A Fatal Alliance between Microglia, Inflammasomes, and Central Pain.

Microglia are the resident immune cells in the CNS, which survey the brain parenchyma for pathogens, initiate inflammatory responses, secrete inflammatory mediators, and phagocyte debris. Besides, they play a role in the regulation of brain ion homeostasis and in pruning synaptic contacts and thereby modulating neural networks. More recent work shows that microglia are embedded in brain response related to stress phenomena, the development of major depressive disorders, and pain-associated neural processing. The microglia phenotype varies between activated-toxic-neuroinflammatory to non-activated-protective-tissue remodeling, depending on the challenges and regulatory signals. Increased inflammatory reactions result from brain damage, such as stroke, encephalitis, as well as chronic dysfunctions, including stress and pain. The dimension of damage/toxic stimuli defines the amplitude of inflammation, ranging from an on-off event to low but continuous simmering to uncontrollable. Pain, either acute or chronic, involves inflammasome activation at the point of origin, the different relay stations, and the sensory and processing cortical areas. This short review aimed at identifying a sinister role of the microglia-inflammasome platform for the development and perpetuation of acute and chronic central pain and its association with changes in CNS physiology.

Structural and functional characterization of SiiA, an auxiliary protein from the SPI4-encoded type 1 secretion system from Salmonella enterica.

Salmonella invasion is mediated by a concerted action of the Salmonella pathogenicity island 4 (SPI4)-encoded type one secretion system (T1SS) and the SPI1-encoded type three secretion system (T3SS-1). The SPI4-encoded T1SS consists of five proteins (SiiABCDF) and secretes the giant adhesin SiiE. Here, we investigated structure-function relationships in SiiA, a non-canonical T1SS subunit. We show that SiiA consists of a membrane domain, an intrinsically disordered periplasmic linker region and a folded globular periplasmic domain (SiiA-PD). The crystal structure of SiiA-PD displays homology to that of MotB and other peptidoglycan (PG)-binding domains. SiiA-PD binds PG in vitro, albeit at an acidic pH, only. Mutation of Arg162 impedes PG binding of SiiA and reduces Salmonella invasion efficacy. SiiA forms a complex with SiiB at the inner membrane (IM), and the observed SiiA-MotB homology is paralleled by a predicted SiiB-MotA homology. We show that, similar to MotAB, SiiAB translocates protons across the IM. Mutating Asp13 in SiiA impairs proton translocation. Overall, SiiA shares numerous properties with MotB. However, MotAB uses the proton motif force (PMF) to energize the bacterial flagellum, it remains to be shown how usage of the PMF by SiiAB assists T1SS function and Salmonella invasion.

High-Throughput Quantification of Bacterial-Cell Interactions Using Virtual Colony Counts.

The quantification of bacteria in cell culture infection models is of paramount importance for the characterization of host-pathogen interactions and pathogenicity factors involved. The standard to enumerate bacteria in these assays is plating of a dilution series on solid agar and counting of the resulting colony forming units (CFU). In contrast, the virtual colony count (VCC) method is a high-throughput compatible alternative with minimized manual input. Based on the recording of quantitative growth kinetics, VCC relates the time to reach a given absorbance threshold to the initial cell count using a series of calibration curves. Here, we adapted the VCC method using the model organism Salmonella enterica sv. Typhimurium (S. Typhimurium) in combination with established cell culture-based infection models. For HeLa infections, a direct side-by-side comparison showed a good correlation of VCC with CFU counting after plating. For MDCK cells and RAW macrophages we found that VCC reproduced the expected phenotypes of different S. Typhimurium mutants. Furthermore, we demonstrated the use of VCC to test the inhibition of Salmonella invasion by the probiotic E. coli strain Nissle 1917. Taken together, VCC provides a flexible, label-free, automation-compatible methodology to quantify bacteria in in vitro infection assays.

Estrogen serum concentration affects blood immune cell composition and polarization in human females under controlled ovarian stimulation.

Estrogens modulate the immune system and possess anti-inflammatory properties. In line, immune cells express a variety of estrogen receptors (ER) including ER-alpha and -beta. In the present study, we examined the influence of 17beta-estradiol (E2) serum concentrations on blood leukocyte composition and their ex vivo polarization/activation status by FACS analysis in sub-fertile human females under controlled ovarian stimulation (COS). Using a set of cell-type and polarization-specific markers, we demonstrate that increased 17ß-estradiol (E2) serum concentrations yield an overall increase in leukocytes, neutrophils and monocytes but decreased lymphocytes. There was a clear ratio shift towards an increase in M2 monocytes with a protective quality and an increase in T-helper cells compared to a decrease in cytotoxic T-cells. These data support experimental findings and clinical trials, i.e. related to multiple sclerosis and other autoimmune-related diseases, that have shown a down-regulation of CD8(+) T cells and up-regulation of T-regulatory cells. Further studies have to pinpoint to which extent the immune system/-responsiveness of otherwise healthy female patients is affected by medium-term systemic E2 variations.

Integrating AlN with GdN Thin Films in an in Situ CVD Process: Influence on the Oxidation and Crystallinity of GdN.

The application potential of rare earth nitride (REN) materials has been limited due to their high sensitivity to air and moisture leading to facile oxidation upon exposure to ambient conditions. For the growth of device quality films, physical vapor deposition methods, such as molecular beam epitaxy, have been established in the past. In this regard, aluminum nitride (AlN) has been employed as a capping layer to protect the functional gadolinium nitride (GdN) from interaction with the atmosphere. In addition, an AlN buffer was employed between a silicon substrate and GdN serving as a seeding layer for epitaxial growth. In pursuit to grow high-quality GdN thin films by chemical vapor deposition (CVD), this successful concept is transferred to an in situ CVD process. Thereby, AlN thin films are included step-wise in the stack starting with Si/GdN/AlN structures to realize long-term stability of the oxophilic GdN layer. As a second strategy, a Si/AlN/GdN/AlN stacked structure was grown, where the additional buffer layer serves as the seeding layer to promote crystalline GdN growth. In addition, chemical interaction between GdN and the Si substrate can be prevented by spatial segregation. The stacked structures grown for the first time with a continuous CVD process were subjected to a detailed investigation in terms of structure, morphology, and composition, revealing an improved GdN purity with respect to earlier grown CVD thin films. Employing thin AlN buffer layers, the crystallinity of the GdN films on Si(100) could additionally be significantly enhanced. Finally, the magnetic properties of the fabricated stacks were evaluated by performing superconducting quantum interference device measurements, both of the as-deposited films and after exposure to ambient conditions, suggesting superparamagnetism of ferromagnetic GdN grains. The consistency of the magnetic properties precludes oxidation of the REN material due to the amorphous AlN capping layer.

Scarless deletion of up to seven methyl-accepting chemotaxis genes with an optimized method highlights key function of CheM in Salmonella Typhimurium.

Site-directed scarless mutagenesis is an essential tool of modern pathogenesis research. We describe an optimized two-step protocol for genome editing in Salmonella enterica serovar Typhimurium to enable multiple sequential mutagenesis steps in a single strain. The system is based on the λ Red recombinase-catalyzed integration of a selectable antibiotics resistance marker followed by replacement of this cassette. Markerless mutants are selected by expressing the meganuclease I-SceI which induces double-strand breaks in bacteria still harboring the resistance locus. Our new dual-functional plasmid pWRG730 allows for heat-inducible expression of the λ Red recombinase and tet-inducible production of I-SceI. Methyl-accepting chemotaxis proteins (MCP) are transmembrane chemoreceptors for a vast set of environmental signals including amino acids, sugars, ions and oxygen. Based on the sensory input of MCPs, chemotaxis is a key component for Salmonella virulence. To determine the contribution of individual MCPs we sequentially deleted seven MCP genes. The individual mutations were validated by PCR and genetic integrity of the final seven MCP mutant WRG279 was confirmed by whole genome sequencing. The successive MCP mutants were functionally tested in a HeLa cell infection model which revealed increased invasion rates for non-chemotactic mutants and strains lacking the MCP CheM (Tar). The phenotype of WRG279 was reversed with plasmid-based expression of CheM. The complemented WRG279 mutant showed also partially restored chemotaxis in swarming assays on semi-solid agar. Our optimized scarless deletion protocol enables efficient and precise manipulation of the Salmonella genome. As demonstrated with whole genome sequencing, multiple subsequent mutagenesis steps can be realized without the introduction of unwanted mutations. The sequential deletion of seven MCP genes revealed a significant role of CheM for the interaction of S. Typhimurium with host cells which might give new insights into mechanisms of Salmonella host cell sensing.

Effect of angular stability and other locking parameters on the mechanical performance of intramedullary nails.

To extend the indications of intramedullary nails for distal or proximal fractures, nails with angle stable locking options have been developed. Studies on the mechanical efficacy of these systems have been inconsistent likely due to confounding variables such as number, geometry, or orientation of the screws, as well as differences in the loading mode. Therefore, the aim of this study was to quantify the effect of angular stability on the mechanical performance of intramedullary nails. The results could then be compared with the effects of various locking screw parameters and loading modes. A generic model was developed consisting of artificial bone material and titanium intramedullary nail that provided the option to systematically modify the locking screw configuration. Using a base configuration, the following parameters were varied: number of screws, distance and orientation between screws, blocking of screws, and simulation of freehand locking. Tension/compression, torsional, and bending loads were applied. Stiffness and clearance around the zero loading point were determined. Angular stability had no effect on stiffness but completely blocked axial clearance (p=0.003). Simulation of freehand locking reduced clearance for all loading modes by at least 70% (p<0.003). The greatest increases in torsional and bending stiffness were obtained by increasing the number of locking screws (up to 80%, p<0.001) and by increasing the distance between them (up to 70%, p<0.001). In conclusion, our results demonstrate that the mechanical performance of IM nailing can be affected by various locking parameters of which angular stability is only one. While angular stability clearly reduces clearance of the screw within the nail, mechanical stiffness depends more on the number of screws and their relative distance. Thus, optimal mechanical performance in IM nailing could potentially be obtained by combining angular stability with optimal arrangement of locking screws.

Comparative analysis of gonadal steroid-mediated neuroprotection after transient focal ischemia in rats: route of application and substrate composition.

Progesterone (P) and 17ß-estradiol (E2) mitigate neuronal damage after experimentally induced traumatic brain injury (TBI) and ischemic stroke. Fish oil components such as omega-3 polyunsaturated fatty acids (PUFA n3) also provide neuroprotection in these traumatic models. Steroids and PUFA n3 dampen neuroinflammatory processes and regulate glial function in the affected brain areas. Using a transient focal ischemic rat model, we demonstrate that the co-application of PUFA n3 and P/E2 and the choice of the application route have a clear impact on the prevention of ischemia-induced infarct volume and behavioral recovery. A combinatory PUFA n3 plus P/E2 emulsion intravenously administered was most effective in reducing the infarct size and in restoring behavioral reconstitution compared to other oil emulsions and subcutaneous depot medication. These data encourage to refining clinical treatment protocols for TBI and stroke with gonadal steroids and to establishing combinatory drugs of steroids and fish oil-enriched emulsions thereby creating a win-win situation with two effective components.

Omega-3 polyunsaturated fatty acids ameliorate neuroinflammation and mitigate ischemic stroke damage through interactions with astrocytes and microglia.

Omega-3 polyunsaturated fatty acids (PUFA n3) provide neuroprotection due to their anti-inflammatory and anti-apoptotic properties as well as their regulatory function on growth factors and neuronal plasticity. These qualities enable PUFA n3 to ameliorate stroke outcome and limit neuronal damage. Young adult male rats received transient middle cerebral artery occlusion (tMCAO). PUFA n3 were intravenously administered into the jugular vein immediately after stroke and 12h later. We analyzed stroke volume and behavioral performance as well as the regulation of functionally-relevant genes in the penumbra. The extent of ischemic damage was reduced and behavioral performance improved subject to applied PUFA n3. Expression of Tau and growth-associated protein-43 genes were likewise restored. Ischemia-induced increase of cytokine mRNA levels was abated by PUFA n3. Using an in vitro approach, we demonstrate that cultured astroglial and microglia directly respond to PUFA n3 administration by preventing ischemia-induced increase of cyclooxygenase 2, hypoxia-inducible factor 1alpha, inducible nitric oxide synthase, and interleukin 1beta. Cultured cortical neurons also appeared as direct targets, since PUFA n3 shifted the Bcl-2-like protein 4 (Bax)/B-cell lymphoma 2 (Bcl 2) ratio towards an anti-apoptotic constellation. Thus, PUFA n3 reveal a high neuroprotective and anti-inflammatory potential in an acute ischemic stroke model by targeting astroglial and microglial function as well as improving neuronal survival strategies. Our findings signify the potential clinical feasibility of PUFA n3 therapeutic treatment in stroke and other acute neurological diseases.

A novel culture device for the evaluation of three-dimensional extracellular matrix materials.

Cell-matrix interactions in a three-dimensional (3D) extracellular matrix (ECM) are of fundamental importance in living tissue, and their in vitro reconstruction in bioartificial structures represents a core target of contemporary tissue engineering concepts. For a detailed analysis of cell-matrix interaction under highly controlled conditions, we developed a novel ECM evaluation culture device (EECD) that allows for a precisely defined surface-seeding of 3D ECM scaffolds, irrespective of their natural geometry. The effectiveness of EECD was evaluated in the context of heart valve tissue engineering. Detergent decellularized pulmonary cusps were mounted in EECD and seeded with endothelial cells (ECs) to study EC adhesion, morphology and function on a 3D ECM after 3, 24, 48 and 96 h. Standard EC monolayers served as controls. Exclusive top-surface-seeding of 3D ECM by viable ECs was demonstrated by laser scanning microscopy (LSM), resulting in a confluent re-endothelialization of the ECM after 96 h. Cell viability and protein expression, as demonstrated by MTS assay and western blot analysis (endothelial nitric oxide synthase, von Willebrand factor), were preserved at maintained levels over time. In conclusion, EECD proves as a highly effective system for a controlled repopulation and in vitro analysis of cell-ECM interactions in 3D ECM.

Biomechanical evaluation of interlocking lag screw design in intramedullary nailing of unstable pertrochanteric fractures.

OBJECTIVES: Intramedullary nails with special lag screw designs may provide improved mechanical performance and alleviate clinical problems. We hypothesize that the proximal design of trochanteric nails affects mechanical performance. METHODS: Ten pairs of human cadaveric femora were implanted with 2 different short intramedullary nails without (Gamma3) and with an interlocking lag screw (Intertan). An unstable, multifragmentary, pertrochanteric fracture was created. Bones were tested in a cyclic testing protocol with increasing loads until failure simulating 1 leg stance. Stiffness, failure load, cycles to failure, and fracture gap movements were measured. RESULTS: Initially stiffness of the interlocking lag screw nail was almost 40% larger (P = 0.005) compared with the noninterlocking nail. During the test, the difference in stiffness gradually decreased. Failure load (13%, P = 0.02) and cycles to failure (18%, P = 0.02) were larger for the interlocking nail construct. Rotation and varus collapse of the head were initially up to 84% lower (P = 0.013) for the interlocking technique. During the test, the rate of rotational instability gradually increased for both techniques. CONCLUSIONS: The interlocking lag screw design reduced movement of the femoral head and relative movement between fracture fragments. Beyond that the trapezoidal nail design of the Intertan reduced toggling within the trochanteric area and prolonged survival. Although this study showed a decrease in the retention of stability over time, failure did not occur until the equivalent of 2-3 months of reduced physical activity in which healing may have occurred under normal clinical conditions.

Biomechanical comparison of intramedullar versus extramedullar stabilization of intra-articular tibial plateau fractures.

BACKGROUND: Fractures of the proximal tibia occur very often and are a great challenge for trauma surgeons to stabilize. Although locked nails were developed to stabilize these fractures, this technique has not been sufficiently investigated. The purpose of this study was to biomechanically assess the stability of locked intramedullary nailing compared to locked plating. METHODS: 16 fresh frozen human cadaveric tibiae were osteotomized in the meta-diaphyseal intersection with an osteotomy gap of 10 mm and a single osteotomy through the medial epicondyle to simulate a 41-C.2 fracture. Stabilization was performed with an angle stable locked Targon-TX nail (n = 8) and two additional canulated screws. The other testing group (n = 8) was treated with two canulated screws and a five-hole LCP-PLT. The bones were tested in a cyclic testing protocol with increasing loads under compression and a load sharing of 60 % through the medial tibial plateau and 40 % to the lateral side. Stiffness and fracture gap movement were measured and failure mode was assessed. RESULTS: No significant differences were found between the two implants regarding load until failure. The stiffness of the intramedullary nailing group (927 N/mm) was statistically significantly higher than the stiffness of the plating group (564 N/mm). No differences were found for fracture gap movement in the z-axis. However, differences were found for dislocation of the proximal-lateral and proximal-medial fragments, with absolute values of 0.099 mm in the plate group and 0.66 mm in the nailing group at 800 N. Prior to failure, fracture gap movement was 0.22 mm for the plating group and 1.66 mm for the nailing group, a difference that was also statistically significantly different. The nailing group failed by screw cut-out while the plating group failed by screw breakage. CONCLUSION: Nailing of proximal tibia fractures leads to a stiffer implant-bone construct than plating. Since no adverse effects were found after nailing it seems to be a good alternative to plating for intra-articular proximal tibia fractures, especially in patients with soft tissue problems.

Auxiliary locking plate improves fracture stability and healing in intertrochanteric fractures fixated by intramedullary nail.

BACKGROUND: Intertrochanteric fractures present a significant management challenge due to their low inherent stability. The objective of this study was to determine whether an auxiliary locking plate decreases interfragmentary motions and improves fracture healing in intertrochanteric fractures treated by intramedullary nail. METHODS: Biomechanical tests and a clinical retrospective study in intertrochanteric to subtrochanteric nonunions were performed. Six synthetic femurs were osteotomized intertrochanterically and fixated with a long gamma nail and an additional locking compression plate. Mechanical tests were conducted that simulated the hip joint force during gait cycle. Following the initial test, the locking compression plate (LCP) was removed from each specimen and the test was repeated. Interfragmentary motions, strains on implants and osteosynthesis stiffness were determined. For the clinical part of the study, 13 intertrochanteric to subtrochanteric nonunions were treated with revisional long gamma nail and additional locking compression plate. Complications and time to union were determined. FINDINGS: Biomechanically, interfragmentary rotation was 48% smaller (P=0.047) and interfragmentary shear movement was 42% smaller (P=0.007) with locking compression plate. Strains on the nail decreased by 20-27% (P<0.027) and the osteosynthesis stiffness increased by 23% (P=0.005) with locking compression plate. Clinically, fracture healing was achieved in eleven out of 13 patients after 9.0months (range 4 to 22months). INTERPRETATION: The findings of our study indicate that auxiliary locked plating considerably improves biomechanical performance and results in successful healing of unstable intertrochanteric to subtrochanteric femur fractures.

RNA aptamers and spiegelmers: synthesis, purification, and post-synthetic PEG conjugation.

This unit describes the solid-phase synthesis and downstream processing for RNA oligonucleotides with a length of up to 40 to 50 nucleotides on a 1- to 4-mmol scale with subsequent conjugation to PEG using the L-RNA spiegelmer NOX-E36 as an example. Following synthesis and two-step deprotection, the crude oligonucleotide is purified by preparative reversed-phase HPLC and desalted by tangential flow ultrafiltration. The resulting intermediate amino-modified oligonucleotide is reacted with NHS-ester-activated PEG, and the oligonucleotide-PEG conjugate is obtained after preparative AX-HPLC purification, followed by ultrafiltration and lyophilization. Critical process parameters are described, as well as time considerations and examples for analytical methods used as in-process and quality controls.

The quest for an optimized protocol for whole-heart decellularization: a comparison of three popular and a novel decellularization technique and their diverse effects on crucial extracellular matrix qualities.

Decellularized cardiac extracellular matrix (ECM) has been introduced as a template for cardiac tissue engineering, providing the advantages of a prevascularized scaffold that mimics native micro- and macroarchitecture to a degree difficult to achieve with synthetic materials. Nonetheless, the decellularization protocols used to create acellular myocardial scaffolds vary widely throughout the literature. In this study we performed a direct comparison of three previously described protocols while introducing and evaluating a novel, specifically developed fourth protocol, by decellularizing whole rat hearts through software-controlled automatic coronary perfusion. Although all protocols preserved the macroarchitecture of the hearts and all resulting scaffolds could successfully be reseeded with C2C12 myoblasts, assessing their biocompatibility for three-dimensional in vitro studies, we found striking differences concerning the microcomposition of the ECM scaffolds on a histological and biochemical level. While laminin could still be detected in all groups, other crucial ECM components, like elastin and collagen IV, were completely removed by at least one of the protocols. Further, only three protocols maintained a glycosaminoglycan content comparable to native tissue, whereas the remaining DNA content within the ECM varied highly throughout all four tested protocols. This study showed that the degree of acellularity and resulting ECM composition of decellularized myocardial scaffolds strongly differs depending on the decellularization protocol.

In vivo functional performance and structural maturation of decellularised allogenic aortic valves in the subcoronary position.

OBJECTIVE: Successful animal and clinical implantation of decellularised heart valves has been performed in the pulmonary position. Comparable results have not yet been achieved for the aortic position with the high haemodynamic demands of the systemic circulation and the challenging implantation procedure. METHODS: Allogenic aortic valves (n=10) were decellularised using detergents (decellularised aortic valves (dAoVs)). Five prostheses were analysed for decellularisation quality and scaffold preservation. Five valves were orthotopically implanted in juvenile sheep in a subcoronary technique. After 5 months, echocardiography, immunohistology, histology, electron microscopy and western blot (WB) were used for analysis. RESULTS: All animals survived the follow-up with increased body weight (38.8 ± 2.8kg vs 56.0 ± 2.6kg, p<0.001). After implantation, three dAoVs showed negligible and two others minor insufficiency (I), which remained unchanged at explantation. Effective orifice area increased slightly (1.1 ± 0.2cm(2) vs 1.6 ± 0.3cm(2), p=0.051). Explanted dAoVs (n=4) showed excellent macroscopy with minor soft-tissue nodules observed at the free cusp margins of only one dAoV. No valve showed any signs of thrombosis or calcification. On microscopic evaluation, the cusp architecture was preserved with an almost complete endothelial repopulation as confirmed by vimentin(+)/von Willebrand factor (vWF(+))-staining, WB of endothelial markers (eNOS/vWF) and scanning electron microscopy (SEM). Partial interstitial reseeding with vimentin(+)/alpha-smooth muscle (αsm(+))-cells was noted. Quantitative measurement of collagen-IV, collagen-I, laminin and elastin (WB) demonstrated preserved scaffold composition as compared to native tissue. CONCLUSION: The dAoVs showed excellent functional outcome at 5 months in a subcoronary model of juvenile sheep. Advanced endothelial and nascent interstitial repopulation, with preserved structural integrity under the high-shear-stress milieu of the aortic valve, encourage further long-term studies.

Cognitive impairment in multiple sclerosis.

PURPOSE OF REVIEW: For a long time, cognitive impairment in multiple sclerosis patients has been considered less important than, for instance, physical disability. This is no longer true because of the crucial role that cognitive deficits play in the good day-to-day adjustment of patients. This review highlights recent progress made in this area. A special focus lies on studies investigating the neural correlates of cognitive impairment in multiple sclerosis patients as detectable by conventional, quantitative and functional magnetic resonance imaging. RECENT FINDINGS: Measures of information-processing speed appear to be the most robust and sensitive markers of cognitive impairment in multiple sclerosis patients. Recent studies demonstrate that single, predominantly speed-related cognitive tests may be superior to extensive and time-consuming test batteries in screening overall cognitive decline. Quantitative magnetic-resonance-imaging findings suggest the extent of subtle tissue damage in normal-appearing white and grey matter to correlate best with the severity of cognitive impairment in multiple sclerosis patients. SUMMARY: From neuropsychological test data, and findings from magnetic resonance imaging and functional magnetic resonance imaging it is evident that cognitive impairment in multiple sclerosis is not just the result of tissue destruction, but rather a balance between tissue destruction, tissue repair, and adaptive functional reorganization.

Phonological activation of category coordinates during speech planning is observable in children but not in adults: evidence for cascaded processing.

There is a long-standing debate in the area of speech production on the question of whether only words selected for articulation are phonologically activated (as maintained by serial-discrete models) or whether this is also true for their semantic competitors (as maintained by forward-cascading and interactive models). Past research has addressed this issue by testing whether retrieval of a target word (e.g., cat) affects--or is affected by--the processing of a word that is phonologically related to a semantic category coordinate of the target (e.g., doll, related to dog) and has consistently failed to obtain such mediated effects in adult speakers. The authors present a series of experiments demonstrating that mediated effects are present in children (around age 7) and diminish with increasing age. This observation provides further evidence for cascaded models of lexical retrieval.