Conventional therapies deplete Brain-Infiltrating adaptive immune cells in a Mouse Model of Group 3 Medulloblastoma implicating Myeloid Cells as favorable immunotherapy targets

Medulloblastoma is the most common childhood brain cancer. Mainstay treatments of radiation and chemotherapy have not changed in decades and new treatment approaches are crucial for the improvement of clinical outcomes. To date, immunotherapies for medulloblastoma have been unsuccessful, and studies investigating the immune microenvironment of the disease and the impact of current therapies are limited. Preclinical models that recapitulate both the disease and immune environment are essential for understanding immune-tumor interactions and to aid the identification of new and effective immunotherapies. Using an immune-competent mouse model of aggressive Myc-driven medulloblastoma, we characterized the brain immune microenvironment and changes induced in response to craniospinal irradiation, or the medulloblastoma chemotherapies cyclophosphamide or gemcitabine. The role of adaptive immunity in disease progression and treatment response was delineated by comparing survival outcomes in wildtype C57Bl/6J and in mice deficient in Rag1 that lack mature T and B cells. We found medulloblastomas in wildtype and Rag1-deficient mice grew equally fast, and that craniospinal irradiation and chemotherapies extended survival equally in wildtype and Rag1-deficient mice, suggesting that tumor growth and treatment response is independent of T and B cells. Medulloblastomas were myeloid dominant, and in wildtype mice, craniospinal irradiation and cyclophosphamide depleted T and B cells in the brain. Gemcitabine treatment was found to minimally alter the immune populations in the brain, resulting only in a depletion of neutrophils. Intratumorally, we observed an abundance of Iba1+ macrophages, and we show that CD45high cells comprise the majority of immune cells within these medulloblastomas but found that existing markers are insufficient to clearly delineate resident microglia from infiltrating macrophages. Ultimately, brain resident and peripheral macrophages dominate the brain and tumor microenvironment and are not depleted by standard-of-care medulloblastoma therapies. These populations therefore present a favorable target for immunotherapy in combination with front-line treatments

Peptide ligands of the cardiac ryanodine receptor as super-resolution imaging probes

To study the structural basis of pathological remodelling and altered calcium channel functional states in the heart, we sought to re-purpose high-affinity ligands of the cardiac calcium channel, the ryanodine receptor (RyR2), into super-resolution imaging probes. Imperacalcin (IpCa), a scorpion toxin peptide which induces channel sub-conduction states, and DPc10, a synthetic peptide corresponding to a sequence of the RyR2, which replicates arrhythmogenic CPVT functional changes, were used in fluorescent imaging experiments. Isolated adult rat ventricular cardiomyocytes were saponin-permeabilised and incubated with each peptide. IpCa-A546 became sequestered into the mitochondria. This was prevented by treatment of the permeabilised cells with the ionophore FCCP, revealing a striated staining pattern in confocal imaging which had weak colocalisation with RyR2 clusters. Poor specificity (as an RyR2 imaging probe) was confirmed at higher resolution with expansion microscopy (proExM) (~70 nm). DPc10-FITC labelled a striated pattern, which had moderate colocalisation with RyR2 cluster labelling in confocal and proExM. There was also widespread non-target labelling of the Z-discs, intercalated discs, and nuclei, which was unaffected by incubation times or 10 mM caffeine. The inactive peptide mut-DPc10-FITC (which causes no functional effects) displayed a similar labelling pattern. Significant labelling of structures unrelated to RyR2 by both peptide conjugates makes their use as highly specific imaging probes of RyR2 in living isolated cardiomyocytes highly challenging. We investigated the native DPc10 sequence within the RyR2 structure to understand the domain interactions and proposed mechanism of peptide binding. The native DPc10 sequence does not directly interact with another domain, and but is downstream of one such domain interface. The rabbit Arg2475 (equivalent to human Arg2474, mutated in CPVT) in the native sequence is the most accessible portion and most likely location for peptide disturbance, suggesting FITC placement does not impact peptide binding

Post-Partum Pituitary Insufficiency and Livedo Reticularis Presenting a Diagnostic Challenge in a Resource Limited Setting in Tanzania: A Case Report, Clinical Discussion and Brief Review of Existing Literature.

Pituitary disorders following pregnancy are an important yet under reported clinical entity in the developing world. Conversely, post partum panhypopituitarism has a more devastating impact on women in such settings due to high fertility rates, poor obstetric care and scarcity of diagnostic and therapeutic resources available. A 37 year old African female presented ten years post partum with features of multiple endocrine deficiencies including hypothyroidism, hypoadrenalism, lactation failure and secondary amenorrhea. In addition she had clinical features of an underlying autoimmune condition. These included a history of post-partum thyroiditis, alopecia areata, livedo reticularis and deranged coagulation indices. A remarkable clinical response followed appropriate hormone replacement therapy including steroids. This constellation has never been reported before; we therefore present an interesting clinical discussion including a brief review of existing literature. Post partum pituitary insufficiency is an under-reported condition of immense clinical importance especially in the developing world. A high clinical index of suspicion is vital to ensure an early and correct diagnosis which will have a direct bearing on management and patient outcome

Reforms: A Quest for Efficiency or an Opportunity for Vested Interests'? A Case Study of Pharmaceutical Policy Reforms in Tanzania.

Regulation of the pharmaceutical sector is a challenging task for most governments in the developing countries. In Tanzania, this task falls under the Food and Drugs Authority and the Pharmacy Council. In 2010, the Pharmacy Council spearheaded policy reforms in the pharmaceutical sector aimed at taking over the control of the regulation of the business of pharmacy from the Tanzania Food and Drugs Authority. This study provides a critical analysis of these reforms. The study employed a qualitative case-study design. Data was collected through in-depth interviews, focus group discussions and document reviews. Data was analyzed thematically using a policy triangle framework. The analysis was done manually. The reforms adopted an incremental model of public policy-making and the process was characterized by lobbying for political support, negotiations and bargaining between the interest groups. These negotiations were largely centred on vested interests and not on the impact of the reforms on the efficiency of pharmaceutical regulations in the country. Stakeholders from the micro and meso levels were minimally involved in the policy reforms. Recent pharmaceutical regulation reforms in Tanzania were overshadowed by vested interests, displacing a critical analysis of optimal policy options that have the potential to increase efficiency in the regulation of the business of pharmacy. Politics influenced decision-making at different levels of the reform process

Nanoscale crystallinity modulates cell proliferation on plasma sprayed surfaces

Calcium phosphate coatings have been applied to the surface of metallic prostheses to mediate hard and soft tissue attachment for more than 40 years. Most coatings are formed of high purity hydroxyapatite, and coating methods are often designed to produce highly crystalline surfaces. It is likely however, that coatings of lower crystallinity can facilitate more rapid tissue attachment since the surface will exhibit a higher specific surface area and will be considerably more reactive than a comparable highly crystalline surface. Here we test this hypothesis by growing a population of MC3T3 osteoblast-like cells on the surface of two types of hip prosthesis with similar composition, but with differing crystallinity. The surfaces with lower crystallinity facilitated more rapid cell attachment and increased proliferation rate, despite having a less heterogeneous surface topography. This work highlights that the influence of the crystallinity of HA at the nano-scale is dominant over macroscale topography for cell adhesion and growth. Furthermore, crystallinity could be easily adjusted by without compromising coating purity. These findings could facilitate designing novel coated calcium phosphate surfaces that more rapidly bond tissue following implantation

Potential effects of oilseed rape expressing oryzacystatin-1 (OC-1) and of purified insecticidal proteins on larvae of the solitary bee Osmia bicornis

Despite their importance as pollinators in crops and wild plants, solitary bees have not previously been included in non-target testing of insect-resistant transgenic crop plants. Larvae of many solitary bees feed almost exclusively on pollen and thus could be highly exposed to transgene products expressed in the pollen. The potential effects of pollen from oilseed rape expressing the cysteine protease inhibitor oryzacystatin-1 (OC-1) were investigated on larvae of the solitary bee Osmia bicornis (= O. rufa). Furthermore, recombinant OC-1 (rOC-1), the Bt toxin Cry1Ab and the snowdrop lectin Galanthus nivalis agglutinin (GNA) were evaluated for effects on the life history parameters of this important pollinator. Pollen provisions from transgenic OC-1 oilseed rape did not affect overall development. Similarly, high doses of rOC-1 and Cry1Ab as well as a low dose of GNA failed to cause any significant effects. However, a high dose of GNA (0.1%) in the larval diet resulted in significantly increased development time and reduced efficiency in conversion of pollen food into larval body weight. Our results suggest that OC-1 and Cry1Ab expressing transgenic crops would pose a negligible risk for O. bicornis larvae, whereas GNA expressing plants could cause detrimental effects, but only if bees were exposed to high levels of the protein. The described bioassay with bee brood is not only suitable for early tier non-target tests of transgenic plants, but also has broader applicability to other crop protection products

Stability analysis of wheat populations and mixtures based on the physical, compositional and processing properties of the seeds

Six cropping populations, three variety mixtures and one diversity population were developed from winter wheat varieties and studied for physical, compositional and end-use quality traits for three years (2011–2013) under different European climatic and management conditions in order to study the stability of these traits resulted by the genetic diversity. The beneficial compositional and nutritional properties of the populations were assessed, while variation and stability of the traits were analysed statistically. No significant differences were found among the populations in low-input and organic management farming systems in the physical, compositional and processing properties, but there was a difference in the stability of these traits. Most of the populations showed higher stability than the control wheat variety, and populations developed earlier had higher stability than those developed later. Furthermore, some populations were found to be especially unstable for some traits at certain sites (mostly at Austrian, Swiss and UK organic sites). Protein content of the populations was high (13.0–14.7%) without significant difference among them, but there was significant variation in their gluten content (28–36%) and arabinoxylan content (14.6–20.3 mg/g). The most outstanding population for both protein and arabinoxylan content was a Hungarian cropping population named ELIT-CCP. It was concluded that the diversity found in the mixtures and CCPs have stabilizing effect on the quality parameters, but a higher stability was observed under low-input than under organic conditions. These results could be beneficial not only for breeders but also for the consumers in the long run

Measurement of the production cross-section of positive pions in the collision of 8.9 GeV/c protons on beryllium

The double-differential production cross-section of positive pions, $d^2\sigma^{\pi^{+}}/dpd\Omega$, measured in the HARP experiment is presented. The incident particles are 8.9 GeV/c protons directed onto a beryllium target with a nominal thickness of 5% of a nuclear interaction length. The measured cross-section has a direct impact on the prediction of neutrino fluxes for the MiniBooNE and SciBooNE experiments at Fermilab. After cuts, 13 million protons on target produced about 96,000 reconstructed secondary tracks which were used in this analysis. Cross-section results are presented in the kinematic range 0.75 GeV/c < $p_{\pi}$ < 6.5 GeV/c and 30 mrad < $\theta_{\pi}$ < 210 mrad in the laboratory frame.Comment: 39 pages, 21 figures. Version accepted for publication by Eur. Phys. J.

Identification of Human Fibroblast Cell Lines as a Feeder Layer for Human Corneal Epithelial Regeneration

There is a great interest in using epithelium generated in vitro for tissue bioengineering. Mouse 3T3 fibroblasts have been used as a feeder layer to cultivate human epithelia including corneal epithelial cells for more than 3 decades. To avoid the use of xeno-components, we evaluated human fibroblasts as an alternative feeder supporting human corneal epithelial regeneration. Five human fibroblast cell lines were used for evaluation with mouse 3T3 fibroblasts as a control. Human epithelial cells isolated from fresh corneal limbal tissue were seeded on these feeders. Colony forming efficiency (CFE) and cell growth capacity were evaluated on days 5–14. The phenotype of the regenerated epithelia was evaluated by morphology and immunostaining with epithelial markers. cDNA microarray was used to analyze the gene expression profile of the supportive human fibroblasts. Among 5 strains of human fibroblasts evaluated, two newborn foreskin fibroblast cell lines, Hs68 and CCD1112Sk, were identified to strongly support human corneal epithelial growth. Tested for 10 passages, these fibroblasts continually showed a comparative efficiency to the 3T3 feeder layer for CFE and growth capacity of human corneal epithelial cells. Limbal epithelial cells seeded at 1×104 in a 35-mm dish (9.6 cm2) grew to confluence (about 1.87–2.41×106 cells) in 12–14 days, representing 187–241 fold expansion with over 7–8 doublings on these human feeders. The regenerated epithelia expressed K3, K12, connexin 43, p63, EGFR and integrin β1, resembling the phenotype of human corneal epithelium. DNA microarray revealed 3 up-regulated and 10 down-regulated genes, which may be involved in the functions of human fibroblast feeders. These findings demonstrate that commercial human fibroblast cell lines support human corneal epithelial regeneration, and have potential use in tissue bioengineering for corneal reconstruction