Selective targeting of gene transfer to vascular endothelial cells by use of peptides isolated by phage display.

BACKGROUND: Gene transfer to vascular cells is a highly inefficient and nonselective process, defined by the lack of specific cell-surface receptors for both nonviral and viral gene delivery vectors. METHODS AND RESULTS: We used filamentous phage display to isolate a panel of peptides that have the ability to bind selectively and efficiently to quiescent human umbilical vein endothelial cells (HUVECs) with reduced or negligible binding to nonendothelial cells, including vascular smooth muscle cells and hepatocytes. By direct biopanning on HUVECs and a second approach involving preclearing steps before panning on HUVECs, we isolated and sequenced 140 individual phages and identified 59 peptides. We selected 7 candidates for further investigation by secondary screening of homogeneous phages on a panel of cell types. Using adenovirus-mediated gene transfer as a model gene delivery system, we cloned the peptide SIGYPLP and the positive control peptide KKKKKKK upstream of the S11e single-chain Fv ("adenobody") directed against the knob domain of the adenovirus to create fusion proteins. Adenovirus-mediated gene transfer via fiber-dependent infection was blocked with S11e, whereas inclusion of the KKKKKKK peptide retargeted gene transfer. The peptide SIGYPLP, however, retargeted gene delivery specifically to endothelial cells with a significantly enhanced efficiency over nontargeted adenovirus and without transduction of nontarget cells. CONCLUSIONS: Our study demonstrates the feasibility of using small, novel peptides isolated via phage display to target gene delivery specifically and efficiently to HUVECs and highlights their use for retargeting both viral and nonviral gene transfer to vascular endothelial cells for future clinical applications.

Cell cycle-related variation in subcellular localization of eIF3e/INT6 in human fibroblasts.

The Int-6 gene is a site of mouse mammary tumour virus (MMTV) integration in murine tumours and INT6 protein has been identified independently as a subunit (eIF3e) of the eukaryotic translation initiation factor eIF3. In addition, the protein can interact with two other multi-subunit complexes: the COP9 signalosome (CSN) and the proteasome. The role of INT6 in tumourigenesis is nonetheless currently unclear. Here, using immunofluorescence microscopy, we show that eIF3e/INT6 is localized in part to the nucleus, while other eIF3 components are cytoplasmic. Primary human fibroblasts, but not their transformed counterparts, showed reduced nuclear INT6 staining in some cells, and this reduction was maximal in early S phase. This variation in eIF3e/INT6 may indicate regulated shuttling between cellular compartments and would be consistent with the presence of a nuclear export signal as well as a nuclear localization signal in the protein sequence. Loss of regulation of eIF3e/INT6 redistribution may therefore be a significant feature of malignancy in human cells.

The effect of workplace health services on sex-specific morbidity rates: a note of warning about a possible artefact in apparent female excess morbidity.

It is suggested that the excess morbidity of women may be accounted for by the greater availability to men of treatment at the place of work, for example by an occupational health nurse, which would diminish their rates of attendance in the official health system, and would also diminish the duration and hence prevalence of minor ailments. This would explain the pattern of female excess morbidity occurring exclusively at lower levels of morbidity and explicable predominantly by excess morbidity in women who do not work. Some evidence exists to support this hypothesis but it remains essentially speculative. The purpose of this paper is to warn that it must be taken into account when studying female excess morbidity.

Public health 2020.

The aim of public health is to improve the health of people in communities and in populations (protection from environmental hazards and provision for health needs). The challenge for public health doctors is to re-establish public health leadership of communities, address social and environmental causes of ill health, and link with primary care (a) to improve the health of neighbourhoods and (b) to combine perspectives in commissioning services. Current threats derive from organisational philosophies. For example, focusing on market development does not allow for population based functions and so neglects the main influences on health. The way forward is a network model of organisation in which small teams collaborate with each other to the common good. For example, successful commissioning authorities would have the public health leadership of the director of public health and the support of the chief executive, treasurer, and representatives of primary care, including a medical adviser from the family health services authority.

Periodontal splinting in general dental practice.

Splinting periodontally involved teeth is a technique that has been in use for centuries. This article gives a brief history and review of the literature concerning periodontal splinting and outlines the rationale and indications for the correct application of periodontal splinting in modern dental practice. The common types of splint and clinical techniques involved are described, addressing some of the clinical problems.

Translation initiation and its deregulation during tumorigenesis.

Regulation of protein synthesis at the level of translation initiation is fundamentally important for the control of cell proliferation under normal physiological conditions. Conversely, misregulation of protein synthesis is emerging as a major contributory factor in cancer development. Most bulk protein synthesis is initiated via recognition of the mRNA 5' cap and subsequent recognition of the initiator AUG codon by a directional scanning mechanism. However, several key regulators of tumour development are translated by a cap-independent pathway. Here we review eukaryotic translation initiation, its regulation and the ways in which this regulation can break down during tumorigenesis.

Unusual indelible enamel staining following fixed appliance treatment.

Two cases are described of indelible enamel staining following fixed appliance therapy. The acquired pigmentation occurred in patients with an identifiable enamel defect prior to treatment. The interaction of factors to cause the staining is discussed and it's prevention in future cases highlighted. Subsequent restoration of the affected teeth is shown.

Winter excess morbidity: is it a summer phenomenon?

BACKGROUND: It was hypothesized that winter excess mortality is a feature of ill health produced by exposure to ambient low temperatures, and will be matched by winter excess morbidity. The aim of the study was to test the prediction that winter excess morbidity would be observable and would show a social class gradient with greater excesses in less affluent groups, who are less able to heat their houses or whose lack of a car exposes them more frequently to outdoor cold exposure. METHODS: The study was set in the Metropolitan Borough of Stockport and documented, from routine health services hospital admissions data, winter and summer differences in ACORN-specific, age- and sex-standardized hospital admission rates and ratios, for ischaemic heart disease, directly and indirectly standardized using the Stockport population as the standard. RESULTS: The expected social class gradient in ischaemic heart disease admissions was more clearly observable in the summer than in the winter. Affluent groups showed winter excess morbidity, less affluent groups showed summer excess morbidity. CONCLUSION: The data serendipitously indicate an alternative hypothesis - that winter excess morbidity is a feature of health benefits derived in the summer and differentially available to the more affluent, such as opportunities for outdoor leisure. This hypothesis deserves testing in a study designed for that purpose, although it is not entirely satisfactory as an explanation of existing data.

The 'adenobody' approach to viral targeting: specific and enhanced adenoviral gene delivery.

Recombinant adenoviruses have enormous potential as vectors for gene therapy. They have evolved an efficient method of infection and a wide host range but this leads to concerns about the specificity of gene delivery. In order to target an adenovirus type 5-based vector we have investigated an antibody approach. A virus neutralising scFv antibody fragment was isolated from a phage library and a C-terminal fusion protein with epidermal growth factor (EGF) constructed. This fusion protein, or 'adenobody', bound both to the fibre protein of the adenovirus and to the EGF receptor (EGFR) on human cells, and was able to direct adenoviral binding to the new receptor. Using this system the efficiency of viral infection was markedly enhanced and was targeted to the EGFR. The adenobody-directed infection correlated with the level of EGF receptor expressed on the cells and could be blocked by competition with pure EGF. Peptide inhibition experiments suggest that infection is mediated directly through attachment to the EGFR and does not require penton-integrin interactions. This work shows that the 'adenobody' approach can enhance the efficiency as well as target adenoviral infection and has numerous potential applications for gene therapy.

Targeting of adenovirus to endothelial cells by a bispecific single-chain diabody directed against the adenovirus fiber knob domain and human endoglin (CD105).

The use of adenoviruses for antivascular cancer gene therapy is limited by their low transduction efficiency for endothelial cells. We have developed a recombinant bispecific antibody as a molecular bridge, linking the adenovirus capsid to the endothelial cell surface protein endoglin, for vascular targeting of adenoviruses. Endoglin (CD105), a component of the transforming growth factor beta receptor complex, represents a promising target for antivascular cancer therapy. Endoglin is expressed predominantly on endothelial cells and is upregulated in angiogenic areas of tumors. We isolated single-chain Fv fragments directed against human endoglin from a human semisynthetic antibody library. One of the isolated scFv fragments (scFv C4) bound specifically to various proliferating primary endothelial cells or cell lines including HUVEC, HDMEC, HMVEC, and HMEC. ScFv C4 was therefore used to construct a bispecific single-chain diabody directed against endoglin and the adenovirus fiber knob domain (scDb EDG-Ad). This bispecific molecule mediated enhanced and selective adenovirus transduction of HUVECs, which was independent from binding to the coxsackievirus and adenovirus receptor (CAR) and alpha(v)-integrins. Thus, adenovirus infection was redirected to a new cellular receptor (CD105) and cell entry pathway. These results demonstrate the utility of bispecific single-chain diabodies, which can be produced in large quantities in bacteria, for the retargeting of adenoviruses in cancer gene therapy.