Explorative immunohistochemical study to evaluate the addition of a topical corticosteroid in the early phase of alefacept treatment for psoriasis.

The aim of this study was to explore the additional effect of betamethasone dipropionate cream in the early phase of an intramuscular (IM) alefacept course, on plaque severity and on modulating T-cell subsets, cells expressing NK-receptors, epidermal proliferation and keratinocyte differentiation in lesional psoriatic skin. Therefore, sixteen patients with moderate-to-severe chronic plaque psoriasis received 15 mg alefacept IM for 12 weeks, followed by a 12-week follow-up period. The first 4 weeks, patients were randomized 1:1 to either betamethasone dipropionate, or the vehicle cream, once daily. Plaque severity (SUM) was assessed and serial biopsies were immunohistochemically stained for T-cell subsets (CD3, CD4, CD8, CD45RO, CD45RA, CD2, CD25, GITR), cells expressing NK-receptors (CD94 and CD161), epidermal proliferation (Ki67) and differentiation (K10), which were quantified using manual and digital image analysis. Alefacept monotherapy resulted in statistically significant improvement in plaque severity. Subsequently, immunohistochemical assessments on T-cell subsets, epidermal proliferation (Ki67) and keratinization (K10) revealed marked time-related improvements with respect to the mentioned parameters, without significant differences between both treatment regimens. Alefacept monotherapy induces improvement of plaque severity, which is accompanied by a reduction in activated (CD2+, CD25+, CD45RO+) dermal CD4+ and activated epidermal CD8+ T cells, epidermal proliferation and differentiation. Once daily treatment with betamethasone dipropionate cream during the first 4 weeks of an intramuscular alefacept course did not provide substantial additional clinical and immunohistochemical improvement.

The role of lesional T cells in recalcitrant psoriasis during infliximab therapy.

With infliximab therapy (anti-TNF-alpha) for plaque psoriasis, over 80% of patients reach > or = 75% PASI improvement in 10 weeks of treatment. We describe a patient with severe recalcitrant psoriasis who was treated with infliximab 5 mg/kg for 22 weeks. Rather than the expected improvement, this patient experienced an initial exacerbation, followed by the lack of efficacy over the entire 22-week period of treatment. Before, during and after treatment we performed immunohistochemical analyses on lesional biopsies, with respect to T cells, NK-T cells, epidermal growth and differentiation. We found a discrepancy between the clinical aggravation and marked reductions of lesional T cell subsets. The most prominent decrease was for CD4+ T cells (72-74%), which suggests that a reduction of T cells in the psoriatic plaque might not be a guarantee for positive clinical outcomes. Remarkably, the number of epidermal CD94+ NK-T cells correlated fairly well with the lack of clinical efficacy, supposing a pathogenic role for these cells in psoriasis. Further studies are needed to clarify the ambiguous role of conventional pathogenic T cells in plaque psoriasis.

Alefacept modifies long-term disease severity and improves the response to other treatments.

Alefacept treatment has been shown in several multicentre studies to result in prolonged post-treatment remission periods (median duration 241 days). This communication describes the clinical responses of 10 patients to multiple courses of 12 weekly intramuscular injections with alefacept given in combination with available antipsoriatic treatments. It was shown that in this group of 10 patients with moderate-severe psoriasis, 8 were no longer candidates for any more long-term continuous systemic treatment with methotrexate or acitretin, 7 had no options for UVB/PUVA courses and 8 were not suitable for treatment with cyclosporine. In 5 out of these 10 patients alefacept had resulted in a considerable long-term improvement with only mild disease expression, which lasted one year or longer. Patients indicated that these responses were outstanding as compared to the troublesome years before. Another patient reported a major improvement in the quality of life by reduction of itch. In two patients alefacept vastly enhanced the efficacy of dithranol treatment, as compared to previous treatments. Another patient was able to discontinue methotrexate without relapsing during alefacept, in contrast to severe rebound episodes following discontinuation of methotrexate in the past. The long-term responses of 8 out of the 10 patients described in this report indicate that alefacept, in the context of individualised care of patients with moderate to severe psoriasis, can have an important contribution. In particular, the long-term response after stopping alefacept makes this treatment a valuable tool, achieving prolonged disease control in at least some patients with moderate to severe psoriasis.

[Health Council report on health claims for foods and food supplements]. / Gezondheidsraadadvies over gezondheidsclaims bij voedingsmiddelen en-supplementen.

The Health Council of the Netherlands has issued a report on the health claims often made for foods and food supplements. It is concluded that the boundary between medical claims and health claims is not clear: there is no scientific or practical distinction between the 'prevention of disease' and the 'promotion and maintenance of health'. Only explicit, specific and evidence-based claims regarding the prevention, and possibly the treatment, of diseases or conditions are of any value to public health. If the impression is correct that the nutrient content of a product, a change in bodily function or a reduction of a disease risk factor reduces the likelihood of developing a certain disease, this effect should be stated explicitly. If the impression is not correct, then the claim is irrelevant and therefore misleading. A claim must always be supported by research involving human subjects and generally accepted scientific criteria must be adhered to.

Alkali-extraction of membranes from mouse erythrocytes infected with Plasmodium berghei.

Alkali-extracted membrane material from hypotonically lysed Plasmodium berghei-infected mouse erythrocytes has been analyzed by dodecyl sulphate polyacrylamide gel electrophoresis and fractionated by discontinuous sucrose gradient centrifugation. Components characteristic for the protein pattern associated with P. berghei infection have been recovered in the alkali-extracted material. Parasite components, free of host cell membrane contamination, have been obtained as a high-density fraction from sucrose gradients. This fraction is characterized by polypeptides with apparent molecular weight of 71,000 and 52,000.

Lipidic particles.

A new type of lipid organization is observed in mixtures of phosphatidylcholine with cardiolipin in the presence of Ca++, monoglucosyldiglyceride and phosphatidylethanolamine (in the presence of cholesterol). This phase is characterized by an isotropic 31P NMR signal and is visualized by freeze fracturing as particles and pits on the fracture faces of the lipid bilayer. As the most favourable model for this phase we propose the inverted micelle sandwiched in between the two monolayers of the lipid bilayer. It will be shown that such particles and pits appear on the fusion site during fusion of unilamellar vesicles of an equimolar mixture of phosphatidylcholine and cardiolipin in the presence of Ca++.

Ca2+-induced changes in the barrier properties of cardiolipin/phosphatidylcholine bilayers.

(1) A selective increase in permeability is induced in cardiolipin/phosphatidylcholine bilayers at Ca2+ concentrations of 1--3 mM. At higher concentrations of Ca2+ the permeability barrier is completely destroyed. (2) The selective increase in permeability is correlated with the formation of lipid particles visualized by freeze-fracture electron microscopy and an isotropic signal in 31P-NMR spectra. (3) Lowering the Ca2+ concentration shows reduction in permeability but the formation of the lipid particles is a non-reversible process. (4) At higher Ca2+ concentrations, 31P-NMR spectra and freeze-fracture results indicate the formation of the hexagonal phase, explaining the disappearance of the permeability barrier.

The lipidic particle as an intermediate structure in membrane fusion processes and bilayer to hexagonal HII transitions.

Small unilamellar vesicles comprised of a mixture of phosphatidylethanolamine/phosphatidylcholine/cholesterol (3 : 1 : 2) fuse to form large multilamellar vesicles on increasing the temperature from 0 to 50 degrees C. This event is associated with the appearance of lipidic particles at the fusion sites, consistent with a role as intermediary structures during the fusion process. Further, for phosphatidylcholine/cardiolipin (1 : 1) liposomes in the presence of Mn2+ a direct relationship between lipidic particles and the hexagonal (HII) phase is demonstrated which suggests that lipidic particles can also occur as intermediaries between bilayer and hexagonal (HII) structures.

The transbilayer movement of phosphatidylcholine in vesicles reconstituted with intrinsic proteins from the human erythrocyte membrane.

Vesicles have been prepared from 18 : 1c/18 : 1c-phosphatidylcholine with or without purified glycophorin or partially purified band 3 (obtained by organomercurial gel chromatography). The vesicles have been characterized by freeze-fracture electron microscopy, binding studies to DEAE-cellulose, 31P-NMR and K+ trap measurements. Pools of phosphatidylcholine available for exchange have been investigated using phosphatidylcholine exchange protein from bovine liver. The protein-containing vesicles both exhibit exchangeable pools larger than the fraction of phosphatidylcholine in the outer monolayer, whereas in the protein-free vesicles the exchangeable pool is consistent with the outer monolayer. The results indicate that both glycophorin and the partially purified band 3 preparation enhance the transbilayer movement of phosphatidylcholine.

Ca2+-induced isotropic motion and phosphatidylcholine flip-flop in phosphatidylcholine-cardiolipin bilayers.

Ca2+ induces a structural change in phosphatidylcholine-cardiolipin bilayers, which is visualised by freeze-fracturing as lipidic particles associated with the bilayer and is detected by 31P-NMR as isotropic motion of the phospholipids. In this structure a rapid transbilayer movement of phosphatidylcholine and a highly increased permeability towards Mn2+ are observed.

Fusion of phospholipid vesicles in association with the appearance of lipidic particles as visualized by freeze fracturing.

The addition of Ca2+ to small unilamellar vesicles of an equimolar mixture of egg phosphatidylcholine and cardiolipin induces fusion of these vesicles in association with the appearance of lipidic particles on the fusion sites.

The occurrence of lipidic particles in lipid bilayers as seen by 31P NMR and freeze-fracture electron-microscopy.

A new type of lipid organization is observed in mixtures of phosphatidyl-choline with cardiolipin (in the presence of Ca2+), monoglycosyldiglyceride and phosphatidylethanolamine (in the presence of cholesterol). This phase is characterised by an isotropic 31P NMR signal and is visualised by freeze-fracturing as particles and pits on the fracture faces of the lipid bilayer. As the most favourable model for this phase we propose the inverted micelle sandwiched in between the two monolayers of the lipid bilayer.

The lateral distribution of intramembrane particles in the erythrocyte membrane and recombinant vesicles.

Triton X-100 (in concentrations which did not cause a significant solubilization of membrane material) caused aggregation of the intramembrane particles of human erythrocyte ghosts. Ghosts from which the extrinsic proteins had been removed by alkali treatment showed a temperature-induced aggregation of the particles. With virtually no spectrin present, the particles in these stripped ghosts could still be aggregated by manipulations with ionic strength and pH, or by the addition of calcium. Recombinant vesicles were made from a Triton X-100 extract and a mixture of phospholipids with a composition which resembled that of the inner monolayer of erythrocyte membrane. In these recombinants the same manipulations with ionic strength and pH and the addition of calcium caused a rearrangement of the particles, resulting in the appearance of particle-free areas. In recombinants prepared from a Triton X-100 extract and egg phosphatidylcholine the lateral distribution of the particles was not altered by these manipulations. It is concluded that in the erythrocyte membrane the intramembrane particles can be aggregated by effects of external agents on lipid components. In this light the role of spectrin in stabilizing the membrane by interactions with lipids in the inner monolayer is discussed.

A 13C NMR method for determination of the transbilayer distribution of phosphatidylcholine in large, unilamellar, protein-free and protein-containing vesicles.

(1) Large unilamellar vesicles have been prepared from N-[Ne3-13C]-18 : 1c/18 : 1c-phosphatidylcholine, both with and without the major intrinsic proteins from the human erythrocyte membrane incorporated in the bilayer. (2) It is shown that the inside-outside distribution of the lipid molecules in these large unilamellar structures can be determined using 13C NMR. (3) Large vesicles of 18 : 1c/18 : 1c-phosphatidylcholine containing glycophorin show an enhanced permeability to Dy3+. It is shown that the permeability barrier of these vesicles can be restored by addition of 10 mol% 18 : 1c/18 : 1c-phosphatidylethanolamine or 1-18 : 1c-lysophosphatidylcholine.

Freeze-fracture appearance and disposition of band 3 protein from the human erythrocyte membrane in lipid vesicles.

Single bilayer lipid vesicles were formed by removal of Triton X-100 with Bio Beads SM-2 from a mixture of egg lecithin and a Triton X-100 extract of human erythrocyte ghosts. Upon freeze-fracturing, these vesicles showed intramembrane particles, similar to those seen in the erythrocyte membrane. Similar particles were also observed when a partially purified band 3 preparation was used instead of the crude Triton X-100 extract. In the reconstituted vesicles an equal distribution of the intramembrane particles between the two fracture faces was observed. This is in contrast to the unequal distribution of the particles in the erythrocyte membrane, which did not seem to be altered by removal of the extrinsic proteins. From digestion studies with trypsin and chymotrypsin of vesicles, reconstituted from the crude X-100 extract, it is concluded that band 3 protein in the vesicle bilayer has a similar orientation as in the native membrane.