Sj Hollingsworth

Antigen gene transfer to cultured human dendritic cells using recombinant avipoxvirus vectors.

Advances in understanding the role of dendritic cells (DCs) as the major antigen (Ag)-presenting cell type of the immune system combined with the recent development of methods for the ex vivo expansion of human DCs have opened the possibility for the transfer of tumor Ags to DCs with a view toward tumor immunotherapy. In this study, we examined the feasibility of Ag transfer to cultured human DCs using the host range-restricted avipoxvirus, fowlpoxvirus (FWPV). FWPV was found to infect and express a lacZ marker gene in a number of mammalian cell lines of fibroblastic, epithelial, and hemopoietic lineage origins. LacZ recombinant FWPV (rFWPV) was found subsequently to infect human DCs that had been cultured ex vivo from peripheral blood monocytes. Using rFWPV containing lacZ under the control of a vaccinia virus (VV) early/late promoter (p7.5K) and a 10 plaque-forming units per cell multiplicity of infection, >80% of cells expressed the lacZ marker gene. Quantitative analysis showed that the level of expression continued to rise for 5 days postinfection, at which point the experiments were terminated. Replication-competent recombinant VV (rVV) was also shown to be capable of transferring the marker gene to primary DC cultures. However, neither rFWPV nor rVV were able to express transgenes under the control of late viral promoters, indicating that both rFWPV and rVV infections are arrested at an early stage in human DCs. Infection of CD83 + DCs by rFWPV was confirmed by double-staining cytochemistry. We conclude that host range-restricted FWPV can be used efficiently to transfer Ag genes to human DCs ex vivo and may have a role in the development of tumor immunotherapy protocols.

Advanced qRT-PCR technology allows detection of the cholecystokinin 1 receptor (CCK1R) expression in human pancreas.

Objectives: To help clarify the controversy over the detection of expression of the cholecystokinin 1 receptor (CCK1R; CCKAR) in human pancreas. Methods: Applied qRT-PCR to detect CCK1R expression using the SYBR™ green/Smart Cycler® II and the QZyme™ oligonucleotide/ABI PRISM 7500™ systems to detect CCK1R expressed message in highly purified cDNAs from human pancreas and other tissues. Samples of normal pancreas were obtained at operation (pancreaticoduodenectomy; Whipples procedure) and used to ascertain the expression of CCK1R in human tissue and investigate donor individual variability in expression levels by semi-quantitative RT-PCR and scanning densitometry. Results: We present molecular evidence obtained with advanced qRT-PCR technology that clearly establishes CCK1R expression in human pancreas. Amplification variation in individual human samples is documented here. By targeting different stretches of the sequence with several primer pairs, it was observed that SYBR™ green qRT-PCR failed to amplify efficiently over GGA-and GAA-rich nucleotide triplet regions, leading to false negative results. The QZyme™ system quantified the expression with the following distribution: stomach > small intestine ∼ colon > brain ∼ kidney > pancreas. CCK1R expression levels varied from undetectable, to high levels of expression, in individual samples collected from surgical specimens. Conclusion: CCK1R message can be conclusively detected and quantified in human pancreas cDNA by targeting the appropriate nucleotide sequence regions of this gene.

In vitro immune modulation by antibodies coupled to tumour cells

Modification of autologous tumour cells to express the immune costimulator B7.1 is a potential strategy for immunotherapy of cancer. Previously, this has involved introduction of genetic material into cells, in vitro culture, and confirmation of the protein product on the cell surface. This is possible only if sufficient tumour is obtainable and efficiently modified in a short time. Whilst progress has been made on ex vivo tumour cell culture and transfection/infection procedures there are still tumour types for which the present means of gene transfer are not efficient enough. We describe a highly efficient in vitro procedure for the modification of over 99% of the cells in a population, allowing the expression of cell surface proteins with potential immune modulatory activities. This procedure, which can be completed in as little as 24 h with no upper limit on cell number, utilizes succinimide esters to label cell surface proteins with biotin covalently. Biotinylated cell membrane proteins then anchor an avidin bridge for immobilizing protein G′-biotin. This can serve to bind immunoglobulin (Ig) molecules via their Fc region such that the variable region of the antibody is freely and functionally available. In the present study the binding of a stimulatory mouse anti-human CD28 monoclonal antibody to the surface of tumour cells is used to show that the modified cells are capable of co-stimulating T cells in vitro. The simplicity of the method, and the use of common reagents, represents a further step towards a realistic, truly ‘off-the-shelf’, nongene immunotherapy protocol.

The effect of combined expression of interleukin 2 and interleukin 4 on the tumorigenicity and treatment of B16F10 melanoma

The recent use of interleukin 2 (IL-2) and interleukin 4 (IL-4) single cytokine modified tumour cells in rodent models has demonstrated a potential use of these cytokines to produce autologous cancer cell vaccines. Here we compare the potential therapeutic benefit of transduction with IL-2 or IL-4 alone, and combined IL-2 + IL-4 in B16F10 cells, a murine malignant melanoma of poor immunogenicity. Transduction of B16F10 cells (MHC class I and II negative) to express either IL-2 or IL-4 alone delays the formation of tumours, IL-4 being more effective than IL-2. However, combined expression of IL-2 + IL-4 reduces tumorigenicity more than either cytokine alone. The eventual formation of tumours may result from loss of gene expression, and preliminary results suggest methylation of the retroviral long terminal repeat (LTR), rather than loss of the transduced DNA sequences. Histological examination of tumours expressing either IL-2 or IL-4 alone shows a non-specific inflammatory reaction with an increased tissue infiltrate of immune effectors (monocytes/macrophages, lymphocytes, granulocytes) localised around the tumour. In comparison, when cells expressing combined IL-2 + IL-4 were injected there were more granulocytes present, and perhaps more importantly, these were mainly localised within the tumour. The benefit of combined IL-2 + IL-4 expression results from a local rather than systemic effect as the growth of tumours from cells expressing IL-2 or IL-4 alone injected at distant sites was comparable with a single inoculation of cells expressing either cytokine alone. However, when cells expressing single cytokines IL-2 or IL-4 were mixed and injected at the same site, in comparison with the clonal population of cells expressing combined IL-2 + IL-4, tumour growth was characteristic of IL-4 alone rather than IL-2 + IL-4. Treatment of established tumours with a single injection of lethally irradiated tumour cells expressing IL-2 + IL-4 was sufficient to either reject tumours, or at least delay further tumour development. Furthermore, treatment stimulated an initial non-specific immune reaction that lead to a systemic immunity. Lethally irradiated wild-type cells were also successful in treating some established tumours, although this did not induce any systemic immunity. However, although successful in treatment studies, neither wild-type nor combined IL-2 + IL-4 expressing cells were able to vaccinate animals against a subsequent challenge with live wild-type tumour. These results indicate a potential therapeutic benefit with the use of combination IL-2 + IL-4 transduction of autologous cancer cells.

Neuromedin U stimulates contraction of human long saphenous vein and gastrointestinal smooth muscle in vitro.

The neuropeptide Neuromedin U (NMU) stimulates smooth muscle contraction, and modulates local blood flow and adrenocortical function via two endogenous receptors, NMU1 and NMU2. Although its amino-acid sequence is highly conserved across species, the physiological effects of NMU are variable between species and little is known of its effects on human tissues. We have examined the contractile effects of NMU-25 on human smooth muscles of the gastrointestinal (GI) tract (ascending colon, gallbladder) and long saphenous vein (LSV) using in vitro organ bath bioassays. From LSV, ileum, gallbladder, caecum and colon, NMU receptor transcripts were amplified by RT-PCR and expression levels were determined by semi-quantitative scanning densitometry. NMU-25 produced a concentration-dependent, sustained contraction of isolated smooth muscle (p[A](50)+/-s.e.m., ascending colon, 8.93+/-0.18; gallbladder, 7.01+/-0.15; LSV, 8.67+/-0.09). NMU1 and NMU2 receptor transcription was detected in all tissues; transcription of both receptors was similar in gallbladder, but NMU1 receptor transcription was predominant in the sigmoid colon and LSV. In summary, these studies indicate that NMU may control tone in the human GI tract and LSV through an action on smooth muscle. Development of NMU receptor subtype-selective ligands will aid the further elucidation of the physiological roles of NMU and its two receptors.

Primary Varicose Veins in the Presence of an Intact Sapheno-Femoral Junction:

Objective: To determine the incidence of primary varicose veins (VVs) occurring in the presence of a competent sapheno-femoral junction. Methods: A retrospective analysis of venous duplex scans was performed for all patients referred to the Vascular Unit, The Middlesex Hospital, London, for assessment of primary VVs, over an 18 month period from 1998 to 2000. Results: One thousand nine hundred and eleven patients with primary VVs referred to hospital for treatment were assessed. Their median age was 52 years (interquartile range 39–64 years). The female:male ratio was 1.92:1. Of these, 43.5% had primary VVs in the presence of a competent sapheno-femoral junction (of whom 70.2% were female). Of those patients with primary VVs in the presence of an incompetent sapheno-femoral junction, 62.4% were female. The incidence of primary VVs did not increase overall with age. The peak incidence for women was between 31 and 60 years of age, whilst that for men was between 51 and 70 years. A similar pattern for age distribution was seen irrespective of sapheno-femoral junction status. Men had a reduced incidence of primary VVs in the presence of an intact sapheno-femoral junction compared with incompetent junctions over the age range of 31–60 years. Conclusions: Primary VVs can readily develop in the presence of a competent sapheno-femoral junction. This occurs most frequently in women below the age of 50 years.

Activity (transcription) of the genes for MLH1, MSH2 and p53 in sporadic colorectal tumours with micro-satellite instability

Micro-satellite instability (MSI) is relevant in the management of colorectal cancers (CRC) and relies on analysis of gene mutations, or production of the proteins involved in DNA mismatch repair (e.g. MLH1, MSH2). p53 mutation is also relevant in MSI, but high-level CRC (MSI-H) demonstrate fewer mutations than low-level (MSI-L) or stable (MSS) cancers. Recently, the importance of gene activity (transcription) in MSI has been identified, where rather than being mutated genes have been downregulated. In this study, 67 sporadic CRC and eight samples of normal bowel were analysed for MSI status (by SSCP) and levels of MLH1, MSH2 and p53 gene transcription (by RT–PCR and scanning densitometry). Micro-satellite instability correlated with gender and site, with more MSI-H CRC in females (P<0.02) and in the right colon (P<0.04). In MSI-H, p53 transcription was markedly reduced (P<0.003). Compared to normal bowel, MLH1 transcription was elevated in all cancers (P<0.01), while MSH2 transcription was elevated only in MSI-H (P<0.04). There was a direct correlation between MLH1 and MSH2 transcription (P<0.001). Although fewer mutations are reported in MSI-H than MSI-L/MSS, these results suggest that reduced p53 transcription might account for decreased tumour suppression in MSI-H. The direct correlation between MLH1 and MSH2 transcription suggests that control of these genes might be coordinated.

Use of a Needleless Injection System for Digital Ring Block Anesthesia

Digital ring block anesthesia, which is frequently used before surgery for ingrown toenails, is often extremely uncomfortable for patients and can be the most distressing aspect of the procedure. The authors used a novel needleless injection device to induce digital anesthesia before surgery and compared it in terms of patient discomfort and preference with use of a standard needle and syringe for injection in individuals undergoing simultaneous bilateral nail procedures. Use of the needleless device significantly reduced the pain associated with this procedure and was preferred over use of a standard needle and syringe by all individuals. Other potential advantages of a needleless injection system are discussed.

The 'Wound Boot': A preliminary assessment of a novel device for the management of leg ulcers

Objective: A preliminary assessment of a novel device, the ‘wound boot’, in the management of leg ulcers. Procedures: Eight subjects with ulcers wore the boot for 5–14 days. Ulcers were cleaned with saline only. No other dressings were applied. Questionnaires examined comfort and ease of use and asked for suggestions for modifications. Additionally, nurses examined ‘time taken to clean and dress ulcers’ in comparison with standard dressings. Although not a primary end-point, ulcer healing was noted. Results: For all, the boot was comfortable, easy to use and preferable to standard dressings. Ulcer-associated odour was eliminated entirely. A significant reduction was seen in the nursing time taken to clean and dress ulcers. Occasionally, excess exudate was problematic, with the boots absorptive materials insufficient. In two cases, where the ‘boot’ was applied for 14 days, significant wound healing was seen. Conclusions: The prototype ‘boot’ helped significantly in leg ulcer management. Modifications based upon this assessment will allow a second prototype to be evaluated fully.

The effects of heparin on cultured explants of varicose long saphenous vein

Objective: To examine the effects of heparin on smooth muscle cells (SMCs) in explants of varicose, long saphenous vein (LSV). Procedures: Explants of varicose LSV were cultured for 7 days either alone, or with heparin at 10, 100 or 1000 IU/ml (Monoparin). At 7 days, cultured explants were analysed for changes in intimal and medial thickness and by immuno-histochemistry. Comparisons were made with explants at initial isolation and with similar, cultured explants of normal LSV. Results: In normal LSV, by day 7, SMC-derived neo-intimal hyperplasia developed (p<0.01) with an increase in intimal thickness (p<0.02) and a decrease in medial thickness (p<0.001). Heparin at 10 and 100 IU/ml further enhanced this neo-intima formation (p<0.001). In contrast, at 1000 IU/ml, heparin inhibited neo-intima formation. In varicose explants, the pattern of intimal and medial changes was different. At isolation, varicose LSVs had substantially thicker intimal layers (p<0.001). When cultured alone, a thicker media developed (p<0.001) but there was little change in intimal thickness. Heparin at all concentrations had no effect on the thicker medial development seen in controls but did, however, reduce intimal thickness (p<0.005). Conclusions: The response to heparin in explants of varicose LSV is different from that of normal LSV, which is biphasic and complex.