Biomedical subjects

L Bingle

Publications and source records attributed to L Bingle.

14 recordsLinked to original sources

PubMed2006

Macrophages promote angiogenesis in human breast tumour spheroids in vivo.

An in vivo model has been established to study the role of macrophages in the initiation of angiogenesis by human breast tumour spheroids in vivo. The extent of the angiogenic response induced by T47D spheroids implanted into the dorsal skinfold chamber in nude mice was measured in vivo and compared to that induced by spheroids infiltrated with human macrophages prior to implantation. Our results indicate that the presence of macrophages in spheroids resulted in at least a three-fold upregulation in the release of vascular endothelial growth factor (VEGF) in vitro when compared with spheroids composed only of tumour cells. The angiogenic response measured around the spheroids, 3 days after in vivo implantation, was significantly greater in the spheroids infiltrated with macrophages. The number of vessels increased (macrophages vs no macrophages 34+/-1.9 vs 26+/-2.5, P<0.01), were shorter in length (macrophages vs no macrophages 116+/-4.92 vs 136+/-6.52, P<0.008) with an increased number of junctions (macrophages vs no macrophages 14+/-0.93 vs 11+/-1.25, P<0.025) all parameters indicative of new vessel formation. This is the first study to demonstrate a role for macrophages in the initiation of tumour angiogenesis in vivo.

Animals↗

PubMed2005

Design and validation of anti-inflammatory peptides from human parotid secretory protein.

Parotid secretory protein (PSP) and palate-lung-nasal epithelium clone (PLUNC) are novel secretory proteins that are expressed in the oral cavity and upper airways. Both proteins are related to bactericidal/permeability increasing protein (BPI). Cationic peptides derived from BPI exhibit anti-inflammatory activity. To test if PSP (C20orf70 gene product) also contains anti-inflammatory peptides, we designed 3 cationic peptides based on the predicted structure of PSP and known active regions of BPI. Each peptide inhibited the lipopolysaccharide (LPS)-stimulated secretion of TNFalpha from RAW 264.7 macrophage cells. At 200 microg/mL, the peptide GK-7 exhibited inhibition similar to that achieved with 10 microg/mL of polymyxin B. PSP peptides directly inhibited the binding of LPS to LPS-binding protein. The cationic peptide Substance P had no inhibitory effect in these assays, confirming the specificity of the PSP peptides. These findings suggest that PSP peptides can serve as templates for the design of novel anti-inflammatory peptides.

Amino Acid Sequence↗

PubMed2004

Cloning and expression of a mouse member of the PLUNC protein family exclusively expressed in tongue epithelium.

Palate, lung, and nasal epithelium clone (Plunc, now renamed Splunc1) is a small secreted protein expressed in the oropharynx and upper airways of humans, mice, rats, and cows. This protein is structurally homologous to known mediators of host defense against gram-negative bacteria. We have characterized the genomic sequence and expression of a novel but closely related gene from rodents, which we call splunc5. Mouse Splunc5 sequence is 60% identical to mouse Splunc1. The genes also share highly conserved genomic elements including intron-exon structure and intronic sequence. Strikingly, splunc5 is expressed exclusively in the interpapillary epithelium of the tongue's dorsal surface. By comparing the expression profiles of splunc5, splunc1, and a third related sequence, lplunc1, in mice, we show that these genes are expressed in unique domains along a continuous corridor of oral, lingual, pharyngeal, and respiratory epithelia. This expression pattern is consistent with the hypothesis that these proteins protect epithelial surfaces colonized by potentially pathogenic microorganisms.

Amino Acid Sequence↗

PubMed2002

The role of tumour-associated macrophages in tumour progression: implications for new anticancer therapies.

The role of macrophages in tumour growth and development is complex and multifaceted. Whilst there is limited evidence that tumour-associated macrophages (TAMs) can be directly tumouricidal and stimulate the anti-tumour activity of T cells, there is now contrasting evidence that tumour cells are able to block or evade the activity of TAMs at the tumour site. In some cases, tumour-derived molecules even redirect TAM activities to promote tumour survival and growth. Indeed, evidence has emerged for a symbiotic relationship between tumour cells and TAMs, in which tumour cells attract TAMs and sustain their survival, with TAMs then responding to micro-environmental factors in tumours such as hypoxia (low oxygen tension) by producing important mitogens as well as various growth factors and enzymes that stimulate tumour angiogenesis. This review presents evidence for the number and/or distribution of TAMs being linked to prognosis in different types of human malignancy. It also outlines the range of pro- and anti-tumour functions performed by TAMs, and the novel therapies recently devised using TAMs to stimulate host immune responses or deliver therapeutic gene constructs to solid tumours.

Cell Count↗

PubMed2001

Genomic organization of the mouse plunc gene and expression in the developing airways and thymus.

Few genes have been isolated which display specific expression in the proximal airways. A recently identified mouse cDNA, plunc, appears to be confined to the upper airways and nasopharyngeal epithelium, and may prove a useful marker for these regions. We now report the genomic cloning and characterization of the mouse plunc gene as well as its developmental expression in the nasal and airway epithelium. We also report the novel finding that plunc is also expressed in the medullary compartment of the murine thymus. The mouse gene contains nine exons and the intron-exon boundaries are conserved with those in the human homologue. At e14.5 plunc is expressed in the nasal epithelium and several days later is seen in the thymic lobes, but not in the lining of the tracheobronchial tree. Expression in the trachea and main-stem bronchi first appears at 1--2 days after birth. Tracheobronchial expression persists at high levels throughout adulthood, as do regional areas of nasal and thymic expression. Finally, we show that the human homologue is expressed in bronchial epithelium, suggesting a transcript that is evolutionarily conserved in the mammalian airway.

Animals↗

PubMed2001

Cooperativity between KorB and TrbA repressors of broad-host-range plasmid RK2.

The KorB and TrbA proteins of broad-host-range plasmid RK2 are key regulators of the plasmid genes required for conjugative transfer. trbBp is the primary promoter responsible for expression of mating pair formation genes. We show that despite the targets for KorB and TrbA at trbBp being about 165 bp apart, 189 bp upstream of the transcription start point and overlapping the -10 region, respectively, these two proteins show up to 10-fold cooperativity for the repression of trbBp. Deletion analysis of TrbA showed that the C-terminal domain (CTD), which has a high degree of sequence conservation with the CTD of KorA, is required for this cooperativity with KorB. Western blotting demonstrated that the apparently mutual enhancement of repression is not due simply to elevation of repressor level by the presence of the second protein, suggesting that the basis for cooperativity is interaction between KorB and TrbA bound at their respective operators.

Amino Acid Sequence↗

PubMed2001

Cytokine-mediated induction of the human elafin gene in pulmonary epithelial cells is regulated by nuclear factor-kappaB.

Elafin, a low molecular-weight proteinase inhibitor, is a member of the recently described trappin gene family. These proteins are thought to play important roles in the regulation of inflammation and are expressed in multiple epithelia. Elafin is found within the lung, and its expression can be induced by inflammatory mediators. The molecular mechanisms that mediate its induction are not understood. In this study we investigated the transcriptional regulation of the elafin gene in pulmonary epithelial cell lines. Transfection of elafin promoter constructs into the elafin-expressing pulmonary epithelial cell line A549 identified a number of positive-acting elements. Cytokine-mediated inducibility of the elafin gene promoter was shown to occur through a nuclear factor (NF)-kappaB site present within the minimal promoter. This site was shown to bind to NF-kappaB proteins within nuclear extracts from cytokine stimulated cell lines as well as to in vitro-translated RelA. Cotransfection with both RelA and NF-kappaB-inducing kinase induced reporter gene activation via this site, and mutagenesis experiments confirmed that it was crucial for induction of elafin gene activity. These results clearly identify a role for elafin in the inflammatory response of the airway epithelium to pathogenic insult and show that this response is mediated by an NF-kappaB site within the proximal promoter.

Base Sequence↗

PubMed2000

Characterisation of the human plunc gene, a gene product with an upper airways and nasopharyngeal restricted expression pattern.

Here we report the cloning and characterization of the human homologue of plunc, a murine gene expressed specifically in the upper airways and nasopharyngeal regions. The human plunc cDNA codes for a leucine-rich protein of 256 amino acids which is 72% identical to the murine protein. RNA blot analysis suggests that expression of plunc is restricted to the trachea, upper airway, nasopharyngeal epithelium and salivary gland. The human plunc gene contains nine exons and is localised to chromosome 20q11.2. The unique expression pattern of the human plunc suggest that it may prove a useful model gene with which to study the regulatory mechanisms which direct expression of genes specifically to the upper airways.

Amino Acid Sequence↗

PubMed2000

Structure and sequence variation of the trypanosome spliced leader transcript.

We have assessed the potential of using the spliced leader (SL) or mini-exon gene as a marker for molecular phylogenetic analysis of genus Trypanosoma. A total of 27 trypanosome sequences were compared, 18 of these being newly reported. In contrast to genus Leishmania, we found the non-transcribed spacer region of the SL locus in trypanosomes to be far too variable for informative comparison of all but the most closely related species. At the other extreme, the short (39 nt) SL exon was usually completely conserved and hence uninformative. The SL RNA showed variation in both length (97-152 nt) and sequence among different trypanosome species, with most variation occurring in stem-loop II. Consequently, this region could not be aligned with confidence in multiple sequence alignment, severely reducing the number of phylogenetically informative nucleotide positions. In computer simulation, most of the SL RNAs readily folded into the 3 stem-loop secondary structure predicted previously, but again stem-loop II was highly variable. No obvious correlation could be seen between the length of this stem-loop and trypanosome biology. We conclude that the SL repeat is not an informative phylogenetic marker for long range evolutionary studies of genus Trypanosoma.

Animals↗

PubMed1996

Secretory leukoprotease inhibitor: partnering alpha 1-proteinase inhibitor to combat pulmonary inflammation.

Secretory leukoprotease inhibitor (SLPI) is a low molecular weight serine proteinase inhibitor, notably of neutrophil elastase (NE), which is synthesised and secreted by the pulmonary epithelium. SLPI plays an important role in limiting NE-induced pulmonary inflammation and, significantly, it also possesses anti-HIV activity. SLPI is a significant component of the anti-NE shield in the lung which has different reactivity from, and is therefore complementary to, the anti-NE action of alpha 1-proteinase inhibitor (alpha 1-PI). Inhaled recombinant SLPI (rSLPI) could prove beneficial in partnership with alpha 1-PI in the treatment of a number of inflammatory lung disorders including emphysema, chronic bronchitis, cystic fibrosis, and adult respiratory distress syndrome.

Animals↗

PubMed1996

Characterization of the human surfactant protein D promoter: transcriptional regulation of SP-D gene expression by glucocorticoids.

We have previously described the characterization of genomic clones encoding the entire translated sequence of human pulmonary surfactant protein D (SP-D). We now describe the characterization of a genomic fragment (H5E7) that encodes the entirety of the first translated exon (Exon 2), Intron 1, a short transcribed untranslated sequence (Exon 1; 39 bp), and approximately 4 kb of sequence upstream from the transcription initiation site. The start site was identified by 5'-RACE-PCR cloning and primer extension. A putative TATA box (CATAAATA) was identified approximately 30 bp upstream of the start site. Complete sequencing of a HindIII/SacI fragment (HS-1674) encoding approximately 1.7 kb of sequence 5' to the TATA demonstrated multiple potential cis-regulatory elements including half-site glucocorticoid response elements (GRE), a canonical AP-1 consensus, several AP-1 like sequences, E-box sequences, NF-IL-6 and PEA3 motifs, and putative interferon response elements. H441 lung adenocarcinoma cells, which express low levels of SP-D mRNA, and liver HepG2 cells, were transiently co-transfected with chloramphenicol acetyl transferase (CAT) reporter constructs containing up to 3,000 base pairs of upstream sequence, and with constructs encoding beta-gal. H441 cells transfected with constructs containing at least 161 bp of upstream sequence gave normalized levels of CAT activity greater than or equal to that obtained for parallel positive control transfections using pTK-CAT. Treatment of the cells for 48 h with 50 nM dexamethasone (Dex) gave a 2- to 5-fold increase in CAT activity. Interestingly, a 5'-deletion construct containing 161 bp of upstream sequence (pFS161-CAT) conferred both cell type-restricted and dexamethasone-responsive expression. These studies emphasize the potential complexity of SP-D gene regulation, and further support the hypothesis that the effects of glucocorticoids on SP-D production in vivo are regulated at the level of transcription.

Base Sequence↗

PubMed1990

Type II pneumocytes in mixed cell culture of human lung: a light and electron microscopic study.

Alveolar Type II epithelial cells dedifferentiate rapidly in vitro. Studies with animal tissue suggest that cell-cell and extracellular matrix-cell interactions are important in the retention of Type II cell morphology in vitro. Thus, in this study with human tissue, alveolar Type II cells, alveolar macrophages, and spindle cells were prepared from the same sample of lung (obtained following lobectomy for cancer, n = 3), cocultured on glass cover slips or tissue culture plastic, and studied by light microscopy with scanning (SEM) and transmission (TEM) electron microscopy for 8 days. The primary cell isolates contained approximately 45% Type II cells; the remainder were macrophages or unidentifiable cells. Clusters, made up of a single layer of cuboidal Type II cells around a central core of connective tissue (largely collagen and some elastic tissue), formed above a monolayer of spindle cells. The Type II cells were morphologically similar to those seen in vivo. The cells were still cuboidal at 8 days but had lost their lamellar bodies, which were released into the medium via the apical surface. The clusters increased in size with time (area, microns 2: day 1, 29(5-143) x 10(2); day 8, 63(10-311) x 10(2); mean(range); p less than 0.02) without changing in number per culture, suggesting Type II cell proliferation. This may have been due to factors produced by the other cells and adherence to the extracellular matrix (ECM); (free collagen fibers, present in the original preparation, spindle cells, and/or Type II cells could be responsible for presence of ECM). We propose this as a useful model for the study of human Type II epithelial cells in vitro.

Cell Differentiation↗

PubMed1989

High intrinsic radiosensitivity of a newly established and characterised human embryonal rhabdomyosarcoma cell line.

A new human rhabdomyosarcoma cell line (HX170c) has been established from a paratesticular embryonal tumour in a 5-year-old male. The cells grew as an adherent monolayer with a doubling time of 32 h and showed pleomorphic features. Intermediate filament analysis revealed the line to be mesenchymal in origin (reactivity to vimentin and desmin antibodies). The line was tumorigenic in nude mice, possessed elevated levels of creatine phosphokinase (mainly of the MM isoenzyme form) and had a near diploid mean chromosome number of 50. In vitro cell cloning determinations gave colony forming efficiencies of 0.01% in soft agar and 24% in a monolayer anchorage-dependent assay. Radiosensitivity determinations using a monolayer clonogenic assay with feeder layer support showed the cells to be among the more radiosensitive human tumour cell types (surviving fraction at 2 Gy of 0.26) that have been investigated. Furthermore, experiments utilising continuous low dose rate radiation at 3.2 cGy min-1, showed that, under these experimental conditions, the cells possessed only a very low capacity to recover from radiation-induced damage (dose reduction factor at 1% cell survival of 1.07 for 150 versus 3.2 cGy min-1). As other human tumour cells of an embryonal cell origin (e.g. neuroblastoma and germ cell tumours of the testis) have also been shown to be radiosensitive it appears that sensitivity to radiation may be a common property of this group of tumours.

Animals↗

PubMed1988

Radiosensitivity and characterisation of a newly established cell line from an epithelioid sarcoma.

A new human tumour cell line (designated HX 165c) has been established from an epithelioid sarcoma presenting in a 28 year old male. The cells grew as an adherent monolayer with a doubling time of 38 h and had mainly epithelial morphology but with areas of mesenchymal-like cytoplasmic extensions. The mixed epithelial-mesenchymal phenotype was also apparent by intermediate filament analysis which showed reactivity to vimentin and keratin. The cells were tumorigenic in nude mice and aneuploid, possessing a mean chromosome number of 65. In vitro cloning determinations gave colony-forming efficiencies of 0.01% in an anchorage-independent soft agar assay and 34% in a monolayer anchorage-dependent assay. The cells were in the mid-range for radiosensitivity of human tumour cells (surviving fraction at 2 Gy of 0.39). In addition, experiments utilising continuous low dose rate irradiation at 3.2 cGy min-1, showed that the cells possessed only a small capacity to recover from radiation damage (dose reduction factor at 1% cell survival of 1.15 for 150 versus 3.2 cGy min-1). This cell line, being only the second we are aware of to be established from this rare soft tissue sarcoma, should be useful in helping to ascertain the histogenesis of epithelioid sarcoma.

Adult↗