Immunomodulation with romiplostim as a second-line strategy in primary immune thrombocytopenia: The iROM study.

Thrombopoietin receptor agonists (TPO-RAs) stimulate platelet production, which might restore immunological tolerance in primary immune thrombocytopenia (ITP). The iROM study investigated romiplostim's immunomodulatory effects. Thirteen patients (median age, 31 years) who previously received first-line treatment received romiplostim for 22 weeks, followed by monitoring until week 52. In addition to immunological data, secondary end-points included the sustained remission off-treatment (SROT) rate at 1 year, romiplostim dose, platelet count and bleedings. Scheduled discontinuation of romiplostim and SROT were achieved in six patients with newly diagnosed ITP, whereas the remaining seven patients relapsed. Romiplostim dose titration was lower and platelet count response was stronger in patients with SROT than in relapsed patients. In all patients, regulatory T lymphocyte (Treg) counts increased until study completion and the counts were higher in patients with SROT. Interleukin (IL)-4, IL-9 and IL-17F levels decreased significantly in all patients. FOXP3 (Treg), GATA3 (Th2) mRNA expression and transforming growth factor-ß levels increased in patients with SROT. Treatment with romiplostim modulates the immune system and possibly influences ITP prognosis. A rapid increase in platelet counts is likely important for inducing immune tolerance. Better outcomes might be achieved at an early stage of autoimmunity, but clinical studies are needed for confirmation.

Insect exoskeletons react to hypergravity.

A typical feature of biological materials is their ability to adapt to mechanical load. However, it is not known whether the cuticle exoskeleton, one of the most common biological structures, also shares this trait. Here, we show direct experimental evidence that prolonged exposure to hypergravity conditions affects the morphology and biomechanics of an insect exoskeleton. Locusts were raised for several weeks in a custom-designed centrifuge at various levels of hypergravity. Biomechanical measurements and X-ray microtomography show that up to 3 g load Young's modulus of the tibiae increased by about 67%. Higher gravitational loads however decreased the survival rate, body mass and endocuticle thickness. These results directly show that cuticle exoskeletons can react to hypergravity. This ability has so far only been known for bone endoskeletons and plants. Our findings thus add important context to the discussion on general ultimate factors in the evolution of adaptive biological materials and skeletal systems.

3D escape: an alternative paradigm for spatial orientation studies in insects.

Arthropods and in particular insects show a great variety of different exoskeletal sensors. For most arthropods, spatial orientation and gravity perception is not fully understood. In particular, the interaction of the different sensors is still a subject of ongoing research. A disadvantage of most of the experimental methods used to date to study the spatial orientation of arthropods in behavioral experiments is that the body or individual body parts are fixed partly in a non-natural manner. Therefore, often only the movement of individual body segments can be used to evaluate the experiments. We here present a novel experimental method to easily study 3D-escape movements in insects and analyze whole-body reaction. The animals are placed in a transparent container, filled with a lightweight substrate and rotating around two axes. To verify our setup, house crickets (Acheta domesticus) with selectively manipulated gravity-perceiving structures were analyzed. The spatial orientation behavior was quantified by measuring the time individuals took to escape toward the surface and the angular deviation toward the gravitational vector. These experiments confirm earlier results and therefore validated our experimental setup. Our new approach thus allows to investigate several comprehensive questions regarding the spatial orientation of insects and other animals.

Insect wing damage: causes, consequences and compensatory mechanisms.

The evolution of wings has played a key role in the success of insect species, allowing them to diversify to fill many niches. Insect wings are complex multifunctional structures, which not only have to withstand aerodynamic forces but also need to resist excessive stresses caused by accidental collisions. This Commentary provides a summary of the literature on damage-reducing morphological adaptations in wings, covering natural causes of wing collisions, their impact on the structural integrity of wings and associated consequences for both insect flight performance and life expectancy. Data from the literature and our own observations suggest that insects have evolved strategies that (i) reduce the likelihood of wing damage and (ii) allow them to cope with damage when it occurs: damage-related fractures are minimized because wings evolved to be damage tolerant and, in the case of wing damage, insects compensate for the reduced aerodynamic efficiency with dedicated changes in flight kinematics.

Morphology, shape variation and movement of skeletal elements in starfish (Asterias rubens).

Starfish (order: Asteroidea) possess a complex endoskeleton composed of thousands of calcareous ossicles. These ossicles are embedded in a body wall mostly consisting of a complex collagen fiber array. The combination of soft and hard tissue provides a challenge for detailed morphological and histological studies. As a consequence, very little is known about the general biomechanics of echinoderm endoskeletons and the possible role of ossicle shape in enabling or limiting skeletal movements. In this study, we used high-resolution X-ray microscopy to investigate individual ossicle shape in unprecedented detail. Our results show the variation of ossicle shape within ossicles of marginal, reticular and carinal type. Based on these results we propose an additional classification to categorize ossicles not only by shape but also by function into 'connecting' and 'node' ossicles. We also used soft tissue staining with phosphotungstic acid successfully and were able to visualize the ossicle ultrastructure at 2-µm resolution. We also identified two new joint types in the aboral skeleton (groove-on-groove joint) and between adambulacral ossicles (ball-and-socket joint). To demonstrate the possibilities of micro-computed tomographic methods in analyzing the biomechanics of echinoderm skeletons we exemplarily quantified changes in ossicle orientation for a bent ray for ambulacral ossicles. This study provides a first step for future biomechanical studies focusing on the interaction of ossicles and soft tissues during ray movements.

What goes up must come down: biomechanical impact analysis of falling locusts.

Many insects are able to precisely control their jumping movements. Once in the air, the properties of the actual landing site, however, are almost impossible to predict. Falling insects thus have to cope with the situation at impact. In particular, for insects jumping to escape predators, a controlled landing movement appears to be a major evolutionary advantage. A quick recovery into an upright and stable body posture minimizes the time to prepare for the next escape jump. In this study, we used high-speed recordings to investigate the falling and in particular the impact behavior of Schistocerca gregaria locusts, a common model organism for studies on the biomechanics of jumping. Detailed impact analyses of free-falling locusts show that most insects typically crashed onto the substrate. Although free-falling locusts tended to spread their legs, they mostly fell onto the head and thorax first. The presence of wings did not significantly reduce impact speed; however, it did affect the orientation of the body at impact and significantly reduced the time to recover. Our results also show that alive warm locusts fell significantly faster than inactive or dead locusts. This indicates a possible tradeoff between active control versus reduced speed. Interestingly, alive insects also tended to perform a characteristic bending movement of the body at impact. This biomechanical adaptation might reduce the rebound and shorten the time to recover. The adhesive pads also play an important role in reducing the time to recover by allowing the insect to anchor itself to the substrate.

RUNX1 Mutations Can Lead to Aberrant Expression of CD79a and PAX5 in Acute Myelogenous Leukemias: A Potential Diagnostic Pitfall.

BACKGROUND: RUNX1 is a crucial transcription factor for hematological stem cells and well-known for its association with acute lymphoblastic leukemia (ALL) and acute myelogenous leukemia (AML). Besides the translocation t(8; 21) that leads to the RUNX1-RUNX1T1 fusion, somatic mutations of RUNX1 have been discovered. METHODS: Four bone marrow trephine biopsies of patients with CD79a-positive and/or PAX5-positive acute leukemias were investigated by immunohistochemistry (IHC), karyotyping, and next-generation sequencing-based genetic analysis. Data were then compared to a historical collective of AML (n = 42) and 42 cases of AML newly diagnosed at our institution between June 2017 and May 2018. RESULTS: We report on 4 cases of acute leukemia with an equivocal immunophenotype showing expression of CD79a and/or PAX5, which led to a preliminary histopathologic classification as probable ALL/unclassifiable acute leukemia. All cases were positive for CD34 and TdT but negative for several myeloid markers on IHC. Mutational analysis revealed point mutations and indels of RUNX1 and further mutations typical for AML such as TET2, DNMT3A, and SRSF2, and 2 cases had tetrasomy 13 characteristic of RUNX1 mutant AML. CONCLUSION: Aberrant CD79a and/or PAX5 expression can be found in AML cases with RUNX1 mutations even without the translocation t(8; 21). Our series shows the expression of CD79a and PAX5 to be a potential pitfall in the classification of RUNX1 mutant acute leukemia.

Fucose-Functionalized Precision Glycomacromolecules Targeting Human Norovirus Capsid Protein.

Norovirus infection is the major cause of nonbacterial gastroenteritis in humans and has been the subject of numerous studies investigating the virus's biophysical properties and biochemical function with the aim of deriving novel and highly potent entry inhibitors to prevent infection. Recently, it has been shown that the protruding P domain dimer (P-dimer) of a GII.10 Norovirus strain exhibits two new binding sites for l-fucose in addition to the canonical binding sites. Thus, these sites provide a novel target for the design of multivalent fucose ligands as entry inhibitors of norovirus infections. In this current study, a first generation of multivalent fucose-functionalized glycomacromolecules was synthesized and applied as model structures to investigate the potential targeting of fucose binding sites in human norovirus P-dimer. Following previously established solid phase polymer synthesis, eight precision glycomacromolecules varying in number and position of fucose ligands along an oligo(amidoamine) backbone were obtained and then used in a series of binding studies applying native MS, NMR, and X-ray crystallography. We observed only one fucose per glycomacromolecule binding to one P-dimer resulting in similar binding affinities for all fucose-functionalized glycomacromolecules, which based on our current findings we attribute to the overall size of macromolecular ligands and possibly to steric hindrance.

Different Munc13 isoforms function as priming factors in lytic granule release from murine cytotoxic T lymphocytes.

In order to fuse lytic granules (LGs) with the plasma membrane at the immunological synapse, cytotoxic T lymphocytes (CTLs) have to render these LGs fusion-competent through the priming process. In secretory tissues such as brain and neuroendocrine glands, this process is mediated by members of the Munc13 protein family. In human CTLs, mutations in the Munc13-4 gene cause a severe loss in killing efficiency, resulting in familial hemophagocytic lymphohistiocytosis type 3, suggesting a similar role of other Munc13 isoforms in the immune system. Here, we investigate the contribution of different Munc13 isoforms to the priming process of murine CTLs at both the mRNA and protein level. We demonstrate that Munc13-1 and Munc13-4 are the only Munc13 isoforms present in mouse CTLs. Both isoforms rescue the drastical secretion defect of CTLs derived from Munc13-4-deficient Jinx mice. Mobility studies using total internal reflection fluorescence microscopy indicate that Munc13-4 and Munc13-1 are responsible for the priming process of LGs. Furthermore, the domains of the Munc13 protein, which is responsible for functional fusion, could be identified. We conclude from these data that both isoforms of the Munc13 family, Munc13-1 and Munc13-4, are functionally redundant in murine CTLs.

An RNA sequencing transcriptome analysis reveals novel insights into molecular aspects of the nitrate impact on the nodule activity of Medicago truncatula.

The mechanism through which nitrate reduces the activity of legume nodules is controversial. The objective of the study was to follow Medicago truncatula nodule activity after nitrate provision continuously and to identify molecular mechanisms, which down-regulate the activity of the nodules. Nodule H2 evolution started to decline after about 4 h of nitrate application. At that point in time, a strong shift in nodule gene expression (RNA sequencing) had occurred (1,120 differentially expressed genes). The most pronounced effect was the down-regulation of 127 genes for nodule-specific cysteine-rich peptides. Various other nodulins were also strongly down-regulated, in particular all the genes for leghemoglobins. In addition, shifts in the expression of genes involved in cellular iron allocation and mitochondrial ATP synthesis were observed. Furthermore, the expression of numerous genes for the formation of proteins and glycoproteins with no obvious function in nodules (e.g. germins, patatin, and thaumatin) was strongly increased. This occurred in conjunction with an up-regulation of genes for proteinase inhibitors, in particular those containing the Kunitz domain. The additionally formed proteins might possibly be involved in reducing nodule oxygen permeability. Between 4 and 28 h of nitrate exposure, a further reduction in nodule activity occurred, and the number of differentially expressed genes almost tripled. In particular, there was a differential expression of genes connected with emerging senescence. It is concluded that nitrate exerts rapid and manifold effects on nitrogenase activity. A certain degree of nitrate tolerance might be achieved when the down-regulatory effect on late nodulins can be alleviated.

Cytomegalovirus-specific T cells are detectable in early childhood and allow assignment of the infection status in children with passive maternal antibodies.

Serological identification of the cytomegalovirus (CMV) status in children less than 18 months of age is complicated by the variable persistence of maternal antibodies. As T cells are not passively transferred, we attempted to assess whether CMV-specific cellular immunity may be superior to determine the actual CMV status; we also performed a functional characterization of T-cell immunity in childhood. Antibodies from 59 mothers and 168 children were determined, and specific CD4(+) T cells were identified by induction of IFN-γ, IL-2, TNF-α, IL-4, and IL-17 after CMV-specific and polyclonal stimulation. Agreement between both tests was perfect for mothers and children more than 18 months. Among infants less than 18 months, 17/30 were concordantly negative. Interestingly, 8/13 seropositive children had detectable CMV-specific T cells, whereas only 5/13 were T-cell negative, indicating passive immunity. CMV-specific T cells from young infants differed in cytokine profiles from that of older age groups, and polyclonal effector T-cell frequencies were higher in young infants with detectable CMV-specific T cells compared with those without. In conclusion, the majority of young infants with CMV-specific antibodies show evidence of true infection, which indicates that passive immunity is overestimated. Our data may have important implications for improved risk stratification and CMV management in infants in the setting of transplantation.

CD4+ T-cell immunity after pandemic influenza vaccination cross-reacts with seasonal antigens and functionally differs from active influenza infection.

Antigen-specific antibodies are well characterized after vaccination with pandemic H1N1 or seasonal influenza vaccines. However, knowledge on cellular immunity toward pandemic H1N1 after vaccination and infection and cross-reactivities toward seasonal antigens is limited. Nineteen individuals were vaccinated with the pandemic H1N1 vaccine. Among those, ten had been prevaccinated against seasonal influenza. CD4(+) T cells specific for pandemic H1N1 and for seasonal vaccine, and antibodies were monitored using flow cytometry and ELISA/neutralization assays, respectively. In addition, seven patients with acute pandemic influenza infection were analyzed. Pandemic H1N1 vaccination induced a strong 4.63-fold (IQR 4.16) increase in antigen-specific CD4(+) T cells that was more pronounced in individuals not prevaccinated with seasonal influenza (p = 0.01). T-cell levels toward seasonal vaccine concomitantly rose by 2.71-fold (IQR 2.26). Likewise, prevaccination with seasonal influenza induced a less pronounced increase in specific antibodies. Influenza-specific T cells in vaccinees had a Th1 phenotype mainly coexpressing IFN-γ and IL-2, whereas patients with active pandemic influenza showed a shift toward cells predominantly expressing IFN-γ. In conclusion, T cells toward seasonal influenza antigens cross-react with pandemic H1N1 antigens and affect induction of specific T cells after pandemic influenza vaccination. In addition, the cytokine patterns of specific T cells during acute H1N1 infection and after vaccination differ, and the predominantly dual-positive cytokine profile of vaccine-induced T cells suggests sufficient functionality to confer successful virus control.

Fatigue of insect cuticle.

Many parts of the insect exoskeleton experience repeated cyclic loading. Although the cuticle of insects and other arthropods is the second most common natural composite material in the world, so far nothing is known about its fatigue properties, despite the fact that fatigue undoubtedly limits the durability of body parts in vivo. For the first time, we here present experimental fatigue data of insect cuticle. Using force-controlled cyclic loading, we determined the number of cycles to failure for hind legs (tibiae) and hind wings of the locust Schistocerca gregaria, as a function of the applied cyclic stress. Our results show that, although both are made from cuticle, these two body parts behave very differently. Wing samples showed a large fatigue range, failing after 100,000 cycles when we applied 46% of the stress needed for instantaneous failure [the ultimate tensile strength (UTS)]. Legs, in contrast, were able to sustain a stress of 76% of the UTS for the same number of cycles to failure. This can be explained by the difference in the composition and structure of the material, two factors that, amongst others, also affect the well-known behaviour of engineering composites. Final failure of the tibiae occurred via one of two different failure modes--propagation in tension or buckling in compression--indicating that the tibia is 'optimized' by evolution to resist both failure modes equally. These results are further discussed in relation to the evolution and normal use of these two body parts.

Fracture toughness of locust cuticle.

Insect cuticle is one of the most common biological materials, yet very little is known about its mechanical properties. Many parts of the insect exoskeleton, such as the jumping legs of locusts, have to withstand high and repeated loading without failure. This paper presents the first measurements of fracture toughness for insect cuticle using a standard engineering approach. Our results show that the fracture toughness of cuticle in locust hind legs is 4.12 MPa m(1/2) and decreases with desiccation of the cuticle. Stiffness and strength of the tibia cuticle were measured using buckling and cantilever bending and increased with desiccation. A combination of the cuticle's high toughness with a relatively low stiffness of 3.05 GPa results in a work of fracture of 5.56 kJ m(-2), which is amongst the highest of any biological material, giving the insect leg an exceptional ability to tolerate defects such as cracks and damage. Interestingly, insect cuticle achieves these unique properties without using reinforcement by a mineral phase, which is often found in other biological composite materials. These findings thus might inspire the development of new biomimetic composite materials.

Semi-automated differentiation of insect exo- and endocuticle in X-ray microtomography.

One of the most versatile and complex biological composite materials, the insect exoskeleton shows a huge range in biomechanical properties. The cuticle exoskeleton can be differentiated into two main histologically different layers with distinct properties: the outer, more sclerotized exocuticle and inner, softer endocuticle. For most biomechanical research questions, it is of great importance to be able to selectively characterize geometrical features of these layers. However, most conventional preparation methods (cross-sections, histological staining, SEM) require complex and destructive sample preparation, which provides only two-dimensional information. Here, we present a novel, simple staining method using X-ray microtomography to distinguish between exo- and endocuticle in a 3D environment without sample destruction. We illustrate the power of our method using locust (Locusta migratoria) hindleg tibia, a well characterized biomechanical sample. Our method allows an easy and direct measurement of exo- and endocuticle and their respective geometric features. Applying our method will help to understand the biomechanical role of exo- and endocuticle within an insect exoskeleton and will allow us to understand its composition and morphological features in more detail.

Dasatinib for treatment of CAR T-cell therapy-related complications.

Cytokine release syndrome (CRS) and immune effector cell-associated neurotoxicity syndrome (ICANS) are severe, potentially life-threatening side effects of chimeric antigen receptor T-cell (CAR T) therapy caused by the release of cytokines by proliferating and activated CAR T-cells. Current mainstay treatment includes interleukin-1 and interleukin-6 (IL-6) blockade and steroids. The use of steroids is still controversial, since they may have the potential to irreversibly damage CAR T-cells and thus increase the risk of relapse. Therefore, additional treatment options need to be explored. We report the successful treatment of a patient with a grade 3 CRS and grade 4 ICANS refractory to IL-6 blockade and steroids with the tyrosine kinase inhibitor dasatinib. The use of dasatinib for treatment of CAR T-cell therapy-related severe complications warrants further studies.

Blast counts are lower in the aspirate as compared to trephine biopsy in acute myeloid leukemia and myelodysplastic syndrome expressing CD56.

INTRODUCTION: CD56 is aberrantly expressed in myeloid neoplasms including myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML). Considering the adhesion effects of CD56, blast quantification in bone marrow might depend on the technique used to obtain respective diagnostic specimens. Therefore, the objective of our study was to investigate the impact of CD56-expression on blast counts in myeloid neoplasms comparing bone marrow aspirates to biopsies. METHODS: We retrospectively analyzed 75 patients diagnosed with MDS and AML. We compared patients with (n = 36) and without (n = 39) CD56-expression by flow cytometry with respect to their blast quantities assessed on bone marrow aspirates versus biopsies. RESULTS: The frequency of CD56-expression on blasts correlated with higher blast counts on biopsies vs. aspirate smears (rs  = 0.52; P = .001). This difference in blast counts was only significant in the CD56 high expressing subgroup (median 68%, 5.5%-95% in biopsy compared to median 32.5%, 1.5%-90% in aspirate; P < .01). The percentage of CD56-positive blasts among the total blast population was lower in the peripheral blood compared to bone marrow (median 31%, 6%-88% vs. 55%, 14%-98%; P = .016). The discrepancy in the blast count between the aspirate and trephine biopsy would have led to misclassification of four cases as MDS instead of AML, if diagnosis had based on the bone marrow aspirate blast count alone. CONCLUSION: Counting blasts in bone marrow aspirates of CD56-positive AML and MDS may be linked to underestimation, potentially leading to misclassification of these myeloid neoplasms, and should therefore be adjusted considering the results obtained on trephine biopsies for reliable diagnosis.

SAUV-A Bio-Inspired Soft-Robotic Autonomous Underwater Vehicle.

Autonomous and remotely operated underwater vehicles allow us to reach places which have previously been inaccessible and perform complex repair, exploration and analysis tasks. As their navigation is not infallible, they may cause severe damage to themselves and their often fragile surroundings, such as flooded caves, coral reefs, or even accompanying divers in case of a collision. In this study, we used a shallow neural network, consisting of interlinking PID controllers, and trained by a genetic algorithm, to control a biologically inspired AUV with a soft and compliant exoskeleton. Such a compliant structure is a versatile and passive solution which reduces the accelerations induced by collisions to 56% of the original mean value acting upon the system, thus, notably reducing the stress on its components and resulting reaction forces on its surroundings. The segmented structure of this spherical exoskeleton protects the encased system without limiting the use of cameras, sensors or manipulators.

Epidermal Cell Surface Structure and Chitin-Protein Co-assembly Determine Fiber Architecture in the Locust Cuticle.

The geometrical similarity of helicoidal fiber arrangement in many biological fibrous extracellular matrices, such as bone, plant cell wall, or arthropod cuticle, to that of cholesteric liquid mesophases has led to the hypothesis that they may form passively through a mesophase precursor rather than by direct cellular control. In search of direct evidence to support or refute this hypothesis, here, we studied the process of cuticle formation in the tibia of the migratory locust, Locusta migratoria, where daily growth layers arise by the deposition of fiber arrangements alternating between unidirectional and helicoidal structures. Using focused ion beam/scanning electron microscopy (FIB/SEM) volume imaging and scanning X-ray scattering, we show that the epidermal cells determine an initial fiber orientation, from which the final architecture emerges by the self-organized co-assembly of chitin and proteins. Fiber orientation in the locust cuticle is therefore determined by both active and passive processes.

Friction ridges in cockroach climbing pads: anisotropy of shear stress measured on transparent, microstructured substrates.

The contact of adhesive structures to rough surfaces has been difficult to investigate as rough surfaces are usually irregular and opaque. Here we use transparent, microstructured surfaces to investigate the performance of tarsal euplantulae in cockroaches (Nauphoeta cinerea). These pads are mainly used for generating pushing forces away from the body. Despite this biological function, shear stress (force per unit area) measurements in immobilized pads showed no significant difference between pushing and pulling on smooth surfaces and on 1-microm high microstructured substrates, where pads made full contact. In contrast, on 4-mum high microstructured substrates, where pads made contact only to the top of the microstructures, shear stress was maximal during a push. This specific direction dependence is explained by the interlocking of the microstructures with nanometre-sized "friction ridges" on the euplantulae. Scanning electron microscopy and atomic force microscopy revealed that these ridges are anisotropic, with steep slopes facing distally and shallow slopes proximally. The absence of a significant direction dependence on smooth and 1-microm high microstructured surfaces suggests the effect of interlocking is masked by the stronger influence of adhesion on friction, which acts equally in both directions. Our findings show that cockroach euplantulae generate friction using both interlocking and adhesion.