A Glutaraldehyde-Free Crosslinking Method for the Treatment of Collagen-Based Biomaterials for Clinical Application.

Biological bioprostheses such as grafts, patches, and heart valves are often derived from biological tissue like the pericardium. These bioprostheses can be of xenogenic, allogeneic, or autologous origin. Irrespective of their origin, all types are pre-treated via crosslinking to render the tissue non-antigenic and mechanically strong or to minimize degradation. The most widely used crosslinking agent is glutaraldehyde. However, glutaraldehyde-treated tissue is prone to calcification, inflammatory degradation, and mechanical injury, and it is incapable of matrix regeneration, leading to structural degeneration over time. In this work, we are investigating an alternative crosslinking method for an intraoperative application. The treated tissue's crosslinking degree was evaluated by differential scanning calorimetry. To confirm the findings, a collagenase assay was conducted. Uniaxial tensile testing was used to assess the tissue's mechanical properties. To support the findings, the treated tissue was visualized using two-photon microscopy. Additionally, fourier transform infrared spectroscopy was performed to study the overall protein secondary structure. Finally, a crosslinking procedure was identified for intraoperative processing. The samples showed a significant increase in thermal and enzymatic stability after treatment compared to the control, with a difference of up to 22.2 °C and 100%, respectively. Also, the tissue showed similar biomechanics to glutaraldehyde-treated tissue, showing greater extensibility, a higher failure strain, and a lower ultimate tensile strength than the control. The significant difference in the structure band ratio after treatment is proof of the introduction of additional crosslinks compared to the untreated control with regard to differences in the amide-I region. The microscopic images support these findings, showing an alteration of the fiber orientation after treatment. For collagen-based biomaterials, such as pericardial tissue, the novel phenolic crosslinking agent proved to be an equivalent alternative to glutaraldehyde regarding tissue characteristics. Although long-term studies must be performed to investigate superiority in terms of longevity and calcification, our novel crosslinking agent can be applied in concentrations of 1.5% or 2.0% for the treatment of biomaterials.

Routine practice data of three cancer entities: Comparison among cancer registry and health insurance data.

INTRODUCTION: Claims data and cancer registry data are valuable secondary data sources for addressing health service research questions. This study provides a thorough insight into the comparability of data from health insurance companies and cancer registries in Germany regarding breast, prostate, and lung cancer patients and their treatment. METHODS: For this study claims data of the InGef database and data of the Cancer Registry of Rhineland-Palatinate were used to identify patients living in Rhineland-Palatinate with an incident breast, prostate, or lung cancer diagnosis between Jan. 1, 2018 and Dec. 31, 2019. Both datasets were compared for patient and tumour characteristics as well as treatment strategy. For the descriptive analysis of tumour localisation and treatment all patients were followed up for a maximum of two years. RESULTS: A total of 1,470 incident cancer cases were identified in the InGef database and 1,694 in the Cancer Registry. Data on sex, age, and tumour localisation matched well for all cancer entities in the cohorts. Data for early UICC stages I+II varied between the cohorts for prostate cancer (84% InGef, 66% Cancer Registry) and lung cancer (29% InGef, 20% Cancer Registry). Larger deviations were found for antihormonal treatment (breast 54% vs. 44%, prostate 32% vs. 18%). Significant differences were found for surgery (breast and lung) and radiation (breast and prostate), respectively. DISCUSSION: Age at diagnosis, tumour localisation, and treatment for breast cancer was well documented in both databases. Tumour-specific deviations were observed for tumour localisations (lung cancer), UICC stage (prostate and lung cancer) and treatment options. CONCLUSION: Both databases show very good completeness across cancer entities, but at the same time have minor limitations where they could readily complement each other. Individual linkage of claims and registry data could be an important step to improve oncological studies with routine practice data and to overcome the limitations identified.

Early Detection of Subsurface Fatigue Cracks in Rolling Element Bearings by the Knowledge-Based Analysis of Acoustic Emission.

Aiming at early detection of subsurface cracks induced by contact fatigue in rotating machinery, the knowledge-based data analysis algorithm is proposed for health condition monitoring through the analysis of acoustic emission (AE) time series. A robust fault detector is proposed, and its effectiveness was demonstrated for the long-term durability test of a roller made of case-hardened steel. The reliability of subsurface crack detection was proven using independent ultrasonic inspections carried out periodically during the test. Subsurface cracks as small as 0.5 mm were identified, and their steady growth was tracked by the proposed AE technique. Challenges and perspectives of the proposed methodology are unveiled and discussed.

Correction: Lange et al. Performance of a Piezoelectric Energy Harvesting System for an Energy-Autonomous Instrumented Total Hip Replacement: Experimental and Numerical Evaluation. Materials 2021, 14, 5151.

The authors wish to make the following corrections to their paper [...].

Performance of a Piezoelectric Energy Harvesting System for an Energy-Autonomous Instrumented Total Hip Replacement: Experimental and Numerical Evaluation.

Instrumented implants can improve the clinical outcome of total hip replacements (THRs). To overcome the drawbacks of external energy supply and batteries, energy harvesting is a promising approach to power energy-autonomous implants. Therefore, we recently presented a new piezoelectric-based energy harvesting concept for THRs. In this study, the performance of the proposed energy harvesting system was numerically and experimentally investigated. First, we numerically reproduced our previous results for the physiologically based loading situation in a simplified setup. Thereafter, this configuration was experimentally realised by the implantation of a functional model of the energy harvesting concept into an artificial bone segment. Additionally, the piezoelectric element alone was investigated to analyse the predictive power of the numerical model. We measured the generated voltage for a load profile for walking and calculated the power output. The maximum power for the directly loaded piezoelectric element and the functional model were 28.6 and 10.2 µW, respectively. Numerically, 72.7 µW was calculated. The curve progressions were qualitatively in good accordance with the numerical data. The deviations were explained by sensitivity analysis and model simplifications, e.g., material data or lower acting force levels by malalignment and differences between virtual and experimental implantation. The findings verify the feasibility of the proposed energy harvesting concept and form the basis for design optimisations with increased power output.

Endurance testing and finite element simulation of a modified hip stem for integration of an energy harvesting system.

Instrumented implants are a promising approach to further improve the clinical outcome of total hip arthroplasties. For the integrated sensors or active functions, an electrical power supply is required. Energy harvesting concepts can provide autonomous power with unlimited lifetime and are independent from external equipment. However, those systems occupy space within the mechanically loaded total hip replacement and can decrease the life span due to fatigue failure in the altered implant. We previously presented a piezoelectric energy harvesting system for an energy-autonomous instrumented total hip stem that notably changes the original implant geometry. The aim of this study was to investigate the remaining structural fatigue failure strength of the metallic femoral implant component in a worst-case scenario. Therefore, the modified hip stem was tested under load conditions based on ISO 7206-4:2010. The required five million cycles were completed twice by all samples (n = 3). Additionally applied cycles with incrementally increased load levels up to 4.7 kN did not induce implant failure. In total, 18 million cycles were endured, outperforming the requirements of the ISO standard. Supplementary finite element analysis was conducted to determine stress distribution within the implant. A high stress concentration was found in the region of modification. The stress level showed an increase compared to the previously evaluated physiological loading situation and was close to the fatigue data from the literature. The stress concentration factor compared to the original geometry amounted to 2.56. The assessed stress level in accordance with the experimental fatigue testing can serve as a maximum reference value for further implant design modifications and optimisations.

Reporting checklist for verification and validation of finite element analysis in orthopedic and trauma biomechanics.

Finite element analysis (FEA) has become a fundamental tool for biomechanical investigations in the last decades. Despite several existing initiatives and guidelines for reporting on research methods and results, there are still numerous issues that arise when using computational models in biomechanical investigations. According to our knowledge, these problems and controversies lie mainly in the verification and validation (V&V) process as well as in the set-up and evaluation of FEA. This work aims to introduce a checklist including a report form defining recommendations for FEA in the field of Orthopedic and Trauma (O&T) biomechanics. Therefore, a checklist was elaborated which summarizes and explains the crucial methodologies for the V&V process. In addition, a report form has been developed which contains the most important steps for reporting future FEA. An example of the report form is shown, and a template is provided, which can be used as a uniform basis for future documentation. The future application of the presented report form will show whether serious errors in biomechanical investigations using FEA can be minimized by this checklist. Finally, the credibility of the FEA in the clinical area and the scientific exchange in the community regarding reproducibility and exchangeability can be improved.

Pluripotency gene expression and growth control in cultures of peripheral blood monocytes during their conversion into programmable cells of monocytic origin (PCMO): evidence for a regulatory role of autocrine activin and TGF-ß.

Previous studies have shown that peripheral blood monocytes can be converted in vitro to a stem cell-like cell termed PCMO as evidenced by the re-expression of pluripotency-associated genes, transient proliferation, and the ability to adopt the phenotype of hepatocytes and insulin-producing cells upon tissue-specific differentiation. However, the regulatory interactions between cultured cells governing pluripotency and mitotic activity have remained elusive. Here we asked whether activin(s) and TGF-ß(s), are involved in PCMO generation. De novo proliferation of PCMO was higher under adherent vs. suspended culture conditions as revealed by the appearance of a subset of Ki67-positive monocytes and correlated with down-regulation of p21WAF1 beyond day 2 of culture. Realtime-PCR analysis showed that PCMO express ActRIIA, ALK4, TßRII, ALK5 as well as TGF-ß1 and the ßA subunit of activin. Interestingly, expression of ActRIIA and ALK4, and activin A levels in the culture supernatants increased until day 4 of culture, while levels of total and active TGF-ß1 strongly declined. PCMO responded to both growth factors in an autocrine fashion with intracellular signaling as evidenced by a rise in the levels of phospho-Smad2 and a drop in those of phospho-Smad3. Stimulation of PCMO with recombinant activins (A, B, AB) and TGF-ß1 induced phosphorylation of Smad2 but not Smad3. Inhibition of autocrine activin signaling by either SB431542 or follistatin reduced both Smad2 activation and Oct4A/Nanog upregulation. Inhibition of autocrine TGF-ß signaling by either SB431542 or anti-TGF-ß antibody reduced Smad3 activation and strongly increased the number of Ki67-positive cells. Furthermore, anti-TGF-ß antibody moderately enhanced Oct4A/Nanog expression. Our data show that during PCMO generation pluripotency marker expression is controlled positively by activin/Smad2 and negatively by TGF-ß/Smad3 signaling, while relief from growth inhibition is primarily the result of reduced TGF-ß/Smad3, and to a lesser extent, activin/Smad2 signaling.

The genetic prehistory of the New World Arctic.

The New World Arctic, the last region of the Americas to be populated by humans, has a relatively well-researched archaeology, but an understanding of its genetic history is lacking. We present genome-wide sequence data from ancient and present-day humans from Greenland, Arctic Canada, Alaska, Aleutian Islands, and Siberia. We show that Paleo-Eskimos (~3000 BCE to 1300 CE) represent a migration pulse into the Americas independent of both Native American and Inuit expansions. Furthermore, the genetic continuity characterizing the Paleo-Eskimo period was interrupted by the arrival of a new population, representing the ancestors of present-day Inuit, with evidence of past gene flow between these lineages. Despite periodic abandonment of major Arctic regions, a single Paleo-Eskimo metapopulation likely survived in near-isolation for more than 4000 years, only to vanish around 700 years ago.

Clonal Progression during the T Cell-Dependent B Cell Antibody Response Depends on the Immunoglobulin DH Gene Segment Repertoire.

The diversity of the third complementarity determining region of the IgH chain is constrained by natural selection of immunoglobulin diversity (DH) sequence. To test the functional significance of this constraint in the context of thymus-dependent (TD) immune responses, we immunized BALB/c mice with WT or altered DH sequence with 2-phenyloxazolone-coupled chicken serum albumin (phOx-CSA). We chose this antigen because studies of the humoral immune response to the hapten phOx were instrumental in the development of the current theoretical framework on which our understanding of the forces driving TD responses is based. To allow direct comparison, we used the classic approach of generating monoclonal Ab (mAb) from various stages of the immune response to phOx to assess the effect of changing the sequence of the DH on clonal expansion, class switching, and affinity maturation, which are hallmarks of TD responses. Compared to WT, TD-induced humoral IgM as well as IgG antibody production in the D-altered ΔD-DµFS and ΔD-iD strains were significantly reduced. An increased prevalence of IgM-producing hybridomas from late primary, secondary, and tertiary memory responses suggested either impaired class switch recombination (CSR) or impaired clonal expansion of class switched B cells with phOx reactivity. Neither of the D-altered strains demonstrated the restriction in the VH/VL repertoire, the elimination of VH1 family-encoded antibodies, the focusing of the distribution of CDR-H3 lengths, or the selection for the normally dominant Ox1 clonotype, which all are hallmarks of the anti-phOx response in WT mice. These changes in clonal selection and expansion, as well as CSR indicate that the genetic constitution of the DH locus, which has been selected by evolution, can strongly influence the functional outcome of a TD humoral response.

The effect of erythropoiesis-stimulating agents in patients with therapy-refractory autoimmune hemolytic anemia.

BACKGROUND: Many patients with autoimmune hemolytic anemia (AIHA) do not respond to standard therapy and/or may develop severe complications which can be of fatal outcome. There is some evidence that erythropoiesis-stimulating agents (ESAs) may be helpful in the management of such patients. METHODS: We describe the effect of ESAs in 12 new patients with therapy-refractory AIHA (7 of warm type and 5 of cold type) and review 5 previously reported cases. Serological testing was performed using standard methods. RESULTS: All patients responded well to treatment with ESAs. At least 5 of the 17 patients demonstrated complete recovery, and none of the patients developed significant adverse reactions due to treatment with ESAs. CONCLUSION: The mechanism by which ESAs improves hemolysis in AIHA is not completely clear. In addition to increased production and prolonged RBC survival, it may inhibit eryptosis (programmed cell death). ESAs represent a new option in the treatment of decompensated and/or refractory AIHA of warm and cold type. However, more information is required to assess which patients can be treated with ESAs.

Immunoglobulin class switching appears to be regulated by B-cell antigen receptor-specific T-cell action.

Antigen affinity is commonly viewed as the driving force behind the selection for dominant clonotypes that can occur during the T-cell-dependent processes of class switch recombination (CSR) and immune maturation. To test this view, we analyzed the variable gene repertoires of natural monoclonal antibodies to the hapten 2-phenyloxazolone (phOx) as well as those generated after phOx protein carrier-induced thymus-dependent or Ficoll-induced thymus-independent antigen stimulation. In contrast to expectations, the extent of IgM heterogeneity proved similar and many IgM from these three populations exhibited similar or even greater affinities than the classic Ox1 clonotype that dominates only after CSR among primary and memory IgG. The population of clones that were selected during CSR exhibited a reduced VH/VL repertoire that was enriched for variable domains with shorter and more uniform CDR-H3 lengths and almost completely stripped of variable domains encoded by the large VH1 family. Thus, contrary to the current paradigm, T-cell-dependent clonal selection during CSR appeared to select for VH family and CDR-H3 loop content even when the affinity provided by alternative clones exhibited similar to increased affinity for antigen.

Benefits and burden of the maternally-mediated immunological imprinting.

The ontogenetic development of both the immune and the nervous system entirely depend on external environmental signals that induce a lifelong learning process. The resulting collective immunological knowledge about the external world is transmitted in an epi-genetic fashion to the offspring, but only from the maternal and not the paternal side, with maternal IgG as the main transgenerational vector. As products of thymus-dependent responses, maternal IgG have undergone immune maturation by somatic hypermutations and are, therefore, acquired immunological phenotypes representing a great deal of the mother's immunological experience. During a limited neonatal imprinting period, maternal antibodies induce T cell-dependent idiotypic responses. These exert up to life-long determinative influences which may even be dominant over seemingly genetic predispositions. Such long-term immunological imprinting effects can be detected as (a) selection of the adult T and B cell repertoires, (b) anti-microbial protection by antigen-reactive antibodies (idiotypes) and anti-idiotypes, (c) allergen-specific suppression of IgE responsiveness by allergen-reactive IgG idiotype or corresponding anti-idiotype and (d) induction of autoimmune diseases by maternally-derived autoantibodies. Hence, immunological imprinting by maternal IgG antibodies will mostly be beneficial, but in case of autoantibodies can also be a burden for the initial development of the nascent immune system.

Thymus-independent type 2 antigen induces a long-term IgG-related network memory.

Thymus-independent type 2 (TI-2) antigens occasionally induce long-lasting IgM memory, but do not prime for typical secondary IgG responses. However, contrary to current understanding, we detected several TI-2-induced long-term memory effects in subsequent thymus-dependent (TD) responses to the hapten 2-phenyloxazolone coupled to a protein carrier. The early primary TD response, even 3 months after TI-2 immunization, included non-mutated IgM as well as IgG antibodies exhibiting higher affinities than the Ox1 idiotype which dominates and has highest affinity in sole TD responses. The secondary exclusive IgG response 8 weeks later contained major hitherto non-observed clones. Somatic hypermutation on the normally dominant V(H)Ox1 gene was largely silenced while the associated VkappaOx1 exhibited the classical affinity-enhancing mutations, thus suggesting a separate regulation of this process for V(H) and V(L) genes. Mutations accumulated in genes which normally are rarely or non-expressed or non-mutating. First evidence is presented that receptor revision by V(H) replacement may occur during immune maturation in genetically non-engineered wildtype mice. We conclude that the TI-2 antigen-induced altered selection of TD Ag-inducible clones and its severe gene-specific influence on further somatic mutations and affinity maturation represents a network memory, which we hypothesize to be mediated by anti-idiotypic regulatory T cells.

Apoptosis is a natural stimulus of IL6R shedding and contributes to the proinflammatory trans-signaling function of neutrophils.

Interleukin 6 (IL6) trans-signaling has emerged as a prominent regulator of immune responses during both innate and acquired immunity. Regulation of IL6 trans-signaling is reliant upon the release of soluble IL6 receptor (sIL6R), which binds IL6 to create an agonistic IL6/sIL6R complex capable of activating cell types that would not normally respond to IL6 itself. Here we show that intrinsic and extrinsic apoptotic stimulation by DNA damage, cytokine deprivation, and Fas stimulation promotes shedding of sIL6R. Apoptosis-induced shedding of the IL6R was caspase dependent but PKC independent, with inhibition of ADAM17 preventing IL6R shedding. Such insight is relevant to the control of acute inflammation, where transition from the initial neutrophil infiltration to a more sustained population of mononuclear cells is essential for the resolution of the inflammatory process. This transitional event is governed by IL6 trans-signaling. This study demonstrates that IL6R is shed from apoptotic human neutrophils. In vivo studies in a murine inflammation model showed that neutrophil depletion resulted in reduced local sIL6R levels and a concomitant decrease in mononuclear cells, suggesting that apoptosis-induced IL6R shedding from neutrophils promotes IL6 trans-signaling and regulates the attraction of monocytic cells involved in the clearance of apoptotic neutrophils.

Antimicrobial psoriasin (S100A7) protects human skin from Escherichia coli infection.

Human healthy skin is continuously exposed to bacteria, but is particularly resistant to the common gut bacterium Escherichia coli. We show here that keratinocytes secrete, as the main E. coli-killing compound, the S100 protein psoriasin in vitro and in vivo in a site-dependent way. In vivo treatment of human skin with antibodies to psoriasin inhibited its E. coli-killing properties. Psoriasin was induced in keratinocytes in vitro and in vivo by E. coli, indicating that its focal expression in skin may derive from local microbial induction. Zn(2+)-saturated psoriasin showed diminished antimicrobial activity, suggesting that Zn(2+) sequestration could be a possible antimicrobial mechanism. Thus, psoriasin may be key to the resistance of skin against E. coli.

Metalloproteinase inhibition augments antitumor efficacy of the anti-CD30 immunotoxin Ki-3(scFv)-ETA' against human lymphomas in vivo.

There is increasing evidence that the shedding of extracellular antigen domains impedes selective immunotherapy. One example is CD30, which is overexpressed on the surface of malignant lymphoma cells and has been identified as a promising target for antibody-based immunotherapy. However, CD30 is cleaved from the surface of target cells and the resulting soluble ectodomain (sCD30) is protecting the cells from antibody binding. Shedding can be inhibited by hydroxamate inhibitors of metalloproteinases such as BB-3644. We thus evaluated the influence of BB-3644 on the efficacy of the anti-CD30 single-chain immunotoxin Ki-3(scFv)-ETA'. In vitro, the addition of BB-3644 augmented the antitumor effect of Ki-3(scFv)-ETA' against Hodgkin-derived L540Cy cells by a factor of 2.75. Severe combined immunodeficiency (SCID) mice challenged with CD30-positive L540Cy cells were treated with the immunotoxin. One single nontoxic dose of BB-3644 increased the mean survival time of animals treated concomitantly with Ki-3(scFv)-ETA' to 93 days as compared with 35 days in the control (p = 0.0017). When BB-3644 was continuously delivered using subcutaneously implanted pumps, this effect was even more pronounced with no observed tumor growth in the animals within 200 days. Thus, concomitant application of metalloproteinase inhibitors might become clinically relevant in antibody-based immunotherapy against targets known to be shed from tumor cells.

Stem cell-mediated tolerance inducing strategies in organ transplantation.

The scope of possible tools to modulate the recipients immune response towards tolerance induction basically includes deletional and non deletional mechanisms, which are currently targeted by various strategies including monoclonal antibodies, cytokine deviation, chimerism induction and the support of regulating T-cells. Here we summarize the main findings in the field derived from experimental animal studies and currently performed clinical studies. This review focuses to give a clinically relevant overview over relevant tolerance inducing concepts, taking into consideration risk profiles and clinical efficacy associated with specific immunosuppressive regiments currently applied in the clinical setting of transplantation.

The ectodomain shedding of CD30 is specifically regulated by peptide motifs in its cysteine-rich domains 2 and 5.

Tumor necrosis factor (TNF)-alpha converting enzyme (TACE) is responsible for the ectodomain release of various membrane proteins by proteolytic cleavage in close proximity to the cell membrane. Despite the wide spectrum of possible substrates, selective cleavage can be achieved by substrate cross-linking. To explore the underlying mechanism, we studied the TACE-mediated shedding of CD30. Whereas the constitutive release of the soluble ectodomain of CD30 (sCD30) from the lymphoma cell line Karpas 299 was enhanced by most anti-CD30 antibodies, it was inhibited by antibodies Ber-H2 and Ki-4. On the basis of the recognized epitopes, shedding seemed to depend on the availability of the cysteine-rich domains (CRD) 2 and 5 of the CD30 ectodomain. CRD2 and 5 have almost identical amino acid sequences and are localized distant from the TACE-targeted cleavage site. Soluble CD30, the product of this enzyme reaction, did not inhibit, but on the contrary, it stimulated CD30 shedding in a CRD2/5-dependent manner. This process could also be induced by CRD2/5-derived peptides but not by a CRD1-derived control peptide. This example of a product-activation was CD30 selective since other TACE substrates such as TNFR1 or TNF-alpha were not affected. These data suggest that CD30 shedding is stimulated by an elevated local availability of CRD2 or 5, possibly by forming a docking station for the releasing enzyme through substrate aggregation.