Inhibition of tissue factor activity reduces the density of cellular network formation in an in vitro model of angiogenesis.

Tissue factor (TF) is a transmembrane glycoprotein that was originally recognized for its ability to initiate the extrinsic pathway of coagulation. More recently, additional functions of TF in cellular signalling have emerged, notably the role of TF in vasculogenesis and angiogenesis. We have described previously the ability of a peptide derived from the apolipoprotein B100 (apoB100) moiety of low-density lipoproteins (KRAD14) to inhibit the procoagulant function of TF. In this study, we demonstrate the ability of the KRAD14 peptide to attenuate the density of cellular network structures of T24 cells grown on specialized matrix (Matrigel). In addition, an alternative inhibitor of TF activity, the TF8 5G9 antibody, also reduces the density of cellular network formation. Targeted use of a stable structural equivalent of the KRAD14 peptide may thus prove useful in the prophylactic treatment of diseases whose pathologies feature the formation of neovascular tissue, e.g. tumour growth and metastasis, rupture of atherosclerotic plaques and retinopathy secondary to diabetes.

The role of the C-terminal domain in the inhibitory functions of tissue factor pathway inhibitor.

Tissue factor pathway inhibitor (TFPI) inhibits the activity of coagulation factors VIIa and Xa through Kunitz domains, thereby inhibiting the activity of tissue factor. However, it has been shown that the C-terminal of this inhibitor is essential for the maximal anticoagulant activity of TFPI. We have investigated the endogenous ability of the C-terminal of TFPI to influence coagulation. A synthetic peptide corresponding to residues 254-265 within the C-terminal of TFPI was prepared and shown to be capable of inhibiting tissue factor pathway by preventing the activation of factor VII. Mutational analysis of the peptide revealed the identity of the key lysine residues.

A collaborative approach to management of chlamydial infection among teenagers seeking contraceptive care in a community setting.

OBJECTIVES: To develop and assess a coordinated model of care for effective management of genital chlamydial infection in young women, identified through a selective screening programme in a community based teenage health clinic. METHODS: Selective screening for genital Chlamydia trachomatis was undertaken among young women aged 13-19 years who were having a routine cervical smear test, being referred for termination of pregnancy, or who reported behavioural risk factors, for, and/or symptoms of, genital infection. Collaboration among family planning, genitourinary medicine (GUM), and public health staff was used to enhance management of infected individuals, with particular focus on partner notification. RESULTS: 94 young women had confirmed genital chlamydial infection, representing 11% of those tested. All index patients received appropriate antibiotic therapy and follow up; 93 (99%) of these were counselled by a health adviser, of whom 62 (66%) were able to provide sufficient details for partner notification, resulting in treatment of male partners associated with 51 (82%) of these young women. Younger age (< or = 16 years) was significantly associated with delay in attending for treatment. CONCLUSIONS: Effective management of genital chlamydial infection is achievable in settings outside GUM clinics using a collaborative approach which incorporates cross referencing between community based services and GUM clinics.

Comparison of the inhibitory effects of ApoB100 and tissue factor pathway inhibitor on tissue factor and the influence of lipoprotein oxidation.

The procoagulant activity of tissue factor is regulated by circulating inhibitors such as tissue factor pathway inhibitor (TFPI) and LDL. These 2 inhibitors also readily associate making the distinction between their activities difficult. We have examined the relative contributions of intact and C-terminal truncated TFPI and ApoB100. By following the inhibitory potential of the preparations, over a period of 120 minutes, it was demonstrated that TFPI and LDL-resembling particles inhibited tissue factor at different rates. TFPI was found to be a short, fast-acting inhibitor, whereas the action of LDL-resembling particles was more prolonged but slower. The oxidation of LDL has been closely associated with the development of cardiovascular disease, including atherosclerosis and thrombosis. Positively charged amino acids, particularly lysine residues, are prone to alterations via the formation of adducts by lipid peroxidation products. These residues are important in the inhibition of tissue factor activity by ApoB100. They also play an important role in the inhibitory Kunitz domains of TFPI. We have shown that the decline in the ability of LDL to inhibit tissue factor was as a result of modifications in LDL arising from oxidation. By examining the effects of oxidation on full-length and C-terminal truncated TFPI bound to LDL-resembling particles, we found that TFPI is only affected when in close association with ApoB100. C-terminal truncated TFPI was not affected significantly by oxidation. Finally, chemical modification of lysine and arginine residues reduced the overall inhibition of tissue factor by TFPI. We propose that TFPI and LDL act separately to inhibit tissue factor in vivo. However, the oxidation of LDL can alter both the endogenous activity of ApoB100 and reduce that of closely associated TFPI, compromising normal hemostasis.

Identification of a domain in apolipoprotein B-100 that inhibits the procoagulant activity of tissue factor.

The ability of low-density lipoprotein (LDL) to inhibit the procoagulant activity of tissue factor is mediated by a direct protein-protein interaction involving apolipoprotein (apo) B-100. A lysine-rich sequence within apo B-100 (residues 3121-3217), which we have termed lysine-rich apo B-100-derived (KRAD)-98 peptide, may be responsible for its activity. Within this region, residues 3147-3160 (KRAD-14) contain an exceptionally high proportion of positive amino acids. Both recombinant KRAD-98 and KRAD-14 peptides inhibited the procoagulant activity of tissue factor by preventing the activation of factor VII. KRAD-14 also inhibited the prothrombinase components, factors Xa and V. In comparison with the parent protein (apo B-100), KRAD-14 peptide displayed a 20-fold enhancement in the rate of inhibition, whereas KRAD-98 peptide exhibited a rate closer to that of apo B-100. Mutational analysis of KRAD-14 peptide revealed three adjacent amino acids, alteration of which greatly reduced the inhibitory potential of this peptide. A peptide derived from tissue factor (residues 58-66) was found to act co-operatively with tissue factor itself, but also augmented the inhibition of tissue-factor activity by apo B-100. In conclusion, LDL may be a physiological regulator of haemostatic mechanisms through the interactions of lysine-rich domains of apo B-100 with tissue factor.

Evaluation of a randomized controlled trial of HIV and sexually transmitted disease prevention in a genitourinary medicine clinic setting.

OBJECTIVE: To evaluate outcomes of a sexual health promotion intervention to prevent transmission of sexually transmitted diseases (STD) including HIV among genitourinary medicine clinic attenders. DESIGN: A randomized controlled trial using pre-test and post-test measurements after 4 months, with clinical follow-up at 18 months. SETTING: A genitourinary medicine clinic in Nottingham, UK. PATIENTS: A total of 492 patients were randomly assigned either to an individually focused counselling and skills training intervention, including written materials (n = 148), to receive written materials only (n = 162), or to usual clinic procedure (n = 182). INTERVENTION: Social learning theory provided the theoretical framework for the intervention, which was informed by previous research in this setting, and aimed to alter perception of risk for HIV infection, increase knowledge and attitudes to condoms, and increase condom use. MAIN OUTCOME MEASURES: The main outcomes of interest were self-reported condom use and behaviour change. Other outcomes of interest were knowledge and attitudes to condoms, and re-attendance at the clinic with a diagnosis of STD. RESULTS: Intervention subjects were significantly more likely than controls to report carrying condoms when anticipating sexual intercourse with a new sexual partner (P < or = 0.05), and were more likely to perceive themselves at risk of HIV infection (P < or = 0.001). There were no significant effects of the intervention on levels of knowledge about correct condom use, attitudes to condoms, self-reported condom use or incidence of STD. CONCLUSIONS: The limited effectiveness of the intervention suggests that condom promotion should continue, but that additional investment in clinic-based health promotion is unlikely to result in consequent health gain.

Modification of tissue factor by peroxynitrite influences its procoagulant activity.

Peroxynitrite, a reactive oxidising species resulting from a reaction between nitric oxide and the superoxide anion, modifies proteins by nitration of certain amino acids such as tyrosine. Tissue factor (TF), a transmembrane protein, is expressed on cells under inflammatory conditions and initiates the coagulation cascade. The extracellular domain of TF is rich in tyrosine. Exposure of recombinant TF and cellular TF to peroxynitrite was associated with a reduction in procoagulant activity. This was accompanied by an elevated level of nitrotyrosine residues. Peroxynitrite may have a protective role by attenuation of the thrombotic properties of TF.

Alterations in the structure of apolipoprotein B-100 determine the behaviour of LDL towards thromboplastin.

Apolipoprotein B-100 acts as an inhibitor of thromboplastin activity independently of the tissue factor pathway inhibitor (TFPI) associated with plasma lipoproteins. Analysis of the primary structure of Apo B-100 showed a higher than expected occurrence of lysine groups in the receptor-binding region. In order to demonstrate the participation of lysine groups of Apo B-100 in the inhibition of thromboplastin, thromboplastin and Apo B-100 were incubated together in the presence of poly-L-lysine, poly-L-arginine, lysine and arginine monomers. The inhibition of thromboplastin by Apo B-100 was completely suppressed in the presence of poly-L-lysine. Poly-L-arginine was found to be less effective and neither lysine or arginine monomers had any significant effect on the inhibitory effect of Apo B-100. Alterations in the structure of Apo B-100 reconstituted in lipid vesicles resembling LDL, brought about by lipid peroxidation and lipid loading were examined by means of Fourier transform infra-red spectroscopy. It was found that, upon oxidation without the addition of cupric ions, the apolipoprotein attains a more exposed conformation with an increase in alpha-helical structure. This increase occurred at the expense of beta-structure. On lipid loading, an increase in beta-structure at the expense of the alpha-helix, was demonstrated. It is therefore proposed that the variable action of LDL towards thromboplastin derives from alterations in the secondary structure of the Apo B-100, particularly the receptor-binding region.

The mechanism of inhibition of factor III (thromboplastin) activity by apolipoprotein B-100. Protein-protein interactions.

Factor III (thromboplastin) activity is inhibited by apoB-100, but the mechanism of inhibition is unknown. By examining the effect of purified apoB-100 on factor III activity, we showed that apoB-100 can inhibit factor III via a different mechanism from that caused by the issue-factor pathway-inhibitor, which is mainly carried on the surface of lipoproteins. Although the presence of calcium ions and factors X and VII may enhance the rate of inhibition, they are not a prerequisite for the inhibition of factor III by apoB-100. In addition, by investigating the changes in the UV spectra of apoB-100 on interaction with factor III and factors X and VII and by assigning the shifts in absorption spectra to particular amino acids, we showed that these interactions involve negative and positive residues within these proteins. By following the rates of interactions between apoB-100 and either factors III, X, VII, a two-step mechanism for the inhibition process involving factors X and VII was postulated. In this mechanism, the primary interaction of apoB-100 with factor III is followed by a rate-limiting step that can be accelerated by the presence of either factor X or VII and leads to the inhibition of factor III. Furthermore, a computer-based analysis of the sequences of factor III revealed a possible binding site for apoB-100.

Physicians' perceived value of international AIDS conferences and attitudes towards patient attendance.

OBJECTIVE: To determine the perceived value of attendance at an International AIDS Conference and attitudes towards the effect of patient attendance on the conference. DESIGN: A confidential, self-administered questionnaire. PARTICIPANTS: 102 physicians from the United Kingdom who attended the VIII International AIDS Conference in Amsterdam. RESULTS: There was an 84% response rate. 50% reported increased motivation for clinical work and 57% for research. Physicians with a lower HIV positive patient workload found the conference more valuable for finding out the latest information on HIV, compared with those with a higher workload (p = 0.04). Those with a higher patient workload found the conference more useful for increasing motivation for research than those with a lower HIV workload (p = 0.047). Conference attendance was felt to reduce burnout by 48% of respondents. The majority (55%) would prefer a more traditional meeting. Patient attendance was seen as improving the standard of discussion of ethical and political issues but not on medical or scientific issues. CONCLUSIONS: The International AIDS Conferences are perceived as useful by those UK physicians who attend, but most would prefer a more "traditional" scientific meeting. Whilst patient participation was not seen as useful for medical or scientific discussions, it was felt to improve discussion of ethical and political issues. A smaller more focused conference may be equally useful to UK physicians.

AIDS-related risk perception and sexual behaviour among sexually transmitted disease clinic attenders.

Perception of risk has been suggested as an important element of sexual behaviour change among people who engage in behaviours which place them at risk of HIV infection. A study of the relationship between perception of risk of HIV infection and risk-related sexual behaviours was conducted in a genitourinary medicine clinic. The sample comprised 767 patients attending over a 3-month period; data collection was by self-completed questionnaire. A total of 574 questionnaires were suitable for analysis, representing a response rate of 75%. The majority of people in the sample reported behaviours which increased their risk of HIV infection, but only 19% (n = 112) of the sample perceived themselves to be personally 'at risk', despite adequate knowledge of HIV transmission and methods of risk reduction. Significant differences between social class groups were found for knowledge scores, with highest scores among professionals and lowest among unemployed subjects (Kruskal Wallis test chi 2 = 24.6, P less than or equal to 0.001). Increasing age was significantly associated with better knowledge; significantly more young people aged 16-20 years who did not perceive themselves 'at risk' (64%), had lower knowledge scores than older people who did not perceive themselves 'at risk' (41%). Among heterosexuals who reported having sex with other people in addition to their regular partner, 79% did not perceive themselves as 'at risk' of HIV infection, and of these, 64% reported only infrequent condom use with casual sexual partners. Significantly more heterosexual men (67%) than women (44%) reported multiple sex partners.(ABSTRACT TRUNCATED AT 250 WORDS)

The reliability of self-reported sexual behaviour.

The quality of data collected by surveys of sexual behaviour may be subject to sources of error involving reliability, validity, and non-participation bias. A preliminary study of sexual behaviour, including perception of risk for HIV infection, knowledge of HIV transmission and attitudes towards AIDS, was conducted in a genitourinary medicine clinic. The sample comprised 767 patients attending clinic over a 3-month period; comparisons were made between different methods of data collection (questionnaires and interviews) and between responders and non-responders. High levels of agreement were found between questionnaire and interview. Some sexual behaviours were more frequently reported at interview. No significant differences were found between responders and non-responders to either questionnaire or interview.

Standardised trunk asymmetry scores. A study of back contour in healthy school children.

This paper reports a new method for expressing numerically asymmetry of the contour of the back in a forward-bending position. Information is given at three spinal levels (T8, T12 and L3) for 636 schoolchildren aged 8 to 15 years. Rib-hump and lumbar-hump scores were standardised to create trunk asymmetry scores (TASs) making comparison possible between children of different age, size and sex. Two groups of children were defined: those with clinically straight spines (585 children); and those with clinical evidence of lateral spinal curves (51 children). In the children with clinically straight spines the main findings were: about 1:4 had objectively detectable rib and lumbar humps; female-to-male ratios were 1.2:1 for the thoracic region and 1.4:1 for the lumbar region; right humps were about 10 times more common than left; TASs in the boys and girls at each spinal level had normal distributions about means to the right of zero (where zero represents perfect symmetry); at T8 and T12, a wider scatter of TASs in girls than in boys; at L3, larger TASs in girls than in boys; a relation between shortening of one lower limb and a contralateral hump on the back; and no relation to age (except at L3), stature (corrected for age) or handedness. The findings are discussed in relation to possible causes of back contour asymmetry, early diagnosis of scoliosis by screening, sexual dimorphism and significance for the pathogenesis of idiopathic scoliosis. Ten children with clinically straight spines and larger TASs, and 42 out of 51 children with clinical evidence of lateral spinal curves in the forward-bending position attended for radiographic examination. Twelve children had "scoliosis curves" of 11 degrees or more as defined by the Scoliosis Research Society. The results are reported in relation to TASs, spinal curve angle (Cobb) and vertebral rotation.

Café-au-lait spots in schoolchildren.

This paper reports a study of café-au-lait spots of a minimum diameter of 1 cm in 732 white schoolchildren. Three groups were identified, according to the number of café-au-lait spots on each child: (1) those with none (74%), (2) those with fewer than 5 (25%), and (3) those with at least 5 (5 children, 2 considered to be normal, and 3 siblings each presumed to have neurofibromatosis, one having died from leukaemia). Excluding the last group, the number of café-au-lait spots in the sample was not significantly related to age or sex. Some support is given for using the number of café-au-lait spots as an empirical threshold to diagnose neurofibromatosis.

Survival of large replanted segment of upper lip and nose. Case report.

A child had essentially her entire lip and most of her nose bitten off in one piece by a dog. This was replanted by microvascular anastomoses. Most of the fragment survived.