. Poly(ether)urethane was taken as the base material and the net charge altered by introducing sulphonate ionic groups to the polymer backbone. Three differently charged poly(ether)urethanes were made with 10, 20, and 30% sulphonate substitution, giving a range of negative charge, with unmodified poly(ether)urethane used as a control. The polymers were implanted intramuscularly into rats for 2 days, and for 1, 2, and 12 weeks. After explantation, the cellular infiltration in the tissue surrounding the implants was evaluated using immunohistochemistry to stain for specific cell types: macrophages, neutrophils, lymphocytes, and the cytokine TNF alpha. In situ hybridization was used to detect expression of mRNA encoding TNF alpha. Stained sections were analyzed and the cellular response quantified using image analysis. Initially macrophages and neutrophils were observed around all the materials, but neutrophils were absent in all samples at 12 weeks. The 2-day time point had significantly more macrophages than the later time points. By 2 weeks the 20%-charged polymer elicited significantly less neutrophil infiltration than the other three polymers. In all samples where macrophages were observed, cells staining positive fore TNF alpha protein and message also were observed. No T or B lymphocytes were observed in the infiltrates around the materials at any time point. The results indicate that surface charge can influence the early phase acute inflammatory response to an implanted material.

Animals↗
PubMed1996

Detection of interleukin-8 mRNA and protein in human colorectal carcinoma cells.

Interleukin-8 (IL-8) is a member of the chemokine family of pro-inflammatory chemotactic cytokines and is secreted by some human colorectal carcinoma cell lines. We have used in situ hybridisation and immunohistochemistry to determine whether IL-8 mRNA and protein, respectively, are produced by human colorectal carcinoma cells in vivo. IL-8 mRNA was detected within the cytoplasm of tumour cells in all nine samples tested, including that of a tumour which had metastasised to a lymph node. Non-involved colonic mucosa within the same tissue blocks showed much weaker labelling. IL-8 protein was detected in 74% (23/31) of tumour samples and was mainly localised to the tumour cell cytoplasm. In 30% of cases, staining was heterogeneous, with between 1 and 30% of cells being positive. In some tumour cells, IL-8 showed a perinuclear distribution resembling that found by in situ hybridisation. Some infiltrating leucocytes, endothelial cells and fibroblast-like cells within the tumour sections were also positive for IL-8 mRNA and protein. The possibilities that colorectal tumours produce IL-8 to aid invasion and/or metastasis or as a tumour growth factor are discussed.

Adenocarcinoma↗
PubMed1995

Extensive TCR junctional diversity of V gamma 9/V delta 2 clones from human female reproductive tissues.

Panels of gamma delta T cell clones bearing the V gamma 9/V delta 2 form of TCR were derived from human first trimester decidualized endometrium and cervix. Seventy-three percent of these clones expressed the human mucosal lymphocyte Ag HML-1 compared with only 14% of PBL V gamma 9/V delta 2 clones, indicating that most clones were derived from the tissue itself rather than contaminating peripheral blood. All 13 clones isolated expressed V gamma 9JPC gamma 1- and V delta 2(D)J delta 1-encoded receptors; TCR gamma and delta junctional regions from most of these were sequenced and analyzed, together with the TCR-delta junctional region of a sequence obtained from bulk CD3+ decidual leukocytes. There was considerable junctional diversity of both gamma- and delta-chains with a similar extent of germline V and J gene trimming and N-region nucleotide addition to that found in PBL V gamma 9/V delta 2 cells. Eight of eleven TCR-delta junctional sequences contained a strongly hydrophobic amino acid in position 97, as has been found in > 90% o V gamma 9/V delta 2 clones. Thymic V gamma 9/V delta 2 cells show much less junctional diversity and less pronounced selection at residue 97 of the delta-chain. Thus, unlike the mouse, gamma delta T cells from human female reproductive tissues exhibit extensive TCR junctional as well as combinatorial diversity. This suggests that V gamma 9/V delta 2 cells in these human tissues have undergone selective but diverse peripheral expansion in response to antigenic stimuli in a similar manner to those in peripheral blood.

Amino Acid Sequence↗
PubMed1993

T cell receptor junctional regions of V gamma 9+/V delta 2+ T cell clones in relation to non-MHC restricted cytotoxic activity.

Human gamma delta T cell clones having V gamma 9JP and V delta 2DJ1 T cell receptor (TCR) gene rearrangements were isolated form an individual donor and tested for non-MHC restricted cytotoxicity against the B lymphoblastoid cell line, BSM. Most clones were highly cytotoxic but 3/9 clones had very low activity, comparable to that of CD4+ alpha beta T cell clones. Although there was a tendency for clones with low cytotoxic function to produce high levels of interferon-gamma and tumor necrosis factor-alpha, this correlation was not complete. TCR gamma and delta junctional sequences were obtained and were found to be different for all clones. There were no consistent structural differences between gamma delta TCRs of cytotoxic and non-cytotoxic clones, but gamma or delta junctional regions of all three non-cytotoxic clones had unusual features. One clone had a particularly short gamma chain junctional sequence, one had a short delta chain junctional sequence and the third clone was the only one of the panel which failed to utilise the D delta 3 segment. If the gamma delta TCR is involved in target cell recognition in this model of non-MHC restricted killing, such variations in receptor structure may be sufficient to inhibit recognition and thereby reduce the cytotoxic capacity of a minority of V gamma 9+/V delta 2+ clones. Also, a panel of gamma delta T cell clones expressing V gamma 8/V delta 3 isolated from a different donor, were all highly cytotoxic against BSM, indicating that these target cells can be recognised by effector cells expressing a TCR other than the V gamma 9/V delta 2 receptor. The possible influence of other cell surface molecules on non-MHC restricted cytotoxic function is discussed.

Amino Acid Sequence↗
PubMed1992

T-cell receptor Vbeta gene usage by lymphocytes infiltrating human renal allografts.

T cell lines have been derived from human kidney allograft biopsies using mitogenic stimulation. Southern blotting using a T-cell receptor (TCR) Cbeta probe revealed an oligoclonal pattern of rearranged bands in all 12 samples analysed. In some cases, differences in band patterns were noted between independent cultures from the same biopsy. Most T-cell clones derived from 2 biopsies showed different patterns of rearranged bands. The polymerase chain reaction (PCR) was used to study TCR Vbeta gene usage in allograft-derived T-cell cultures. This was more sensitive and more informative than Southern blotting and revealed that most TCR Vbeta genes were expressed in T cells from biopsies showing cellular rejection. The potential usefulness of this technique to quantify TCR V gene usage in allospecific T-cell populations is discussed.

DNA↗
PubMed1991

Polymorphism of the human CD46 gene in normal individuals and in recurrent spontaneous abortion.

Restriction fragment length polymorphism analysis of the human CD46 gene, which encodes a cell surface complement regulatory protein, has been performed using a 1.5-kb cDNA probe containing a complete CD46 coding sequence. The sum of the invariant band sizes indicates that this gene encompasses at least 35-kb genomic DNA. Polymorphisms were detected in normal individuals with both HindIII and EcoRI. Several of the polymorphic bands occurred significantly less frequently in recurrent spontaneous abortion individuals. Northern blot analysis has shown the predominant RNA transcript size to be 4.2 kb in trophoblast-derived cell lines and peripheral blood mononuclear cells, indicating these transcripts may contain an unusually long 3'-untranslated region.

Abortion, Habitual↗
PubMed1990

DNase hypersensitivity and methylation of the human CD3G and D genes during T-cell development.

The mouse and human CD3G and D genes are organized in opposite transcriptional orientation, their 5' ends being separated by about 1.6 kilobases (kb) of DNA. The molecular basis of the tissue-specific regulation of expression of the human CD3G and D genes were examined using DNase I hypersensitivity and CpG methylation analysis. Two T cell-specific DNase I hypersensitivity sites were defined within the intergenic region. A third hypersensitive site (DHS3) was detected 0.4 kb 3' to the CD3D gene. This latter site was present in all T cells, but was absent in all other committed cell types examined. DHS3 was also detected in the lympho-myeloid progenitor cell KG1, but was absent when this line was induced to differentiate to the macrophage lineage. The intergenic region was undermethylated in T cells expressing CD3, but was in general more extensively methylated in other cell types. Importantly, however, in KG1 sublines which express the CD3 genes the intergenic region remains extensively methylated. These results define areas 3' to the D gene and within the intergenic region which contain regulatory elements that influence both CD3D and G expression. They further show that transcription from the CD3D and G genes may occur initially from a methylated promoter. Significantly, the 3' regulatory region was shown to adopt an open chromatin structure prior to lineage commitment and before CD3 transcription.

Antigens, Differentiation, T-Lymphocyte↗
PubMed1989

Chromatin configuration of the human CD2 gene locus during T-cell development.

To investigate the molecular basis for the tissue-specific expression of the human CD2 gene, its chromatin configuration was assessed by determining DNase I hypersensitivity and the degree of methylation during T-cell lineage commitment and development. Tissue-specific DNase I-hypersensitive sites were found within the 5' promoter region and a region 3' of the gene essential for gene expression. DNase I hypersensitivity of the 5' region correlated strictly with transcriptional activity, whereas hypersensitivity of the 3' region correlated with T-cell progenitor activity or lineage commitment but not necessarily with transcription. Hha I and Hpa II sites around the 5' and 3' regions were undermethylated in CD2-expressing T cells but were more extensively methylated in other cell types. These results define likely regulatory elements both upstream and downstream of the CD2 gene that control its tissue-specific expression. Further, they show that the 3' regulatory region adopts an open chromatin configuration prior to lineage commitment and during early stages of T-cell development before the CD2 gene is transcribed.

Antigens, Differentiation, T-Lymphocyte↗
PubMed1989

Postnatal development and possible ligand function of the CNS-specific membrane glycoprotein CNSgp130.

CNSgp 130 is a CNS-specific membrane glycoprotein present in large amounts in the adult mammalian CNS. Using immunohistological techniques, we demonstrated that CNSgp130 is not detectable in the rat cerebellum at birth, and does not appear in the cerebellum until the tenth day of postnatal life. It is expressed first in the white matter of the cerebellar folia, and subsequently (by day 14) it is expressed also in the molecular layer. Expression in the granular layer is not seen until the 18th day of postnatal life, by which time the adult pattern of expression is established. CNSgp130 is also not detectable in the cerebrum at birth. However, it is expressed weakly but diffusely in the cerebrum by the fourth day of life. By the 10th day, there is strong expression in the cerebrum, in marked contrast to its virtual absence from the cerebellum at this stage. By quantitative absorption analysis, CNSgp 130 was undetectable on the day of birth, and increased steadily to 80% of adult values by the 22nd day of postnatal life. Binding studies with pure CNSgp130 demonstrated a Pronase-sensitive ligand in adult chicken brain. This ligand was absent from neonatal rat brain and non-CNS tissues.

Animals↗
PubMed1989

Chemical composition and tissue distribution of the human CDw44 glycoprotein.

The CDw44 glycoprotein was purified from 2.3 x 10(11) CD3+ CD4+ CD8- T-chronic lymphocytic leukaemia (CLL) cells using F10-44-2 monoclonal antibody affinity chromatography, DEAE-Sepharose anion-exchange chromatography, passage down carboxymethyl (CM)-Sepharose cation-exchange columns, wheat germ lectin affinity chromatography and gel-permeation chromatography. On elution in non-ionic detergents from the DEAE column, two distinct peaks of antigen activity were obtained. The CDw44 glycoprotein in each peak was a glycoprotein of 85,000 MW, but the amino acid composition of the peaks was noticeably different. Carbohydrate compositions showed that each peak contained approximately 30% (w/w) carbohydrate, the composition suggesting both O-linked and complex N-linked glycans. Modulation studies with the F10-44-2 antibody on normal peripheral blood mononuclear cells (PBMC) demonstrated that the CDw44 glycoprotein of T cells consisted of one fraction that was readily modulated, and the other which was resistant to modulation. Detailed tissue distribution studies for CDw44 were performed using the F10-44-2 antibody on frozen sections of human tissues. CDw44 has a restricted tissue distribution, but is found on many highly diverse cell types (e.g. T lymphocytes, smooth muscle cells, some secretory glands, skin epithelial cells).

Amino Acid Sequence↗
PubMed1986

Structural implications of the location and stability to proteolytic enzymes of immunodominant determinants of the human leukocyte common molecule.

In this report, we use 8 different mouse monoclonal antibodies to define 3 immunodominant determinants of the human leukocyte common (LC) antigen, and by defining the relative location and the stability to proteolytic enzymes of these determinants we make some structural predictions about the LC family of molecules. One lineage-restricted determinant is expressed predominantly on B lymphocytes and is located on the two higher (210 and 195) kDa bands of LC, with possibly some expression on the 180-kDa band. The two other determinants, one of which is a complex of 3 partially overlapping sites, are expressed on all leukocytes and found on all four (210, 195, 180, 160 kDa) LC bands. Pronase and trypsin treatment of peripheral blood lymphocytes and of Daudi cells gave complete cleavage of the lineage-restricted determinant from the cells, but the two common determinants were, surprisingly, left intact on the cell surface. By contrast, pronase and trypsin treatment of pure, detergent-solubilized LC resulted in rapid degradation of the common determinants, while the restricted determinant was sensitive to pronase but not to trypsin. Purification of the LC molecule from pronase-treated Daudi cells yielded a 130-kDa lentil lectin-binding protein, while undegraded LC from Daudi cells has a molecular mass of 210 kDa. Our results demonstrate that both common and restricted determinants are at least partly protein in nature and that the restricted determinant is external to and therefore on the presumed amino terminal side of the common determinants. Moreover, because LC has been reported to have a large (approximately 100 kDa) intracellular component, our data suggest the presence of a relatively small (approximately 25 kDa) membrane proximal domain containing both of the common LC determinants and at least one N-linked oligosaccharide chain. This domain is potentially susceptible to proteolytic cleavage, but it is interesting that on the intact cell it is both protected from proteolytic degradation and unable to be cleaved from the cell surface.

Antibodies, Monoclonal↗
PubMed1986

Unusual phylogenetic conservation of the N-terminal amino acid sequence of the central nervous system-specific membrane glycoprotein F3-87-8 (CNSgp130).

CNSgp130 is a CNS-specific membrane glycoprotein abundantly expressed throughout the mature mammalian CNS. The molecule is recognised by the mouse monoclonal antibody F3-87-8, which reacts with a determinant of CNSgp130 common to all mammals tested to date. Rat and human CNSgp130 were purified by a combination of F3-87-8 monoclonal antibody affinity and gel permeation chromatography, and the N-terminal amino acid sequence was determined by gas-phase sequencing techniques. The results show a remarkable conservation of the N terminus of the CNSgp130 polypeptide between rats and humans, with complete identity of the first 20 amino acid residues. There was an unusually high and phylogenetically conserved number of cysteines in this region. The sequence showed no homology to other known sequences and should prove useful in precisely identifying the relationship of CNSgp130 to other CNS membrane molecules.

Amino Acid Sequence↗
PubMed1982

Streptobacillus actinoides (Bacillus actinoides): isolation from pneumonic lungs of calves and pathogenicity studies in gnotobiotic calves.

Pneumonic lungs of 56 calves were examined and 12 (21 per cent) of them yielded Streptobacillus moniliformis-like organisms. These organisms resembled those previously described as Bacillus actinoides or Actinobacillus actinoides. After intratracheal inoculation of cultures of two strains of these organisms, pneumonic consolidation developed in five out of six gnotobiotic calves and involved up to 16 per cent of the lung surface. Histological lesions of interstitial pneumonia were observed in the lungs of all six calves. Swellings at the site of the infection followed intradermal and subcutaneous inoculation of cultures of all strains in calves. Mice showed no signs of illness following intraperitoneal injection of three stains. The bacteriological findings suggested that a more appropriate name for these organisms would be Streptobacillus actinoides.

Actinobacillus↗