We describe a two-photon microscopy-based method to evaluate the in vivo systemic transport of compounds. This method comprises imaging of the intact liver, kidney and intestine, the main organs ...responsible for uptake and elimination of xenobiotics and endogenous molecules. The image quality of the acquired movies was sufficient to distinguish subcellular structures like organelles and vesicles. Quantification of the movement of fluorescent dextran and fluorescent cholic acid derivatives in different organs and their sub-compartments over time revealed significant dynamic differences. Calculated half-lives were similar in the capillaries of all investigated organs but differed in the specific sub-compartments, such as parenchymal cells and bile canaliculi of the liver, glomeruli, proximal and distal tubules of the kidney and lymph vessels (lacteals) of the small intestine. Moreover, tools to image immune cells, which can influence transport processes in inflamed tissues, are described. This powerful approach provides new possibilities for the analysis of compound transport in multiple organs and can support physiologically based pharmacokinetic modeling, in order to obtain more precise predictions at the whole body scale.
Characterisation of bioaerosols has important implications within environment and public health sectors. Recent developments in ultraviolet light-induced fluorescence (UV-LIF) detectors such as the ...Wideband Integrated Bioaerosol Spectrometer (WIBS) and the newly introduced Multiparameter Bioaerosol Spectrometer (MBS) have allowed for the real-time collection of fluorescence, size and morphology measurements for the purpose of discriminating between bacteria, fungal spores and pollen.This new generation of instruments has enabled ever larger data sets to be compiled with the aim of studying more complex environments. In real world data sets, particularly those from an urban environment, the population may be dominated by non-biological fluorescent interferents, bringing into question the accuracy of measurements of quantities such as concentrations. It is therefore imperative that we validate the performance of different algorithms which can be used for the task of classification.For unsupervised learning we tested hierarchical agglomerative clustering with various different linkages. For supervised learning, 11 methods were tested, including decision trees, ensemble methods (random forests, gradient boosting and AdaBoost), two implementations for support vector machines (libsvm and liblinear) and Gaussian methods (Gaussian naïve Bayesian, quadratic and linear discriminant analysis, the k-nearest neighbours algorithm and artificial neural networks).The methods were applied to two different data sets produced using the new MBS, which provides multichannel UV-LIF fluorescence signatures for single airborne biological particles. The first data set contained mixed PSLs and the second contained a variety of laboratory-generated aerosol.Clustering in general performs slightly worse than the supervised learning methods, correctly classifying, at best, only 67. 6 and 91. 1 % for the two data sets respectively. For supervised learning the gradient boosting algorithm was found to be the most effective, on average correctly classifying 82. 8 and 98. 27 % of the testing data, respectively, across the two data sets.A possible alternative to gradient boosting is neural networks. We do however note that this method requires much more user input than the other methods, and we suggest that further research should be conducted using this method, especially using parallelised hardware such as the GPU, which would allow for larger networks to be trained, which could possibly yield better results.We also saw that some methods, such as clustering, failed to utilise the additional shape information provided by the instrument, whilst for others, such as the decision trees, ensemble methods and neural networks, improved performance could be attained with the inclusion of such information.
Nonhematopoietic stromal cells of secondary lymphoid organs form important scaffold and fluid transport structures, such as lymph node (LN) trabeculae, lymph vessels, and conduits. Furthermore, ...through the production of chemokines and cytokines, these cells generate a particular microenvironment that determines lymphocyte positioning and supports lymphocyte homeostasis. IL-7 is an important stromal cell-derived cytokine that has been considered to be derived mainly from T-cell zone fibroblastic reticular cells. We show here that lymphatic endothelial cells (LECs) are a prominent source of IL-7 both in human and murine LNs. Using bacterial artificial chromosome transgenic IL-7–Cre mice, we found that fibroblastic reticular cells and LECs strongly up-regulated IL-7 expression during LN remodeling after viral infection and LN reconstruction after avascular transplantation. Furthermore, IL-7–producing stromal cells contributed to de novo formation of LyveI-positive lymphatic structures connecting reconstructed LNs with the surrounding tissue. Importantly, diphtheria toxin–mediated depletion of IL-7–producing stromal cells completely abolished LN reconstruction. Taken together, this study identifies LN LECs as a major source of IL-7 and shows that IL-7–producing stromal cells are critical for reconstruction and remodeling of the distinct LN microenvironment.
CD11c
hi dendritic cells (DC) play an essential role during the initiation of cell-mediated immunity. Recently, CD11c
loCD45RB
hi DC with regulatory properties have been described. However, the ...origins of regulatory DC are poorly understood. Here, we show that spleen-derived stromal cells promote selective development of CD11c
loCD45RB
+ IL-10-producing regulatory DC from lineage-negative c-kit
+ progenitor cells. These DC have the capacity to suppress T cell responses and induce IL-10-producing regulatory T cells in vitro and to induce antigen-specific tolerance in vivo. Furthermore, stromal cells from mice infected with
Leishmania donovani more effectively supported differentiation of these highly potent regulatory DC. The ability of tissue stromal cells to direct the development of DC with a regulatory phenotype thus provides a new mechanism for local immune regulation.
An emerging problem in the treatment of breast cancer is the increasing incidence of metastases to the brain. Metastatic brain tumours are incurable and can cause epileptic seizures and cognitive ...impairment, so better understanding of this niche, and the cellular mechanisms, is urgently required. Microglia are the resident brain macrophage population, becoming "activated" by neuronal injury, eliciting an inflammatory response. Microglia promote proliferation, angiogenesis and invasion in brain tumours and metastases. However, the mechanisms underlying microglial involvement appear complex and better models are required to improve understanding of function.
Here, we sought to address this need by developing a model to study metastatic breast cancer cell-microglial interactions using intravital imaging combined with ex vivo electrophysiology. We implanted an optical window on the parietal bone to facilitate observation of cellular behaviour in situ in the outer cortex of heterozygous Cx3cr1
mice.
We detected GFP-expressing microglia in Cx3cr1
mice up to 350 μm below the window without significant loss of resolution. When DsRed-expressing metastatic MDA-MB-231 breast cancer cells were implanted in Matrigel under the optical window, significant accumulation of activated microglia around invading tumour cells could be observed. This inflammatory response resulted in significant cortical disorganisation and aberrant spontaneously-occurring local field potential spike events around the metastatic site.
These data suggest that peritumoral microglial activation and accumulation may play a critical role in local tissue changes underpinning aberrant cortical activity, which offers a possible mechanism for the disrupted cognitive performance and seizures seen in patients with metastatic breast cancer.
IL-10 is a critical regulatory cytokine involved in the pathogenesis of visceral leishmaniasis caused by Leishmania donovani and clinical and experimental data indicate that disease progression is ...associated with expanded numbers of CD4⁺ IFNγ⁺ T cells committed to IL-10 production. Here, combining conditional cell-specific depletion with adoptive transfer, we demonstrate that only conventional CD11c(hi) DCs that produce both IL-10 and IL-27 are capable of inducing IL-10-producing Th1 cells in vivo. In contrast, CD11c(hi) as well as CD11c(int/lo) cells isolated from infected mice were capable of reversing the host protective effect of diphtheria toxin-mediated CD11c⁺ cell depletion. This was reflected by increased splenomegaly, inhibition of NO production and increased parasite burden. Thus during chronic infection, multiple CD11c⁺ cell populations can actively suppress host resistance and enhance immunopathology, through mechanisms that do not necessarily involve IL-10-producing Th1 cells.
The neurotrophic tyrosine kinase receptor type 2 (Ntrk2, also known as TrkB) and its ligands brain derived neurotrophic factor (Bdnf), neurotrophin-4 (NT-4/5), and neurotrophin-3 (NT-3) are known ...primarily for their multiple effects on neuronal differentiation and survival. Here, we provide evidence that Ntrk2 plays a role in the pathologic remodeling of the spleen that accompanies chronic infection. We show that in Leishmania donovani-infected mice, Ntrk2 is aberrantly expressed on splenic endothelial cells and that new maturing blood vessels within the white pulp are intimately associated with F4/80(hi)CD11b(lo)CD11c(+) macrophages that express Bdnf and NT-4/5 and have pro-angiogenic potential in vitro. Furthermore, administration of the small molecule Ntrk2 antagonist ANA-12 to infected mice significantly inhibited white pulp neovascularization but had no effect on red pulp vascular remodeling. We believe this to be the first evidence of the Ntrk2/neurotrophin pathway driving pathogen-induced vascular remodeling in lymphoid tissue. These studies highlight the therapeutic potential of modulating this pathway to inhibit pathological angiogenesis.
Post-kala-azar dermal leishmaniasis (PKDL) is a chronic, stigmatizing skin condition occurring frequently after apparent clinical cure from visceral leishmaniasis. Given an urgent need for new ...treatments, we conducted a phase IIa safety and immunogenicity trial of ChAd63-KH vaccine in Sudanese patients with persistent PKDL. LEISH2a (ClinicalTrials.gov: NCT02894008) was an open-label three-phase clinical trial involving sixteen adult and eight adolescent patients with persistent PKDL (median duration, 30 months; range, 6–180 months). Patients received a single intramuscular vaccination of 1 × 1010 viral particles (v.p.; adults only) or 7.5 × 1010 v.p. (adults and adolescents), with primary (safety) and secondary (clinical response and immunogenicity) endpoints evaluated over 42–120 days follow-up. AmBisome was provided to patients with significant remaining disease at their last visit. ChAd63-KH vaccine showed minimal adverse reactions in PKDL patients and induced potent innate and cell-mediated immune responses measured by whole-blood transcriptomics and ELISpot. 7/23 patients (30.4%) monitored to study completion showed >90% clinical improvement, and 5/23 (21.7%) showed partial improvement. A logistic regression model applied to blood transcriptomic data identified immune modules predictive of patients with >90% clinical improvement. A randomized controlled trial to determine whether these clinical responses were vaccine-related and whether ChAd63-KH vaccine has clinical utility is underway.
Display omitted
Post-kala-azar dermal leishmaniasis (PKDL) is a chronic parasitic disease with limited treatment options. Here, Musa and colleagues report on a phase IIa therapeutic trial of the adenoviral-vectored leishmaniasis vaccine ChAd63-KH. Single-dose vaccination with ChAd63-KH was safe and induced innate and T cell responses in Sudanese patients with PKDL.
The Amazon basin is a vast continental area in which atmospheric composition is relatively unaffected by anthropogenic aerosol particles. Understanding the properties of the natural biogenic aerosol ...particles over the Amazon rainforest is key to understanding their influence on regional and global climate. While there have been a number of studies during the wet season, and of biomass burning particles in the dry season, there has been relatively little work on the transition period – the start of the dry season in the absence of biomass burning. As part of the Brazil–UK Network for Investigation of Amazonian Atmospheric Composition and Impacts on Climate (BUNIAACIC) project, aerosol measurements, focussing on unpolluted biogenic air masses, were conducted at a remote rainforest site in the central Amazon during the transition from wet to dry season in July 2013. This period marks the start of the dry season but before significant biomass burning occurs in the region. Median particle number concentrations were 266 cm−3, with size distributions dominated by an accumulation mode of 130–150 nm. During periods of low particle counts, a smaller Aitken mode could also be seen around 80 nm. While the concentrations were similar in magnitude to those seen during the wet season, the size distributions suggest an enhancement in the accumulation mode compared to the wet season, but not yet to the extent seen later in the dry season, when significant biomass burning takes place. Submicron nonrefractory aerosol composition, as measured by an aerosol chemical speciation monitor (ACSM), was dominated by organic material (around 81 %). Aerosol hygroscopicity was probed using measurements from a hygroscopicity tandem differential mobility analyser (HTDMA), and a quasi-monodisperse cloud condensation nuclei counter (CCNc). The hygroscopicity parameter, κ, was found to be low, ranging from 0.12 for Aitken-mode particles to 0.18 for accumulation-mode particles. This was consistent with previous studies in the region, but lower than similar measurements conducted in Borneo, where κ ranged 0.17–0.37. A wide issue bioaerosol sensor (WIBS-3M) was deployed at ground level to probe the coarse mode, detecting primary biological aerosol by fluorescence (fluorescent biological aerosol particles, or FBAPs). The mean FBAP number concentration was 400 ± 242 L−1; however, this ranged from around 200 L−1 during the day to as much as 1200 L−1 at night. FBAPs dominated the coarse-mode particles, comprising between 55 and 75 % of particles during the day to more than 90 % at night. Non-FBAPs did not show a strong diurnal pattern. Comparison with previous FBAP measurements above canopy at the same location suggests there is a strong vertical gradient in FBAP concentrations through the canopy. Cluster analysis of the data suggests that FBAPs were dominated (around 70 %) by fungal spores. Further, long-term measurements will be required in order to fully examine the seasonal variability and distribution of primary biological aerosol particles through the canopy. This is the first time that such a suite of measurements has been deployed at this site to investigate the chemical composition and properties of the biogenic contributions to Amazonian aerosol during the transition period from the wet to the dry season, and thus provides a unique comparison to the aerosol properties observed during the wet season in previous similar campaigns. This was also the first deployment of a WIBS in the Amazon rainforest to study coarse-mode particles, particularly primary biological aerosol particles, which are likely to play an important role as ice nuclei in the region.
PDF
