Chondrocyte suspension in fibrin glue

Autologous chondrocyte implantation has been shown to be a promising method for treatment of deep articular cartilage defects. The hyaline cartilage formed by implanted autologous chondrocytes has biomechanical properties similar to those of natural articular cartilage. Between June 2006 and September 2008 we performed Autologous chondrocyte implantation (ACI) in 50 patients and the chondrocytes were supported in fibrin glue. The cartilage biopsy samples were taken from the non-weight bearing area of the patient’s femoral condyle and the samples were transferred to the cell culture laboratory. Chondrocyte were kept in culture about 20 days. Fibrin glue was used as a three dimensional carrier for chondrocyte implantation. A 450 ml of patient’s own blood was collected prior to transplantation to produce autologous fibrinogen. Alternatively the allogenic fibrinogen was prepared from Regional Blood Center voluntary donors. Before surgery the chondrocyte suspension was mixed with fibrin glue and gel—like fibrograft was prepared. The total number of cells and the size of fibrograft depended on the defect size in the knee. Our results suggest that ACI technique with fibrin glue is a promising method for treatment of cartilage defect.     

Prognostic Significance of ESR1 Amplification and ESR1 PvuII,CYP2C19*2, UGT2B15*2 Polymorphisms in Breast Cancer Patients

Introduction

Amplification of the ESR1 gene, coding for estrogen receptor alpha, was shown to predict responsiveness to tamoxifen, however its prognostic impact in breast cancer patients has not been thoroughly investigated. Other factors that could contribute to responsiveness to tamoxifen treatment are polymorphisms in ESR1 gene and genes involved in tamoxifen metabolism.The aim of this study was to assess the prognostic role of ESR1 gene dosage in a consecutive group of breast cancer patients and to correlate this feature with clinico-pathological factors. Additionally, ESR1 PvuII, CYP2C19*2 and UGT2B15*2 polymorphisms were analyzed in the tamoxifen-treated subgroup of patients.

Materials and Methods

Primary tumor samples from 281 stage I-III consecutive breast cancer patients were analyzed for ESR1 gene dosage using real-time PCR with locked nucleic acids hydrolysis probes. In the tamoxifen-treated subgroup of patients, ESR1 PvuII, CYP2C19*2 and UGT2B15*2 polymorphism in leukocytes genomic DNA were analyzed. Results were correlated with clinico-pathological factors and with disease-free survival (DFS) and overall survival (OS).

Results

ESR1 amplification (with a cut-off level of 2.0) was found in 12% of the entire group of breast cancer patients, and in 18% of the ER-negative subgroup. This feature was associated with decreased DFS both in the entire group (P=0.007) and in the ER-negative subgroup (P=0.03), but not in the tamoxifen-treated patients.Patients with ESR1 PvuII wt/wt genotype and at least one UGT2B15 wt allele had a worse DFS (P=0.03) and showed a trend towards decreased Os (P=0.08) in comparison to patients with ESR1 PvuII wt/vt or vt/vt genotype and UGT2B15 *2/*2 genotype.

Conclusions

ESR1 amplification can occur in ER-negative tumors and may carry poor prognosis. In the tamoxifen-treated subgroup, poor prognosis was related to the combined presence of ESR1 PvuII wt/wt and UGT2B15wt/wt or wt/*2 genotype.     

The Comparison of the Effects of Three Physiotherapy Techniques on Hamstring Flexibility in Children: A Prospective,Randomized, Single-Blind Study

The aim of the study was to evaluate changes in hamstring flexibility in 120 asymptomatic children who participated in a 6-week program consisting of one physiotherapy session per week and daily home exercises. The recruitment criteria included age (10–13 years), no pain, injury or musculoskeletal disorder throughout the previous year, physical activity limited to school sport. Subjects were randomly assigned to one of the three groups: (1) post-isometric relaxation – PIR (n = 40), (2) static stretch combined with stabilizing exercises – SS (n = 40) and (3) stabilizing exercises – SE (n = 40). Hamstring flexibility was assessed with straight leg raise (SLR), popliteal angle (PA) and finger-to-floor (FTF) tests. The examinations were conducted by blinded observers twice, prior to the program and a week after the last session with the physiotherapist. Twenty-six children who did not participate in all six exercise sessions with physiotherapists were excluded from the analysis. The results obtained by 94 children were analyzed (PIR, n = 32; SS, n = 31; SE, n = 31). In the PIR and SS groups, a significant (P<0.01) increase in SLR, PA, FTF results was observed. In the SE group, a significant (P<0.001) increase was observed in the SLR but not in the PA and FTF (P>0.05). SLR result in the PIR and SS groups was significantly (P<0.001) higher than in the SE group. As far as PA results are concerned, a significant difference was observed only between the SS and SE groups (P = 0.014). There were no significant (P = 0.15) differences regarding FTF results between the three groups. Post-isometric muscle relaxation and static stretch with stabilizing exercises led to a similar increase in hamstring flexibility and trunk forward bend in healthy 10–13-year-old children. The exercises limited to straightening gluteus maximus improved the SLR result, but did not change the PA and FTF results.     

Limited Promiscuity of HLA-DRB1 Presented Peptides Derived of Blood Coagulation Factor VIII

The formation of inhibitory antibodies directed against coagulation factor VIII (FVIII) is a severe complication in the treatment of hemophilia A patients. The induction of anti-FVIII antibodies is a CD4⁺ T cell-dependent process. Activation of FVIII-specific CD4⁺ T cells is dependent on the presentation of FVIII-derived peptides on MHC class II by antigen-presenting cells. Previously, we have shown that FVIII-pulsed human monocyte-derived dendritic cells can present peptides from several FVIII domains. In this study we show that FVIII peptides are presented on immature as well as mature dendritic cells. In immature dendritic cells half of the FVIII-loaded MHC class II molecules are retained within the cell, whereas in LPS-matured dendritic cells the majority of MHC class II/peptide complexes is present on the plasma membrane. Time-course studies revealed that presentation of FVIII-derived peptides was optimal between 12 and 24 hours after maturation but persisted for at least 96 hours. We also show that macrophages are able to internalize FVIII as efficiently as dendritic cells, however FVIII was presented on MHC class II with a lower efficiency and with different epitopes compared to dendritic cells. In total, 48 FVIII core-peptides were identified using a DCs derived of 8 different donors. Five HLA-promiscuous FVIII peptide regions were found – these were presented by at least 4 out of 8 donors. The remaining 42 peptide core regions in FVIII were presented by DCs derived from a single (30 peptides) or two to three donors (12 peptides). Overall, our findings show that a broad repertoire of FVIII peptides can be presented on HLA-DR.     

From macro to lab-scale: Changes in bacterial community led to deterioration of EBPR in lab reactor

A laboratory scale sequencing batch reactor (SBR), fed with synthetic wastewater containing a mixture of organic compounds, was operated for nearly six months. Despite maintaining the same operational conditions, a deterioration of enhanced biological phosphorus removal (EBPR) occurred after 40 days of SBR operation. The P_rel/C_upt ratio decreased from 0.28 to 0.06 P-mol C-mol^?1, and C requirements increased from 11 to 32 mg C h^?1 g^?1 of mixed liquor suspended solids. A FISH analysis showed that the percentage of Accumulibacter in an overall community of polyphosphate accumulating organisms (PAOs) and glycogen accumulating organisms (GAOs) dropped from 93% to 13%. An increase in abundance of Gammaproteobacteria (from 2.6% to 22%) and Alphaproteobacteria (from 1.8% to 10%) was observed. The number of Competibacter increased from 0.5% to nearly 9%. Clusters 1 and 2 of Defluviicoccus-related GAOs, not detected before deterioration, constituted 35% and 27% of Alphaproteobacteria, respectively. We concluded that lab-scale experiments should not be extended implicitly to full-scale EBPR systems because some bacterial groups are detected mainlyin lab-scale reactors. Well-defined, lab-scale operational conditions reduce the number of ecological niches available to bacteria.     

Manufacturing of peptides exhibiting biological activity

Numerous studies have shown that food proteins may be a source of bioactive peptides. Those peptides are encrypted in the protein sequence. They stay inactive within the parental protein until release by proteolytic enzymes (Mine and Kovacs-Nolan in Worlds Poult Sci J 62(1):87–95, 2006; Hartman and Miesel in Curr Opin Biotechnol 18:163–169, 2007). Once released the bioactive peptides exhibit several biofunctionalities and may serve therapeutic roles in body systems. Opioid peptides, peptides lowering high blood pressure, inhibiting platelet aggregation as well as being carriers of metal ions and peptides with immunostimulatory, antimicrobial and antioxidant activities have been described (Hartman and Miesel in Curr Opin Biotechnol 18:163–169, 2007). The biofunctional abilities of the peptides have therefore aroused a lot of scientific, technological and consumer interest with respect to the role of dietary proteins in controlling and influencing health (Möller et al. in Eur J Nutr 47(4):171–182, 2008). Biopeptides may find wide application in food production, the cosmetics industry as well as in the prevention and treatment of various medical conditions. They are manufactured by chemical and biotechnological methods (Marx in Chem Eng News 83(11):17–24. 2005; Hancock and Sahl in Nat Biotechnol 24(12):1551–1557, 2006). Depending on specific needs (food or pharmaceutical industry) different degrees of peptide purifications are required. This paper discusses the practicability of manufacturing bioactive peptides, especially from food proteins.     

Nutrigenomics-based personalised nutritional advice: in search of a business model?

Nutritional advice has mainly focused on population-level recommendations. Recent developments in nutrition, communication, and marketing sciences have enabled potential deviations from this dominant business model in the direction of personalisation of nutrition advice. Such personalisation efforts can take on many forms, but these have in common that they can only be effective if they are supported by a viable business model. The present paper takes an inventory of approaches to personalised nutrition currently available in the market place as its starting point to arrive at an identification of their underlying business models. This analysis is presented as a unifying framework against which the potential of nutrigenomics-based personalised advice can be assessed. It has uncovered nine archetypical approaches to personalised nutrition advice in terms of their dominant underlying business models. Differentiating features among such business models are the type of information that is used as a basis for personalisation, the definition of the target group, the communication channels that are being adopted, and the partnerships that are built as a part of the business model. Future research should explore the consumer responses to the diversity of “archetypical” business models for personalised nutrition advice as a source of market information on which the delivery of nutrigenomics-based personalised nutrition advice may further build.     

Guanylate Cyclase-Activating Protein-2 Undergoes Structural Changes upon Binding to Detergent Micelles and Bicelles

GCAPs are neuronal Ca^2 +-sensors playing a central role in light adaptation. GCAPs are N-terminally myristoylated membrane-associated proteins. Although, the myristoylation of GCAPs plays an important role in light adaptation its structural and physiological roles are not yet clearly understood. The crystal-structure of GCAP-1 shows the myristoyl moiety inside the hydrophobic core of the protein, stabilizing the protein structure; but ²H-solid-state NMR investigations on the deuterated myristoyl moiety of GCAP-2 in the presence of liposomes showed that it is inserted into the lipid bilayer. In this study, we address the question of the localization of the myristoyl group of Ca^2 +-bound GCAP-2, and the influence of CHAPS-, DPC-micelles and DMPC/DHPC-bicelles on the structure, and on the localization of the myristoyl group, of GCAP-2 by solution-state NMR. We also carried out the backbone assignment. Characteristic chemical shift differences have been observed between the myristoylated and the non-myristoylated forms of the protein. Our results support the view that in the absence of membrane forming substances the myristoyl moiety is buried inside a hydrophobic pocket of GCAP-2 similar to the crystal structure of GCAP-1. Addition of CHAPS-micelles and DMPC/DHPC-bicelles cause specific structural changes localized in and around the myristoyl binding pocket. We interpret these changes as an indication for the extrusion of the myristoyl moiety from its binding pocket and its insertion into the hydrophobic interior of the membrane mimic. On the basis of the backbone chemical shifts, we propose a structural model of myristoylated GCAP-2 in the presence of Ca^2 + and membrane mimetics.