Intestinal epithelial cell-intrinsic deletion of Setd7 identifies role for developmental pathways in immunity to helminth infection

The intestine is a common site for a variety of pathogenic infections. Helminth infections continue to be major causes of disease worldwide, and are a significant burden on health care systems. Lysine methyltransferases are part of a family of novel attractive targets for drug discovery. SETD7 is a member of the Suppressor of variegation 3-9-Enhancer of zeste-Trithorax (SET) domain-containing family of lysine methyltransferases, and has been shown to methylate and alter the function of a wide variety of proteins in vitro. A few of these putative methylation targets have been shown to be important in resistance against pathogens. We therefore sought to study the role of SETD7 during parasitic infections. We find that Setd7-/- mice display increased resistance to infection with the helminth Trichuris muris but not Heligmosomoides polygyrus bakeri. Resistance to T. muris relies on an appropriate type 2 immune response that in turn prompts intestinal epithelial cells (IECs) to alter differentiation and proliferation kinetics. Here we show that SETD7 does not affect immune cell responses during infection. Instead, we found that IEC-specific deletion of Setd7 renders mice resistant to T. muris by controlling IEC turnover, an important aspect of anti-helminth immune responses. We further show that SETD7 controls IEC turnover by modulating developmental signaling pathways such as Hippo/YAP and Wnt/β-Catenin. We show that the Hippo pathway specifically is relevant during T. muris infection as verteporfin (a YAP inhibitor) treated mice became susceptible to T. muris. We conclude that SETD7 plays an important role in IEC biology during infection

Acoustic cell concentration, washing & perfusion for cellular therapy manufacturing

A major wave of promising cellular therapies is progressing through clinical trials, such that engineers and scientists need to address the challenges of economically ensuring the manufacture of safe and efficacious cell therapy products. These processes often depend on devices and methods that were developed for only related blood cell processing or vaccine manufacturing. Thus, we are in a window of opportunity to tailor innovative technologies to address the emerging specialized needs of cell therapy manufacturing. Concentrating and washing cells between stages is a repeated bioprocess unit operation, such as to transfer cells from culture medium to cryopreservation medium. Especially for small-scale autologous therapies, cell washing is not well performed by closed-system centrifuges or filters, including due to the loss of potentially lifesaving cells. We previously developed an acoustic cell separation device that has been widely used for 20 years as part of mammalian cell perfusion bioreactors. This non-fouling filter technology uses gentle ultrasonic standing wave forces to separate cells from medium based on density and compressibility differences. We have now adapted this technology to concentrate and then wash cells at high concentrations, so as to reduce the wash volumes by an order of magnitude, thereby reducing the process cost of goods compared to centrifuge washing. The device operation has been optimized to obtain greater than 99.9% washing with 95% washed cell recoveries, such that this acoustic technology could become the method of choice for cell therapy bioprocessing. We also have recently enhanced automated acoustic devices to perfuse over 140 million cell/mL cultures, maintaining \u3e99% cell separation efficiencies. With the simplicity of no physical filter barrier or mechanical moving parts, this tailored technology provides a high performance closed manufacturing device, to perfuse, concentrate and wash cells. The development of robust and economical means of mammalian cell manufacturing is on the critical path to ensuring that promising innovative therapies can become widely available to innumerable patients in dire need

Acoustic cell washing and raman spectroscopy technologies To address cell therapy bioprocess challenges

Many organizations are confronting the challenges of economically ensuring the manufacture of safe and efficacious cell therapy products. These processes often depend on devices and methods that were developed for only related applications, such as blood cell processing or scientific research. Thus, we are in a window of opportunity to tailor innovative technologies to address the emerging specialized needs of cell therapy manufacturing. The most frequent unit operation is to wash cells between process stages, such as from DMSO containing cryopreservation medium to culture expansion medium. In particular for relatively small-scale autologous cell therapy processing, cell washing is imperfectly performed by closed system blood cell centrifuges or filters. We previously developed an acoustic cell separation device, widely used for over 15 years in CHO cell perfusion cultures. This technology acts as a non-fouling filter for months of operation, by using the forces generated in ultrasonic standing wave fields. These forces separate cells from medium based on differences in density and compressibility. Greater than 99.9% cell washing with 95% washed cell recovery efficiencies have been provided by our device. We also have recently enhanced the acoustic technology to perfuse 100 million cell/mL cultures, maintaining \u3e99% cell separation efficiencies. This provides an alternative high performance closed manufacturing system, to perfuse, concentrate and wash cells, with no physical filter barrier or mechanical moving parts. While many clinical trials have had few adverse events, the great promise of cellular therapies comes with grave risks, such as from potentially oncogenic pluripotent cells present in embryonic stem cell derived populations. There is an urgent need for process analytical technologies to non-invasively monitor mammalian cell populations and improve the reliability of manufactured cell products. This includes to monitor both the expected differentiation as well as to detect unexpected cells in the process. Recently, technological advances have led to an explosive growth in the capabilities of Raman spectroscopy, increasing the potential for novel applications. We are developing the use of this spectroscopic technique to track cell development, by measuring macromolecular changes in cell samples from cultures where stem cells are differentiated towards insulin-producing cells for the treatment of diabetes. Raman spectroscopy has great potential to provide continuous on-line assessment of cell quality during the manufacture of cell-derived therapeutic cells

The relationship between religious involvement and clinical status of patients with bipolar disorder

Cruz M, Pincus HA, Welsh DE, Greenwald D, Lasky E, Kilbourne AM. The relationship between religious involvement and clinical status of patients with bipolar disorder. Bipolar Disord 2010: 12: 68–76. © 2010 The Authors. Journal compilation © 2010 John Wiley & Sons A/S.Religion and spirituality are important coping strategies in depression but have been rarely studied within the context of bipolar disorder. The present study assessed the association between different forms of religious involvement and the clinical status of individuals treated for bipolar disorder.A cross-sectional observation study of follow-up data from a large cohort study of patients receiving care for bipolar disorder (n = 334) at an urban Veterans Affairs mental health clinic was conducted. Bivariate and multivariate analyses were performed to assess the association between public (frequency of church attendance), private (frequency of prayer/meditation), as well as subjective forms (influence of beliefs on life) of religious involvement and mixed, manic, depressed, and euthymic states when demographic, anxiety, alcohol abuse, and health indicators were controlled.Multivariate analyses found significant associations between higher rates of prayer/meditation and participants in a mixed state [odds ratio (OR) = 1.29; 95% confidence interval (CI) = 1.10–1.52, chi square = 9.42, df = 14, p < 0.05], as well as lower rates of prayer/meditation and participants who were euthymic (OR = 0.84; 95% CI = 0.72–0.99, chi square = 4.60, df = 14, p < 0.05). Depression and mania were not associated with religious involvement.Compared to patients with bipolar disorder in depressed, manic, or euthymic states, patients in mixed states have more active private religious lives. Providers should assess the religious activities of individuals with bipolar disorder in mixed states and how they may complement/deter ongoing treatment. Future longitudinal studies linking bipolar states, religious activities, and treatment-seeking behaviors are needed.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/78616/1/j.1399-5618.2009.00772.x.pd

Moyal star product approach to the Bohr-Sommerfeld approximation

The Bohr-Sommerfeld approximation to the eigenvalues of a one-dimensional quantum Hamiltonian is derived through order $\hbar^2$ (i.e., including the first correction term beyond the usual result) by means of the Moyal star product. The Hamiltonian need only have a Weyl transform (or symbol) that is a power series in $\hbar$, starting with $\hbar^0$, with a generic fixed point in phase space. The Hamiltonian is not restricted to the kinetic-plus-potential form. The method involves transforming the Hamiltonian to a normal form, in which it becomes a function of the harmonic oscillator Hamiltonian. Diagrammatic and other techniques with potential applications to other normal form problems are presented for manipulating higher order terms in the Moyal series.Comment: 27 pages, no figure

AT1 Receptor Blockade Prevents the Increase in Blood Pressure and the Augmentation of Intrarenal ANG II Levels in Hypertensive Cyp1a1-Ren2 Transgenic Rats Fed With a High-Salt Diet

BACKGROUND: The present study was performed to determine the effects of high-salt diet on the magnitude of the increases in systolic blood pressure (SBP) and kidney tissue ANG II levels that occur following induction of ANG II-dependent malignant hypertension in Cyp1a1-Ren2 transgenic rats with inducible expression of the mouse Ren2 renin gene [strain name: TGR (Cyp1a1Ren2)]. METHODS: Cyp1a1-Ren2 rats (n=6) were fed a normal diet containing 0.3% indole-3-carbinol (I3C) for 10 days to induce ANG II-dependent malignant hypertension. RESULTS: Rats induced with I3C exhibited increases in (SBP) and elevations of ANG II levels in kidney cortex and medulla. In a second group of rats (n=6), high salt intake alone did not alter basal SBP; however, subsequent dietary administration of 0.3% I3C during continued high salt intake elicited a substantially greater increase in SBP than observed in rats fed a normal salt diet. ANG II levels in kidney cortex and medulla of rats induced with I3C and fed a high salt diet were elevated similarly to those in rats induced with I3C alone. Chronic administration of the AT(1) receptor antagonist, losartan (100 mg/L in drinking water, n=6), markedly attenuated the I3C-induced increase in SBP and prevented the augmentation of ANG II levels in kidney cortex and medulla in rats induced with I3C and maintained on a high salt diet. CONCLUSIONS: Activation of AT(1) receptors contributes to the augmented blood pressure and elevated kidney tissue ANG II levels that occur in Cyp1a1-Ren2 transgenic rats with malignant hypertension maintained on a high salt diet

G9a regulates group 2 innate lymphoid cell development by repressing the group 3 innate lymphoid cell program.

Innate lymphoid cells (ILCs) are emerging as important regulators of homeostatic and disease-associated immune processes. Despite recent advances in defining the molecular pathways that control development and function of ILCs, the epigenetic mechanisms that regulate ILC biology are unknown. Here, we identify a role for the lysine methyltransferase G9a in regulating ILC2 development and function. Mice with a hematopoietic cell-specific deletion of G9a (Vav.G9a(-/-) mice) have a severe reduction in ILC2s in peripheral sites, associated with impaired development of immature ILC2s in the bone marrow. Accordingly, Vav.G9a(-/-) mice are resistant to the development of allergic lung inflammation. G9a-dependent dimethylation of histone 3 lysine 9 (H3K9me2) is a repressive histone mark that is associated with gene silencing. Genome-wide expression analysis demonstrated that the absence of G9a led to increased expression of ILC3-associated genes in developing ILC2 populations. Further, we found high levels of G9a-dependent H3K9me2 at ILC3-specific genetic loci, demonstrating that G9a-mediated repression of ILC3-associated genes is critical for the optimal development of ILC2s. Together, these results provide the first identification of an epigenetic regulatory mechanism in ILC development and function

Bioprocess and cell line engineering to improve pancreatic endocrine differentiation outcomes and increase safety of pluripotent stem cells

Diabetes is a debilitating disorder that affects nearly 500 million people worldwide and is characterized by chronic hyperglycemia caused by pancreatic β-cell dysfunction or death. Those that are affected need to frequently monitor their blood glucose levels and administer exogenous insulin, but this strategy does not match the tight regulation maintained by healthy β-cells. Progress in the field has demonstrated an effective clinical path to treat type 1 diabetes through the success of islet transplants. More recently, groups have demonstrated that human pluripotent stem cells, differentiated into pancreatic progenitors or beyond, can prevent or reverse diabetes in rodents and have shown promising results in human clinical trials. Here I develop a differentiation protocol to further differentiate pancreatic progenitors generated with a commercially available kit into endocrine cells. I identified FGF7 signalling as being important for aggregate survival and Notch-inhibition improved endocrine commitment at the expense of ductal cells. Combining in vitro β-cell differentiation strategies with newly developed precision gene editing techniques allows for disease modelling in a dish and transgene elements can be introduced into cells to provide new functionalities. To this end, I used a novel gene editing technique to correct a single-base mutation in pluripotent stem cells derived from an individual living with a severe form of neonatal diabetes. Using stem cell-derived cell therapies does not come without risk, given the potential for teratoma outgrowth, inappropriate differentiation and function, or an immune response to the graft. Here, I generated cell lines containing an inducible safety-switch, which was shown to eliminate pluripotent and pancreatic progenitor cells in vitro and teratomas in vivo. Finally, I targeted the B2M locus for silencing using an inducible CRISPRi platform, preventing HLA-ABC formation in a reversible manner. This strategy could both allow cells to avoid immune detection as well as act as a safety-switch by deactivating CRISPRi and re-expressing the B2M gene. Overall, the projects described in this dissertation demonstrate the combined potential of pluripotent stem cells, gene editing, and cell differentiation with the goal of advancing the cell therapy field, particularly for diabetes.Science, Faculty ofGraduat