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Biomedical subjects

P Stephens

Publications and source records attributed to P Stephens.

At least 37 records · Page 2Linked to original sources
PubMed1998

Molecular characterisation of tumour infiltrating lymphocytes in oral squamous cell carcinoma.

Oral squamous cell carcinoma (OSCC) is often associated with a lymphocytic infiltrate that is believed to represent an in vivo immune reaction to the tumour cells. In this study, the tumour-infiltrating lymphocytes (TIL) associated with primary OSCC were characterised molecularly in six newly-diagnosed patients to determine the nature of the immune response to the primary tumour. The primary tumours in three of the six patients were associated with a moderate to dense CD8+ T cell infiltrate whilst the cellular infiltrate in the other primary tumours was sparse. The CD3+ T cells were also HLA-DR+. In all cases, there were few CD56+ cells, suggesting that TIL were predominantly T cells bearing the alpha/beta T cell receptors (TCR). The TCR Vbeta repertoire of TIL in these six cases was analysed. TCR Vbeta gene usage by TIL was heterogeneous. A restricted usage of TCR Vbeta genes by TIL was evident in two tumours associated with a dense CD3+ T cell infiltrate. In one of these, there was histological evidence of tumour cell destruction by TIL. Further analysis of the predominant TCR Vbeta gene family used by TIL in this individual showed a unique in-frame nucleotide sequence in 100% of the transcripts. This recurrent transcript was not detected in the peripheral blood of this patient, indicating a local T cell clonal expansion in the vicinity of the tumour. Overall, these results suggest that activation and clonal expansion of T cells occurs and may play a role in local tumour destruction in OSCC.

Adult↗
PubMed1998

Assessment of bacterial viability status by flow cytometry and single cell sorting.

Rapid bacterial detection and viability measurements have been greatly enhanced by recent advances in the use of fluorescent stains in cytometry. It has previously been shown that four physiological states can be distinguished: reproductively viable, metabolically active, intact and permeabilized. Previous sorting experiments have shown that not all intact cells readily grow, but some intact cells can grow even when they fail to show metabolic activity, as determined by esterase turnover. To circumvent the limitations imposed by active dye extrusion or cell dormancy on viability measurements used to date (e.g., enzyme activity or cell polarization), a fast triple fluorochrome staining procedure has been developed that takes account of these problems. This allows further cellular characterization of intact cells by: active exclusion of ethidium bromide (EB) (metabolically active cells), uptake of EB but exclusion of bis-oxonol (BOX) (de-energized but with a polarized cell membrane) and uptake of both dyes (depolarized). Permeabilized cells were identified by propidium iodide (PI) uptake. The method was validated using an electronically programmable single cell sorter (EPICS Elite) and aged Salmonella typhimurium cells. Reproductive viability was determined by sorting single cells to their staining pattern directly onto agar plates. Most polarized cells could be recovered as well as a significant fraction of the depolarized cells, demonstrating that depolarization is a sensitive measure of cell damage but a poor indicator of cell death.

Bacteriological Techniques↗
PubMed1997

Integrin receptor involvement in actin cable formation in an in vitro model of events associated with wound contraction.

Actin cables have been reported to act in vivo as contractile 'purse strings' capable of closing embryonic wounds through generation of circumferential tension. Furthermore, their involvement in wounds within in vitro model systems suggests that actin cable contraction may be an important mechanism involved in the process of wound closure. The aim of this study therefore, was to investigate the appearance of actin cables in a contracting fibroblast populated collagen lattice, an in vitro model of events associated with wound contraction. Utilising this in vitro model, the time-course of actin cable production was investigated and the involvement of integrin receptors analysed using immunofluorescent labelling techniques. Over a period of hours distinct cellular cable-like structures developed at the edges of collagen lattices coinciding with the onset of contraction. Cellular organisation within the cable was evident as was polymerisation of actin microfilaments into elongated stress fibres forming a continuous cell-cell 'actin cable' around the circumference of the lattice. Immunolocalisation demonstrated that integrin receptor subunits beta 1 and alpha 2 but not alpha 5 were involved in apparent intimate cell-cell contact between juxtaposed fibroblasts within this actin cable. This study demonstrates the involvement of integrin receptors in actin cable formation within collagen lattice systems undergoing reorganisation. Such integrin involvement may enable participating cells to respond to the tensional status of their surrounding environment and via cell-cell communication, to permit a co-ordinated contraction of the cable. It is concluded that integrin receptor involvement in active actin cable contraction may be involved in the process of wound contraction.

Actins↗
PubMed1997

Neonatal cleft lip repair: a retrospective review of anaesthetic complications.

A retrospective analysis of the first 50 cases of neonatal cleft lip repair performed at the Hospital for Sick Children is presented. The patient population included 11 expremature infants of less than 45 weeks postconceptual age. There was no mortality at the time of follow-up. There was one case of peroperative hypoxaemia. There was one case of postoperative laryngospasm requiring reintubation. Postoperatively there were four cases of mild hypoxaemia and one patient with transient apnoea. No patients required blood transfusion. Seventy-six percent of patients did not require opioid analgesia. The remainder received a single dose of intramuscular codeine phosphate. The advantages and risks of anaesthesia for cleft lip in the neonatal period are reviewed. Recommendations for safe practice include the selection of gestationally mature infants with no intercurrent illness, avoidance of opioid analgesia, adequate staffing ratios of experienced postoperative nursing care, appropriate monitoring including oximetry and apnoea detectors.

Anesthesia, General↗
PubMed1997

T-cell receptor V beta usage by lesional lymphocytes in oral lichen planus.

To determine whether the T-cell inflammatory infiltrate in oral lichen planus (OLP) represents a selective activation and expansion of a limited repertoire of T-cell receptor (TCR) specific T-cells, V beta gene expression was investigated in lesional T-lymphocytes in OLP. A reverse transcriptase-polymerase chain reaction (RT-PCR) technique was used to amplify the 24 major V beta gene sub-families of infiltrating mucosal lymphocytes and peripheral blood mononuclear cells (PMNC) in seven patients with reticular OLP and four healthy control patients. Specificity of amplified products was confirmed by Southern blotting with a C beta internal probe. TCR V beta usage by lesional T-cells in OLP was markedly heterogeneous 5-23 V beta sub-families). In 6/8 patients with OLP, V beta usage was restricted with < or = 20/25 sub-families detected; only one of the V beta sub-families (V beta 8) was present in all of the OLP patients demonstrating TCR V beta restriction. In contrast, TCR V beta usage was unrestricted in PMNC from OLP patients and controls (> or = 23/ 25 sub-families detected). In three patients, certain V beta sub-families (V beta 13, V beta 14 & V beta 15) were present in the lesional T-cell population but were under-represented in PMNC. These results suggest a selective V beta gene usage by lesional infiltrating T-cells in oral lichen planus. The non-uniformity of V beta restriction in lesional T-cells does not support the concept of a common superantigen in OLP and reflects the heterogeneity of the disease.

Adult↗
PubMed1997

Molecular analysis of T cell receptor beta variability in a patient with orofacial granulomatosis.

BACKGROUND: Orofacial granulomatosis (OFG) is a rare chronic inflammatory disorder of unknown causation and is characterised histologically by non-caseating granulomas and aggregates of small lymphocytes. The molecular nature of these T cells is, however, unclear. AIMS: To determine the T cell receptor (TCR) V beta gene usage of the T cell infiltrate associated with the primary lesions in a patient with OFG. METHODS: A molecular method involving reverse transcriptase (RT)-polymerase chain reaction (PCR), DNA cloning, single strand conformation polymorphism (SSCP), length analysis, and nucleotide sequencing was used. RESULTS: Compared with peripheral blood lymphocytes from the same patient, notably restricted TCRV beta gene usage was observed in the T cell infiltrate. Only three of the 24 major TCRV beta gene families were represented in the repertoire. There was preferential usage of the V beta 6 gene. In addition, more than 20% of the V beta 6 TCR transcripts exhibited an identical unique V-D-J junctional sequence, suggesting a local antigen driven V beta 6 T cell clonal expansion in vivo, a phenomenon not observed in normal oral mucosa. CONCLUSIONS: The TCRV beta repertoire of T cells associated with OFG is restricted. It is also associated with a local T cell clonal expansion. The results, therefore, provide a new perspective on the immunopathology of OFG.

Amino Acid Sequence↗
PubMed1997

An investigation of preferential fibroblast wound repopulation using a novel in vitro wound model.

To overcome the difficulties of studying wounding and wound repopulation in monolayer systems, a 3-dimensional model of wound repopulation has been developed which allows the in vitro investigation of fibroblast migration in response to experimental wounding. This model was utilized to determine whether fibroblasts derived from sites which demonstrate preferential healing (child and oral mucosal fibroblasts) possessed an increased ability to repopulate experimental wounds when compared to adult dermal fibroblasts. Fibroblasts were established from specimens derived from healthy donors undergoing minor elective surgery. Standard wounds were created in fibroblast populated collagen lattices (FPCLs) which were then overlaid upon an extracellular wound matrix. Fibroblast repopulation of the wounds was studied over 12 days using light- and scanning electron microscopy and quantified using computerized image analysis. Wound repopulation by fibroblasts derived from child donors (n = 3) was significantly (P < 0.001) more rapid than their adult tissue-matched counterparts (n = 3). Wound repopulation by oral mucosal fibroblasts (n = 3) was significantly greater than that exhibited by age-matched dermal fibroblasts (n = 3; P < 0.05). These differences were not reflected in differences in DNA synthesis (P > 0.5) or cell number (P > 0.5) within similar attached FPCL systems. These findings further support the concept of a gradual transition from the fetal to adult phenotype in wound healing. The potential applications of the model are discussed.

Adult↗
PubMed1996

T cell receptor Vbeta repertoire of tumour-infiltrating lymphocytes in oral squamous-cell carcinoma.

Oral squamous cell carcinoma (OSCC) are frequently infiltrated by tumour infiltrating lymphocytes (TILs) which may demonstrate specific anti-tumour cytotoxicity. We have used a panel of family-specific primers in reverse-transcriptase (RT)-polymerase chain reactions (PCR) to determine the TCR Vbeta repertoire of TILs in the primary and metastatic tumours in patients with OSCC. We observed a diverse and heterogenous usage of TCR Vbeta genes by TILs in the primary tumours, a scenario not unlike normal oral mucosal lymphocytes. In one patient with a longstanding history of OSCC, the repertoire was restricted. Restricted TCR Vbeta gene usage also occurred in TILs of metastatic tumours. Within individual patients, discrepancies of TCR Vbeta gene usage were also observed between TILs in the primary OSCC and those in metastatic lymph nodes. Work is ongoing to determine the clonality and functional significance of these TILs.

Biopsy↗
PubMed1996

An investigation of the interaction between alcohol and fibroblasts in wound healing.

This study investigated the effect of clinical concentrations of alcohol on fibroblast function (proliferation and ECM synthesis) in vitro. Basal and TGF-beta-induced collagen synthesis was assayed in confluent cultures in serum-free medium at 48 h with a commercial collagen assay system. At concentrations of alcohol > 5%, fibroblast proliferation was significantly inhibited. Although noninhibitory, subclinical concentrations of alcohol failed to inhibit basal collagen synthesis (P > 0.1), they significantly decreased TGF-beta-induced collagen synthesis (P < 0.03). These data support the notion that the local, as well as the systemic, effects of alcohol are important in mediating delayed healing in alcoholic patients.

Cell Division↗
PubMed1996

A comparison of the ability of intra-oral and extra-oral fibroblasts to stimulate extracellular matrix reorganization in a model of wound contraction.

Intra-oral wounds, like wounds in children, demonstrate privileged healing when compared with adult wounds at extra-oral sites. This study investigated whether this preferential healing is related to an increased ability of oral mucosal fibroblasts to reorganize extracellular matrix (ECM) when compared with their dermal counterparts. ECM reorganization was investigated by means of a fibroblast-populated collagen lattice (FPCL) system. The effect of donor age was also investigated in this system. Differences in ECM reorganization and FPCL contraction were evident: FPCL contraction was more rapid by oral mucosal fibroblasts than dermal fibroblasts (p < 0.01). FPCL contraction was also greater in child (donor < 10 years) than adult (donor > 18 years) oral mucosal fibroblasts (p < 0.01). These differences were not related to phenotypic differences in cell viability (p > 0.5), DNA synthesis (p > 0.05), and cell number (p > 0.5) within the FPCLs, or cellular attachment to collagen (p > 0.07). FPCL contraction was not stimulated by the addition of conditioned medium from oral mucosal or dermal fibroblasts (p > 0.05). These data show that the significantly increased ability of oral mucosal fibroblasts to reorganize ECM in vitro, when compared with dermal fibroblasts, represents a distinct phenotypic contractile difference, rather than differences in their production of soluble mediators or cell attachment to ECM.

Adolescent↗
PubMed1995

Iron supplying systems of Salmonella in diagnostics, epidemiology and infection.

Well-known and newly characterised mechanisms, both endogenous and exogenous, for the uptake of iron by Salmonella are outlined, and their possible roles at various stages in infection are discussed. The contributions of a detailed understanding of iron supplying systems to techniques for diagnosis, epidemiology and disease management are described.

Bacterial Outer Membrane Proteins↗
PubMed1995

Temperature dependence of cell-cell fusion induced by the envelope glycoprotein of human immunodeficiency virus type 1.

We investigated cell-cell fusion induced by the envelope glycoprotein of human immunodeficiency virus type 1 strain IIIB expressed on the surface of CHO cells. These cells formed syncytia when incubated together with CD4-positive human lymphoblastoid SupT1 cells or HeLa-CD4 cells but not when incubated with CD4-negative cell lines. A new assay for binding and fusion was developed by using fluorescent phospholipid analogs that were produced in SupT1 cells by metabolic incorporation of BODIPY-labeled fatty acids. Fusion occurred as early as 10 min after mixing of labeled SupT1 cells with unlabeled CHO-gp160 cells at 37 degrees C. When both the fluorescence assay and formation of syncytia were used, fusion of SupT1 and HeLa-CD4 cells with CHO-gp160 cells was observed only at temperatures above 25 degrees C, confirming recent observations (Y.-K. Fu, T.K. Hart, Z.L. Jonak, and P.J. Bugelski, J. Virol. 67:3818-3825, 1993). This temperature dependence was not observed with influenza virus-induced cell-cell fusion, which was quantitatively similar at both 20 and 37 degrees C, indicating that cell-cell fusion in general is not temperature dependent in this range. gp120-CD4-specific cell-cell binding was found over the entire 0 to 37 degrees C range but increased markedly above 25 degrees C. The enhanced binding and fusion were reduced by cytochalasins B and D. Binding of soluble gp120 to CD4-expressing cells was equivalent at 37 and 16 degrees C. Together, these data indicate that during gp120-gp41-induced syncytium formation, initial cell-cell binding is followed by a cytoskeleton-dependent increase in the number of gp120-CD4 complexes, leading to an increase in the avidity of cell-cell binding. The increased number of gp120-CD4 complexes is required for fusion, which suggests that the formation of a fusion complex consisting of multiple CD4 and gp120-gp41 molecules is a step in the fusion mechanism.

Animals↗
PubMed1995

KIM127, an antibody that promotes adhesion, maps to a region of CD18 that includes cysteine-rich repeats.

A series of fusion proteins have been generated between human and mouse CD18. These proteins have been used to carry out preliminary mapping studies on a number of anti-CD18 antibodies including KIM127 an antibody that promotes CD18-dependent adhesion. This antibody maps to a region of the CD18 molecule between amino acids 406 and 570 in a region containing cysteine-rich repeats.

Animals↗