Blockade of CD40-CD154 pathway interactions suppresses ectopic lymphoid structures and inhibits pathology in the NOD/ShiLtJ mouse model of Sjögren's syndrome.

OBJECTIVE: To examine the role of CD40-CD154 costimulation and effects of therapeutic pathway blockade in the non-obese diabetic (NOD/ShiLtJ) model of Sjögren's syndrome (SS). METHODS: We assessed leucocyte infiltration in salivary glands (SGs) from NOD/ShiLtJ mice by immunohistochemistry and examined transcriptomics data of SG tissue from these animals for evidence of a CD40 pathway gene signature. Additionally, we dosed MR1 (anti-CD154 antibody) in NOD mice after the onset of SS-like disease and examined the effects of MR1 treatment on sialadenitis, autoantibody production, SG leucocyte infiltration, gene expression downstream of CD40 and acquaporin 5 (AQP5) expression. RESULTS: We could detect evidence of CD40 expression and pathway activation in SG tissue from NOD mice. Additionally, therapeutic treatment with MR1 suppressed CD40 pathway genes and sialadenitis, inhibited ectopic lymphoid structure formation and autoantibody production, as well as decreased the frequency of antibody-secreting cells in SGs but had minimal effects on AQP5 expression in NOD/ShiLtJ SGs. CONCLUSION: CD40-CD154 interactions play an important role in key pathological processes in a mouse model of SS, suggesting that blockade of this costimulatory pathway in the clinic may have beneficial therapeutic effects in patients suffering from this autoimmune exocrinopathy.

Developmental self-construction and -configuration of functional neocortical neuronal networks.

The prenatal development of neural circuits must provide sufficient configuration to support at least a set of core postnatal behaviors. Although knowledge of various genetic and cellular aspects of development is accumulating rapidly, there is less systematic understanding of how these various processes play together in order to construct such functional networks. Here we make some steps toward such understanding by demonstrating through detailed simulations how a competitive co-operative ('winner-take-all', WTA) network architecture can arise by development from a single precursor cell. This precursor is granted a simplified gene regulatory network that directs cell mitosis, differentiation, migration, neurite outgrowth and synaptogenesis. Once initial axonal connection patterns are established, their synaptic weights undergo homeostatic unsupervised learning that is shaped by wave-like input patterns. We demonstrate how this autonomous genetically directed developmental sequence can give rise to self-calibrated WTA networks, and compare our simulation results with biological data.

Simulating cortical development as a self constructing process: a novel multi-scale approach combining molecular and physical aspects.

Current models of embryological development focus on intracellular processes such as gene expression and protein networks, rather than on the complex relationship between subcellular processes and the collective cellular organization these processes support. We have explored this collective behavior in the context of neocortical development, by modeling the expansion of a small number of progenitor cells into a laminated cortex with layer and cell type specific projections. The developmental process is steered by a formal language analogous to genomic instructions, and takes place in a physically realistic three-dimensional environment. A common genome inserted into individual cells control their individual behaviors, and thereby gives rise to collective developmental sequences in a biologically plausible manner. The simulation begins with a single progenitor cell containing the artificial genome. This progenitor then gives rise through a lineage of offspring to distinct populations of neuronal precursors that migrate to form the cortical laminae. The precursors differentiate by extending dendrites and axons, which reproduce the experimentally determined branching patterns of a number of different neuronal cell types observed in the cat visual cortex. This result is the first comprehensive demonstration of the principles of self-construction whereby the cortical architecture develops. In addition, our model makes several testable predictions concerning cell migration and branching mechanisms.

Forced G1-phase reduction alters mode of division, neuron number, and laminar phenotype in the cerebral cortex.

The link between cortical precursors G1 duration (TG1) and their mode of division remains a major unresolved issue of potential importance for regulating corticogenesis. Here, we induced a 25% reduction in TG1 in mouse cortical precursors via forced expression of cyclin D1 and cyclin E1. We found that in utero electroporation-mediated gene transfer transfects a cohort of synchronously cycling precursors, necessitating alternative methods of measuring cell-cycle phases to those classical used. TG1 reduction promotes cell-cycle reentry at the expense of differentiation and increases the self-renewal capacities of Pax6 precursors as well as of Tbr2 basal precursors (BPs). A population level analysis reveals sequential and lineage-specific effects, showing that TG1 reduction: (i) promotes Pax6 self-renewing proliferative divisions before promoting divisions wherein Pax6 precursors generate Tbr2 BPs and (ii) promotes self-renewing proliferative divisions of Tbr2 precursors at the expense of neurogenesis, thus leading to an amplification of the BPs pool in the subventricular zone and the dispersed mitotic compartment of the intermediate zone. These results point to the G1 mode of division relationship as an essential control mechanism of corticogenesis. This is further supported by long-term studies showing that TG1 reduction results in cytoarchitectural modifications including supernumerary supragranular neuron production. Modeling confirms that the TG1-induced changes in neuron production and laminar fate are mediated via the changes in the mode of division. These findings also have implications for understanding the mechanisms that have contributed to brain enlargement and complexity during evolution.

Time-dependent changes of zinc speciation in four soils contaminated with zincite or sphalerite.

The long-term speciation of Zn in contaminated soils is strongly influenced by soil pH, clay, and organic matter content as well as Zn loading. In addition, the type of Zn-bearing contaminant entering the soil may influence the subsequent formation of pedogenic Zn species, but systematic studies on such effects are currently lacking. We therefore conducted a soil incubation study in which four soils, ranging from strongly acidic to calcareous, were spiked with 2000 mg/kg Zn using either ZnO (zincite) or ZnS (sphalerite) as the contamination source. The soils were incubated under aerated conditions in moist state for up to four years. The extractability and speciation of Zn were assessed after one, two, and four years using extractions with 0.01 M CaCl(2) and Zn K-edge X-ray absorption fine structure (XAFS) spectroscopy, respectively. After four years, more than 90% of the added ZnO were dissolved in all soils, with the fastest dissolution occurring in the acidic soils. Contamination with ZnO favored the formation of Zn-bearing layered double hydroxides (LDH), even in acidic soils, and to a lesser degree Zn-phyllosilicates and adsorbed Zn species. This was explained by locally elevated pH and high Zn concentrations around dissolving ZnO particles. Except for the calcareous soil, ZnS dissolved more slowly than ZnO, reaching only 26 to 75% of the added ZnS after four years. ZnS dissolved more slowly in the two acidic soils than in the near-neutral and the calcareous soil. Also, the resulting Zn speciation was markedly different between these two pairs of soils: Whereas Zn bound to hydroxy-interlayered clay minerals (HIM) and octahedrally coordinated Zn sorption complexes prevailed in the two acidic soils, Zn speciation in the neutral and the calcareous soil was dominated by Zn-LDH and tetrahedrally coordinated inner-sphere Zn complexes. Our results show that the type of Zn-bearing contaminant phase can have a significant influence on the formation of pedogenic Zn species in soils. Important factors include the rate of Zn release from the contaminant phases and effects of the contaminant phase on bulk soil properties and on local chemical conditions around weathering contaminant particles.

Immune cell landscaping reveals a protective role for regulatory T cells during kidney injury and fibrosis.

Acute kidney injury (AKI) and chronic kidney diseases are associated with high mortality and morbidity. Although the underlying mechanisms determining the transition from acute to chronic injury are not completely understood, immune-mediated processes are critical in renal injury. We have performed a comparison of 2 mouse models leading to either kidney regeneration or fibrosis. Using global gene expression profiling we could identify immune-related pathways accounting for the majority of the observed transcriptional changes during fibrosis. Unbiased examination of the immune cell composition, using single-cell RNA sequencing, revealed major changes in tissue-resident macrophages and T cells. Following injury, there was a marked increase in tissue-resident IL-33R+ and IL-2Ra+ regulatory T cells (Tregs). Expansion of this population before injury protected the kidney from injury and fibrosis. Transcriptional profiling of Tregs showed a differential upregulation of regenerative and proangiogenic pathways during regeneration, whereas in the fibrotic environment they expressed markers of hyperactivation and fibrosis. Our data point to a hitherto underappreciated plasticity in Treg function within the same tissue, dictated by environmental cues. Overall, we provide a detailed cellular and molecular characterization of the immunological changes during kidney injury, regeneration, and fibrosis.

Interleukin-17A Inhibition Diminishes Inflammation and New Bone Formation in Experimental Spondyloarthritis.

OBJECTIVE: It remains unclear if and how inflammation and new bone formation in spondyloarthritis (SpA) are coupled. We undertook this study to assess the hypothesis that interleukin-17A (IL-17A) is a pivotal driver of both processes. METHODS: The effect of tumor necrosis factor (TNF) and IL-17A on osteogenesis was tested in an osteoblastic differentiation assay using SpA fibroblast-like synoviocytes (FLS) differentiated with dexamethasone, ß-glycophosphatase, and ascorbic acid. IL-17A blockade was performed in HLA-B27/human ß2 -microglobulin (hß2 m)-transgenic rats, which served as a model for SpA in both prophylactic and therapeutic settings. Inflammation and new bone formation were evaluated by micro-computed tomography imaging, histologic analysis, and gene expression profiling. RESULTS: TNF and IL-17A significantly increased in vitro osteoblastic differentiation. In vivo, prophylactic blockade of IL-17A significantly delayed spondylitis and arthritis development and decreased arthritis severity. Anti-IL-17A treatment was also associated with prevention of bone loss and periosteal new bone formation. Therapeutic targeting of IL-17A after the initial inflammatory insult also significantly reduced axial and peripheral joint inflammation. This treatment was again associated with a marked reduction in spinal and peripheral structural damage, including new bone formation. RNA sequencing of target tissue confirmed that IL-17A is a key driver of the molecular signature of disease in this model and that therapeutic anti-IL-17A treatment reversed the inflammatory signature and the selected gene expression related to bone damage. CONCLUSION: Both prophylactic and therapeutic inhibition of IL-17A diminished inflammation and new bone formation in HLA-B27/hß2 m-transgenic rats. Taken together with the ability of IL-17A to promote osteoblastic differentiation of human SpA FLS, these data suggest a direct link between IL-17A-driven inflammation and pathologic new bone formation in SpA.

Unsupervised lineage-based characterization of primate precursors reveals high proliferative and morphological diversity in the OSVZ.

Generation of the primate cortex is characterized by the diversity of cortical precursors and the complexity of their lineage relationships. Recent studies have reported miscellaneous precursor types based on observer classification of cell biology features including morphology, stemness, and proliferative behavior. Here we use an unsupervised machine learning method for Hidden Markov Trees (HMTs), which can be applied to large datasets to classify precursors on the basis of morphology, cell-cycle length, and behavior during mitosis. The unbiased lineage analysis automatically identifies cell types by applying a lineage-based clustering and model-learning algorithm to a macaque corticogenesis dataset. The algorithmic results validate previously reported observer classification of precursor types and show numerous advantages: It predicts a higher diversity of progenitors and numerous potential transitions between precursor types. The HMT model can be initialized to learn a user-defined number of distinct classes of precursors. This makes it possible to 1) reveal as yet undetected precursor types in view of exploring the significant features of precursors with respect to specific cellular processes; and 2) explore specific lineage features. For example, most precursors in the experimental dataset exhibit bidirectional transitions. Constraining the directionality in the HMT model leads to a reduction in precursor diversity following multiple divisions, thereby suggesting that one impact of bidirectionality in corticogenesis is to maintain precursor diversity. In this way we show that unsupervised lineage analysis provides a valuable methodology for investigating fundamental features of corticogenesis.

Precursor diversity and complexity of lineage relationships in the outer subventricular zone of the primate.

Long-term ex vivo live imaging combined with unbiased sampling of cycling precursors shows that macaque outer subventricular zone (OSVZ) includes four distinct basal radial glial (bRG) cell morphotypes, bearing apical and/or basal processes in addition to nonpolar intermediate progenitors (IPs). Each of the five precursor types exhibits extensive self-renewal and proliferative capacities as well as the ability to directly generate neurons, albeit with different frequencies. Cell-cycle parameters exhibited an unusual stage-specific regulation with short cell-cycle duration and increased rates of proliferative divisions during supragranular layer production at late corticogenesis. State transition analysis of an extensive clonal database reveals bidirectional transitions between OSVZ precursor types as well as stage-specific differences in their progeny and topology of the lineage relationships. These results explore rodent-primate differences and show that primate cortical neurons are generated through complex lineages by a mosaic of precursors, thereby providing an innovative framework for understanding specific features of primate corticogenesis.

An instruction language for self-construction in the context of neural networks.

Biological systems are based on an entirely different concept of construction than human artifacts. They construct themselves by a process of self-organization that is a systematic spatio-temporal generation of, and interaction between, various specialized cell types. We propose a framework for designing gene-like codes for guiding the self-construction of neural networks. The description of neural development is formalized by defining a set of primitive actions taken locally by neural precursors during corticogenesis. These primitives can be combined into networks of instructions similar to biochemical pathways, capable of reproducing complex developmental sequences in a biologically plausible way. Moreover, the conditional activation and deactivation of these instruction networks can also be controlled by these primitives, allowing for the design of a "genetic code" containing both coding and regulating elements. We demonstrate in a simulation of physical cell development how this code can be incorporated into a single progenitor, which then by replication and differentiation, reproduces important aspects of corticogenesis.

Changes in zinc speciation in field soil after contamination with zinc oxide.

Recent studies on the speciation of Zn in contaminated soils confirmed the formation of Zn-layered double hydroxide (LDH) and Zn-phyllosilicate phases. However, no information on the kinetics of the formation of those phases under field conditions is currently available. In the present study, the transformation of Zn in a field soil artificially contaminated with ZnO containing filter dust from a brass foundry was monitored during 4 years using extended X-ray absorption fine structure (EXAFS) spectroscopy. Soil sections were studied by micro-X-ray fluorescence (micro-XRF) and micro-EXAFS spectroscopy. EXAFS spectra were analyzed by principal component analysis (PCA) and linear combination fitting (LCF). The results show that ZnO dissolved within 9 months and that half of the total Zn reprecipitated. The precipitate was mainly of the Zn-LDH type (>75%). Only a minor fraction (<25%) may be of Zn-phyllosilicate type. The remaining Zn was adsorbed to soil organic and inorganic particles. No significant changes in Zn speciation occurred from 9 to 47 months after the contamination. Thermodynamic calculations show that both Zn-LDH and Zn-phyllosilicate may form in the presence of ZnO but that the formation of Zn-phyllosilicate would be thermodynamically favored. Thus, the dominance of Zn-LDH found by spectroscopy suggests that the formation of the Zn precipitates was not solely controlled bythermodynamics but also contained a kinetic component. The rate-limiting step could be the supply of Al and Si from soil minerals to the Zn-rich solutions around dissolving ZnO grains.

Combining selective sequential extractions, X-ray absorption spectroscopy, and principal component analysis for quantitative zinc speciation in soil.

Selective sequential extractions (SSE) and, more recently, X-ray absorption fine-structure IXAFS) spectroscopy have been used to characterize the speciation of metal contaminants in soils and sediments. However, both methods have specific limitations when multiple metal species coexist in soils and sediments. In this study, we tested a combined approach, in which XAFS spectra were collected after each of 6 SSE steps, and then analyzed by multishell fitting, principal component analysis (PCA) and linear combination fits (LCF), to determine the Zn speciation in a smelter-contaminated, strongly acidic soil. In the topsoil, Zn was predominately found in the smelter-emitted minerals franklinite (60%) and sphalerite (30%) and as aqueous or outer-sphere Zn2+ (10%). In the subsoil, aqueous or outer-sphere Zn2+ prevailed (55%), but 45% of Zn was incorporated by hydroxy-Al interlayers of phyllosilicates. Formation of such Zn-bearing hydroxy-interlayers, which has been observed here for the first time, may be an important mechanism to reduce the solubility of Zn in those soils, which are too acidic to retain Zn by formation of inner-sphere sorption complexes, layered double hydroxides or phyllosilicates. The stepwise removal of Zn fractions by SSE significantly improved the identification of species by XAFS and PCA and their subsequent quantification by LCF. While SSE alone provided excellent estimates of the amount of mobile Zn species, it failed to identify and quantify Zn associated with mineral phases because of nonspecific dissolution and the precipitation of Zn oxalate. The systematic combination of chemical extraction, spectroscopy, and advanced statistical analysis allowed us to identify and quantify both mobile and recalcitrant species with high reliability and precision.