Sav1 Loss Induces Senescence and Stat3 Activation Coinciding with Tubulointerstitial Fibrosis

Tubulointerstitial fibrosis (TIF) is recognized as a final phenotypic manifestation in the transition from chronic kidney disease (CKD) to end-stage renal disease (ESRD). Here we show that conditional inactivation of Sav1 in the mouse renal epithelium resulted in upregulated expression of profibrotic genes and TIF. Loss of Sav1 induced Stat3 activation and a senescence-associated secretory phenotype (SASP) that coincided with the development of tubulointerstitial fibrosis. Treatment of mice with the YAP inhibitor verteporfin (VP) inhibited activation of genes associated with senescence, SASPs, and activation of Stat3 as well as impeded the development of fibrosis. Collectively, our studies offer novel insights into molecular events that are linked to fibrosis development from Sav1 loss and implicate VP as a potential pharmacological inhibitor to treat patients at risk for developing CKD and TIF

Nutrient regulation by continuous feeding removes limitations on cell yield in the large-scale expansion of Mammalian cell spheroids.

Cellular therapies are emerging as a standard approach for the treatment of several diseases. However, realizing the promise of cellular therapies across the full range of treatable disorders will require large-scale, controlled, reproducible culture methods. Bioreactor systems offer the scale-up and monitoring needed, but standard stirred bioreactor cultures do not allow for the real-time regulation of key nutrients in the medium. In this study, β-TC6 insulinoma cells were aggregated and cultured for 3 weeks as a model of manufacturing a mammalian cell product. Cell expansion rates and medium nutrient levels were compared in static, stirred suspension bioreactors (SSB), and continuously fed (CF) SSB. While SSB cultures facilitated increased culture volumes, no increase in cell yields were observed, partly due to limitations in key nutrients, which were consumed by the cultures between feedings, such as glucose. Even when glucose levels were increased to prevent depletion between feedings, dramatic fluctuations in glucose levels were observed. Continuous feeding eliminated fluctuations and improved cell expansion when compared with both static and SSB culture methods. Further improvements in growth rates were observed after adjusting the feed rate based on calculated nutrient depletion, which maintained physiological glucose levels for the duration of the expansion. Adjusting the feed rate in a continuous medium replacement system can maintain the consistent nutrient levels required for the large-scale application of many cell products. Continuously fed bioreactor systems combined with nutrient regulation can be used to improve the yield and reproducibility of mammalian cells for biological products and cellular therapies and will facilitate the translation of cell culture from the research lab to clinical applications

Fluctuations in medium nutrient levels contributed to limitations in cell expansion.

<p>Glucose measurements from β-TC6 cell spheroids cultured in (A) Static cultures, and (B) stirred suspension bioreactors using high (4.5 g/L), intermediate (2.75 g/L), and low (1.0 g/L) glucose medium, as depicted by their position on the Y axis. The physiological glucose range is indicated by the grey bar. Error bars for glucose measurements are too small to be visible on the scale shown (Standard Error ≤4% for all measurements). (C) No difference was seen comparing static to SSB cultures with any of the glucose levels. Comparison of expansion of β-TC6 spheroid cultures indicated that changing the glucose in the medium to achieve levels closer to the physiological range did not significantly improve cell expansion. (*indicates a p value of 0.027 compared with the same culture method using high glucose medium.) SSB: stirred suspension bioreactor.</p

Adjusting culture medium feed rate regulates glucose concentrations and improves cell growth compared to continuous feeding at a constant rate.

<p>(<b>A</b>) Cell counts comparing constant feed rate to adjusted feed rate from the same cultures (*represents a p value <0.05 indicating a significant difference in culture expansion). (<b>B</b>) Average culture medium glucose levels from 21 day constant feeding, and adjusted feeding bioreactor cultures. The physiological glucose range is indicated by the grey bar. Error bars for glucose measurements are too small to be visible on the scale shown (Standard Error ≤4% for all measurements).</p

Continuously fed SSB eliminated glucose fluctuations and improved cell expansion.

<p>(<b>A</b>) Glucose measurements for β-TC6 spheroid culture medium using static, SSB, and CF-SSB culture methods and feeding with standard high glucose medium. The physiological glucose range is indicated by the grey bar. Error bars for glucose measurements are too small to be visible on the scale shown (Standard Error ≤4% for all measurements). (<b>B</b>) Fold Expansion of β-TC6 spheroids over 21 days of culture comparing static, SSB, and CF culture methods. SSB: stirred suspension bioreactor. CF: Continuously fed stirred suspension bioreactor.</p

Continuous feeding at a constant rate cannot maintain a physiological glucose level.

<p>Culture medium glucose measurements from continuous fed cultures with high, intermediate, or low glucose medium during 21 days of expansion. The physiological glucose range is indicated by the grey bar.</p

Design of continuous feed apparatus.

<p>(<b>A</b>) Schematic diagram of continuous feeding perfusion circuit with fresh medium, perfusion pump, outflow tube, and waste medium. Solid blue lines are medium connections, dashed green lines are fiber optic connections, and dotted black lines are electronic connections. (<b>B</b>) Exploded illustration of outflow tube showing small pores, which do not allow spheroids to pass through, but allow the removal of waste medium. The continuous feeding system was designed to help improve consistency of nutrient and supplement concentrations making β-TC6 cell culture parameters more stable and controlled.</p

Stirred suspension bioreactors offered no growth improvement compared to static cultures.

<p>Fold expansion of β-TC6 spheroids after twenty one days compared stirred suspension bioreactor to static culture using standard high glucose medium.</p

Racial differences in the relationship between tobacco, alcohol, and the risk of head and neck cancer: pooled analysis of US studies in the INHANCE Consortium

There have been few published studies on differences between Blacks and Whites in the estimated effects of alcohol and tobacco use on the incidence of head and neck cancer (HNC) in the United States. Previous studies have been limited by small numbers of Blacks. Using pooled data from 13 US case\u2013control studies of oral, pharyngeal, and laryngeal cancers in the International Head and Neck Cancer Epidemiology Consortium, this study comprised a large number of Black HNC cases (n = 975). Logistic regression was used to estimate adjusted odds ratios (OR) and 95% confidence intervals (CI) for several tobacco and alcohol consumption characteristics. Blacks were found to have consistently stronger associations than Whites for the majority of tobacco consumption variables. For example, compared to never smokers, Blacks who smoked cigarettes for > 30\ua0years had an OR 4.53 (95% CI 3.22\u20136.39), which was larger than that observed in Whites (OR 3.01, 95% CI 2.73\u20133.33; pinteraction < 0.0001). The ORs for alcohol use were also larger among Blacks compared to Whites. Exclusion of oropharyngeal cases attenuated the racial differences in tobacco use associations but not alcohol use associations. These findings suggest modest racial differences exist in the association of HNC risk with tobacco and alcohol consumption. \ua9 2018, Springer International Publishing AG, part of Springer Nature