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Biomedical subjects

B F Flanagan

Publications and source records attributed to B F Flanagan.

At least 19 recordsLinked to original sources
PubMed2006

The complement regulatory proteins CD55 (decay accelerating factor) and CD59 are expressed on the inner acrosomal membrane of human spermatozoa as well as CD46 (membrane cofactor protein).

The complement regulatory proteins CD55 and CD59 are expressed on the plasma membrane of human spermatozoa, whereas CD46 is only on the inner acrosomal membrane (IAM) which becomes surfaced exposed after the acrosome reaction when sperm assume fertilisation-competence. CD55 & CD59, two glycosylphosphatidylinositol (GPI)-anchored proteins, have been detected previously in some studies also in the acrosomal region of chemically fixed spermatozoa but never demonstrated at this site on unfixed spermatozoa. Dual labelling immunofluorescence and confocal microscopy on fresh unfixed spermatozoa, with minimal subsequent time to fixation, has shown CD55 to be markedly expressed on the IAM, more than on the plasma membrane. However, unlike for CD46, CD55 displayed patchy staining over the acrosome, with some variation between individual spermatozoa. All IAM-associated CD55 was localised within GM1-containing lipid rafts. CD59 was expressed also on the IAM, but in a pronounced granular pattern with more variation observed from one spermatozoa to another. Both CD55 & CD59 were released from the IAM by PI-PLC, demonstrating them to be GPI-anchored. Analysis of acrosome-reacted spermatozoal CD55 by Western blotting revealed a novel single 55 kDa protein lacking significant oligosaccharides susceptible to glycosidases. Antibody-induced membrane rafting and release of CD55 & CD59 in vitro may have influenced previous results. Significant coexpression of CD55 & CD46 on the IAM suggests some functional cooperation at this site.

Acrosome↗
PubMed2005

Flow cytometric measurement of phagocytosis reveals a role for C3b in metal particle uptake by phagocytes.

A methodology for the quick and efficient study of phagocytosis has been developed. It uses the flow cytometer to exploit the change in size and granularity that occurs in cells upon the ingestion of particulate material. The numbers of cells that have phagocytosed particles can be calculated from the distinct shift in regions that occurs. The method also allows the factors governing phagocytosis to be studied in detail through the use of blocking agents or antibodies. Blood-derived monocytes were studied to investigate the role of complement in metal particle phagocytosis to further understand aseptic loosening. Factor C3b was found to be fundamental to the opsonization and phagocytosis of metal particles by monocytes.

Antibodies↗
PubMed2004

Differential regulation of mast cell cytokines by both dexamethasone and the p38 mitogen-activated protein kinase (MAPK) inhibitor SB203580.

Activated mast cells generate multiple cytokines but it is not known if these can be differentially regulated by pharmacological agents. We report here that the glucocorticoid dexamethasone (DEX) preferentially inhibited Ag-induced expression of IL-4 and IL-6 mRNA relative to TNF-alpha mRNA in RBL-2H3 cells. Likewise, the drug more readily inhibited release of IL-4 than TNF-alpha protein. SB203580, an inhibitor of p38 mitogen-activated protein kinase (MAPK), enhanced Ag-induced TNF-alpha mRNA expression without affecting IL-4 or IL-6 mRNA. At the protein level, SB203580 exerted little effect on TNF-alpha release but inhibited IL-4 release; notably, the ratio of TNF-alpha : IL-4 increased markedly with the concentration of SB203580, confirming the differential regulation of these cytokines. PD98059, an inhibitor of MAPK kinase (MEK), a component of the p44/42 MAPK pathway, partially inhibited Ag-induced expression of mRNA for all three cytokines while cyclosporin A inhibited Ag-induced IL-4 and IL-6 mRNA more readily than TNF-alpha mRNA. Ag activation of the cells led to phosphorylation of p38 and p44/42 MAPK but this was not influenced by DEX. In conclusion, mast cell cytokines can be differentially regulated pre- and post-translationally by DEX and SB203580 but there does not appear to be a direct mechanistic link between the actions of these two drugs.

Cells, Cultured↗
PubMed2004

Pro- and anti-inflammatory responses in respiratory syncytial virus bronchiolitis.

Respiratory syncytial virus (RSV) bronchiolitis is an important cause of severe respiratory disease in infants. This study aimed to characterise changes in pulmonary pro- and anti-inflammatory responses in infants with RSV bronchiolitis over the course of the illness. On the day of intubation (Day 1) and the day of extubation (Day X), nonbronchoscopic bronchoalveolar lavage was performed on term and preterm infants ventilated for RSV bronchiolitis and on control infants on Day 1. Tumour necrosis factor (TNF)-alpha, soluble TNF receptor (sTNFR) and interleukin (IL)-6 messenger ribonucleic acid (mRNA) and protein were measured. Twenty-four infants, born at term and 23 infants born preterm with RSV bronchiolitis and 10 controls were recruited. TNF-alpha and IL-6 mRNA and protein in infants with bronchiolitis were greater than the control group on Day 1. In preterm infants, who were ventilated for longer than term infants, TNF-alpha and IL-6 proteins decreased between Day 1 and Day X. Concentrations of sTNFRs differed between groups on Day 1, but levels did not change between Day 1 and Day X. Large amounts of tumour necrosis factor-alpha and interleukin-6 in the respiratory syncytial virus-infected lung suggest important roles for these cytokines in the pathogenesis of respiratory syncytial virus bronchiolitis. The decrease in tumour necrosis factor-alpha and interleukin-6 protein in preterm infants may reflect the prolonged clinical course seen in these infants.

Bronchiolitis, Viral↗
PubMed2002

A study of tissue interface membranes from revision accord knee arthroplasty: the role of T lymphocytes.

Despite four decades of advances in the design of orthopaedic devices aseptic loosening remains a major cause for the revision of total joint arthroplasty. This study used the techniques of immunohistochemistry and reverse transcription polymerase chain reaction to identify the inflammatory cell types, cytokines and chemokines within the interface tissue surrounding failed Accord Knee prostheses. Many T cells were identified within the tissue: however, the classical marker of activation, CD25 was expressed on very few cells. Molecular analysis failed to detect the synthesis of either Th1 or Th2 cytokines. These results suggest that the T cells are being actively recruited to the site of inflammation along the chemokine gradients but are not participating in a classical immune response.

Aged↗
PubMed2002

The ratio of 2nd to 4th digit length: a proxy for transactivation activity of the androgen receptor gene?

The androgen receptor gene (AR) contains a domain which includes a variable number of CAG sequences and alleles with low numbers of CAG repeats show high transactivation activity when complexed with testosterone. The ratio of 2nd and 4th digit length (2D:4D) is negatively correlated with phenotypic effects of testosterone. Low numbers of CAG repeats and low 2D:4D are both associated with high sperm numbers and protection against breast cancer. This suggests that CAG number and 2D:4D are correlated i.e. low CAG number and low 2D:4D indicate high activation of androgen-responsive genes. Findings from AR studies predict that low 2D:4D will be associated with prostate and hepatocellular cancer, urolithiasis, ADHD, ankylosing spondylitis, spontaneous abortion, and polycystic ovaries, while high 2D:4D will be associated with motor neuron diseases and endometrial cancer. Findings from 2D:4D studies predict that short CAG length will be common in autism and Asperger's syndrome, while high numbers of CAG repeats will be found in men who are prone to early myocardial infarction.

Breast Neoplasms↗
PubMed2001

Penicillin conjugates to interferon-gamma and reduces its activity: a novel drug-cytokine interaction.

Beta-lactam antibiotics are the class of drug most frequently associated with IgE-mediated allergy but the mechanisms underlying this response are poorly understood. IFN-gamma is a key cytokine in immunity with regulatory actions on monocytes, NK cells, epithelial cells, and T and B lymphocytes. IFN-gamma promotes Th1 responses and inhibits Th2- and IgE-mediated responses. In this study we show, by Western blotting, that the prototype beta-lactam benzylpenicillin (BP) conjugates to human IFN-gamma but not to IL-4. The interaction of BP with IFN-gamma inhibited the cytokine's detection by immunoassay and impaired its activity, as assessed in three different assays: upregulation of MHC molecules on monocytes plus induction of nitric oxide synthesis and expression of monocyte chemoattractant protein-1 mRNA by epithelial cells. This is the first reported example of a direct drug-cytokine interaction and suggests a mechanism by which penicillin may disrupt IFN-gamma-dependent immune responses and promote allergy.

Cell Line↗
PubMed1999

Eotaxin and eotaxin receptor (CCR3) expression in Sephadex particle-induced rat lung inflammation.

The beta chemokine eotaxin is a potent eosinophil activator and chemoattractant. We examined immunohistochemically eotaxin protein expression in a range of normal rat tissues and in rat lung during Sephadex particle-induced pulmonary inflammation. The time course of eotaxin expression in lung at various time points after Sephadex administration was related to the appearance of eosinophils in the bronchoalveolar lavage fluid and tissue distribution of eotaxin receptor (CCR3) positive cells. Results showed that eotaxin protein was constitutively expressed by both lung airway epithelial cells and gut epithelial cells in normal tissues in the absence of inflammation. During Sephadex induced pulmonary inflammation, eotaxin expression increased in alveolar macrophages prior to the major increase in eosinophil numbers which reached a peak at 72 h. The pattern of eotaxin pulmonary expression and the location of CCR3 receptor positive cells suggest a chemoattractant gradient resulting in migration firstly into the tissue and subsequently through the airway epithelium into the airways. Treatment of rats with the glucocorticoid dexamethasone or the immunosuppressant cyclosporin A reduced eosinophil entry into lung tissue and airways but had no apparent effect on eotaxin expression in vivo, indicating that both these drugs inhibit eosinophil recruitment either by an eotaxin-independent mechanism, or by targetting factors that synergise with eotaxin, or an event post eotaxin expression.

Animals↗
PubMed1999

Human decidualized endometrial T lymphocytes do not substantially down-regulate CD3zeta.

PROBLEM: The T-cell antigen receptor (TCR) has been reported to be down-regulated on T-cells in the decidualized endometrium in early pregnancy. METHOD OF STUDY: The expression of CD3zeta, a component of the TCR complex, has been investigated in human first-trimester decidual T-cells using flow cytometric analysis of permeabilized cells. RESULTS: Levels of CD3zeta expression were significantly lower in decidual than in peripheral T-cells from non-pregnant women, as assessed by mean fluorescence intensity (4.2 vs. 5.5, logarithmic scale, P < 0.05). However, when decidual and peripheral T-cells from the same subjects were analyzed (n = 10), mean levels of CD3zeta were slightly, but not significantly, lower in decidual than in peripheral T-cells (P > 0.1). CD3zeta was not substantially down-regulated systemically as mean cytoplasmic CD3zeta levels did not differ significantly between peripheral blood T-cells from pregnant women and non-pregnant controls (P > 0.2). CD8+ cells outnumber CD4+ cells in decidua, but neither the proportions of these two T-cell subsets positive for cytoplasmic CD3zeta nor the mean levels of CD3zeta were significantly different. CONCLUSIONS: These results indicate that human decidual T-cells do not greatly down-regulate CD3zeta, but it is unclear if a small decrease in mean levels may be sufficient to compromise their capacity for activation.

CD3 Complex↗
PubMed1998

A novel isoform of human membrane cofactor protein (CD46) mRNA generated by intron retention.

The reverse transcription polymerase chain reaction (RT-PCR) with primers specific for each of the 14 exons of the human complement regulatory protein membrane cofactor protein (MCP;CD46) has been utilized to determine MCP mRNA transcript expression in peripheral blood mononuclear cells (PBMC). An additional transcript of a larger size than predicted was consistently detected in reactions with a sense primer for exon 7, that encodes the first alternatively spliced serine-threonine-rich region (ST-A), together with an antisense exon 12 primer, RT-PCR with primers for other exons both 5' and 3' of exon 7 further showed that these MCP transcripts contain additional sequences immediately both 5' and 3' to the exon 7-encoded sequence. Comparison of genomic DNA with cDNA by PCR, in combination with sequence analysis, demonstrated the presence of the complete invariant sequences of both introns adjacent to exon 7, i.e. intron 6 (411 bp) and intron 7 (127 bp). RT-PCR using primers specific for the intron 6 sequence, together with Southern and Northern blotting using an intron 6-specific probe, confirmed retention of this intron within a novel 4.8-kb mRNA transcript in human PBMC. Due to the presence of a stop codon within intron 6, translation would result in a novel truncated MCP isoform (MCPi) containing the four invariant short consensus repeat (SCR) regions and a unique C-terminal 39 amino acid transmembrane and cytoplasmic tail region that may promote endoplasmic reticulum retention.

Alternative Splicing↗
PubMed1998

Human eotaxin induces alpha 4 and beta 2 integrin-dependent eosinophil accumulation in rat skin in vivo: delayed generation of eotaxin in response to IL-4.

The CC chemokine eotaxin, originally purified from bronchoalveolar lavage fluid of sensitized guinea pigs following allergen challenge, is a potent eosinophil-selective chemoattractant. In the present study, we have used (111)In-eosinophils and human eotaxin to characterize the profile of chemokine-induced eosinophil accumulation in vivo in rat skin. Intradermally injected eotaxin caused a dose-dependent accumulation of (111)In-eosinophils. Time course studies indicated that the response was rapid, since all the accumulation occurred within the first 1 to 2 h of eotaxin injection. The i.v. administration of anti-intercellular adhesion molecule-1, anti-vascular cell adhesion molecule-1, or anti-alpha4 integrin mAbs significantly inhibited the eosinophil accumulation induced by 100 pmol of human eotaxin by 73, 43, and 67%, respectively. Further, when (111)In-eosinophils were pretreated in vitro with anti-alpha4 integrin or anti-beta2 integrin mAbs, or with a combination of both mAbs, eotaxin-induced responses in vivo were reduced by 52, 49, and 68%, respectively. Eosinophil accumulation induced by intradermal IL-4, but not that induced by TNF-alpha or leukotriene B4, appeared to be mediated in part by endogenously generated eotaxin. Anti-eotaxin Abs significantly inhibited (54%) the later phases (24-28 h) but not the early phase (0-4 h) of the response to IL-4. This was consistent with eotaxin mRNA expression peaking at 18 h after IL-4 injection. Our findings show that human eotaxin is a potent inducer of eosinophil accumulation in vivo, this response being dependent on alpha4 integrin/vascular cell adhesion molecule-1 and beta2 integrin/intercellular adhesion molecule-1 adhesion pathways. Further, the eosinophil accumulation in response to IL-4 is partly mediated by endogenously generated eotaxin.

Animals↗
PubMed1998

TNFB gene polymorphism in insulin-dependent diabetes mellitus: association with HLA-DR alleles.

A polymorphism of the TNF-beta gene can be detected by restriction digestion of a PCR product with NcoI. In this study we look at the risk associated with this polymorphism in a study of 69 insulin-dependent diabetes patients and 119 healthy controls. The risk was further characterized by comparison to the HLA type of the individual, since the TNF polymorphism is in linkage disequilibrium with HLA genes.

Alleles↗
PubMed1997

Techniques to investigate cellular and molecular interactions in the host response to implanted biomaterials.

Evaluation of the host response to implanted materials requires systematic, objective investigations of responses at both the cellular and molecular levels. This article explains the basis behind two technologies: antibody and molecular techniques, which will give valuable information when applied to investigations of cells and molecules involved in the host biomaterial interaction. Such investigations are well underway, and a number of groups are now studying well characterised cell markers or molecules to evaluate the host response to biomaterials. Here we outline current technologies for the development of antibodies as tools to study cell markers or molecules, including those for which reagents are not yet available and DNA based technologies, whose continued application should prove an invaluable adjunct to existing approaches. These technologies may be particularly valuable to investigations focusing on newly characterised cytokines, receptors or cell adhesion molecules and subsequently provide a way forward for the production of advanced biomaterials.

Animals↗
PubMed1997

Tumour necrosis factor-alpha expression and cell recruitment in Sephadex particle-induced lung inflammation: effects of dexamethasone and cyclosporin A.

1. Tumour necrosis factor-alpha (TNF-alpha) is a cytokine with diverse properties consistent with a possible role in inflammatory disease. We investigated whether TNF-alpha is induced during the progression of lung inflammation elicited by a particulate non-antigenic stimulus, and whether pharmacological control of TNF-alpha expression influences recruitment of specific inflammatory cell types. 2. A single intravenous injection of Sephadex particles into rats led to extensive granulomatous inflammation in lung alveolar and bronchial tissue that peaked in intensity after 24-72 h. Mononuclear cells were the principal component of granulomas, but neutrophils and eosinophils were also abundant. Numbers of mononuclear cells, neutrophils and eosinophils recovered by bronchoalveolar lavage (BAL) peaked at 72 h, 48 h and 72 h, respectively. 3. Messenger RNA encoding TNF-alpha was induced in lung epithelial cells, lung granulomas and BAL cells 6 h after Sephadex administration and remained elevated for 72 h before declining to baseline by 7 days. In BAL cell populations TNF-alpha protein was localized to mononuclear cells at all times points pre- and post-Sephadex administration. 4. Treatment of rats with dexamethasone significantly reduced the Sephadex-induced recruitment of mononuclear cells, neutrophils and eosinophils into the bronchoalveolar cavity, and significantly reduced TNF-alpha mRNA expression by BAL cells. 5. Treatment of rats with cyclosporin A was without effect on Sephadex-induced elevations of mononuclear cell numbers and expression of TNF-alpha, but did reduce significantly recruitment of neutrophils and eosinophils to BAL cell populations. 6. These results show that a sequential asthma-like recruitment of neutrophils, eosinophils and mononuclear cells into lung tissue can be induced by single exposure to a non-antigenic stimulus. Pharmacological and histological studies reveal that mononuclear cell mobilization relates closely to induced TNF-alpha expression, whereas mobilization of neutrophils and eosinophils appears secondary to expression of the cytokine.

Animals↗
PubMed1997

Localization of IL-4 and IL-4 receptors in the human term placenta, decidua and amniochorionic membranes.

There has been much recent interest in cytokine expression at the materno-fetal interface. Although T-helper 2 (Th2)-type cytokines have been described in the murine feto-placental unit, few studies have as yet been performed in human pregnancy. We have examined the production of interleukin-4 (IL-4) and expression of IL-4 receptors in the human term placenta, decidua and amniochorionic membranes. Immunohistochemical analyses revealed that cytotrophoblast, decidual macrophages and both maternal and fetal endothelial cells consistently expressed IL-4, whereas syncytiotrophoblast and placental macrophages showed an inconsistent pattern between specimens. High- and low-affinity IL-4 receptors were demonstrated by immunohistochemistry at the same cellular sites as stained for IL-4, and detection of IL-4 receptors was also variable in syncytiotrophoblast. Reverse-transcribed-polymerase chain reaction (RT-PCR) analysis showed that both IL-4 and its alternative splice variant, IL-482, are produced both in placental villi and in amniochorionic and decidual tissue. Ligand-binding assays identified the presence, on isolated term syncytiotrophoblast microvillous plasma membrane vesicle preparations, of functional high-affinity binding sites for IL-4 with a Kd in the range 102-112 pM and an apparent receptor density in the range 99-102 x 10(8) sites/mg protein. Three human choriocarcinoma (BeWo, JEG-3 and Jar) and one amnion-derived (AV3) cell lines expressed IL-4 and both high- and low-affinity IL-4 receptors. The constitutive expression of both IL-4 and IL-4 receptors, together with the novel finding of the alternative splice variant IL-482 in the immediate tissues at the materno fetal interface suggest an immunobiological role for IL-4 in human pregnancy.

Antigens, CD↗