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Development of a human embryonic stem cell (hESC)-based therapy for type 1 diabetes will require the translation of proof-of-principle concepts into a scalable, controlled, and regulated cell manufacturing process. We have previously demonstrated that hESC can be directed to differentiate into pancreatic progenitors that mature into functional glucose-responsive, insulin-secreting cells in vivo. In this study we describe hESC expansion and banking methods and a suspension-based differentiation system, which together underpin an integrated scalable manufacturing process for producing pancreatic progenitors. This system has been optimized for the CyT49 cell line. Accordingly, qualified large-scale single-cell master and working cGMP cell banks of CyT49 have been generated to provide a virtually unlimited starting resource for manufacturing. Upon thaw from these banks, we expanded CyT49 for two weeks in an adherent culture format that achieves 50-100 fold expansion per week. Undifferentiated CyT49 were then aggregated into clusters in dynamic rotational suspension culture, followed by differentiation en masse for two weeks with a four-stage protocol. Numerous scaled differentiation runs generated reproducible and defined population compositions highly enriched for pancreatic cell lineages, as shown by examining mRNA expression at each stage of differentiation and flow cytometry of the final population. Islet-like tissue containing glucose-responsive, insulin-secreting cells was generated upon implantation into mice. By four- to five-months post-engraftment, mature neo-pancreatic tissue was sufficient to protect against streptozotocin (STZ)-induced hyperglycemia. In summary, we have developed a tractable manufacturing process for the generation of functional pancreatic progenitors from hESC on a scale amenable to clinical entry.

Of paramount importance for the development of cell therapies to treat diabetes is the production of sufficient numbers of pancreatic endocrine cells that function similarly to primary islets. We have developed a differentiation process that converts human embryonic stem (hES) cells to endocrine cells capable of synthesizing the pancreatic hormones insulin, glucagon, somatostatin, pancreatic polypeptide and ghrelin. This process mimics in vivo pancreatic organogenesis by directing cells through stages resembling definitive endoderm, gut-tube endoderm, pancreatic endoderm and endocrine precursor—en route to cells that express endocrine hormones. The hES cell–derived insulin-expressing cells have an insulin content approaching that of adult islets. Similar to fetal β-cells, they release C-peptide in response to multiple secretory stimuli, but only minimally to glucose. Production of these hES cell–derived endocrine cells may represent a critical step in the development of a renewable source of cells for diabetes cell therapy.

This paper describes a new protocol for producing insulin‐producing cells in vitro that represents another potential cell source for a diabetes cell therapy. These cells can be loaded into a protective device that is implanted under the skin. The device is designed to protect the cells from immune rejection by the implant recipient. The implant can engraft and respond to glucose by secreting insulin, thus potentially replacing the β cells lost in patients with type 1 diabetes. The PEC‐01 cell population, differentiated from human embryonic stem cells (hESCs), contains pancreatic progenitors (PPs) that, when loaded into macroencapsulation devices (to produce the VC‐01 candidate product) and transplanted into mice, can mature into glucose‐responsive insulin‐secreting cells and other pancreatic endocrine cells involved in glucose metabolism. We modified the protocol for making PEC‐01 cells such that 73%–80% of the cell population consisted of PDX1‐positive (PDX1+) and NKX6.1+ PPs. The PPs were further differentiated to islet‐like cells (ICs) that reproducibly contained 73%–89% endocrine cells, of which approximately 40%–50% expressed insulin. A large fraction of these insulin‐positive cells were single hormone‐positive and expressed the transcription factors PDX1 and NKX6.1. To preclude a significant contribution of progenitors to the in vivo function of ICs, we used a simple enrichment process to remove remaining PPs, yielding aggregates that contained 93%–98% endocrine cells and 1%–3% progenitors. Enriched ICs, when encapsulated and implanted into mice, functioned similarly to the VC‐01 candidate product, demonstrating conclusively that in vitro‐produced hESC‐derived insulin‐producing cells can mature and function in vivo in devices. A scaled version of our suspension culture was used, and the endocrine aggregates could be cryopreserved and retain functionality. Although ICs expressed multiple important β cell genes, the cells contained relatively low levels of several maturity‐associated markers. Correlating with this, the time to function of ICs was similar to PEC‐01 cells, indicating that ICs required cell‐autonomous maturation after delivery in vivo, which would occur concurrently with graft integration into the host. Significance Type 1 diabetes (T1D) affects approximately 1.25 million people in the U.S. alone and is deadly if not managed with insulin injections. This paper describes the production of insulin‐producing cells in vitro and a new protocol for producing the cells, representing another potential cell source for a diabetes cell therapy. These cells can be loaded into a protective device that is implanted under the skin. The device is designed to protect the cells from immune rejection by the implant recipient. The implant can engraft and respond to glucose by secreting insulin, thus potentially replacing the β cells lost in patients with T1D.

Human mesenchymal stem cells are thought to be multipotent cells, which are present in adult marrow, that can replicate as undifferentiated cells and that have the potential to differentiate to lineages of mesenchymal tissues, including bone, cartilage, fat, tendon, muscle, and marrow stroma. Cells that have the characteristics of human mesenchymal stem cells were isolated from marrow aspirates of volunteer donors. These cells displayed a stable phenotype and remained as a monolayer in vitro. These adult stem cells could be induced to differentiate exclusively into the adipocytic, chondrocytic, or osteocytic lineages. Individual stem cells were identified that, when expanded to colonies, retained their multilineage potential.

Experts in food hygiene have long struggled to eliminate microorganisms established within the food-manufacturing environment. Such environments may be contaminated with pathogenic or spoilage organisms that evade sanitation and contaminate food. It was hypothesized that sensitivity of L. innocua to the quaternary ammonium compound sanitizer cetrimide is altered following adaptation to acid, starvation, cold and heat stress, stressors commonly found within the food manufacturing environment and this relates to altered cell hydrophobicity and membrane fluidity. This research demonstrated that exposure of L. innocua to acid and starvation stress diminishes sensitivity to 10 ppm cetrimide while exposure to cold and heat stress enhance sensitivity. Furthermore, acid and starvation stress increased net cell hydrophobicity and reduced cell membrane fluidity. In contrast, decreased hydrophobicity and increased membrane fluidity were observed in cold adapted L. innocua. No significant changes in hydrophobicity or indicators of membrane fluidity, aside from increased C-18 unsaturated fatty acids, were detected in heat adapted L. innocua. That certain environmental conditions within food manufacturing facilities such as acid and starvation could diminish cellular sensitivity to industrial sanitizers suggest the physiological stress response not only diminishes sensitivity to the stress, but also enables persistence upon exposure to low levels of quaternary ammonium compound sanitizers. Conversely, that other modifications of the environment, such as cold temperature, would stress-adapt and concurrently enhance sensitivity of L. innocua to quaternary ammonium compounds suggest interventions exist that enhance sanitation efficacy. The potential exists therefore, for the application of stress conditions to equipment or manufacturing sites persistently testing positive for problematic microorganisms, and thereby diminish the ability the microorganisms to survive sanitizer exposure.

Incorporating genetics into risk-stratification for treatment of childhood B-progenitor acute lymphoblastic leukaemia (B-ALL) has contributed significantly to improved survival. In about 30% B-ALL (B-other-ALL) without well-established chromosomal changes, new genetic subtypes have recently emerged, yet their true prognostic relevance largely remains unclear. We integrated next generation sequencing (NGS): whole genome sequencing (WGS) (n = 157) and bespoke targeted NGS (t-NGS) (n = 175) (overlap n = 36), with existing genetic annotation in a representative cohort of 351 B-other-ALL patients from the childhood ALL trail, UKALL2003. PAX5alt was most frequently observed (n = 91), whereas PAX5 P80R mutations (n = 11) defined a distinct PAX5 subtype. DUX4-r subtype (n = 80) was defined by DUX4 rearrangements and/or ERG deletions. These patients had a low relapse rate and excellent survival. ETV6::RUNX1-like subtype (n = 21) was characterised by multiple abnormalities of ETV6 and IKZF1, with no reported relapses or deaths, indicating their excellent prognosis in this trial. An inferior outcome for patients with ABL-class fusions (n = 25) was confirmed. Integration of NGS into genomic profiling of B-other-ALL within a single childhood ALL trial, UKALL2003, has shown the added clinical value of NGS-based approaches, through improved accuracy in detection and classification into the range of risk stratifying genetic subtypes, while validating their prognostic significance.

Childhood B-cell acute lymphoblastic leukaemia (B-ALL) is characterised by recurrent genetic abnormalities that drive risk-directed treatment strategies. Using current techniques, accurate detection of such aberrations can be challenging, due to the rapidly expanding list of key genetic abnormalities. Whole genome sequencing (WGS) has the potential to improve genetic testing, but requires comprehensive validation. We performed WGS on 210 childhood B-ALL samples annotated with clinical and genetic data. We devised a molecular classification system to subtype these patients based on identification of key genetic changes in tumour-normal and tumour-only analyses. This approach detected 294 subtype-defining genetic abnormalities in 96% (202/210) patients. Novel genetic variants, including fusions involving genes in the MAP kinase pathway, were identified. WGS results were concordant with standard-of-care methods and whole transcriptome sequencing (WTS). We expanded the catalogue of genetic profiles that reliably classify PAX5alt and ETV6::RUNX1-like subtypes. Our novel bioinformatic pipeline improved detection of DUX4 rearrangements (DUX4-r): a good-risk B-ALL subtype with high survival rates. Overall, we have validated that WGS provides a standalone, reliable genetic test to detect all subtype-defining genetic abnormalities in B-ALL, accurately classifying patients for the risk-directed treatment stratification, while simultaneously performing as a research tool to identify novel disease biomarkers.

Chikungunya is a mosquito-borne viral disease that causes fever and severe joint pain for which there is no direct acting drug treatments. Vinyl sulfone SGC-NSP2PRO-1 ( 3 ) was identified as a potent inhibitor of the nsP2 cysteine protease (nsP2pro) that reduced viral titer against infectious isolates of Chikungunya and other alphaviruses. The covalent warhead in 3 captured the active site C478 and inactivated nsP2pro with a k inact / K i ratio of 5950 M –1  s –1 . The vinyl sulfone 3 was inactive across a panel of 23 other cysteine proteases and demonstrated remarkable proteome-wide selectivity by two chemoproteomic methods. A negative control analog SGC-NSP2PRO-1N ( 4 ) retained the isoxazole core and covalent warhead but demonstrated > 100-fold decrease in enzyme inhibition. Both 3 and 4 were stable across a wide range of pH in solution and upon prolonged storage as solids. Vinyl sulfone 3 and its negative control 4 will find utility as high-quality chemical probes to study the role of the nsP2pro in cellular studies of alphaviral replication and virulence.

Background Tolerogenic vaccines represent antigen-specific interventions designed to re-establish self-tolerance and thereby alleviate autoimmune diseases, which collectively comprise over 100 chronic inflammatory diseases afflicting more than 20 million Americans. Tolerogenic vaccines comprised of single-chain GM-CSF-neuroantigen (GMCSF-NAg) fusion proteins were shown in previous studies to prevent and reverse disease in multiple rodent models of experimental autoimmune encephalomyelitis (EAE) by a mechanism contingent upon the function of CD4 + CD25 + FOXP3 + regulatory T cells (Tregs). GMCSF-NAg vaccines inhibited EAE in both quiescent and inflammatory environments in association with low-efficiency T cell receptor (TCR) signaling events that elicited clonal expansion of immunosuppressive Tregs. Methods This study focused on two vaccines, including GMCSF-MOG (myelin oligodendrocyte glycoprotein 35–55/MOG 35–55 ) and GMCSF-NFM (neurofilament medium peptide 13–37/NFM 13–37 ), that engaged the transgenic 2D2 TCR with either low or high efficiencies, respectively. 2D2 mice were crossed with FOXP3 IRES eGFP (FIG) mice to track Tregs and further crossed with Rag −/− mice to reduce pre-existing Treg populations. Results This study provided evidence that low and high efficiency TCR interactions were integrated via CD40L expression levels to control the Treg/Tcon balance. The high-efficiency GMCSF-NFM vaccine elicited memory Tcon responses in association with activation of the CD40L costimulatory system. Conversely, the low-efficiency GMCSF-MOG vaccine lacked adequate TCR signal strength to elicit CD40L expression and instead elicited Tregs by a mechanism that was impaired by a CD40 agonist. When combined, the low- and high-efficiency GMCSF-NAg vaccines resulted in a balanced outcome and elicited both Tregs and Tcon responses without the predominance of a dominant immunogenic Tcon response. Aside from Treg expansion in 2D2-FIG mice, GMCSF-MOG caused a sustained decrease in TCR-β, CD3, and CD62L expression and a sustained increase in CD44 expression in Tcon subsets. Subcutaneous administration of GMCSF-MOG without adjuvants inhibited EAE in wildtype mice, which had a replete Treg repertoire, but was pathogenic rather than tolerogenic in 2D2-FIG- Rag1 −/− mice, which lacked pre-existing Tregs. Conclusions This study provided evidence that the GMCSF-MOG vaccine elicited antigenic responses beneath the CD40L triggering threshold, which defined an antigenic niche that drove dominant expansion of tolerogenic myelin-specific Tregs that inhibited EAE.

Iowa's South Fork watershed is dominated by corn (Zea mays L.) and soybean [Glycine max L. (Merr.)] rotations, and animal feeding operations are common. Artificial subsurface (tile) drainage is extensive; hydric soils cover 54% of the watershed. During spring and early summer, NO3-N concentrations in tile and stream discharge often exceed 20 mg L-1. Total N loads during 2002 to 2005 ranged from 16 to 26 kg NO3-N ha-1 y-1 (14 to 23 lb ac-1 yr-1). Nitrate concentrations increased linearly with log baseflow, effectively a surrogate measure of tile discharge. Phosphorus loads were only 0.4 to 0.7 kg P ha-1 y-1 (0.4 to 0.6 lb ac-1 yr-1), but concentrations commonly exceeded 0.1 mg L-1, a eutrophication-risk threshold. Mean E. coli populations in the stream exceeded 500 cells 100 ml-1 during summer. Statistical comparison of actual nitrate records with independent records generated using regression equations provided modeling efficiencies of 0.91 or less, suggesting performance targets for watershed model validation. Tile drainage is more important in transport of nitrate and dissolved phosphorus than E. coli. Variations in nitrate, phosphorus, and E. coli are uniquely timed, highlighting the complexity of integrated water quality assessments.