Long-term survivorship of stemless anatomical shoulder replacement.

PURPOSE: Like in many other joints, current shoulder replacement designs aim at bone preservation. According to the literature available, stemless total shoulder arthroplasty (TSA) compares favourably with stemmed designs in terms of function and survivorship of the implant. However, long-term results of stemless shoulder arthroplasty are still missing. Therefore, the aim of the present study was to evaluate long-term results of stemless anatomical TSA. METHODS: Between 2006 and 2009, 51 shoulders in 46 patients were resurfaced using the Biomet Total Evolutive Shoulder System (TESS). Thirty-one shoulders in 26 patients who were aged 66.7 ± 10.0 (range 34-82) years were available for review at a mean follow-up of 94.7 ± 11.3 (76-124) months. RESULTS: The implant survival rate was 93.5% at eight years. The overall revision rate of the TESS implant was 9.7%. Radiolucent lines were found on the glenoid side of the TESS arthroplasty in 90.9% of the cases. All stemless humeral corolla implants showed solid fixation at follow-up. Clinical scores significantly improved at long-term follow-up (VAS from 8.1 ± 0.9 to 1.0 ± 1.2, p < 0.001; Quick-DASH from 67.9 ± 13.5 to 18.7 ± 16.5, p < 0.001 and Constant score from 14.7 ± 6.1 to 68.8 ± 13.2, p < 0.001). CONCLUSIONS: Stemless TSA has stood the test of time at eight years in terms of clinical scores, radiographic loosening, complication rates and implant survivorship.

Late onset Krabbe disease due to the new GALC p.Ala543Pro mutation, with intriguingly high residual GALC activity in vitro.

BACKGROUND: Krabbe disease (KD) is an inherited leukodystrophy due to a defect in the GALC gene which encodes the lysosomal galactosylceramide ß-galactosidase (GALC). About two thirds of patients show the early onset form of KD dominated by cerebral demyelination leading to death in early infancy. Late onset forms include a spectrum of late infantile, juvenile and adult clinical courses. The deficiency of GALC leads to a galactosylceramide lipidosis in which lysosomal storage phenomena are seen almost only at the ultrastructural level. RESULTS: In a 4-year-old boy, the clinical suspicion of KD was high according to neurologic and neuroimaging findings. However, laboratory results were inconclusive; white blood cell GALC activity being at 23 to 25% of the normal level, and GALC genotyping revealing the new homozygous p.Ala543Pro variant which, ex silico, was of unclear significance. Studying a skin biopsy, cultured fibroblasts showed the GALC activity at 21 to 30% of the normal level; ultrastructurally, clearly KD-specific inclusions were seen in the eccrine sweat gland cells, confirming a KD diagnosis. CONCLUSION: The high clinical suspicion combined with the morphologic evidence for KD predict that the p.Ala543Pro variant is pathogenic for (late onset) KD. A hypothesis linked to the proline in the mutant GALC may explain the in vitro effect with high residual GALC activity. This patient would not have been correctly diagnosed, despite the strong clinical criteria of KD, if the electron microscopic results had not been available. The detailed knowledge of neurologic and neuroimaging signs is important in diagnostically problematic KD patients in which also an electron microscopic approach can be crucial.

PPARα activation by culinary herbs and spices.

Hyper- and dyslipidemia are risk factors for cardiovascular disease, the primary cause of death in industrialized countries. Peroxisome proliferators-activated receptor (PPAR)α activation is involved in various mechanisms that improve the lipid profile. We tested various plant extracts and their compounds to determine whether they stimulated PPARα activity in vitro. Out of 34 tested plant extracts, nine exhibited low to moderate PPARα transactivation, including caraway, chili pepper, nutmeg, licorice, black and white pepper, paprika, coriander, saffron, and stevia tea. The active components of black pepper and chili pepper, piperine, and capsaicin exerted the highest transactivational activities with EC50 values of 84 µM and 49 µM, respectively. The chalcones, including 2-hydroxychalcone, 2'-hydroxychalcone, 4-hydroxychalcone, and 4-methoxychalcone, moderately transactivated PPARα. Resveratrol and apigenin only slightly transactivated PPARα. These results suggest that a diet rich in fruit, herbs, and spices provides a number of PPARα agonists that might contribute to an improved lipid profile.

Isoflavones are safe compounds for therapeutical applications - evaluation of in vitro data.

Isoflavone-rich food and food supplements have gained increasing popularity also in the Western world. Their weak estrogenic effect has been considered as a potential risk, although all epidemiological studies and clinical trials show a significant cancer protection and decreased risk of cardiovascular diseases. In vitro data suggest that the concerted action of the isoflavones and their metabolites show antiproliferative behaviour, reduce angiogenesis, reduce tumor progression and exert antiinflammatory effects. For the evaluation of the biological effects, special emphasis has to be put on the concerted action between the isoflavones and their metabolites. For instance, while isolated genistein shows some growth promoting effect at low concentrations, the metabolite equol or soy extract show growth retardation as well as higher concentrations of genistein do. The isoflavones have multiple affinities to other members of the steroid hormone receptor superfamily. The beneficial effect on metabolic diseases and weight reduction by isoflavone consumption can be partly explained by its affinity for the PPAR family. In light of the in vitro experiments, together with the epidemiological observations and the clinical experience, isoflavones can be considered as safe compounds and their consumption as food and food supplements has to be promoted.

Comparison of hormonal activity of isoflavone-containing supplements used to treat menopausal complaints.

OBJECTIVES: The isoflavones present in red clover and soy are used as an alternative treatment for menopausal complaints and are commercially available as high-dose food supplements. These preparations contain varying amounts of active ingredients, often without detailed specifications. Thus, it is difficult to derive a recommended daily dose, and the reliability of these products is rather low. METHODS: We quantified the isoflavone content of 19 different isoflavone-containing preparations and compared their binding and transactivational activities with regard to estrogen receptor alpha, estrogen receptor beta, androgen receptor, progesterone receptor, peroxisome-proliferator-activated receptor, and aryl hydrocarbon receptor. RESULTS: The food supplements that we tested bound to and transactivated both the estrogen receptors and the other receptors. After comparing the isoflavone content quantified by us with the isoflavone content specified on the package labels, we found that at least the specified isoflavone content or more could be detected in only 5 of the 19 food supplements that we tested. CONCLUSIONS: Preparations containing isoflavones should be standardized for the isoflavone aglycone content to facilitate the prediction of theoretical hormonal activity, facilitate the intake of a controlled amount of isoflavones, and ensure greater product reliability.

Androgen receptor transactivation assay using green fluorescent protein as a reporter.

For screening of a large number of samples for androgenic activity, a robust system with minimal handling is required. The coding sequence for human androgen receptor (AR) was inserted into expression plasmid YEpBUbi-FLAG1, resulting in the plasmid YEpBUbiFLAG-AR, and the estrogen response element (ERE) on the reporter vector YRpE2 was replaced by an androgen response element (ARE), resulting in the plasmid YRpE2-ARE. Thus, a fully functional transactivation assay system with beta-galactosidase as a reporter gene could be created. Furthermore, green fluorescent protein (GFP) was introduced as an alternative reporter gene that resulted in a simplification of the whole assay procedure. For evaluation of both reporter systems, seven steroidal compounds with known AR agonistic properties (5 alpha-dihydrotestosterone, testosterone, androstenedione, 17 alpha-methyltestosterone, progesterone, epitestosterone, and d-norgestrel) were tested, and their potencies obtained in the different assays were compared. Furthermore, potencies from the transactivation assays were compared with IC(50) values obtained in radioligand binding assays. The newly developed androgen receptor transactivation assay is a useful tool for characterizing compounds with androgenic activity.

A new way to generate cytolytic tumor-specific T cells: electroporation of RNA coding for a T cell receptor into T lymphocytes.

Effective T cell receptor (TCR) transfer until now required stable retroviral transduction. However, retroviral transduction poses the threat of irreversible genetic manipulation of autologous cells. We, therefore, used optimized RNA transfection for transient manipulation. The transfection efficiency, using EGFP RNA, was >90%. The electroporation of primary T cells, isolated from blood, with TCR-coding RNA resulted in functional cytotoxic T lymphocytes (CTLs) (>60% killing at an effector to target ratio of 20:1) with the same HLA-A2/gp100-specificity as the parental CTL clone. The TCR-transfected T cells specifically recognized peptide-pulsed T2 cells, or dendritic cells electroporated with gp100-coding RNA, in an IFNgamma-secretion assay and retained this ability, even after cryopreservation, over 3 days. Most importantly, we show here for the first time that the electroporated T cells also displayed cytotoxicity, and specifically lysed peptide-loaded T2 cells and HLA-A2+/gp100+ melanoma cells over a period of at least 72 h. Peptide-titration studies showed that the lytic efficiency of the RNA-transfected T cells was similar to that of retrovirally transduced T cells, and approximated that of the parental CTL clone. Functional TCR transfer by RNA electroporation is now possible without the disadvantages of retroviral transduction, and forms a new strategy for the immunotherapy of cancer.

GFP-reporter for a high throughput assay to monitor estrogenic compounds.

In vitro test systems using yeast cells are a useful tool for the determination of the estrogenic activity of estrogens, phyto- and xeno-estrogens and can be used for monitoring large sample numbers in a routine analysis procedure. Our conventional transactivation assay functions with an expression plasmid expressing estrogen receptor alpha (ERalpha) under the control of a copper-inducible CUP1 promoter and a reporter plasmid expressing beta-galactosidase under the control of the vitellogenin estrogen response element (ERE). In the novel yeast screen system the lacZ gene in the reporter plasmid was substituted by a gene for green fluorescent protein (GFP). Incubation of yeast with various concentrations of estrogenically active substances led to expression of the reporter gene product GFP in a dose dependent manner. The yeast transactivation assay was further down-scaled to be performed in a microplate scale, which is an important step to facilitate handling of large sample numbers. The sensitivity and reproducibility of the novel test system could be confirmed by analysis of the potencies of various estrogenically active substances. Thus, the newly developed yeast estrogen screen using GFP as a reporter can substitute the assay that has been used for a period of several years.

Generation of an optimized polyvalent monocyte-derived dendritic cell vaccine by transfecting defined RNAs after rather than before maturation.

Transfection with RNA is an attractive method of Ag delivery to dendritic cells (DCs), but has not yet been standardized. We describe in this study the methods to efficiently generate an optimized mature monocyte-derived DC vaccine at clinical scale based on the electroporation of several RNAs either into immature DC followed by maturation or, alternatively, directly into mature DCs, which has not been possible so far with such high efficiency. Electroporation of DCs resulted in high yield, high transfection efficiency (>90%), and high migration capacity. Intracellular staining allowed the study of the expression kinetics of Ags encoded by the transfected RNAs (MelanA, MAGE-3, and survivin) and a validation of the vaccine (>/=90% transfection efficiency). Expression of all three Ags peaked 3-4 h after electroporation in DC transfected either before or after maturation, but decreased differently. The DC vaccine can also be cryopreserved and nevertheless retains its viability, stimulatory capacity as well as migratory activity. In addition, we uncover that DC transfected after rather than before maturation appear to be preferable vaccines not only from a production point of view but also because they appear to be immunologically superior for CTL induction in sharp contrast to common belief. DCs transfected after maturation not only more effectively generate and present the Mage-3.A1 and MelanA.A2.1 epitopes to T cell clones, but they even are superior in priming to the standard proteasome-dependent MelanA.A2.1 wild-type prototype tumor epitope, both in terms of T cell expansion and effector function on a per cell basis.

Estrogenic activity in white and red wine extracts.

Red wine is enriched in resveratrol, trans-3,5,4'-trihydroxystilbene, a compound in grape skin that inhibits the development of pre-neoplastic lesions in mouse mammary tumor cells in culture and inhibits cancer cell proliferation in vitro. Grapes also contain other bioactive compounds including flavonoids, flavans, and anthocyanins. The estrogenic activities of extracts prepared from one white (Freie Weingärtner Wachau, Grüner Veltliner, Austria) and two red wines (Woodbridge, Cabernet Sauvignon, California; and Lenz Moser Prestige, Blaufränkisch Barrique, Austria) were examined and compared with those induced by estradiol (E(2)) and trans-resveratrol. First, the estrogenic activity of the wine extracts was evaluated in a yeast estrogen screen (YES) assay, in which yeast express copper-inducible estrogen receptor alpha (ERalpha) and an estrogen-response-element (ERE)-driven beta-galactosidase reporter. In YES, the white wine extract showed no estrogenic activity. In contrast, both of the red wine extracts showed estrogenic activity equivalent to that of 0.2 nM E(2). Similarly, the white wine extract showed no transcriptional activity with either ERalpha and ERbeta in transiently transfected CHO-K1 cells. In contrast, both red wine extracts stimulated ERE-reporter activity in a concentration-dependent manner that was inhibited by 4-hydroxytamoxifen (4-OHT), indicating that the observed transcriptional activity was ER-mediated. The red wine extracts showed significantly higher ERbeta versus ERalpha agonist activity. Resveratrol showed no agonist activity in YES but activated ERalpha and ERbeta in CHO-K1 cells in a concentration-dependent manner that was inhibited by 4-OHT. This indicates that resveratrol requires mammalian cell components that are absent in yeast for estrogen agonist activity, whereas the estrogenic activity of wine extracts is directly through ERalpha and does not require mammalian cell factors such as coactivators. The estrogenic activity in red wine found by using YES indicates that estrogenic compounds other than resveratrol are present. Chemical analysis clearly showed that the trans-resveratrol content of the red wine extracts was 1 order of magnitude below the detection limit for YES assay.

Yeast reporter system for rapid determination of estrogenic activity.

An in vitro test system for the determination of estrogens, xeno- and phytoestrogens, based on the activation of human estrogen receptor-alpha, has been examined for ability in monitoring environmental estrogens. The system consists of an expression plasmid for the human estrogen receptor-alpha and a reporter plasmid containing the lacZ gene under the control of the vitellogenin hormone response element. These plasmids have been transformed into S. cerevisae. Cultivation of yeast in the presence of estrogenic substances leads to activation of the estrogen receptor and induces the expression of the reporter lacZ. beta-Galactosidase activity of the translated gene lacZ is a measure of the estrogenic activity of a compound. First, the selectivity of the system was compared to data available in the literature. Then the sensitivity of the system was checked. The detection limit is 0.1 ng 17-beta estradiol or an equivalent activity per liter, if a sample can be concentrated 1000-fold. The system has been further characterized by selected compounds with known and unknown estrogenic activity.