Introduction to placebo effects in medicine: mechanisms and clinical implications

The field of placebo research has made considerable progress in the last years and it has become a major focus of interest. We know now that the placebo effect is a real neurobiological phenomenon and that the brain's 'inner pharmacy' is a critical determinant for the occurrence of psychobiological and behavioural changes relevant to healing processes and well-being. However, harnessing the advantages of placebo effects in healthcare is still a challenge. The first part of the theme issue summarizes and discusses the various kinds of placebo mechanisms across medical fields, thereby not only focusing on two main explanatory models-expectation and conditioning theory-but also taking into account empathy and social learning, emotion and motivation, spirituality and the healing ritual. The second part of the issue focuses on questions related to transferring knowledge from placebo research into clinical practice and discusses implications for the design and interpretation of clinical trials, for the therapeutic settings in daily patient care, and for future translational placebo research.     

Mixed Magnetism for Refrigeration and Energy Conversion

The efficient coupling between lattice degrees of freedom and spin degrees of freedom in magnetic materials can be used for refrigeration and energy conversion. This coupling is enhanced in materials exhibiting the giant magnetocaloric effect. First principle electronic structure calculations on hexagonal MnFe(P, Si) reveal a new form of magnetism: the coexistence of strong and weak magnetism in alternate atomic layers. The weak magnetism of Fe layers (disappearance of local magnetic moments at the Curie temperature) is responsible for a strong coupling with the crystal lattice while the strong magnetism in adjacent Mn‐layers ensures Curie temperatures high enough to enable operation at and above room temperature. Varying the composition on these magnetic sublattices gives a handle to tune the working temperature and to achieve a strong reduction of the undesired thermal hysteresis. In this way we design novel materials based on abundantly available elements with properties matched to the requirements of an efficient refrigeration or energy‐conversion cycle.     

Variation in macrophage-migration-inhibitory-factor immunoreactivity during porcine gestation

The localization and activity of macrophage migration inhibitory factor (MIF) was investigated in the interhemal region of the noninvasive, diffuse, folded epitheliochorial placenta and in the nonpregnant uterus of the pig. MIF, a proinflammatory cytokine with many actions on macrophages and monocytes, may play an important role in materno-fetal immuno-tolerance during placental establishment, modulation, and growth. Immunohistochemical staining with anti-human MIF polyclonal antibodies was carried out on placental sections from 11 stages of gestation (16-95 days postcoitus) and on nonpregnant uterus at 13 days postestrus. Western blot analysis confirmed the specificity of the anti-human MIF polyclonal antibodies on pig tissues. MIF staining was intense in both the trophoblast and maternal epithelium in the early stages; in the later stages, it decreased dramatically in the maternal epithelium but remained high in the trophoblast. The uterine glands showed immunoreactivity at all stages, and the maternal and fetal epithelial linings of the areolar cavity showed high reactivity at Day 25. The vasculature also showed staining for MIF, and an intense to moderate staining was shown in the nonpregnant uterus, mostly in the surface and glandular epithelium. The high activity of MIF in the maternal and fetal tissues throughout placentation and its expression in the nonpregnant uterus indicate a regulatory role for MIF during embryo receptivity and epitheliochorial placentation.     

Exploring the genome of Trypanosoma vivax through GSS and in silico comparative analysis

A survey of the Trypanosoma vivax genome was carried out by the genome sequence survey (GSS) approach resulting in 1,086 genomic sequences. A total of 455 high-quality GSS sequences were generated, consisting of 331 non-redundant sequences distributed in 264 singlets and 67 clusters in a total of 135.5 Kb of the T. vivax genome. The estimation of the overall G+C content, and the prediction of the presence of ORFs and putative genes were carried out using the Glimmer and Jemboss packages. Analysis of the obtained sequences was carried out by BLAST programs against 12 different databases and also using the Conserved Domain Database, InterProScan, and tRNAscan-SE. Along with the existing 23 T. vivax entries in the GenBank, the 32 putative genes predicted and the 331 non-redundant GSS sequences reported herein represent new potential markers for the development of PCRbased assays for specific diagnosis and typing of Trypanosoma vivax.     

Cytosolic prion protein (PrP) is not toxic in N2a cells and primary neurons expressing pathogenic PrP mutations

Inherited prion diseases are linked to mutations in the prion protein (PrP) gene, which favor conversion of PrP into a conformationally altered, pathogenic isoform. The cellular mechanism by which this process causes neurological dysfunction is unknown. It has been proposed that neuronal death can be triggered by accumulation of PrP in the cytosol because of impairment of proteasomal degradation of misfolded PrP molecules retrotranslocated from the endoplasmic reticulum (Ma, J., Wollmann, R., and Lindquist, S. (2002) Science 298, 1781-1785). To test whether this neurotoxic mechanism is operative in inherited prion diseases, we evaluated the effect of proteasome inhibitors on the viability of transfected N2a cells and primary neurons expressing mouse PrP homologues of the D178N and nine octapeptide mutations. We found that the inhibitors caused accumulation of an unglycosylated, aggregated form of PrP exclusively in transfected N2a expressing PrP from the cytomegalovirus promoter. This form contained an uncleaved signal peptide, indicating that it represented polypeptide chains that had failed to translocate into the ER lumen during synthesis, rather than retrogradely translocated PrP. Quantification of N2a viability in the presence of proteasome inhibitors demonstrated that accumulation of this form was not toxic. No evidence of cytosolic PrP was found in cerebellar granule neurons from transgenic mice expressing wild-type or mutant PrPs from the endogenous promoter, nor were these neurons more susceptible to proteasome inhibitor toxicity than neurons from PrP knock-out mice. Our analysis fails to confirm the previous observation that mislocation of PrP in the cytosol is neurotoxic, and argues against the hypothesis that perturbation of PrP metabolism through the proteasomal pathway plays a pathogenic role in prion diseases.     

The antioxidant effect of tannic acid on the in vitro copper-mediated formation of free radicals

Tannic acid (TA) has well-described antimutagenic and antioxidant activities. The antioxidant activity of TA has been previously attributed to its capacity to form a complex with iron ions, interfering with the Fenton reaction [Biochim. Biophys. Acta 1472, 1999, 142]. In this work, we observed that TA inhibits, in the micromolar range, in vitro Cu(II) plus ascorbate-mediated hydroxyl radical (*OH) formation (determined as 2-deoxyribose degradation) and oxygen uptake, as well as copper-mediated ascorbate oxidation and ascorbate radical formation (quantified in EPR studies). The effect of TA against 2-deoxyribose degradation was three orders of magnitude higher than classic *OH scavengers, but was similar to several other metal chelators. Moreover, the inhibitory effectiveness of TA, by the four techniques used herein, was inversely proportional to the Cu(II) concentration in the media. These results and the observation of copper-induced changes in the UV spectra of TA are indications that the antioxidant activity of TA relates to its copper chelating ability. Thus, copper ions complexed to TA are less capable of inducing ascorbate oxidation, inhibiting the sequence of reactions that lead to 2-deoxyribose degradation. On the other hand, the efficiency of TA against 2-deoxyribose degradation declined considerably with increasing concentrations of the *OH detector molecule, 2-deoxyribose, suggesting that the copper-TA complex also possesses an *OH trapping activity.     

Purification of a lectin from the marine red alga Gracilaria ornata and its effect on the development of the cowpea weevil Callosobruchus maculatus (Coleoptera: Bruchidae)

A lectin from the marine red alga Gracilaria ornata (Gracilariaceae, Rodophyta) was purified and characterized. The purification procedure consisted of extracting soluble proteins in 0.025 M Tris-HCl buffer, pH 7.5, followed by ammonium sulfate precipitation (70% saturation), ion exchange chromatography on DEAE-cellulose and affinity chromatography on mucin-Sepharose 4B. The purified G. ornata lectin (GOL) showed a single protein band with an apparent molecular mass of 17 kDa when submitted to SDS-polyacrylamide gel electrophoresis under reducing conditions. The native molecular mass of GOL determined by gel filtration on a Sephadex G-100 column was 17.4 kDa and its carbohydrate content was estimated to be 2.9%. Therefore, GOL is a monomeric glycoprotein. The purified lectin agglutinated trypsin-treated erythrocytes from rabbit and chicken but not from human. Its activity was not inhibited by any of the mono- and disaccharides tested but by the complex glycoproteins porcine stomach mucin, lactotransferrin, asialofetuin and bovine and porcine thyroglobulins. Isoelectric focusing showed that GOL is an acidic protein with a pI of 5.4 with analysis of its amino acid composition revealing high contents of Asx, Glx, Ser, Glu, Ala and Cys. When incorporated in artificial seeds, GOL significantly affected the development of Callosobruchus maculatus larvae, indicating the possibility of using this lectin in a biotechnological strategy for insect management of stored cowpea seeds.     

Identification and relevance of the CD95-binding domain in the N-terminal region of ezrin

The CD95 (Fas/APO-1) linkage to the actin cytoskeleton through ezrin is an essential requirement for susceptibility to the CD95-mediated apoptosis in CD4+ T cells. We have previously shown that moesin was not involved in the binding to CD95. Here we further support the specificity of the ezrin/CD95 binding, showing that radixin did not bind CD95. The ezrin region specifically and directly involved in the binding to CD95 was located in the middle lobe of the ezrin FERM domain, between amino acids 149 and 168. In this region, ezrin, radixin, and moesin show 60-65% identity, as compared with the 86% identity in the whole FERM domain. Transfection of two different human cell lines with a green fluorescent protein-tagged ezrin mutated in the CD95-binding epitope, induced a marked inhibition of CD95-mediated apoptosis. In these cells, the mutated ezrin did not co-localize or co-immunoprecipitate with CD95. Further analysis showed that the mutated ezrin, while unable to bind CD95, was fully able to bind actin, thus preventing the actin linkage to CD95. Altogether, our results support the specificity of ezrin in the association to CD95 and the importance of the ezrin-to-CD95 linkage in CD95-mediated apoptosis. Moreover, this study suggests that a major role of ezrin is to connect CD95 to actin, thus allowing the CD95 polarization on the cells and the occurrence of the following multiple cascades of the CD95 pathway.