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Biomedical subjects

Sally Roberts

Publications and source records attributed to Sally Roberts.

At least 19 recordsLinked to original sources
PubMed2025

Repression of CADM1 transcription by HPV type 18 is mediated by three-dimensional rearrangement of promoter-enhancer interactions.

Upon infection, human papillomavirus (HPV) manipulates host cell gene expression to create an environment that is supportive of a productive and persistent infection. The virus-induced changes to the host cell's transcriptome are thought to contribute to carcinogenesis. Here, we show by RNA-sequencing that oncogenic HPV18 episome replication in primary human foreskin keratinocytes (HFKs) drives host transcriptional changes that are consistent between multiple HFK donors. We have previously shown that HPV18 recruits the host protein CTCF to viral episomes to control the differentiation-dependent viral transcriptional programme. Since CTCF is an important regulator of host cell transcription via coordination of epigenetic boundaries and long-range chromosomal interactions, we hypothesised that HPV18 may also manipulate CTCF to contribute to host transcription reprogramming. Analysis of CTCF binding in the host cell genome by ChIP-Seq revealed that while the total number of CTCF binding sites is not altered by the virus, there are a sub-set of CTCF binding sites that are either enriched or depleted of CTCF. Many of these altered sites are clustered within regulatory elements of differentially expressed genes, including the tumour suppressor gene cell adhesion molecule 1 (CADM1), which supresses epithelial cell growth and invasion. We show that HPV18 establishment results in reduced CTCF binding at the CADM1 promoter and upstream enhancer. Loss of CTCF binding is coincident with epigenetic repression of CADM1, in the absence of CpG hypermethylation, while adjacent genes including the transcriptional regulator ZBTB16 are activated. These data indicate that the CADM1 locus is subject to topological rearrangement following HPV18 establishment. We tested this hypothesis using 4C-Seq (circular chromosome confirmation capture-sequencing) and show that HPV18 establishment causes a loss of long-range chromosomal interactions between the CADM1 transcriptional start site and the upstream transcriptional enhancer. These data show that HPV18 manipulates host cell promoter-enhancer interactions to drive transcriptional reprogramming that may contribute to HPV-induced disease progression.

Humans↗
PubMed2006

Human intervertebral disc cells promote nerve growth over substrata of human intervertebral disc aggrecan.

STUDY DESIGN: Coculture assays of the migration and interaction of human intervertebral disc cells and chick sensory nerves on alternate substrata of collagen and aggrecan. OBJECTIVE: To examine the effects of aggrecan on disc cell migration, how disc cells and sensory nerves interact, and whether disc cells affect previously reported inhibitory effects of aggrecan on sensory nerve growth. SUMMARY OF BACKGROUND DATA: Human intervertebral disc aggrecan is inhibitory to sensory nerve growth in vitro, suggesting that a loss of aggrecan from the disc may have a role in the increased innervation seen in disc degeneration. Endothelial cells that appear to co-migrate with nerves into degenerated intervertebral disc express neurotrophic factors, but the effects of disc cells on nerve growth are not known. METHODS: Human disc cells were seeded onto tissue culture plates that had been coated with type I collagen and human intervertebral disc aggrecan. Explants of chick dorsal root ganglions (DRGs) were subsequently added to the plates and sensory neurite outgrowth stimulated by the addition of nerve growth factor. Time-lapse video and fluorescence microscopy were used to examine the migration and interaction of the disc cells and sensory neurites, in the context of the different matrix substrata. The effects of disc cell conditioned medium on nerve growth were also examined. RESULTS: Disc cells spread and migrated on collagen until they encountered the aggrecan substrata, where some cells, but not all, were repelled. In coculture, DRG neurites extended onto the collagen/disc cells until they encountered the aggrecan, where, like the disc cells, many were repelled. However, in the presence of disc cells, some neurites were able to cross onto this normally inhibitory substratum. The number of neurite crossings onto aggrecan correlated significantly with the number of disc cells present on the aggrecan. In control experiments using DRG alone, all extending neurites were repelled at the collagen/aggrecan border. Conditioned medium from disc cell cultures stimulated DRG neurite outgrowth on collagen but did not increase neurite crossing onto aggrecan substrata. CONCLUSIONS: Human disc cells migrate across aggrecan substrata that are repellent to sensory DRG neurites. Disc cells synthesize neurotrophic factors in vitro that promote neurite outgrowth. Furthermore, the presence of disc cells in coculture with DRG partially abrogates the inhibitory effects of aggrecan on nerve growth. These findings have important implications for the regulation of nerve growth into the intervertebral disc, but whether disc cells promote nerve growth in vivo remains to be determined.

Aggrecans↗
PubMed2006

Topographical guidance of intervertebral disc cell growth in vitro: towards the development of tissue repair strategies for the anulus fibrosus.

The anulus fibrosus (AF) of the intervertebral disc consists of concentric sheets of collagenous matrix that is synthesised during embryogenesis by aligned disc cells. This highly organised structure may be severely disrupted during disc degeneration and/or herniation. Cell scaffolds that incorporate topographical cues as contact guidance have been used successfully to promote the healing of injured tendons. Therefore, we have investigated the effects of topography on disc cell growth. We show that disc cells from the AF and nucleus pulposus (NP) behaved differently in monolayer culture on micro-grooved membranes of polycaprolactone (PCL). Both cell types aligned to and migrated along the membrane's micro-grooves and ridges, but AF cells were smaller (or less spread), more bipolar and better aligned to the micro-grooves than NP cells. In addition, AF cells were markedly more immunopositive for type I collagen, but less immunopositive for chondroitin-6-sulphated proteoglycans than NP cells. There was no evidence of extracellular matrix (ECM) deposition. Disc cells cultured on non-grooved PCL did not show any preferential alignment at sub-confluence and did not differ in their pattern of immunopositivity to those on grooved PCL. We conclude that substratum topography is effective in aligning disc cell growth and may be useful in tissue engineering for the AF. However, there is a need to optimise cell sources and/or environmental conditions (e.g. mechanical influences) to promote the synthesis of an aligned ECM.

Animals↗
PubMed2006

Aggrecan turnover in human intervertebral disc as determined by the racemization of aspartic acid.

We have used the racemization of aspartic acid as a marker for the "molecular age" of aggrecan components of the human intervertebral disc matrix (aggregating and non-aggregating proteoglycans as well as the different buoyant density fractions of aggrecan). By measuring the D/L(Asp) ratio of the various aggrecan species as a function of age and using the values of the racemization constant, k(i), found earlier for aggrecan in articular cartilage, we were able to establish directly the relative residence time of these molecules in human intervertebral disc matrix. For A1 preparations taken from normal tissue, turnover rates of 0.059 +/- 0.01 and 0.063 +/- 0.01/year correspond to half-life values of 12 +/- 2.0 and 11.23 +/- 1.9 years for nucleus pulposus and annulus fibrosus, respectively; the turnover rates of 0.084 +/- 0.022 and 0.092 +/- 0.034/year for degenerate tissue correspond to half-life values of 8.77 +/- 2.2 and 8.41 +/- 2.8 years, suggesting increased rate of removal of small aggrecan fragments. For the large monomer, fraction A1D1, turnover is 0.13 +/- 0.04/year, corresponding to a half-life of 5.56 +/- 1.58 years, similar to 3.4 years in human articular cartilage. For the binding region (A1D6), turnover is 0.033 +/- 0.0012/year, corresponding to a half-life of 21.53 +/- 0.6 years, similar to 23.5 years in articular cartilage. A1 preparations from nucleus pulposus contain a lower proportion of aggregating proteoglycans as compared with annulus fibrosus, suggesting increased proteolytic modification in the nucleus pulposus. D/L(Asp) values in aggregating and non-aggregating proteoglycans of a 24-year-old individual show similar results, suggesting that the non-aggregating molecules are synthesized initially as aggregating proteoglycans, which thereafter undergo cleavage and detachment from hyaluronan.

Adolescent↗
PubMed2006

Role for Wee1 in inhibition of G2-to-M transition through the cooperation of distinct human papillomavirus type 1 E4 proteins.

The infectious cycle of human papillomavirus type 1 (HPV1) is accompanied by abundant expression of the full-length E1;E4 protein (17-kDa) and smaller E4 polypeptides (16-, 11-, and 10-kDa) that arise by sequential loss of N-terminal E1;E4 sequences. HPV1 E4 inhibits G(2)-to-M transition of the cell cycle. Here, we show that HPV1 E4 proteins mediate inhibition of cell division by more than one mechanism. Cells arrested by coexpression of E1;E4 (E4-17K) and a truncated protein equivalent to the 16-kDa species (E4-16K) contain inactive cyclin B1-cdk1 complexes. Inactivation of cdk1 is through inhibitory Tyr(15) phosphorylation, with cells containing elevated levels of Wee1, the kinase responsible for inhibitory cdk1 phosphorylation. Consistent with these findings, overexpression of Wee1 enhanced the extent to which E4-17K/16K-expressing cells arrest in G(2), indicating that maintenance of Wee1 activity is necessary for inhibition of cell division induced by coexpression of the two E4 proteins. Moreover, we have determined that depletion of Wee1 by small interfering RNA (siRNA) alleviates the G(2) block imposed by E4-17K/16K. In contrast however, maintenance of Wee1 activity is not necessary for G(2)-to-M inhibition mediated by E4-16K alone, as overexpression or depletion of Wee1 does not influence the G(2) arrest function of E4-16K. Cells arrested by E4-16K expression contain low levels of active cyclin B1-cdk1 complexes. We hypothesize that differential expression of HPV1 E4 proteins during the viral life cycle determines the host cell cycle status. Different mechanisms of inhibition of G(2)-to-M transition reinforce the supposition that distinct E4 functions are important for HPV replication.

CDC2 Protein Kinase↗
PubMed2006

Histology and pathology of the human intervertebral disc.

The intervertebral disc is a highly organized matrix laid down by relatively few cells in a specific manner. The central gelatinous nucleus pulposus is contained within the more collagenous anulus fibrosus laterally and the cartilage end plates inferiorly and superiorly. The anulus consists of concentric rings or lamellae, with fibers in the outer lamellae continuing into the longitudinal ligaments and vertebral bodies. This arrangement allows the discs to facilitate movement and flexibility within what would be an otherwise rigid spine. At birth, the human disc has some vascular supply within both the cartilage end plates and the anulus fibrosus, but these vessels soon recede, leaving the disc with little direct blood supply in the healthy adult. With increasing age, water is lost from the matrix, and the proteoglycan content also changes and diminishes. The disc-particularly the nucleus-becomes less gelatinous and more fibrous, and cracks and fissures eventually form. More blood vessels begin to grow into the disc from the outer areas of the anulus. There is an increase in cell proliferation and formation of cell clusters as well as an increase in cell death. The cartilage end plate undergoes thinning, altered cell density, formation of fissures, and sclerosis of the subchondral bone. These changes are similar to those seen in degenerative disc disease, causing discussion as to whether aging and degeneration are separate processes or the same process occurring over a different timescale. Additional disorders involving the intervertebral disc can demonstrate other changes in morphology. Discs from patients with spinal deformities such as scoliosis have ectopic calcification in the cartilage end plate and sometimes in the disc itself. Cells in these discs and cells from patients with spondylolisthesis have been found to have very long cell processes. Cells in herniated discs appear to have a higher degree of cellular senescence than cells in nonherniated discs and produce a greater abundance of matrix metalloproteinases. The role that abnormalities play in the etiopathogenesis of different disorders is not always clear. Disorders may be caused by a genetic predisposition or a tissue response to an insult or altered mechanical environment. Whatever the initial cause, a change in the morphology of the tissue is likely to alter the physiologic and mechanical functioning of the tissue.

Adolescent↗
PubMed2006

Promoting adequate nutrition: using artificial feeding.

An earlier article in this series (Holman et al 2005a) identified that in some circumstances the decision is made to administer artificial feeding. Here, Cheryl Holman, Sally Roberts and Maggie Nicol describe the reasons why artificial feeding may be necessary in older people and how it can be administered.

Aged↗
PubMed2005

The elastic fiber network of the anulus fibrosus of the normal and scoliotic human intervertebral disc.

STUDY DESIGN: Immunohistochemical study of elastic fibers in human intervertebral discs (IVD) collected at surgery from patients with scoliosis. OBJECTIVES: To compare the elastic fiber network in scoliotic discs (idiopathic scoliosis or neuromuscular scoliosis) to that of control (normal) discs. To study whether the change in elastic fiber organization could contribute to the progression of spinal deformity. SUMMARY OF BACKGROUND DATA: Elastin and elastic fibers have been identified previously in human IVD but were believed to contribute little to the tissue's mechanical properties. However, a recent immunohistochemical study has revealed an abundant and organized elastic fiber network in bovine IVD, indicating that elastic fibers could play an important mechanical role. This article reports the organization of elastic fibers in human IVD and the changes of elastic fiber organization in scoliosis. METHODS: Intact wedges of IVD were obtained from patients undergoing surgery for scoliosis (aged 12-22 years). Control discs were obtained from a patient (aged 12 years) with a spinal tumor and a trauma patient (aged 17 years). The discs were dissected to give radial slices and were snap frozen. Frozen sections were cut and digested with hyaluronidase to remove glycosaminoglycans. Micrographs of the sections were examined by polarized light to visualize collagen organization. The elastic fiber network was visualized immunohistochemically or by histochemical staining with orcein. RESULTS: A highly organized elastic fiber network, similar to that described in bovine discs, was revealed in the control human discs. In the anulus fibrosus of control discs, dense elastic fibers were located between adjacent lamellae, with fibers also present within individual lamellae. Elastic fibers appeared to be long (>200 microm) and straight in outer anulus, whereas in inner anulus, they nearly ran parallel to each other and at an angle of approximately 60 degrees or 120 degrees to those in adjacent lamellae. However, in scoliotic discs, elastic fibers were sparse, and the collagen and elastic fiber networks were disorganized with loss of lamellar structure. Cell clusters, one of typical degenerative feature, were seen in scoliotic discs but not in age-matched control discs. CONCLUSIONS: Our results reveal an abundant and organized network of elastic fibers in the adolescent (12 and 17-year-olds) human IVD, and suggest that elastic fiber network plays a significant biomechanical role. This network is sparse and disrupted in scoliotic discs, and could be involved in the progression of the spinal deformity.

Adolescent↗
PubMed2005

Human intervertebral disc aggrecan inhibits endothelial cell adhesion and cell migration in vitro.

STUDY DESIGN: The effect of human intervertebral disc aggrecan on endothelial cell growth was examined using cell culture assays. OBJECTIVE: To determine the response of endothelial cells to human intervertebral disc aggrecan, and whether the amount and type of aggrecan present in the intervertebral disc may be implicated in disc vascularization. SUMMARY OF BACKGROUND DATA: Intervertebral disc degeneration has been associated with a loss of proteoglycan, and the ingrowth of blood vessels and nerves. Neovascularization is a common feature also of disc herniation. Intervertebral disc aggrecan is inhibitory to sensory nerve growth, but the effects of disc aggrecan on endothelial cell growth are not known. METHODS: Aggrecan monomers were isolated separately from the anulus fibrosus and nucleus pulposus of human lumbar intervertebral discs, and characterized to determine the amount and type of sulfated glycosaminoglycan side chains present. The effects of these aggrecan isolates on the cellular adhesion and migration of the human endothelial cell lines, HMEC-1 and EAhy-926, were examined in vitro. RESULTS: Homogenous substrata of disc aggrecan inhibited endothelial cell adhesion and cell spreading in a concentration dependent manner. In substrata choice assays, endothelial cells seeded onto collagen type I migrated over the collagen until they encountered substrata of disc aggrecan, where they either stopped migrating, retreated onto the collagen, or, more commonly, changed direction to align along the collagen-aggrecan border. The inhibitory effect of aggrecan on endothelial cell migration was concentration dependent, and reduced by enzymatic treatment of the aggrecan monomers with a combination of chondroitinase ABC and keratinase/keratinase II. Anulus fibrosus aggrecan was more inhibitory to endothelial cell adhesion than nucleus pulposus aggrecan. However, this difference did not relate to the extent to which the different aggrecan isolates were charged, as determined by colorimetric assay with 1,9-dimethylmethylene blue, or to marked differences in the distribution of chondroitin sulfated and keratan sulfated side chains. CONCLUSIONS: Human intervertebral disc aggrecan is inhibitory to endothelial cell migration, and this inhibitory effect appears to depend, in part, on the presence of glycosaminoglycan side chains on the aggrecan monomer.

Aggrecans↗
PubMed2005

Excess cost associated with Staphylococcus aureus poststernotomy mediastinitis.

AIM: To determine the additional cost attributable to Staphylococcus aureus poststernotomy mediastinitis (PSM) following sternotomy for cardiac surgery at Green Lane Hospital. METHODS: A retrospective case-control study was undertaken. Nine patients with S. aureus PSM (cases) were matched with nine patients without PSM (controls) for gender, age, type of surgical procedure, time of procedure, and presence of diabetes mellitus. Patients' length and cost of hospital stay (for the admission associated with the initial surgery and any subsequent admissions associated with complications of that surgery) were obtained from the hospital's clinical costing system. RESULTS: S. aureus PSM was associated with longer average length of hospital stay, 42.6+/-18.7 vs 10.4+/-4.0 days (p=0.005). The mean cost per patient in New Zealand dollars was 30,527 dollars +/-10,489 dollars for controls and 76,104 dollars +/- 31,460 dollars for cases, and the mean excess cost associated with S. aureus PSM was 45,677 dollars per patient. CONCLUSIONS: This study illustrates the significant cost of deep surgical site infection, both in terms of length of hospital stay and hospital revenue, and highlights the potential cost benefit of successful strategies to reduce surgical site infection such as S. aureus PSM.

Aged↗
PubMed2005

A review of emerging flea-borne bacterial pathogens in New Zealand.

Recent studies have shown that Rickettsia typhi, R. felis, Bartonella henselae, and B. clarridgeiae occur in New Zealand. To raise awareness of these emerging and re-emerging flea-borne bacterial pathogens among New Zealand health workers, we review the clinical features, diagnosis, and treatment of infections, and the biology of the major flea vectors.

Animals↗
PubMed2005

Managing student transitions into primary care.

In response to its specific recruitment needs, Walsall teaching primary care trust has implemented a new final placement programme for community nursing students that focuses on providing students with hands-on experience in the community setting. This article presents the results of an evaluation of the programme. Outcomes of the programme are discussed and analysis is drawn from all key participants. This has been the first stage of a career pathway for 80% of these students, who are now successfully employed as community staff nurses within the PCT.

Education, Nursing↗
PubMed2005

Inflammatory mediators as potential therapeutic targets in the spine.

Inflammation plays a variable part in the pathogenesis of several spinal disorders. Ankylosing spondylitis is a chronic inflammatory arthropathy of the spine and rheumatoid arthritis, whilst affecting predominantly limb joints, also affects the cervical spine in a significant proportion of people. Inflammation is also involved in disorders such as disc herniation and sciatica, which have previously been thought of as being primarily mechanical or degenerative. Anti-inflammatory agents which have been shown to be effective elsewhere in the body are discussed in this review as possible therapeutic agents in the spine. As the inflammatory cascade and immunopathology of these conditions continue to be elucidated, it has become apparent that individual molecules may be potential targets for inactivation or down-regulation. Candidates include pro-inflammatory cytokines, such as TNF-alpha, cytokines, e.g. IL-1 and IL-15, or enzymes enhancing the inflammation pathway such as the cyclooxygenases. Hence treatments based on inactivation of these molecules by various mechanisms, including antibodies, receptor antagonists, enzyme inhibitors or gene therapy, are being introduced. However, the mode of action of a particular molecule can be complex and sometimes apparently contradictory. For example, TNF-alpha is known to play an important role in promoting inflammation by upregulating expression of cell adhesion molecules on endothelial cells and stimulating the production of reactive oxygen intermediates, nitric oxide and prostaglandins. However, it can also have an immunosuppressive and anti-inflammatory role after prolonged release. Therefore, although inhibitors of many of these molecules are now in clinical application and trials (many with promising results in rheumatoid arthritis), it is important to remain vigilant and monitor long-term outcomes particularly when these treatments are used in clinical syndromes with relatively poorly defined immunopathology such as spinal disorders.

Animals↗