Accuracy measurement of different marker based motion analysis systems for biomechanical applications: A round robin study.

INTRODUCTION: Multiple camera systems are widely used for 3D-motion analysis. Due to increasing accuracies these camera systems gained interest in biomechanical research areas, where high precision measurements are desirable. In the current study different measurement systems were compared regarding their measurement accuracy. MATERIALS AND METHODS: Translational and rotational accuracy measurements as well as the zero offset measurements of seven different measurement systems were performed using two reference devices and two different evaluation algorithms. All measurements were performed in the same room with constant temperature at the same laboratory. Equal positions were measured with the systems according to a standardized protocol. Measurement errors were determined and compared. RESULTS: The highest measurement errors were seen for a measurement system using active ultrasonic markers, followed by another active marker measurement system (infrared) having measurement errors up to several hundred micrometers. The highest accuracies were achieved by three stereo camera systems, using passive 2D marker points having errors typically below 20 µm. CONCLUSIONS: This study can help to better assess the results obtained with different measurement systems. With the focus on the measurement accuracy, only one aspect in the selection of a system was considered. Depending on the requirements of the user, other factors like measurement frequency, the maximum analyzable volume, the marker type or the costs are important factors as well.

Functionalization of screw implants with superelastic structured Nitinol anchoring elements.

BACKGROUND: Demographic change is leading to an increase in the number of osteoporotic patients, so a rethink is required in implantology in order to be able to guarantee adequate anchoring stability in the bone. The functional modification of conventional standard screw implants using superelastic, structured Ti6Al4V anchoring elements promises great potential for increasing anchoring stability. METHODS: For this purpose, conventional screw implants were mechanically machined and extended so that structured-superelastic-positionable-Ti6Al4V anchoring elements could be used. The novel implants were investigated with three tests. The setup of the anchoring elements was investigated in CT studies in an artificial bone. In a subsequent simplified handling test, the handling of the functional samples was evaluated under surgical conditions. The anchorage stability compared to standard screw implants was investigated in a final pullout test according to ASTM F543-the international for the standard specification and test methods for metallic medical bone screws. RESULTS: The functionalization of conventional screw implants with structured superelastic Ti6Al4V anchoring elements is technically realizable. It was demonstrated that the anchoring elements can be set up in the artificial bone without any problems. The anchorage mechanism is easy to handle under operating conditions. The first simplified handling test showed that at the current point of the investigations, the anchoring elements have no negative influence on the surgical procedure (especially under the focus of screw implantation). Compared to conventional standard screws, more mechanical work is required to remove the functional patterns completely from the bone. CONCLUSION: In summary, it was shown that conventional standard screw implants can be functionalized with Ti6Al4V-structured NiTi anchoring elements and the new type of screws are suitable for orthopedic and neurosurgical use. A first biomechanical test showed that the anchoring stability could be increased by the anchoring elements.

Investigation into the Hybrid Production of a Superelastic Shape Memory Alloy with Additively Manufactured Structures for Medical Implants.

The demographic change in and the higher incidence of degenerative bone disease have resulted in an increase in the number of patients with osteoporotic bone tissue causing. amongst other issues, implant loosening. Revision surgery to treat and correct the loosenings should be avoided, because of the additional patient stress and high treatment costs. Shape memory alloys (SMA) can help to increase the anchorage stability of implants due to their superelastic behavior. The present study investigates the potential of hybridizing NiTi SMA sheets with additively manufactured Ti6Al4V anchoring structures using laser powder bed fusion (LPBF) technology to functionalize a pedicle screw. Different scanning strategies are evaluated, aiming for minimized warpage of the NiTi SMA sheet. For biomechanical tests, functional samples were manufactured. A good connection between the additively manufactured Ti6Al4V anchoring structures and NiTi SMA substrate could be observed though crack formation occurring at the transition area between the two materials. These cracks do not propagate during biomechanical testing, nor do they lead to flaking structures. In summary, the hybrid manufacturing of a NiTi SMA substrate with additively manufactured Ti6Al4V structures is suitable for medical implants.

Biological Cell Investigation of Structured Nitinol Surfaces for the Functionalization of Implants.

Expandable implants including shape memory alloy (SMA) elements have great potential to minimize the risk of implant loosening and to increase the primary stability of bone anchoring. Surface structuring of such elements may further improve these properties and support osteointegration and bone healing. In this given study, SMA sheets were processed by deploying additive and removal manufacturing technologies for 3D-printed surgical implants. The additive technology was realized by applying a new laser beam melting technology to print titanium structures on the SMA sheets. The removal step was realized as a standard process with an ultrashort-pulse laser. The morphology, metabolic activity, and mineralization patterns of human bone marrow stromal cells were examined to evaluate the biocompatibility of the new surface structures. It was shown that both surface structures support cell adhesion and the formation of a cytoskeleton. The examination of the metabolic activity of the marrow stromal cells on the samples showed that the number of cells on the laser-structured samples was lower when compared to the 3D-printed ones. The calcium phosphate accumulation, which was used to examine the mineralization of marrow stromal cells, was higher in the laser-structured samples than in the 3D-printed ones. These results indicate that the additive- and laser-structured SAM sheets seem biocompatible and that the macrostructure surface and manufacturing technology may have positive influences on the behavior of the bone formation. The use of the new additive technique and the resulting macrostructures seems to be a promising approach to combine increased anchorage stability with simultaneously enhanced osteointegration.

CX3CR1 knockout aggravates Coxsackievirus B3-induced myocarditis.

Studies on inflammatory disorders elucidated the pivotal role of the CX3CL1/CX3CR1 axis with respect to the pathophysiology and diseases progression. Coxsackievirus B3 (CVB3)-induced myocarditis is associated with severe cardiac inflammation, which may progress to heart failure. We therefore investigated the influence of CX3CR1 ablation in the model of acute myocarditis, which was induced by inoculation with 5x105 plaque forming units of CVB3 (Nancy strain) in either CX3CR1-/- or C57BL6/j (WT) mice. Seven days after infection, myocardial inflammation, remodeling, and titin expression and phosphorylation were examined by immunohistochemistry, real-time PCR and Pro-Q diamond stain. Cardiac function was assessed by tip catheter. Compared to WT CVB3 mice, CX3CR1-/- CVB3 mice exhibited enhanced left ventricular expression of inflammatory cytokines and chemokines, which was associated with an increase of immune cell infiltration/presence. This shift towards a pro-inflammatory immune response further resulted in increased cardiac fibrosis and cardiomyocyte apoptosis, which was reflected by an impaired cardiac function in CX3CR1-/- CVB3 compared to WT CVB3 mice. These findings demonstrate a cardioprotective role of CX3CR1 in CVB3-infected mice and indicate the relevance of the CX3CL1/CX3CR1 system in CVB3-induced myocarditis.

CX3CR1-dependent recruitment of mature NK cells into the central nervous system contributes to control autoimmune neuroinflammation.

Fractalkine receptor (CX3CR1)-deficient mice develop very severe experimental autoimmune encephalomyelitis (EAE), associated with impaired NK cell recruitment into the CNS. Yet, the precise implications of NK cells in autoimmune neuroinflammation remain elusive. Here, we investigated the pattern of NK cell mobilization and the contribution of CX3CR1 to NK cell dynamics in the EAE. We show that in both wild-type and CX3CR1-deficient EAE mice, NK cells are mobilized from the periphery and accumulate in the inflamed CNS. However, in CX3CR1-deficient mice, the infiltrated NK cells displayed an immature phenotype contrasting with the mature infiltrates in WT mice. This shift in the immature/mature CNS ratio contributes to EAE exacerbation in CX3CR1-deficient mice, since transfer of mature WT NK cells prior to immunization exerted a protective effect and normalized the CNS NK cell ratio. Moreover, mature CD11b(+) NK cells show higher degranulation in the presence of autoreactive 2D2 transgenic CD4(+) T cells and kill these autoreactive cells more efficiently than the immature CD11b(-) fraction. Together, these data suggest a protective role of mature NK cells in EAE, possibly through direct modulation of T cells inside the CNS, and demonstrate that mature and immature NK cells are recruited into the CNS by distinct chemotactic signals.

Deep transcriptome profiling of clinical Klebsiella pneumoniae isolates reveals strain and sequence type-specific adaptation.

Health-care-associated infections by multi-drug-resistant bacteria constitute one of the greatest challenges to modern medicine. Bacterial pathogens devise various mechanisms to withstand the activity of a wide range of antimicrobial compounds, among which the acquisition of carbapenemases is one of the most concerning. In Klebsiella pneumoniae, the dissemination of the K. pneumoniae carbapenemase is tightly connected to the global spread of certain clonal lineages. Although antibiotic resistance is a key driver for the global distribution of epidemic high-risk clones, there seem to be other adaptive traits that may explain their success. Here, we exploited the power of deep transcriptome profiling (RNA-seq) to shed light on the transcriptomic landscape of 37 clinical K. pneumoniae isolates of diverse phylogenetic origins. We identified a large set of 3346 genes which was expressed in all isolates. While the core-transcriptome profiles varied substantially between groups of different sequence types, they were more homogenous among isolates of the same sequence type. We furthermore linked the detailed information on differentially expressed genes with the clinically relevant phenotypes of biofilm formation and bacterial virulence. This allowed for the identification of a diminished expression of biofilm-specific genes within the low biofilm producing ST258 isolates as a sequence type-specific trait.

Treatment of Chronic Experimental Autoimmune Encephalomyelitis with Epigallocatechin-3-Gallate and Glatiramer Acetate Alters Expression of Heme-Oxygenase-1.

We previously demonstrated that epigallocatechin-3-gallate (EGCG) synergizes with the immunomodulatory agent glatiramer acetate (GA) in eliciting anti-inflammatory and neuroprotective effects in the relapsing-remitting EAE model. Thus, we hypothesized that mice with chronic EAE may also benefit from this combination therapy. We first assessed how a treatment with a single dose of GA together with daily application of EGCG may modulate EAE. Although single therapies with a suboptimal dose of GA or EGCG led to disease amelioration and reduced CNS inflammation, the combination therapy had no effects. While EGCG appeared to preserve axons and myelin, the single GA dose did not improve axonal damage or demyelination. Interestingly, the neuroprotective effect of EGCG was abolished when GA was applied in combination. To elucidate how a single dose of GA may interfere with EGCG, we focused on the anti-inflammatory, iron chelating and anti-oxidant properties of EGCG. Surprisingly, we observed that while EGCG induced a downregulation of the gene expression of heme oxygenase-1 (HO-1) in affected CNS areas, the combined therapy of GA+EGCG seems to promote an increased HO-1 expression. These data suggest that upregulation of HO-1 may contribute to diminish the neuroprotective benefits of EGCG alone in this EAE model. Altogether, our data indicate that neuroprotection by EGCG in chronic EAE may involve regulation of oxidative processes, including downmodulation of HO-1. Further investigation of the re-dox balance in chronic neuroinflammation and in particular functional studies on HO-1 are warranted to understand its role in disease progression.

Characterization of natural killer cells in paired CSF and blood samples during neuroinflammation.

Natural killer (NK) cells from paired CSF and blood samples of patients with multiple sclerosis (MS), other neuroinflammatory diseases (IND), and non-inflammatory neurological diseases (NIND) were characterized using flow cytometry. NK cell frequency in CSF was overall decreased compared to blood, particularly in MS patients. In contrast to blood NK cells, during neuroinflammation, CSF NK cells display an immature phenotype with bright expression of CD56 and CD27 and reduced CX3CR1 expression. Our findings suggest that, as for central memory T cells, CSF may represent an intermediary compartment for NK cell trafficking and differentiation before entering the CNS parenchyma.

Mitoxantrone induces natural killer cell maturation in patients with secondary progressive multiple sclerosis.

Mitoxantrone is one of the few drugs approved for the treatment of progressive multiple sclerosis (MS). However, the prolonged use of this potent immunosuppressive agent is limited by the appearance of severe side effects. Apart from its general cytotoxic effect, the mode of action of mitoxantrone on the immune system is poorly understood. Thus, to develop safe therapeutic approaches for patients with progressive MS, it is essential to elucidate how mitoxantrone exerts it benefits. Accordingly, we initiated a prospective single-arm open-label study with 19 secondary progressive MS patients. We investigated long-term effects of mitoxantrone on patient peripheral immune subsets using flow cytometry. While we corroborate that mitoxantrone persistently suppresses B cells in vivo, we show for the first time that treatment led to an enrichment of neutrophils and immunomodulatory CD8(low) T cells. Moreover, sustained mitoxantrone applications promoted not only persistent NK cell enrichment but also NK cell maturation. Importantly, this mitoxantrone-induced NK cell maturation was seen only in patients that showed a clinical response to treatment. Our data emphasize the complex immunomodulatory role of mitoxantrone, which may account for its benefit in MS. In particular, these results highlight the contribution of NK cells to mitoxantrone efficacy in progressive MS.

Magnetic resonance elastography reveals altered brain viscoelasticity in experimental autoimmune encephalomyelitis.

Cerebral magnetic resonance elastography (MRE) measures the viscoelastic properties of brain tissues in vivo. It was recently shown that brain viscoelasticity is reduced in patients with multiple sclerosis (MS), highlighting the potential of cerebral MRE to detect tissue pathology during neuroinflammation. To further investigate the relationship between inflammation and brain viscoelasticity, we applied MRE to a mouse model of MS, experimental autoimmune encephalomyelitis (EAE). EAE was induced and monitored by MRE in a 7-tesla animal MRI scanner over 4 weeks. At the peak of the disease (day 14 after immunization), we detected a significant decrease in both the storage modulus (G') and the loss modulus (G″), indicating that both the elasticity and the viscosity of the brain are reduced during acute inflammation. Interestingly, these parameters normalized at a later time point (day 28) corresponding to the clinical recovery phase. Consistent with this, we observed a clear correlation between viscoelastic tissue alteration and the magnitude of perivascular T cell infiltration at both day 14 and day 28. Hence, acute neuroinflammation is associated with reduced mechanical cohesion of brain tissues. Moreover, the reduction of brain viscoelasticity appears to be a reversible process, which is restored when inflammation resolves. For the first time, our study has demonstrated the applicability of cerebral MRE in EAE, and showed that this novel imaging technology is highly sensitive to early tissue alterations resulting from the inflammatory processes. Thus, MRE may serve to monitor early stages of perivascular immune infiltration during neuroinflammation.

Arteriogenesis is modulated by bradykinin receptor signaling.

RATIONALE: Positive outward remodeling of pre-existing collateral arteries into functional conductance arteries, arteriogenesis, is a major endogenous rescue mechanism to prevent cardiovascular ischemia. Collateral arterial growth is accompanied by expression of kinin precursor. However, the role of kinin signaling via the kinin receptors (B1R and B2R) in arteriogenesis is unclear. OBJECTIVE: The purpose of this study was to elucidate the functional role and mechanism of bradykinin receptor signaling in arteriogenesis. METHODS AND RESULTS: Bradykinin receptors positively affected arteriogenesis, with the contribution of B1R being more pronounced than B2R. In mice, arteriogenesis upon femoral artery occlusion was significantly reduced in B1R mutant mice as evidenced by reduced microspheres and laser Doppler flow perfusion measurements. Transplantation of wild-type bone marrow cells into irradiated B1R mutant mice restored arteriogenesis, whereas bone marrow chimeric mice generated by reconstituting wild-type mice with B1R mutant bone marrow showed reduced arteriogenesis after femoral artery occlusion. In the rat brain 3-vessel occlusion arteriogenesis model, pharmacological blockade of B1R inhibited arteriogenesis and stimulation of B1R enhanced arteriogenesis. In the rat, femoral artery ligation combined with arterial venous shunt model resulted in flow-driven arteriogenesis, and treatment with B1R antagonist R715 decreased vascular remodeling and leukocyte invasion (monocytes) into the perivascular tissue. In monocyte migration assays, in vitro B1R agonists enhanced migration of monocytes. CONCLUSIONS: Kinin receptors act as positive modulators of arteriogenesis in mice and rats. B1R can be blocked or therapeutically stimulated by B1R antagonists or agonists, respectively, involving a contribution of peripheral immune cells (monocytes) linking hemodynamic conditions with inflammatory pathways.

Analyses of phenotypic and functional characteristics of CX3CR1-expressing natural killer cells.

We previously demonstrated a correlation between the frequency of CX3CR1-expressing human natural killer (NK) cells and disease activity in multiple sclerosis and showed that CX3CR1(high) NK cells were more cytotoxic than their CX3CR1(neg/low) counterparts. Here we aimed to determine whether human NK cell fractions defined by CX3CR1 represent distinct subtypes. Phenotypic and functional NK cell analyses revealed that, distinct from CX3CR1(high), CX3CR1(neg/low) NK cells expressed high amounts of type 2 cytokines, proliferated robustly in response to interleukin-2 and promoted a strong up-regulation of the key co-stimulatory molecule CD40 on monocytes. Co-expression analyses of CX3CR1 and CD56 demonstrated the existence of different NK cell fractions based on the surface expression of these two surface markers, the CX3CR1(neg) CD56(bright), CX3CR1(neg) CD56(dim) and CX3CR1(high) CD56(dim) fractions. Additional investigations on the expression of NK cell receptors (KIR, NKG2A, NKp30 and NKp46) and the maturation markers CD27, CD62L and CD57 indicated that CX3CR1 expression of CD56(dim) discriminated between an intermediary CX3CR1(neg) CD56(dim) and fully mature CX3CR1(high) CD56(dim) NK cell fractions. Hence, CX3CR1 emerges as an additional differentiation marker that may link NK cell maturation with the ability to migrate to different organs including the central nervous system.

Preproenkephalin expression in peripheral blood mononuclear cells of acutely underweight and recovered patients with anorexia nervosa.

BACKGROUND: The prohormone preproenkephalin (ppE) and its derived peptides are involved in leukocyte functioning as well as in the regulation of hunger and satiety. Various abnormalities of the immune and endocrine systems have been described in states of malnutrition such as anorexia nervosa (AN). We hypothesized that ppE expression in AN patients may vary depending on the state of the disorder and the extent of malnutrition. METHODS: Expression of ppE mRNA was analysed in peripheral blood mononuclear cells of 29 underweight and 29 weight-recovered patients with AN and compared to that in 29 healthy control women. The extent of malnutrition was characterized by BMI and plasma leptin. Psychological distress and eating disorder specific-psychopathology was determined with the Symptom Checklist-90-Revised and the Eating Disorders Inventory-2. RESULTS: ppE gene expression was similar in all 3 groups and was not related to nutritional status or eating disorder symptoms. However, a significant negative correlation was found between ppE expression and obsessive-compulsive, depressive and anxious symptoms. In addition, ppE expression was higher in smokers compared to non-smokers. CONCLUSION: Although malnutrition and hypoleptinaemia as seen in patients with AN were not related to peripheral ppE expression, we demonstrated reduced ppE expression in patients with elevated psychological distress. Similar associations have been shown in animal models of stress. It remains speculative if psychological symptoms and/or stress may augment immune abnormalities in AN patients via a pathway that is independent of nutritional status and involves ppE.

Therapeutic targeting of chemokine signaling in Multiple Sclerosis.

Multiple Sclerosis (MS) is an autoimmune disease of the central nervous system (CNS) that is initiated and maintained by continuous migration of inflammatory immune cells from the periphery into the target organ. However, in autoimmunity, migration of immune cells is not only involved in the pathogenesis but also in the down-modulation of the autoimmune attack, which is probably mediated by the infiltration of certain regulatory immune cell populations inside the affected organs. The migratory activity of both proinflammatory and regulatory leucocytes is controlled by chemokines and their receptors. Thus, targeting the directed migration of immune cells and regulating leukocyte trafficking across the blood-brain barrier (BBB) by means of modulation of chemokine signaling receptors might open up new therapeutic avenues not only for MS but also for other autoimmune diseases. In this review we summarize the chemotactic signaling pathways known to be involved in neuroinflammation to date and the viability of these pathways as targets for therapeutic strategies.

Frequency of blood CX3CR1-positive natural killer cells correlates with disease activity in multiple sclerosis patients.

Multiple sclerosis (MS) is an autoimmune disease of the central nervous system (CNS) characterized by enormous variability in its clinical presentation and course, and for which clear diagnostic parameters are lacking. Here we performed an RNA screen in peripheral mononuclear cells from relapsing-remitting (RR) and primary progressive (PP) MS patients compared with healthy donors (HD) that indicated, among other findings, a role for the chemokine receptor CX3CR1 as a diagnostic marker. Gene expression and flow cytometric analyses demonstrated a significantly lower expression of CX3CR1 in MS patients compared with healthy individuals. The subpopulation of cells responsible for causing this reduced expression of CX3CR1 consisted exclusively of natural killer (NK) cells. Importantly, we found a correlation between disease activity and frequency of CX3CR1-positive NK cells in RRMS patients. These findings emphasize the role of NK cells in the development and course of MS and provide evidence for CX3CR1 expression as a marker for MS patients and disease activity.