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Introduction: The variability in patients' femoral and tibial anatomy requires to use different tibia component sizes with the same femoral component size. These size combinations are allowed by manufacturers, but the clinical impact remains unclear. Therefore, the goals of our study were to investigate whether combining different sizes has an impact on the kinematics for two well-established knee systems and to compare these systems' kinematics to the native kinematics., Materials and Methods: Six fresh frozen knee specimens were tested in a force controlled knee rig before and after implantation of a cruciate retaining (CR) and a posterior-stabilized (PS) implant. Femoro-tibial kinematics were recorded using a ultrasonic-based motion analysis system while performing a loaded squat from 30° to 130°. In each knee, the original best fit inlay was then replaced by different inlays simulating a smaller or bigger tibia component. The kinematics obtained with the simulated sizes were compared to the original inlay kinematics using descriptive statistics., Results: For all size combinations, the difference to the original kinematics reached an average of 1.3 ± 3.3 mm in translation and - 0.1 ± 1.2° in rotation with the CR implant. With the PS implant, the average differences reached 0.4 ± 2.7 mm and  - 0.2 ± 0.8°. Among all knees, no size combination consistently resulted in significantly different kinematics. Each knee showed a singular kinematic pattern. For both knee systems, the rotation was smaller than in the native knee, but the direction of the rotation was preserved. The PS showed more rollback and the CR less rollback than the native knee., Conclusion: TKA systems designed with a constant tibio-femoral congruency among size combinations should enable to combine different sizes without having substantial impact on the kinematics. The rotational pattern was preserved by both TKA systems, while the rollback could only be maintained by the PS design., (© 2021. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Purpose: Third-generation autologous chondrocyte implantation (ACI) is an established and frequently used method and successful method for the treatment of full-thickness cartilage defects in the knee. There are also an increasing number of patients with autologous chondrocyte implantation as a second-line therapy that is used after failed bone marrow stimulation in the patient's history. The purpose of this study is to investigate the effect of previous bone marrow stimulation on subsequent autologous chondrocyte implantation therapy. In this study, the clinical results after the matrix-based autologous chondrocyte implantation in the knee in a follow-up over 3 years postoperatively were analysed., Methods: Forty patients were included in this study. A total of 20 patients with cartilage defects of the knee were treated with third-generation autologous chondrocyte implantation (Novocart® 3D) as first-line therapy. The mean defect size was 5.4 cm 2 (SD 2.6). IKDC subjective score and VAS were used for clinical evaluation after 6, 12, 24 and 36 months postoperatively. The results of these patients were compared with 20 matched patients with autologous chondrocyte implantation as second-line therapy. Matched pair analysis was performed by numbers of treated defects, defect location, defect size, gender, age and BMI., Results: Both the first-line (Group I) and second-line group (Group II) showed significantly better clinical results in IKDC score and VAS score in the follow-up over 3 years compared with the preoperative findings. In addition, Group I showed significantly better results in the IKDC and VAS during the whole postoperative follow-up after 6, 12, 24 and 36 months compared to Group II with second-line autologous chondrocyte implantation (IKDC 6 months p = 0.015, 1 year p = 0.001, 2 years p = 0.001, 3 years p = 0.011). Additionally, we found a lower failure rate in Group I. No revision surgery was performed in Group I. The failure rate in the second-line Group II was 30%., Conclusion: This study showed that third-generation autologous chondrocyte implantation is a suitable method for the treatment of full-thickness cartilage defects. Both, Group I and Group II showed significant improvement in our follow-up. However, in comparing the results of the two groups, autologous chondrocyte implantation after failed bone marrow stimulation leads to worse clinical results., Level of Evidence: III.

Introduction: Low frequency electromagnetic fields (LF-EMF) and simulated microgravity (SMG) have been observed to affect chondrogenesis. A controlled bioreactor system was developed to apply LF-EMF and SMG singly or combined during chondrogenic differentiation of human mesenchymal stem cells (hMSCs) in 3D culture., Material and Methods: An external motor gear SMG bioreactor was combined with magnetic Helmholtz coils for EMF (5 mT; 15 Hz). Pellets of hMSCs (±TGF-β3) were cultured (P5) under SMG, LF-EMF, LF-EMF/SMG and control (1 g) conditions for 3 weeks. Sections were stained with safranin-O and collagen type II. Gene expression was evaluated by microarray and real-time polymerase chain reaction analysis., Results: Simulated microgravity application significantly changed gene expression; specifically, COLXA1 but also COL2A1, which represents the chondrogenic potential, were reduced ( p < 0.05). Low frequency electromagnetic fields application showed no gene expression changes on a microarray basis. LF-EMF/SMG application obtained significant different expression values from cultures obtained under SMG conditions with a re-increase of COL2A1, therefore rescuing the chondrogenic potential, which had been lowered by SMG., Conclusions: Simulated microgravity lowered hypertrophy but also the chondrogenic potential of hMSCs. Combined LF-EMF/SMG provided a rescue effect of the chondrogenic potential of hMSCs although no LF-EMF effect was observed under optimal conditions. The study provides new insights into how LF-EMF and SMG affect chondrogenesis of hMSCs and how they generate interdependent effects., Competing Interests: The authors declare no conflict of interest.

Introduction: Third generation autologous chondrocyte implantation (ACI) is a suitable method for the treatment of cartilage defects in the knee joint. However, knowledge about the development of graft thickness and the clinical relevance of incomplete defect filling in the postoperative course is low. This prospective study analyses the graft integration into the surrounding cartilage, with special consideration of the graft thickness., Material and Methods: A total of 71 consecutive patients with 79 cartilage defects were treated with third generation autologous chondrocyte implantation (NOVOCART 3D) in the knee. Follow-up magnetic resonance imaging (MRI) was performed at 0.25, 0.5, 1 and 2 years. Graft thickness was measured compared to the surrounding healthy cartilage. The International Knee Documentation Committee (IKDC) scoring system and the visual analogue scale (VAS) were used for clinical evaluation. Cartilage defect filling was classified as the percentage of the surrounding cartilage., Results: The average graft thickness showed a significant increase between 3 and 6 months after autologous chondrocyte implantation. Incomplete defect filling occurred in 44 (55.7%) cases. Of these, 33 cases showed incomplete defect filling grade I (> 75%), 10 cases were grade II (> 50%) and one case grade III (> 25%). Incomplete defect filling grade IV (< 25%) was not observed. Incomplete defect filling occurred significantly more often in women (p = 0.021), without worse clinical results., Conclusions: Graft thickness after third generation autologous chondrocyte implantation shows increasing graft thickness over the period of 2 years postoperatively. A high rate of incomplete defect filling in the surrounding cartilage was observed, without worse clinical results.

Purpose: During in vitro chondrogenesis of human mesenchymal stem cells (hMSCs) hypertrophy is an inadvertent event associated with cell differentiation toward the osteogenic lineage. Up to now, there is no stringent experimental control mechanism to prevent hypertrophy of MSCs. Microgravity is known to have an impact on osteogenesis. In this study, the influence of simulated microgravity (SMG) on both chondrogenesis and hypertrophy of hMSCs was evaluated., Methods: A bioreactor using a rotating wall vessel was constructed to simulate microgravity. Pellet cultures formed from hMSCs (P5) were supplemented with human transforming growth factor-β3 (TGF-β3). The hMSC pellet cultures treated with TGF-β3 were either kept in SMG or in a control system. After three weeks of culture, the chondrogenic differentiation status and level of hypertrophy were examined by safranin-O staining, immunohistochemistry and quantitative real-time PCR., Results: SMG reduced the staining for safranin-O and collagen type II. The expression of collagen type X α1 chain (COL10A1) and collagen type II α1 chain (COL2A1) were both significantly reduced. There was a higher decrease in COL2A1 than in COL10A1 expression, resulting in a low COL2A1/COL10A1 ratio., Conclusions: SMG reduced hypertrophy of hMSCs during chondrogenic differentiation. However, the expression of COL2A1 was likewise reduced. Even more, the COL2A1/COL10A1 ratio decreased under SMG conditions. We therefore assume that SMG has a significant impact on the chondrogenic differentiation of hMSCs. However, due to the high COL2A1 suppression under SMG, this culture system does not yet seem to be suitable for a potential application in cartilage repair.

Interleukin-4 (IL-4) is the major factor promoting the development of T helper type 2 (Th2) cells from naive precursor T cells. Minute amounts of IL-4 produced by naive T cells seem to be sufficient; however, the molecular mechanisms explaining this efficient utilization of IL-4 are not yet known. Here, we show that human CD4+ CD45RA+ naive T cells, in contrast to CD4+ CD45R0+ effector T cells, show responsiveness to endogenous as well as exogenous IL-4 to proliferate and differentiate towards Th2 cells in vitro. Despite production levels of IL-4 below conventional detection limits, CD45RA+ T cell-derived IL-4 could clearly activate STAT6. Although the expression levels of IL-4R and STAT6 were not different between naive and effector T cells, only naive T cells responded to IL-4 in a STAT6-dependent reporter gene assay. Transfecting a trans-dominant negative form of STAT6 abrogated IL-4-induced proliferation in CD45RA+ cells. A significantly higher protein tyrosine phosphatase (PTPase) activity was detected in CD45R0+ T cells as compared to CD45RA+ T cells. Cross-linking CD45 potently reduced PTPase activity in CD45R0+ T cells and restored their ability to proliferate in response to IL-4. Thus, CD45 PTPase activity contributes to the susceptibility of naive and memory T cells to respond to IL-4.

Identification of new target proteins is a novel paradigm in drug discovery. A major bottleneck of this strategy is the rapid and simultaneous expression of proteins from differential gene expression to identify eligible candidates. By searching for a generic system enabling high throughput expression analysis and purification of unknown cDNAs, we evaluated the YEpFLAG-1 yeast expression system. We have selected cDNAs encoding model proteins (eukaryotic initiation factor-5A [eIF-5A] and Homo sapiens differentiation-dependent protein-A4) and cDNA encoding an unknown protein (UP-1) for overexpression in Saccharomyces cerevisiae using fusions with a peptide that changes its conformation in the presence of Ca2+ ions, the FLAG tag (Eastman Kodak, Rochester, NY). The cDNAs encoding unknown proteins originating from a directionally cloned cDNA library were expressed in all three possible reading frames. The expressed proteins were detected by an antibody directed against the FLAG tag and/or by antibodies against the model proteins. The alpha-leader sequence, encoding a yeast mating pheromone, upstream of the gene fusion site facilitates secretion into the culture supernatant. EIF-5A could be highly overexpressed and was secreted into the culture supernatant. In contrast, the Homo sapiens differentiation-dependent protein-A4 as well as the protein UP-1, whose cDNA did not match to any known gene, could not be detected in the culture supernatant. The expression product of the correct frame remained in the cells, whereas the FLAG-tagged proteins secreted into the supernatant were short, out-of-frame products. The presence of transmembrane domains or patches of hydrophobic amino acids may preclude secretion of these proteins into the culture supernatant. Subsequently, isolation and purification of the various proteins was accomplished by affinity chromatography or affinity extraction using magnetizable beads coated with the anti-FLAG monoclonal antibody. The purity of isolated proteins was in the range of 90%. In the case of unknown cDNAs, the expression product with the highest molecular mass was assumed to represent the correct reading frame. In summary, we consider the YEpFLAG-1 system to be a very efficient tool to overexpress and isolate recombinant proteins in yeast. The expression system enables high throughput production and purification of proteins under physiological conditions, and allows miniaturization into microtiter formats.

One novel approach for the biological delivery of peptide drugs is to incorporate the sequence of the peptide into the structure of a natural transport protein such as human serum transferrin (HST). However, a potential drawback is that the HST may increase the immunoreactivity of the peptide, in the same way that carrier proteins can be used to generate highly immunogenic peptide hapten conjugates. In this study we have generated a recombinant HST carrier protein that contains a peptide substrate of HIV-1 protease (VSQNYPIVL). The protein retained native HST function, and the peptide was surface exposed since it was immunoreactive in native dot blots, and was cleaved by HIV-1 protease. Immunisation of rabbits with the recombinant protein elicited only a very poor anti-peptide immune response. In contrast, strong anti-peptide immune responses were raised against both the peptide alone, and a chemical conjugate of the peptide with HST. These data demonstrate that it is possible to attenuate the immune response normally directed against an immunogenic peptide sequence by engineering into a surface exposed loop of HST. These findings may have an important impact on the future design of peptide delivery systems.

One novel approach for the biological delivery of peptide drugs is to incorporate the sequence of the peptide into the structure of a natural transport protein, such as human serum transferrin. To examine whether this is feasible, a peptide sequence cleavable by the human immunodeficiency virus type 1 protease (VSQNYPIVL) was inserted into various regions of human serum transferrin, and the resultant proteins were tested for function. Experimentally, molecular modeling was used to identify five candidate insertion sites in surface exposed loops of human serum transferrin that were distant from biologically active domains. These insertions were cloned using polymerase chain reaction mutagenesis, and the proteins were expressed using a baculovirus expression vector system. Analysis of the mutant proteins provided a number of important findings: (a) they retained native human serum transferrin function, (b) the inserted peptide sequence was surface exposed, and most importantly, (c) two of these mutants could be cleaved by human immunodeficiency virus-1 protease. In conclusion, this investigation has validated the use of human serum transferrin as a carrier protein for functional peptide domains introduced into its structure using protein engineering. These findings will be useful for developing a novel class of therapeutic agents for a broad spectrum of diseases.

Cyclic AMP phosphodiesterase (PDE) type 4 has been the subject of considerable interest as a potential molecular target for treating cutaneous inflammatory and allergic disorders; however, little is known regarding the expression of PDE 4 isogenes in human keratinocytes, the predominant cell type present in the epidermis. In this study, we investigated the expression of PDE 4 subtypes in epidermal cells (primary keratinocytes derived from breast skin, epidermoid cell lines A431, KB, and HaCaT) using reverse transcriptase polymerase chain reaction. Analysis of the amplified cDNA showed that there were differences in the expression of PDE 4 isogenes in the epidermal cells. Constitutive expression of the PDE 4 isozymes was detected in untreated epidermal cells at different levels. Transcripts for PDE 4B and 4D were abundantly expressed in breast skin-derived keratinocytes, whereas transcripts for PDE 4A, 4C, and 4D were present in HaCaT cells and transcripts for all the PDE 4 isotypes were present in A431 and KB. PDE 4A and 4C were detectable in HaCaT cells in equal amounts, but PDE 4C was only marginally expressed in the other cells. Of the four isogenes, PDE 4D was abundantly expressed in all the cells. Elevation of intracellular levels of cAMP by forskolin increased the expression of all the hPDE 4 isogenes 2-3-fold as revealed by semiquantitative reverse transcriptase polymerase chain reaction. Western blot analysis of extracts from control and forskolin or dibutyryl cAMP-treated A431 and KB cells demonstrated the presence of proteins with a molecular mass identical to that corresponding to recombinant human PDE 4B. Together, these findings show that PDE 4 isogenes are expressed in keratinocytes to a different degree and that their expression can be modulated by intracellular levels of cAMP.

Anti-FcepsilonRIalpha autoantibodies (autoAbs) occur and may be of pathogenetic relevance in a subset of chronic urticaria (CU) patients. To analyze the prevalence and magnitude of the humoral anti-FcepsilonRIalpha response in cohorts of CU patients compared with individuals suffering from classic skin- related (auto)immune diseases, we developed an ELISA system for the measurement of anti-FcepsilonRIalpha autoAbs in nonfractionated serum samples. Results obtained using this assay correlated well with those generated by Western blotting. We found IgG anti-FcepsilonRIalpha autoreactivity in 38% of CU patients but not in atopic dermatitis patients, psoriatics, or healthy individuals. We frequently detected anti-FcepsilonRIalpha autoAbs in pemphigus vulgaris (PV, 39%), dermatomyositis (DM, 36%), systemic lupus erythematosus (SLE, 20%), and bullous pemphigoid (BP, 13%). While the autoAb titers in DM, SLE, BP, and PV were similar to those encountered in CU patients, only anti-FcepsilonRIalpha+ CU serum specimens displayed pronounced histamine-releasing activity. The anti-FcepsilonRIalpha autoAbs in CU patients belong predominantly to the complement-fixing subtypes IgG1 and IgG3, whereas in DM, PV, and BP, they were found to be mainly of the IgG2 or IgG4 subtype. Complement-activating properties of anti-FcepsilonRIalpha autoAbs can indeed be of pathogenetic relevance, because C5a receptor blockade on basophils as well as decomplementation reduced drastically the histamine-releasing capacity of most anti-FcepsilonRIalpha-reactive CU sera. As a consequence, therapeutic efforts in CU should aim at altering not only the quantity but also the complement-activating properties of IgG anti-FcepsilonRIalpha autoAbs.

Recently, there has been much interest in expressing recombinant human serum transferrin (HST) and mutants thereof for structural and functional studies. We have developed a baculovirus expression system for the rapid and efficient production of large quantities of HST (> 20 mg/l). Like native HST, the recombinant protein can bind two ferric ions in the presence of bicarbonate, and is actively taken up by receptor-mediated endocytosis. Secondary structure calculations from CD measurements indicate a content of 42% alpha-helix and 28% beta-sheet. This is the first reported use of a non-mammalian expression system to produce functional HST, and will provide a practical tool to allow expression of a wide range of HST variants for mutagenesis studies.

A set of chimeric antibodies with identical F(ab')2 fragments specific for the hapten 5-iodo-4-hydroxyl-3-nitrophenacetyl (NIP), but with different human Fc parts (gamma 1, gamma 2, gamma 3, gamma 4, epsilon), was used to compare the role of IgG and IgE antibodies in antigen presentation by human Epstein-Barr virus (EBV) B cells. Two or three molecules of NIP were coupled to one molecule of Der pI (Der pI-(3)NIP), a major allergen of Dermatophagoides pteronyssinus. Both monomeric IgG and performed complexes of various Der pI/IgG ratios failed to bind significantly to the Fc receptor for IgG on B cells (Fc gamma RII; CD32). Binding of IgG3 (> IgG1)-containing complexes (optimal ratio of antigen to antibody = 1:1) could be enhanced by increasing the number of haptens per Der pI molecule to nine or more. However, antigen presentation mediated by IgG and CD32 was not seen with either pulsed B cells or B cells that were allowed to capture the IgG complexes during the whole stimulation period. IgE binding to CD23 and subsequent IgE-mediated antigen presentation was seen under all conditions tested. Even monomeric immune complexes (IC) (Der pI-(3)NIP/IgE), in the absence of CD23 cross-linking, induced an immune response. As the number of natural epitopes for human antibodies on Der pI was less than five, we conclude that, in vivo, complexes consisting of Der pI/IgG will be directed to antigen-presenting cells expressing the high-affinity receptor for IgG (CD64), whereas IgE will allow antigen presentation by CD23-expressing cells, including B cells.

SDZ NIM 811 is a cyclosporin A analog that is completely devoid of immunosuppressive capacity but exhibits potent and selective anti-human immunodeficiency virus type 1 (HIV-1) activity. The mechanism of action of SDZ NIM 811 is clearly different from those of all other anti-HIV agents described so far. In cell-free assays, it is not an inhibitor of reverse transcriptase, protease, integrase, and it does not interfere with Rev or Tat function. SDZ NIM 811 does not down-regulate CD4 or inhibit fusion between infected and uninfected, CD4-expressing cells. p24 production from chronically HIV-infected cells is not impaired either. To elucidate the mode of action of SDZ NIM 811, we performed DNA PCR analysis in HIV-1 IIIB-infected MT4 cells in one cycle of virus replication. The effects of SDZ NIM 811 on the kinetics of viral DNA synthesis, appearance of two-long terminal repeat circles (2-LTR circles), and integration of DNA were studied. SDZ NIM 811 inhibited 2-LTR circle formation in a concentration-dependent manner, which is indicative of nuclear localization of preintegration complexes. Half-maximal inhibition was achieved at 0.17 microgram/ml; this concentration is close to the 50% inhibitory concentrations (0.01 to 0.2 microgram/ml) for viral growth inhibition. As expected, integration of proviral DNA into cellular DNA was also inhibited by SDZ NIM 811. Analysis of the viral particles produced by SDZ NIM 811-treated, chronically infected cells revealed amounts of capsid proteins, reverse transcriptase activity, and viral RNA comparable to those of the untreated control. However, these particles showed a dose-dependent reduction in infectivity (50% inhibitory concentration of 0.028 microgram/ml) which indicates that the assembly process is also impaired by SDZ NIM 811. Gag proteins are postulated to play a role not only in assembly but also in early steps of viral replication, e.g., nuclear localization of the preintegration complex. Recently, it was reported that HIV-1 Gag protein binds to cyclophilin A, the intracellular receptor for cyclosporin A. Interference with Gag-cyclophilin interaction may be the molecular basis for the antiviral activity of cyclosporin A and its analogs.

1) The aspartic proteinase of the human immunodeficiency virus type 1 (HIV-1) was purified from cultures of recombinant E. coli. The enzyme preparation is homogeneous as judged by SDS-polyacrylamide gel electrophoresis and isoelectric focusing. 2) A rapid assay procedure for the proteinase was established which makes use of the cleavage of a radiolabeled decapeptide and the separation of substrate and labeled product by ion-exchange resin. 3) Activity of the enzyme is optimal at an ionic strength of 2.5-3.5M; also, the inhibitor pepstatin is a more potent inhibitor at higher ionic strength. This can be attributed to a tighter binding of both substrate and inhibitor in high-salt buffer. 4) The Km value of the decapeptide substrate is independent of the pH in the range of 3.5-7.5, while kcat shows a bell-shaped curve with a maximum at pH 5.2. The shape of the curve can be attributed to pKa values of 4.2 and 6.2 of groups on the enzyme. Pepstatin inhibition is optimal below pH 5.5, but becomes weak above pH 6.

An unusual type of teichoic acid has been isolated from Streptococcus agalactiae type III. It has the same backbone as the lipoteichoic acid from Streptococcus faecalis, but is devoid of fatty acid residues, a phosphatidyl group, and substituents in the poly(glycerol phosphate) side-chain. The following structure, with n approximately 20, was determined mainly with the aid of n.m.r. spectroscopy. (Formula: see text)