Oral propranolol in the treatment of proliferating infantile haemangiomas: British Society for Paediatric Dermatology consensus guidelines.

BACKGROUND: Infantile haemangiomas (IH) are the most common vascular tumours of infancy. Despite their frequency and potential complications, there are currently no unified U.K. guidelines for the treatment of IH with propranolol. There are still uncertainties and diverse opinions regarding indications, pretreatment investigations, its use in PHACES (posterior fossa malformations-haemangiomas-arterial anomalies-cardiac defects-eye abnormalities-sternal cleft and supraumbilical raphe) syndrome and cessation of treatment. OBJECTIVES: To provide unified guidelines for the treatment of IH with propranolol. METHODS: This study used a modified Delphi technique, which involved an international treatment survey, a systematic evidence review of the literature, a face-to-face multidisciplinary panel meeting and anonymous voting. RESULTS: The expert panel achieved consensus on 47 statements in eight categories, including indications and contraindications for starting propranolol, pretreatment investigations, starting and target dose, monitoring of adverse effects, the use of propranolol in PHACES syndrome and how to stop treatment. CONCLUSIONS: These consensus guidelines will help to standardize and simplify the treatment of IH with oral propranolol across the U.K. and assist in clinical decision-making.

The European TREatment of severe Atopic eczema in children Taskforce (TREAT) survey.

BACKGROUND: There is a paucity of evidence for the use of systemic agents in children with atopic eczema refractory to conventional therapy, resulting in considerable variation in patient management. OBJECTIVES: The European TREatment of severe Atopic eczema in children Taskforce (TREAT) survey was established to collect data on current prescribing practice, to identify factors influencing the use of specific systemic agents, and to inform the design of a clinically relevant intervention study. METHODS: Consultant physician members of the paediatric dermatology societies and interest groups of eight European countries were invited to participate in a web-based survey. The multiple-response format questionnaire collated data on clinical practice in general, as well as detailed information on the use of systemic agents in refractory paediatric atopic eczema. RESULTS: In total, 343/765 members (44·8%) responded to the invitational emails; 89·2% were dermatologists and 71% initiate systemic immunosuppression for children with severe atopic eczema. The first-line drugs of choice were ciclosporin (43·0%), oral corticosteroids (30·7%) and azathioprine (21·7%). Ciclosporin was also the most commonly used second-line medication (33·6%), with methotrexate ranked as most popular third choice (26·2%). Around half of the respondents (53·7%) replied that they routinely test and treat reservoirs of cutaneous infection prior to starting systemic treatment. Across the eight countries, penicillins were the first-line antibiotic of choice (78·3%). CONCLUSIONS: In the absence of a clear evidence base, the European TREAT survey confirms the wide variation in prescribing practice of systemic immunosuppression in refractory paediatric atopic eczema. The results will be used to inform the design of a randomized controlled trial relevant to patient management across Europe.

Effect of Nippostrongylus brasiliensis L3 ES on inflammatory mediator gene transcription in lipopolysaccharide lung inflammation.

The anti-inflammatory properties of parasitic helminths have been largely linked to their excretory-secretory (ES) products. Some studies have noted a lack of TNF-alpha production and limited recruitment of neutrophils into the lungs after Nippostrongylus brasiliensis infection. We previously reported that instillation of ES from L3 larvae of N. brasiliensis to the lungs could inhibit the recruitment of neutrophils on a background of LPS-induced inflammation. A similar reduction in neutrophil recruitment was observed in this study. This reduction was associated with the significant inhibition in gene transcription of the adhesion molecule, ICAM-1, and the chemokine, MIP-2 in bronchoalveolar lavage (BAL) cells. The LPS-stimulated gene transcription of the pro-inflammatory cytokines TNF-alpha and IL-1beta was also significantly reduced by L3 ES. Inducible nitric oxide synthase (iNOS) is normally elevated in classically activated macrophages, however, in this case gene transcription of iNOS was inhibited by L3 ES and may suggest a phenotype change to anti-inflammatory. The general inhibition of pro-inflammatory mediators observed in this study suggests that infective stage L3 larvae excrete and/or secrete inhibitory products capable of modifying the normally potent LPS inflammatory response.

The application of enhanced chemiluminescence to membrane-based nucleic acid detection.

The ECL gene detection system is a novel, sensitive, non-radioactive system for the detection of nucleic acid hybridized on both nylon and nitrocellulose membranes. It is characterized by direct labeling of probe sequences with horseradish peroxidase combined with an enhanced chemiluminescent (ECL) detection reaction; the light output is captured on blue-light sensitive film. The application of the system to a range of standard molecular biology hybridization techniques is described.

The surface lipid of parasitic nematodes: organization, and modifications during transition to the mammalian host environment.

The biophysical properties of the surface lipid of a range of nematode species and their developmental stages were examined, using fluorescent lipid probes and fluorescence recovery after photobleaching (FRAP). These methods can be applied to living, intact parasites, and the analysis confined to lipid on the outermost surface. In all cases, surface lipid was unusual in its selectivity for the insertion of the lipid probes. In addition, a polar lipid probe was generally not free to diffuse in the plane of the surface, in contrast to a non-polar lipid probe which was free to diffuse. This is evidence that the surface lipid layer is heterogeneous, and possibly comprises lipid domains. The infective larvae of Acanthocheilonema viteae, Nippostrongylus brasiliensis, Trichinella spiralis and Ostertagia ostertagi were found to exhibit a rapid change in lipophilicity upon exposure to conditions simulating entry into a mammalian host environment. Parasitic nematodes, therefore, present their hosts not only with a highly unusual biological surface, but also one which can be rapidly re-organised upon a change of environment.

The developmental regulation and biosynthesis of GPI-related structures in Leishmania parasites.

Most macromolecules on the surface of Leishmania parasites, including the major surface proteins and a complex lipophosphoglycan (LPG) are anchored to the plasma membrane via GPI glycolipids. Free glycoinositol-phospholipids (GIPLs) which are not linked to protein or phosphoglycan are also abundant in the plasma membrane. From structural and metabolic labeling studies it is proposed that most Leishmania species express three distinct pathways of GPI biosynthesis. Some of these pathways (i.e. those involved in the protein and LPG anchor biosynthesis) are down-regulated during the differentiation of the insect (promastigote) stage to the mammalian (amastigote) stage. In contrast, the GIPLs are expressed in high copy number in both developmental stages. Based on analysis of the lipid moieties of the different GPI species it is possible that the pathways of GPI anchor and GIPL biosynthesis are located in different subcellular compartments. The relative flux through the GIPL and LPG biosynthetic pathways has been examined in L. major promastigotes. These studies showed that while the rate of synthesis of the GIPLs and LPG is similar, LPG is shed more rapidly from the plasma membrane and has a higher turnover. The possible metabolic relationship between the GIPL and LPG biosynthetic pathways is discussed.

Testing incontinence pads using single-case research designs.

This article is predominantly concerned with the value of single-case research designs for generating nursing knowledge. The argument in favour of single-case research designs is made with reference to the testing of a range of body-worn incontinence pads in a group of cognitively impaired elderly women in two clinical settings.

Glycoinositolphospholipids of Leishmania major inhibit nitric oxide synthesis and reduce leishmanicidal activity in murine macrophages.

Murine macrophages express high levels of inducible nitric oxide (NO) synthase and produce large amounts of nitric oxide when activated with interferon-gamma and lipopolysaccharide in vitro. Nitric oxide is a mediator of a variety of biological functions including microbicidal activity against the protozoan parasite Leishmania species. Glycoinositolphospholipids (GIPL) are the predominant surface glycolipids in both developmental stages of Leishmania major. We report here that GIPL can inhibit the synthesis of NO in a time- and dose-dependent manner. In contrast, lipophosphoglycan, which is present in the promastigote stage did not inhibit NO synthesis. GIPL-treated macrophages also showed markedly reduced leishmanicidal activity. The majority of the inhibitory activity of GIPL was found within the alkylacylglycerol moiety of the GIPL molecule. These data, therefore, suggest that GIPL may contribute towards the survival of the parasite in the immune hosts.

Cytokines and nitric oxide as effector molecules against parasitic infections.

Nitric oxide (NO) derived from L-arginine by the catalytic action of inducible NO synthase (iNOS) plays an important role in killing parasites. Many cell types express high levels of iNOS when activated by a number of immunological stimuli which include interferon-gamma (IFN-gamma), tumour necrosis factor alpha, and lipopolysaccharide. IFN-gamma is typically produced by the Th1 subject of CD4+ T cells, whose differentiation depends on interleukin-12 (IL-12) produced by macrophages. Mice with a disrupted iNOS gene were highly susceptible to Leishmania major infection compared with similarly infected control wild-type mice. The mutant mice developed significantly higher levels of TH1-cell response compared with the control mice, suggesting that NO is likely to be the effector molecule in the immunological control of this and other intracellular parasitic infections. To ensure their survival, the Leishmania parasites have evolved effective means to inhibit NO synthesis. The highly conserved major surface glycolipids, glycoinositol-phospholipids and lipophosphoglycan (LPG), of Leishmania are potent inhibitors of NO synthesis. Furthermore, LPG can also inhibit IL-12 synthesis, thereby indirectly blocking the induction of iNOS. The evolutionary and therapeutic implications of these findings are discussed.

External stimuli and intracellular signalling in the modification of the nematode surface during transition to the mammalian host environment.

Previous work has shown that the surface of infective larvae of parasitic nematodes will not bind the fluorescent lipid analogue 5-N-(octadecanoyl)aminofluorescein (AF18) until after exposure of the parasite to mammalian tissue-culture conditions. In this study, culture media which are permissive or non-permissive for the acquisition of lipophilicity for AF18 were altered in order to examine possible stimuli involved. This showed that external alkaline pH and high sodium ion concentration were highly stimulatory. The internal signalling pathways which may be involved in the surface alteration were then examined using agents which are known to affect intracellular signalling in mammalian cells. The results indicated that elevation of cGMP levels was stimulatory whereas inhibition of a putative Na+/H+ antiporter or calcium mobilization was inhibitory, and it is argued that high intracellular levels of cAMP may be inhibitory. Whilst the precise effects of the agents used on nematode cells remain to be established, these results provide a framework for the examination of the processes involved in the modification of the nematode surface which takes place immediately after the infection event.

Inhibition of neutrophil recruitment by ES of Nippostrongylus brasiliensis.

It has been reported that excretory-secretory (ES) material from the parasitic nematode Nippostrongylus brasiliensis has potential modulatory effects on the host's immune system. We observed that intratracheal instillation of ES from the L3 stage of the parasite reduced neutrophil numbers in LPS-induced inflammation as assessed by bronchoalveolar lavage.

Biosynthesis of the glycolipid anchor of lipophosphoglycan and the structurally related glycoinositolphospholipids from Leishmania major.

The major macromolecule on the surface of the protozoan parasite Leishmania major is a lipophosphoglycan (LPG) which contains a glycosylphosphatidylinositol glycolipid anchor. This parasite also synthesizes a complex family of abundant low-molecular-mass glycoinositolphospholipids (GIPLs) which are structurally related to the LPG anchor. In this study, L. major promastigotes were metabolically labelled with [3H]GlcN, and the kinetics of incorporation into free glycolipids and the LPG anchor followed to elucidate the pathway of GIPL biosynthesis and possible precursor-product relationships between the GIPLs and LPG. Labelled GIPLs were identified by TLC and by liquid chromatography of the released headgroups, before and after enzymic and chemical cleavage. On the basis of the measured specific radioactivities of the GIPLs, and their kinetics of radiolabelling, we suggest the pathway GlcN-PI-->Man1GlcN-PI (M1)-->Man2GlcN-PI (iM2)-->GalfMan2GlcN-PI (GIPL-1)-->Gal1GalfMan2GlcN-PI (GIPL-2)-->Gal2GalfMan2GlcN-PI (GIPL-3). All of the GIPLs were shown to contain alkylacylglycerol or lyso-alkylglycerol lipid moieties with the exception of the earliest intermediate, glucosaminylphosphatidylinositol (GlcN-PI), which contained both alkylacylglycerol and diacylglycerol. A significant proportion (approx. 50%) of GIPL-3 appeared to be selectively modified by the addition of a Glc-1-PO4 residue to one of the mannose residues (P-GIPL-3). On the basis of the specific radioactivity and kinetics of labelling of GIPL-3 and P-GIPL-3 we suggest that both of these low-abundance species are rapidly utilized as LPG precursors. The turnover of LPG and the GIPLs was also studied by [3H]Gal pulse-chase labelling and cell-surface labelling experiments. Whereas LPG was rapidly shed from the cell surface, consistent with previous studies, the GIPLs (both the total cellular and cell-surface pools) had a much slower turnover. These results suggest that the majority of the GIPLs do not act as LPG precursors and indicate that the cellular levels of these molecules is determined, at least in part, by the rate at which they are shed from the cell surface.

Rapid changes in the surface of parasitic nematodes during transition from pre- to post-parasitic forms.

All mammalian-parasitic stages of a range of nematode species investigated (Brugia pahangi, Acanthocheilonema viteae, Strongyloides ratti, Nippostrongylus brasiliensis, Trichinella spiralis and Ostertagia ostertagi) labelled in a surface-restricted manner with the fluorescent lipid analogues 5-N-(octadecanoyl)aminofluorescein (AF18) or nitrobenzoxadiazole-cholesterol (NBD-chol), but failed to bind other similar probes. In contrast, the surfaces of the 'pre-parasitic' infective stages of these species had affinity for neither AF18 nor NBD-chol. This exclusion of lipid analogues changed rapidly upon exposure of the larvae to tissue culture conditions which mimic the mammalian tissue environment (e.g. RPMI 1640/37 degrees C) such that the above probes could then insert into the surface layer of the larvae. The dauer larva of Caenorhabditis elegans also excluded the probes, but became permissive to labelling upon stimulation to emerge from the dauer state. The time taken for the surface transformation to occur ranged from less than 10 min in the vector-borne parasites to approximately 5 h in those which enter by the oral route, with direct skin-penetrators occupying an intermediate position. In all cases, the alteration proceeded too rapidly for it to have been associated with a moult. Fluorescence Recovery After Photobleaching (FRAP) studies of A. viteae larvae showed that approximately 50% of the AF18 probe was free to diffuse within the plane of the surface immediately after transformation. This is only a transitory state because AF18 was found to be highly restricted in its lateral diffusion on the surface of adult parasites. In the larvae of S. ratti, the change in affinity for AF18 was accompanied by the rapid shedding of an otherwise stable surface coat of polyanionic material, here visualized by labelling with fluorescein-conjugated cationized ferritin. Incubation of larvae in lipid-rich host serum during the induction of transformation inhibited subsequent labelling with AF18. This possibly reflects competition for insertion sites and an in vivo propensity towards the acquisition of host lipid by invading parasites.

Regulation of the expression of nitric oxide synthase and leishmanicidal activity by glycoconjugates of Leishmania lipophosphoglycan in murine macrophages.

Lipophosphoglycan (LPG) glycoconjugates from promastigotes of Leishmania were not able to induce the expression of the cytokine-inducible nitric oxide synthase (iNOS) by the murine macrophage cell line, J774. However, they synergize with interferon gamma to stimulate the macrophages to express high levels of iNOS. This synergistic effect was critically time-dependent. Preincubation of J774 cells with the LPG glycans 4-18 h before stimulation with interferon gamma resulted in a significant reduction in the expression of iNOS mRNA and of NO synthesis, compared with cells preincubated with culture medium alone. The regulatory effect on the induction of iNOS by LPG is located in the LPG phosphoglycan disaccharide backbone. Synthetic fragments of this backbone had a similar regulatory effect on NO synthesis. Further, the production of NO by activated macrophages in the present system was correlated directly with the leishmanicidal capacity of the cells. These data therefore demonstrate that LPG glycoconjugates have a profound effect on the survival of Leishmania parasites through their ability to regulate the expression of iNOS by macrophages.

Regulation of macrophage IL-12 synthesis by Leishmania phosphoglycans.

It is now generally accepted that IFN-gamma, secreted by Th1 cells, is the most potent cytokine leading to macrophage activation and host resistance against infection with the intracellular protozoan parasite Leishmania. It is also established that IL-12 is a critical cytokine involved in the differentiation and expansion of Th1 cells. Therefore, the ability of Leishmania parasites to actively suppress IL-12 production by host macrophages may be an important strategy for parasite survival. Here we report that a major parasite cell surface molecule, phosphoglycan (PG), of Leishmania could selectively inhibit the synthesis of IL-12(p40, p70) by activated murine macrophages. Furthermore, synthetic PG (sPG) was able to inhibit IL-12 release in a dose-dependent manner. Inhibition was dependent on the galactose(beta1-4)mannose(alpha1)-PO4 repeating units and not the glycophosphoinositol lipid anchor of lipophosphoglycan. At the concentration used, sPG had no effect on the release of TNF-alpha or IL-6 in activated macrophages. The inhibition of IL-12(p40) production was at the transcriptional level, but was not mediated through NF kappaB inhibition. These data demonstrate that PG may be an important molecule for the establishment and survival of the parasite in permissive hosts.