In Vitro Induction of Human Regulatory T Cells Using Conditions of Low Tryptophan Plus Kynurenines.

Thymic regulatory T cells (tTregs) and induced regulatory T cells (iTregs) suppress murine acute graft-versus-host disease (GVHD). Previously, we demonstrated that the plasmacytoid dendritic cell indoleamine 2,3-dioxygenase (IDO) fosters the in vitro development of human iTregs via tryptophan depletion and kynurenine (Kyn) metabolites. We now show that stimulation of naïve CD4+ T cells in low tryptophan (low Trp) plus Kyn supports human iTreg generation. In vitro, low Trp + Kyn iTregs and tTregs potently suppress T effector cell proliferation equivalently but are phenotypically distinct. Compared with tTregs or T effector cells, bioenergetics profiling reveals that low Trp + Kyn iTregs have increased basal glycolysis and oxidative phosphorylation and use glutaminolysis as an energy source. Low Trp + Kyn iTreg viability was reliant on interleukin (IL)-2 in vitro. Although in vivo IL-2 administration increased low Trp + Kyn iTreg persistence on adoptive transfer into immunodeficient mice given peripheral blood mononuclear cells to induce GVHD, IL-2-supported iTregs did not improve recipient survival. We conclude that low Trp + Kyn create suppressive iTregs that have high metabolic needs that will need to be addressed before clinical translation.

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.

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.

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.

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.

Cytosolic malate dehydrogenase activity helps support glycolysis in actively proliferating cells and cancer.

Increased glucose consumption is a hallmark of cancer cells. The increased consumption and subsequent metabolism of glucose during proliferation creates the need for a constant supply of NAD, a co-factor in glycolysis. Regeneration of the NAD required to support enhanced glycolysis has been attributed to the terminal glycolytic enzyme, lactate dehydrogenase (LDH). However, loss of glucose carbons to biosynthetic pathways early in glycolysis reduces the carbon supply to LDH. Thus, alternative routes for NAD regeneration must exist to support the increased glycolytic rate while allowing for the diversion of glucose to generate biomass and support proliferation. Here we demonstrate, using a variety of cancer cell lines as well as activated primary T cells, that cytosolic malate dehydrogenase 1 (MDH1) is an alternative to LDH as a supplier of NAD. Moreover, our results indicate that MDH1 generates malate with carbons derived from glutamine, thus enabling utilization of glucose carbons for glycolysis and for biomass. Amplification of MDH1 occurs at an impressive frequency in human tumors and correlates with poor prognosis. Together, our findings suggest that proliferating cells rely on both MDH1 and LDH to replenish cytosolic NAD, and that therapies designed at targeting glycolysis must consider both dehydrogenases.

Early onset of inflammation during ontogeny of bipolar disorder: the NLRP2 inflammasome gene distinctly differentiates between patients and healthy controls in the transition between iPS cell and neural stem cell stages.

Neuro-inflammation and neuronal communication are considered as mis-regulated processes in the aetiology and pathology of bipolar disorder (BD). Which and when specific signal pathways become abnormal during the ontogeny of bipolar disorder patients is unknown. To address this question, we applied induced pluripotent stem cell (iPSC) technology followed by cortical neural differentiation on adipocyte-derived cells from BD type I patients (with psychotic episodes in psychiatric history) and healthy volunteers (controls). RNA sequencing in iPSC and cortical neural stem cell (NSC) lines were used to examine alterations between the transcriptomes from BD I and control samples during transition from the pluripotent stage towards the neural developmental stage. At the iPSC stage, the most highly significant differentially expressed gene (DEG) was the NLRP2 inflammasome (P=2.66 × 10-10). Also among 42 DEGs at the NSC stage, NLRP2 showed the strongest statistical significance (P=3.07 × 10-19). In addition, we have also identified several cytoskeleton-associated genes as DEGs from the NSC stage, such as TMP2, TAGLN and ACTA2; the former two genes are recognised for the first time to be associated with BD. Our results also suggest that iPSC-derived BD-cortical NSCs carry several abnormalities in dopamine and GABA receptor canonical pathways, underlining that our in vitro BD model reflects pathology in the central nervous system. This would indicate that mis-regulated gene expression of inflammatory, neurotransmitter and cytoskeletal signalling occurs during early fetal brain development of BD I patients.

Transcription factor C/EBP-ß induces tumor-suppressor phosphatase PHLPP2 through repression of the miR-17-92 cluster in differentiating AML cells.

PHLPP2, a member of the PH-domain leucine-rich repeat protein phosphatase (PHLPP) family, which targets oncogenic kinases, has been actively investigated as a tumor suppressor in solid tumors. Little is known, however, regarding its regulation in hematological malignancies. We observed that PHLPP2 protein expression, but not its mRNA, was suppressed in late differentiation stage acute myeloid leukemia (AML) subtypes. MicroRNAs (miR or miRNAs) from the miR-17-92 cluster, oncomir-1, were shown to inhibit PHLPP2 expression and these miRNAs were highly expressed in AML cells that lacked PHLPP2 protein. Studies showed that miR-17-92 cluster regulation was, surprisingly, independent of transcription factors c-MYC and E2F in these cells; instead all-trans-retinoic acid (ATRA), a drug used for terminally differentiating AML subtypes, markedly suppressed miR-17-92 expression and increased PHLPP2 protein levels and phosphatase activity. Finally, we demonstrate that the effect of ATRA on miR-17-92 expression is mediated through its target, transcription factor C/EBPß, which interacts with the intronic promoter of the miR-17-92 gene to inhibit transactivation of the cluster. These studies reveal a novel mechanism for upregulation of the phosphatase activity of PHLPP2 through C/EBPß-mediated repression of the miR-17-92 cluster in terminally differentiating myeloid cells.

Paired-pulse plasticity at the single release site level: an experimental and computational study.

CA3-CA1 glutamatergic synapses in the hippocampus exhibit a large heterogeneity in release probability (p) and paired-pulse (PP) plasticity, established already in the early neonatal period when the CA3-CA1 connections consist of only a single release site. At such a site two factors decide initial release probability: the number of immediately releasable vesicles (preprimed pool) and the vesicle release probability (P(ves1)). Depletion and replenishment of this pool, an alteration in P(ves), and desensitization of postsynaptic receptors may contribute to PP plasticity. A model based on data from single neonatal CA3-CA1 synapses has been used to address the relative importance of these factors for the heterogeneity in PP plasticity. At a 20 msec PP interval, the PP ratio (P(2)/P(1)) varied from 0.1 to 4.5 among the synapses. At this interval desensitization and replenishment were of little importance. The heterogeneity was explained mostly by the variation in P(ves1), whereas the preprimed pool size was of minor importance. P(ves) altered from the first to the second stimulus such that P(ves2) was rather uniform among the synapses. Its variation thus contributed little to the heterogeneity in PP ratio. The model also shows that the relationship between alterations in release probability and PP ratio is complex. Thus, an increase in release probability can be associated with an increase, a decrease, or no change at all in PP ratio, depending on the original values of P(ves1) and the preprimed pool and on which one of these factors is altered to produce the increase in release probability.

Onset Characteristics of Long-Term Potentiation in the Guinea-Pig Hippocampal CA1 Region in Vitro.

The temporal development of long-term potentiation (LTP) was examined in the CA1 region of the hippocampal slice preparation (bath temperature 30 degrees C). LTP was evoked by a single brief afferent tetanus (3 - 40 impulses at 50 Hz) given in the presence of picrotoxin (to facilitate LTP induction). Short-lasting potentiation processes unrelated to LTP were excluded by comparing the potentiation obtained in picrotoxin solution with that obtained in normal solution or in the presence of the N-methyl-D-aspartate receptor antagonist 2-amino-5-phosphonovalerate. LTP was also evoked by pairing single test volleys with brief (2 - 3 impulses) heterosynaptic tetani in picrotoxin solution. Both methods showed no significant rise of LTP until about 3 s after the induction event. LTP thereafter developed almost linearly towards a peak within 20 - 25 s after the tetanus, the time course being practially independent of the induction method and of the relative amount of LTP evoked. The latency and rise time of LTP depended on bath temperature, being about twice as long at 25 degrees C as at 30 degrees C. Following the peak, LTP rapidly decayed to less than half its peak value in 8 min, the decay tending to be less with longer trains. The LTP component reaching its peak 20 - 25 s after a tetanus was practically occluded after a saturating homosynaptic tetanization, and was only partially recovered 1 h afterwards. The latency to the onset of LTP suggests an indirect coupling between the calcium influx, presumed to trigger the potentiation, and the expression of LTP. The independence of the early time course with respect to the induction strength indicates that the intervening system(s) operates in a linear manner.

Long-term potentiation in the hippocampal CA1 region in the presence of N-methyl-D-aspartate receptor antagonists.

The present study deals with the question of whether L-type voltage-gated calcium channels can support the induction of input specific long-term potentiation. Tetanus-induced potentiation of synaptic transmission was examined in the CA1 region of normal and disinhibited guinea-pig hippocampal slices. It was found that afferent tetanization in the presence of 50 microM of the N-methyl-D-aspartate receptor antagonist D(-)-2-amino-5-phosphonopentanoic acid led to a prolonged input specific potentiation. This potentiation was found only in disinhibited slices, its induction required cooperativity, and it was associated with an increase in the early part of the field excitatory postsynaptic potential initial slope. It was not affected by the L-type voltage-gated calcium channel blocker nifedipine, but it was reduced when D(-)-2-amino-5-phosphonopentanoic acid was supplemented with other N-methyl-D-aspartate receptor antagonists. The present study also examined a potentiation that was not restricted to the activated synapses and that was not associated with an increase in the early part of the field excitatory postsynaptic potential initial slope. This potentiation was blocked by the L-type voltage-gated calcium channel antagonist nifedipine. It is concluded that calcium influx through L-type voltage-gated calcium channels participates in the generation of a prolonged potentiation, but not of the input specific N-methyl-D-aspartate receptor-dependent long-term potentiation. The results also suggest that high concentrations of D(-)-2-amino-5-phosphonopentanoic acid can be insufficient to fully prevent N-methyl-D-aspartate receptor activation by strong afferent tetanization in the disinhibited slice.

Staurosporine impairs both short-term and long-term potentiation in the dentate gyrus in vitro.

The present study shows that the protein kinase inhibitor staurosporine impairs the transient (< 60 min) potentiation (short-term potentiation) evoked by a weak tetanus to about the same extent as the more stable potentiation (long-term potentiation) evoked by a strong tetanus. This effect on short-term and long-term potentiation was seen both as a reduced magnitude and an increased decay rate, the latter being increased by about 50% compared to that seen under normal conditions. Comparison with potentiations evoked at different strengths in control solution suggested that much, but not all, of the increased decay rate observed in the presence of staurosporine could be explained by an impared induction. Staurosporine did not affect the N-methyl-D-aspartate-mediated field excitatory postsynaptic potential evoked by low-frequency stimulation or the magnitude of N-methyl-D-aspartate-mediated currents during high-frequency tetanization. This result suggests that the induction is impaired at a stage not related to the N-methyl-D-aspartate-mediated calcium influx. The present results suggest that short-term and long-term potentiation cannot be separated on the basis of protein kinase dependence. They do not support the common notion that short-term and long-term potentiation are mechanistically separate entities. Instead, the results support the view that long-term potentiation has a variable duration/stability dependent on the induction conditions and that protein kinase activation, via an action on induction mechanisms, contributes to its stabilization.

In vivo evidence for an activity-independent maturation of AMPA/NMDA signaling in the developing hippocampus.

Correlated pre- and postsynaptic activity is thought to promote maturation of excitatory synapses in the developing brain by directing AMPA receptors to pure NMDA synapses. However, this hypothesis has not been tested in vivo. Here, we have performed such test by inhibiting correlated neural activity in vivo using a single injection of tetanus toxin into the rat hippocampal CA1 area at postnatal day 1. When examined in the acute slice preparation (1-7 days post-injection), there was a strong reduction, down to 20% of control level, in the frequency of glutamatergic and GABAergic spontaneous postsynaptic currents (sPSCs). This activity deprivation led to a growth retardation of CA1 pyramidal neurons and to markedly faster decay kinetics of NMDA sPCSs. However, it did not alter the relationship between AMPA and NMDA sPSCs with respect to either their frequency or amplitude. Thus, although critical for certain aspects of neuronal development, correlated neural activity in the neonatal hippocampus does not seem to promote incorporation of AMPA receptors at pure NMDA synapses.

Distinct expressions for synaptic potentiation induced by calcium through voltage-gated calcium and N-methyl-D-aspartate receptor channels in the hippocampal CA1 region.

Brief elevation in postsynaptic calcium in hippocampal CA1 neurons leads to prolonged changes in synaptic strength. The calcium may enter the postsynaptic neuron via different routes, such as voltage-gated calcium channels or glutamate receptor channels of N-methyl-D-aspartate type, and/or be released from intracellular stores. The manner in which the synapse is altered, leading to the expression of an enhanced/depressed synaptic strength, is still unclear. The present study, performed using whole-cell recording from CA1 pyramidal cells of three- to five-week-old guinea-pigs, shows that postsynaptic depolarization alone, allowing for calcium influx through voltage-gated calcium channels, leads to a synaptic potentiation characterized by an altered time-course of the evoked excitatory synaptic response, an unaltered coefficient of variation of that response and a decreased paired-pulse facilitation likely related to a postsynaptic mechanism. These characteristics contrasted with those of long-term potentiation induced via activation of N-methyl-D-aspartate receptor channels, where the time-course was unaltered, the coefficient of variation was decreased and no change in paired-pulse facilitation was observed. Synapses can thus have mechanistically separate, but co-existent, potentiations of synaptic transmission initiated from separate sources for postsynaptic calcium.

Onset and stabilization of NMDA receptor-dependent hippocampal long-term potentiation.

This article discusses recent data concerning the temporal development of N-methyl-D-aspartate (NMDA) receptor-dependent potentiation in the hippocampus. It argues against a mechanistic subdivision of NMDA receptor-dependent potentiation into an early short-term potentiation (STP) and a slowly developing long-term potentiation (LTP). Thus, the article proposes that LTP starts a few seconds after the induction event, that it is fully developed within a minute, and that its subsequent stabilization is controlled by the degree of NMDA receptor activation, and associated increase of calcium concentration in the spine, during the induction event. It is suggested that most biochemical interventions that have been reported to interfere with the LTP process, for example application of protein kinase inhibitors, might have acted through an impairment of the induction mechanism rather than through an impairment of specific stabilization or maintenance mechanisms.

The early decay of long-term potentiation in the hippocampal CA1 region in vitro is reduced by activators of protein kinase C.

The effect of the exogenous protein kinase C (PKC) activator phorbol-12,13-diacetate (PDAc) on the early (0-10 min) time course of long-term potentiation (LTP) has been studied in the CA1 region of the guinea pig hippocampal slice. As shown previously, following a brief tetanus LTP develops almost linearly towards a peak value within 20-25 s, and decays thereafter rapidly to about a third of the peak value within 10 min after tetanization before a more stable level is reached. In the presence of 1.0 microM PDAc the growth phase of LTP is prolonged to 40-50 s, and the subsequent early decay is reduced. This reduction of the early decay resembles that previously found when increasing the number of afferent impulses of the LTP-generating tetanus. Examination of the early time course in solutions with different calcium-magnesium concentration ratios suggests that the observed effect of PDAc is not directly mediated via a change in presynaptic release probability, another effect observed after phorbol ester application. The results show that PKC activity is involved in the early stage of LTP development and support the idea that the early phase of LTP represents the same modification process as that underlying the more sustained phase of LTP.

Long-lasting potentiations evoked by a brief heterosynaptic tetanus in the guinea pig dentate gyrus in vitro.

The heterosynaptic effects induced by a brief afferent tetanization in the molecular layer of the dentate gyrus were investigated in the guinea pig hippocampal slice preparation using extracellular recording technique. At a brief interval (5 ms) between a single stimulation of the test afferents and the tetanus evoked in the conditioning afferents, a long-lasting (greater than 1 h) potentiation of the test field excitatory postsynaptic potential (EPSP) initial slope and amplitude was observed. This potentiation was occluded by prior homosynaptic tetanization of the test afferents, suggesting that it represents long-term potentiation (LTP). Thus, in the dentate gyrus, a single activation of a single test EPSP suffices to induce LTP when coinciding in time with a brief tetanus to other afferents. When not temporally paired with the test stimulation, i.e. at longer test-conditioning intervals (greater than 50 ms), the conditioning tetanus also elicited a long-lasting potentiation of the test field EPSP. This potentiation was, however, seen as a prolongation of the rising phase with no change in the field EPSP initial slope, and may represent a potentiation distinct from LTP.

Synaptic potentiation in the hippocampal CA1 region induced by application of N-methyl-D-aspartate.

The effect of local pressure application of N-methyl-D-aspartate (NMDA) in the synaptic layer of CA1 pyramidal cells was investigated in the guinea pig hippocampal slice preparation using extracellular recording technique. Application of NMDA produced a transient depression and a subsequent 30-60 min potentiation of the field excitatory postsynaptic potential (EPSP) seen as an increase of the initial slope and amplitude of the EPSP. The increase in amplitude was consistently greater than that of the initial slope. Prior tetanization that caused saturation of long-term potentiation prevented the generation of an NMDA-induced potentiation of the initial slope for more than 1-2 h, but not the generation of an increase of the amplitude.