The structural response of the cornea to changes in stromal hydration

The primary aim of this study was to quantify the relationship between corneal structure and hydration in humans and pigs. X-ray scattering data were collected from human and porcine corneas equilibrated with polyethylene glycol (PEG) to varying levels of hydration, to obtain measurements of collagen fibril diameter, interfibrillar spacing and intermolecular spacing. Both species showed a strong positive linear correlation between hydration and interfibrillar spacing2 and a non-linear, bi-phasic relationship between hydration and fibril diameter, whereby fibril diameter increased up to approximately physiological hydration, H = 3.0, with little change thereafter. Above H = 3.0, porcine corneas exhibited a larger fibril diameter than human corneas (p < 0.001). Intermolecular spacing also varied with hydration in a bi-phasic manner but reached a maximum value at a lower hydration (H = 1.5) than fibril diameter. Human corneas displayed a higher intermolecular spacing than porcine corneas at all hydrations (p < 0.0001). Human and porcine corneas required a similar PEG concentration to reach physiological hydration, suggesting that the total fixed charge that gives rise to the swelling pressure is the same. The difference in their structural responses to hydration can be explained by variations in molecular crosslinking and intra/interfibrillar water partitioning

Taking a Swing at TIMP1-Armed Immunosuppressive Astrocytes Unleashes T cell Immunity against Brain Metastases

Priego and colleagues identify a secreted glycoprotein TIMP1, expressed downstream of the transcription factor STAT3, in a subpopulation of STAT3+ reactive astrocytes as a mediator of immunosuppression in late-stage brain metastases. The STAT3 inhibitor silibinin enhances the preclinical efficacy of the combined PD-1/CTLA4 immune checkpoint blockade, providing a rationale to translate the combination therapy into clinical use for this underserved patient group with poor prognosis

Progress on transverse beam profile measurement using the heterodyne near field speckles method at Alba

We present the recent developments of a study aimed at measuring the transverse beam profile using the Heterodyne Near Field Speckles (HNFS) method. The HNFS technique works by illuminating a suspension of Brownian nanoparticles with synchrotron radiation and studying the resulting interference pattern. The transverse coherence of the source, and therefore, under the conditions of validity of the Van Cittert and Zernike theorem, the transverse electron beam size is retrieved from the interference between the transmitted beam and the spherical waves scattered by each nanoparticle. We here describe the fundamentals of this technique, as well as the recent experimental results obtained with 12 keV undulator radiation at the NCD beamline at the ALBA synchrotron. The applicability of such a technique for future accelerators (e.g. CLIC or FCC) is also discussed

Effects on collagen orientation in the cornea after trephine injury

Purpose: Structural changes are well known to occur in the cornea after injury. The aim of this study was to investigate collagen orientation changes in the cornea during a short-term wound healing process. Methods: Seven bovine corneas were injured using a penetrating 5 mm biopsy punch and were subsequently organ cultured for up to two weeks. Six uninjured corneas acted as controls. The trephine wounded samples were snap frozen in liquid nitrogen either immediately after injury (0 h) or after 1 or 2 weeks in culture. Control/uninjured samples were snap frozen on arrival (0 h) or after 1 or 2 weeks in culture. Wide angle X-ray diffraction data were collected from each cornea at the UK Synchrotron Radiation Source or at the European Synchrotron Radiation Facility. Data analysis revealed information about collagen orientation and distribution in the corneal stroma during wound healing. For histology, two trephine wounded corneas at 0 h and 1 week and one control/uninjured cornea at 0 h were fixed in 10% neutral buffered formalin and processed for wax embedding. Wax sections were subsequently counterstained with haematoxylin and eosin to observe tissue morphology and the time course of complete re-epithelialization. Results: Immediately after injury, collagen organization was altered in a small area inside the wound but remained similar to the control/uninjured sample in the remainder of the tissue. After one week, the trephine wounded corneas showed complete re-epithelialization and evidence of swelling while collagen adopted a radial arrangement inside and outside the wound. Conclusions: Remarkable changes in collagen fibril orientation were observed in trephine wounded corneas. Orientation changes immediately after wounding are likely to be due to the mechanical deformation of the tissue during the wounding process. However, tissue swelling and changes in collagen orientation at later stages probably reflect the processes of tissue repair. These differences will determine corneal stability and strength following trauma and possibly refractive surgery

Electron Beam Size Measurements Using the Heterodyne Near Field Speckles at ALBA

Experiments using the heterodyne near field speckle method (HNFS) have been performed at ALBA to characterize the spatial coherence of the synchrotron radiation, with the ultimate goal of measuring both the horizontal and vertical electron beam sizes. The HNFS technique consists on the analysis of the interference between the radiation scattered by a colloidal suspension of nanoparticles and the synchrotron radiation, which in this case corresponds to the hard x-rays (12keV) produced by the in-vacuum undulator of the NCD-Sweet beamline. This paper describes the fundamentals of the technique, possible limitations, and shows the first experimental results changing the beam coupling of the storage ring

Coordination of asparagine uptake and asparagine synthetase expression modulates CD8+ T cell activation

T cell receptor (TCR) triggering by antigen results in metabolic reprogramming that, in turn, facilitates the exit of T cells from quiescence. The increased nutrient requirements of activated lymphocytes are met, in part, by upregulation of cell surface transporters and enhanced uptake of amino acids, fatty acids, and glucose from the environment. However, the role of intracellular pathways of amino acid biosynthesis in T cell activation is relatively unexplored. Asparagine is a nonessential amino acid that can be synthesized intracellularly through the glutamine-hydrolyzing enzyme asparagine synthetase (ASNS). We set out to define the requirements for uptake of extracellular asparagine and ASNS activity in CD8+ T cell activation. At early time points of activation in vitro, CD8+ T cells expressed little or no ASNS, and, as a consequence, viability and TCR-stimulated growth, activation, and metabolic reprogramming were substantially impaired under conditions of asparagine deprivation. At later time points (more than 24 hours of activation), TCR-induced mTOR-dependent signals resulted in ASNS upregulation that endowed CD8+ T cells with the capacity to function independently of extracellular asparagine. Thus, our data suggest that the coordinated upregulation of ASNS expression and uptake of extracellular asparagine is involved in optimal T cell effector responses

The effect of vitamin C deficiency and chronic ultraviolet-B exposure on corneal ultrastructure: a preliminary investigation

Purpose: In the visually debilitating condition of climatic droplet keratopathy, corneal transparency is progressively lost. Although the precise cause of the disease and the mechanism by which it progresses are not known, a lifetime exposure to high solar radiation and a vitamin C–deficient diet may be involved in its development. This study examines the effect of dietary ascorbate levels and ultraviolet (UV)-B exposure on corneal stromal structure. Methods: Eight guinea pigs were divided into four treatment groups (A, B, C, and D). For 15 weeks, Groups A and C were fed an ascorbate-rich diet (2 mg/100 g bodyweight/day), while Groups B and D received an ascorbate-deficient diet (0.07 mg/100 g bodyweight/day). For the last 12 weeks of the study, Groups C and D also experienced chronic UVB exposure (0.12 J/cm2 for 40 min/day). Following euthanasia, the corneas were enucleated and their stromal ultrastructure examined using X-ray scattering and electron microscopy. Results: UVB exposure resulted in an increased corneal thickness (p<0.001), but this was not accompanied by a widespread expansion of the collagen fibrillar array, and in the case of ascorbate-deficient animals, stromal thickening was associated with the compaction of collagen fibrils (p<0.01). Neither UVB exposure nor ascorbic acid deficiency caused any change in the average diameter or D-periodicity of the stromal collagen fibrils. Conclusions: UVB-induced changes in the corneal ultrastructure were most pronounced in animals fed an ascorbic acid–deficient diet. This suggests that ascorbic acid may play a vital role in protecting the corneal stroma from the harmful effects of UVB

Hematopoietic stem cell gene therapy for brain metastases using myeloid cell-specific gene promoters

Background: Brain metastases (BrM) develop in 20-40% of cancer patients and represent an unmet clinical need. Limited access of drugs into the brain due to the blood-brain barrier is at least partially responsible for therapeutic failure, necessitating improved drug delivery systems. Methods: Green fluorescent protein (GFP)-transduced murine and non-transduced human hematopoietic stem cells (HSCs) were administered into mice (n = 10 and 3). The HSC progeny in mouse BrM and in patient-derived BrM tissue (n = 6) was characterized by flow cytometry and immunofluorescence. Promoters driving gene expression, specifically within the BrM-infiltrating HSC progeny, were identified through differential gene expression analysis and subsequent validation of a series of promoter-GFP-reporter constructs in mice (n = 5). One of the promoters was used to deliver TNF-related apoptosis-inducing ligand (TRAIL) to BrM in mice (n = 17/21 for TRAIL versus control group). Results: HSC progeny (consisting mostly of macrophages) efficiently homed to macrometastases (37.6% [SD = 7.2%] of all infiltrating cells for murine HSC progeny; 27.9% [SD = 4.9%] of infiltrating CD45+ hematopoietic cells for human HSC progeny) and micrometastases in mice (19.3-53.3% of all macrophages for murine HSCs). Macrophages were also abundant in patient-derived BrM tissue (8.8%, SD = 7.8%). Collectively, this provided a rationale to optimize the delivery of gene therapy to BrM within myeloid cells. MMP14 promoter emerged as the strongest promoter construct capable of limiting gene expression to BrM-infiltrating myeloid cells in mice TRAIL delivered under MMP14 promoter statistically significantly prolonged survival in mice (19.0 [SD = 3.4] versus 15.0 [SD = 2.0] days for TRAIL versus control group; two-sided p = 0.006), demonstrating therapeutic and translational potential of our approach. Conclusions: Our study establishes HSC gene therapy using a myeloid cell-specific promoter as a new strategy to target BrM. This approach, with strong translational value, has potential to overcome the blood-brain barrier, target micrometastases, and control multifocal lesions

Hematopoietic stem cell gene therapy targeting TGFβ enhances the efficacy of irradiation therapy in a preclinical glioblastoma model

Patients with glioblastoma (GBM) have a poor prognosis, and inefficient delivery of drugs to tumors represents a major therapeutic hurdle. Hematopoietic stem cell (HSC)-derived myeloid cells efficiently home to GBM and constitute up to 50% of intratumoral cells, making them highly appropriate therapeutic delivery vehicles. Because myeloid cells are ubiquitously present in the body, we recently established a lentiviral vector containing matrix metalloproteinase 14 (MMP14) promoter, which is active specifically in tumor-infiltrating myeloid cells as opposed to myeloid cells in other tissues, and resulted in a specific delivery of transgenes to brain metastases in HSC gene therapy. Here, we used this novel approach to target transforming growth factor beta (TGFβ) as a key tumor-promoting factor in GBM. Transplantation of HSCs transduced with lentiviral vector expressing green fluorescent protein (GFP) into lethally irradiated recipient mice was followed by intracranial implantation of GBM cells. Tumor-infiltrating HSC progeny was characterized by flow cytometry. In therapy studies, mice were transplanted with HSCs transduced with lentiviral vector expressing soluble TGFβ receptor II–Fc fusion protein under MMP14 promoter. This TGFβ-blocking therapy was compared with the targeted tumor irradiation, the combination of the two therapies, and control. Tumor growth and survival were quantified (statistical significance determined by t-test and log-rank test). T cell memory response was probed through a repeated tumor challenge. Myeloid cells were the most abundant HSC-derived population infiltrating GBM. TGFβ-blocking HSC gene therapy in combination with irradiation significantly reduced tumor burden as compared with monotherapies and the control, and significantly prolonged survival as compared with the control and TGFβ-blocking monotherapy. Long-term protection from GBM was achieved only with the combination treatment (25% of the mice) and was accompanied by a significant increase in CD8+ T cells at the tumor implantation site following tumor rechallenge. We demonstrated a preclinical proof-of-principle for tumor myeloid cell-specific HSC gene therapy in GBM. In the clinic, HSC gene therapy is being successfully used in non-cancerous brain disorders and the feasibility of HSC gene therapy in patients with glioma has been demonstrated in the context of bone marrow protection. This indicates an opportunity for clinical translation of our therapeutic approach

In vitro selection of RNA aptamers against a conserved region of the Plasmodium falciparum erythrocyte membrane protein 1

The var-gene encoding Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) is known to play a major role in the pathogenicity of the P. falciparum parasite. The protein enables the parasite to adhere to the endothelial linings of small blood vessels (cytoadherence) as well as to non-infected erythrocytes (rosetting), thus preventing clearance from the bloodstream. The development and spread of resistance towards most anti-malarial drugs used for treatment and prevention of the most severe form of malaria truly emphasise the importance of a continuous research and development of new drugs. In this study we use Systematic Evolution of Ligands by EXponential enrichment (SELEX) methodology to isolate high-affinity ligands (aptamers). To validate the results from the SELEX in vitro selection, different aptamers have been selected against PfEMP1 in a live cell assay of P. falciparum strain FCR3S1.2, a highly rosetting strain. We have been able to show the rosette disrupting capacity of these SELEX-aptamers at concentrations of 33 nM and with 100% disruption at 387 nM. The described results show that RNA aptamers are promising candidates for adjunct therapy in severe malaria