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Abstract Objective Glomerular filtration rate (GFR) is a key indicator of renal function. In both clinical practice and pre-clinical research, serum levels of endogenous filtration markers, such as creatinine, are often used to estimate GFR. However, these markers often do not reflect minor changes in renal function. In this study, we therefore set out to evaluate the applicability of transcutaneous GFR (tGFR) measurements to monitor the changes in renal function, as compared to plasma creatinine (pCreatinine), in two models of obstructive nephropathy, namely unilateral ureteral obstruction (UUO) or bilateral ureteral obstruction followed by release (BUO-R) in male Wistar rats. Results UUO animals showed a significant reduction in tGFR compared to baseline; whereas pCreatinine levels were not significantly changed. In BUO animals, tGFR drops 24 h post BUO and remains lower upon release of the obstruction until day 11. Concomitantly, pCreatinine levels were also increased 24 h after obstruction and 24 h post release, however after 4 days, pCreatinine returned to baseline levels. In conclusion, this study revealed that the tGFR method is superior at detecting minor changes in renal function as compared to pCreatinine measurements.

Abstract Objective Renal fibrosis is one of the main pathophysiological processes underlying the progression of chronic kidney disease and kidney allograft failure. In the past decades, overwhelming efforts have been undertaken to find druggable targets for the treatment of renal fibrosis, mainly using cell- and animal models. However, the latter often do not adequately reflect human pathogenesis, obtained results differ per strain within a given species, and the models are associated with considerable discomfort for the animals. Therefore, the objective of this study is to implement the 3Rs in renal fibrosis research by establishing an animal-free drug screening platform for renal fibrosis based on human precision-cut kidney slices (PCKS) and by limiting the use of reagents that are associated with significant animal welfare concerns. Results Using Western blotting and gene expression arrays, we show that transforming growth factor-β (TGF-β) induced fibrosis in human PCKS. In addition, our results demonstrated that butaprost, SC-19220 and tamoxifen – all putative anti-fibrotic compounds – altered TGF-β-induced pro-fibrotic gene expression in human PCKS. Moreover, we observed that all compounds modulated fairly distinct sets of genes, however they all impacted TGF-β/SMAD signaling. In conclusion, this study revealed that it is feasible to use an animal-free approach to test drug efficacy and elucidate mechanisms of action.

Abstract Background Chimeric antigen receptor (CAR)-based T cell therapy is in early clinical trials to target the neuroectodermal tumor, neuroblastoma. No preclinical or clinical efficacy data are available for retinoblastoma to date. Whereas unilateral intraocular retinoblastoma is cured by enucleation of the eye, infiltration of the optic nerve indicates potential diffuse scattering and tumor spread leading to a major therapeutic challenge. CAR-T cell therapy could improve the currently limited therapeutic strategies for metastasized retinoblastoma by simultaneously killing both primary tumor and metastasizing malignant cells and by reducing chemotherapy-related late effects. Methods CD171 and GD2 expression was flow cytometrically analyzed in 11 retinoblastoma cell lines. CD171 expression and T cell infiltration (CD3+) was immunohistochemically assessed in retrospectively collected primary retinoblastomas. The efficacy of CAR-T cells targeting the CD171 and GD2 tumor-associated antigens was preclinically tested against three antigen-expressing retinoblastoma cell lines. CAR-T cell activation and exhaustion were assessed by cytokine release assays and flow cytometric detection of cell surface markers, and killing ability was assessed in cytotoxic assays. CAR constructs harboring different extracellular spacer lengths (short/long) and intracellular co-stimulatory domains (CD28/4-1BB) were compared to select the most potent constructs. Results All retinoblastoma cell lines investigated expressed CD171 and GD2. CD171 was expressed in 15/30 primary retinoblastomas. Retinoblastoma cell encounter strongly activated both CD171-specific and GD2-specific CAR-T cells. Targeting either CD171 or GD2 effectively killed all retinoblastoma cell lines examined. Similar activation and killing ability for either target was achieved by all CAR constructs irrespective of the length of the extracellular spacers and the co-stimulatory domain. Cell lines differentially lost tumor antigen expression upon CAR-T cell encounter, with CD171 being completely lost by all tested cell lines and GD2 further down-regulated in cell lines expressing low GD2 levels before CAR-T cell challenge. Alternating the CAR-T cell target in sequential challenges enhanced retinoblastoma cell killing. Conclusion Both CD171 and GD2 are effective targets on human retinoblastoma cell lines, and CAR-T cell therapy is highly effective against retinoblastoma in vitro. Targeting of two different antigens by sequential CAR-T cell applications enhanced tumor cell killing and preempted tumor antigen loss in preclinical testing.

Abstract Background There are links between obesity and inflammation that may relate activation of pro-inflammatory pathways by dietary factors. Because dietary fat intake of vegetarians is thought to be more beneficial than that of omnivores, we hypothesized that obese vegetarians would have less adipose tissue inflammation and lower intramyocellular ceramide concentrations than equally obese omnivores. Methods Eight obese vegetarian (1 male) and 8 obese omnivore volunteers (1 male) completed a Food Frequency Questionnaire, underwent body composition measures, subcutaneous adipose tissue and muscle biopsies. We used immunohistochemistry to measure adipose macrophage (ATM) and senescent cells. Plasma free fatty acid (FFA), adipose FA and muscle ceramide profiles were measured using liquid chromatography/mass spectrometry. Student t tests were used for the comparison of primary outcomes; univariate regression analysis was used to test for associations between dietary patterns and ATMs (secondary analysis). Results There were no differences in age (38 ± 8 vs. 39 ± 8 years), BMI (32.2 ± 2.6 vs. 33.3 ± 1.9 kg/m2) or percent body fat (44 ± 8 vs. 45 ± 4) between the vegetarians and omnivores. Vegetarians consumed 42% (P = 0.02) less saturated fat and 50% (P = 0.04) less cholesterol than the omnivores. Plasma FFA of vegetarians had lesser proportions of palmitic acid (24 ± 3 vs. 29 ± 4%, P = 0.02) and vegetarians had fewer femoral pro-inflammatory ATMs than omnivores (3.6 ± 2.8 vs. 7.9 ± 4.4 per 100 adipocytes, respectively; P = 0.02). Omnivores had 50% greater (P = 0.01) expression of TNF mRNA in abdominal fat. We found no significant between group differences in muscle ceramide concentrations. Conclusions Although the sample size is small, these results may indicate that dietary patterns play a role in adipose tissue inflammation, as reflected by reduced number of femoral ATMs in obese vegetarians than obese omnivores.

Abstract Background The physiological characterization of microorganisms provides valuable information for bioprocess development. Chemostat cultivations are a powerful tool for this purpose, as they allow defined changes to one single parameter at a time, which is most commonly the growth rate. The subsequent establishment of a steady state then permits constant variables enabling the acquisition of reproducible data sets for comparing microbial performance under different conditions. We performed physiological characterizations of a 3-hydroxypropionic acid (3-HP) producing Saccharomyces cerevisiae strain in a miniaturized and parallelized chemostat cultivation system. The physiological conditions under investigation were various growth rates controlled by different nutrient limitations (C, N, P). Based on the cultivation parameters obtained subsequent fed-batch cultivations were designed. Results We report technical advancements of a small-scale chemostat cultivation system and its applicability for reliable strain screening under different physiological conditions, i.e. varying dilution rates and different substrate limitations (C, N, P). Exploring the performance of an engineered 3-HP producing S. cerevisiae strain under carbon-limiting conditions revealed the highest 3-HP yields per substrate and biomass of 16.6 %C-mol and 0.43 g gCDW−1, respectively, at the lowest set dilution rate of 0.04 h−1. 3-HP production was further optimized by applying N- and P-limiting conditions, which resulted in a further increase in 3-HP yields revealing values of 21.1 %C-mol and 0.50 g gCDW−1 under phosphate-limiting conditions. The corresponding parameters favoring an increased 3-HP production, i.e. dilution rate as well as C- and P-limiting conditions, were transferred from the small-scale chemostat cultivation system to 1-L bench-top fermenters operating in fed-batch conditions, revealing 3-HP yields of 15.9 %C-mol and 0.45 g gCDW−1 under C-limiting, as well as 25.6 %C-mol and 0.50 g gCDW−1 under phosphate-limiting conditions. Conclusions Small-scale chemostat cultures are well suited for the physiological characterization of microorganisms, particularly for investigating the effect of changing cultivation parameters on microbial performance. In our study, optimal conditions for 3-HP production comprised (i) a low dilution rate of 0.04 h−1 under carbon-limiting conditions and (ii) the use of phosphate-limiting conditions. Similar 3-HP yields were achieved in chemostat and fed-batch cultures under both C- and P-limiting conditions proving the growth rate as robust parameter for process transfer and thus the small-scale chemostat system as powerful tool for process optimization.

Abstract Background Brain metastasis is becoming increasingly prevalent in breast cancer due to improved extra-cranial disease control. With emerging availability of modern image-guided radiation platforms, mouse models of brain metastases and small animal magnetic resonance imaging (MRI), we examined brain metastases’ responses from radiotherapy in the pre-clinical setting. In this study, we employed half brain irradiation to reduce inter-subject variability in metastases dose-response evaluations. Methods Half brain irradiation was performed on a micro-CT/RT system in a human breast cancer (MDA-MB-231-BR) brain metastasis mouse model. Radiation induced DNA double stranded breaks in tumors and normal mouse brain tissue were quantified using γ-H2AX immunohistochemistry at 30 min (acute) and 11 days (longitudinal) after half-brain treatment for doses of 8, 16 and 24 Gy. In addition, tumor responses were assessed volumetrically with in-vivo longitudinal MRI and histologically for tumor cell density and nuclear size. Results In the acute setting, γ-H2AX staining in tumors saturated at higher doses while normal mouse brain tissue continued to increase linearly in the phosphorylation of H2AX. While γ-H2AX fluorescence intensities returned to the background level in the brain 11 days after treatment, the residual γ-H2AX phosphorylation in the radiated tumors remained elevated compared to un-irradiated contralateral tumors. With radiation, MRI-derived relative tumor growth was significantly reduced compared to the un-irradiated side. While there was no difference in MRI tumor volume growth between 16 and 24 Gy, there was a significant reduction in tumor cell density from histology with increasing dose. In the longitudinal study, nuclear size in the residual tumor cells increased significantly as the radiation dose was increased. Conclusions Radiation damages to the DNAs in the normal brain parenchyma are resolved over time, but remain unrepaired in the treated tumors. Furthermore, there is a radiation dose response in nuclear size of surviving tumor cells. Increase in nuclear size together with unrepaired DNA damage indicated that the surviving tumor cells post radiation had continued to progress in the cell cycle with DNA replication, but failed cytokinesis. Half brain irradiation provides efficient evaluation of dose-response for cancer cell lines, a pre-requisite to perform experiments to understand radio-resistance in brain metastases.

Abstract Background Several inhaled drugs are dependent on organic cation transporters to cross cell membranes. To further evaluate their potential to impact on inhaled drug disposition, the localization of MATE1, P-gp, OCTN1 and OCTN2 were investigated in human lung. Methods Transporter proteins were analysed by immunohistochemistry in lung tissue from healthy subjects and COPD patients. Transporter mRNA was analysed by qPCR in lung tissue and in bronchoalveolar lavage (BAL) cells from smokers and non-smokers. Results We demonstrate for the first time MATE1 protein expression in the lung with localization to the apical side of bronchial and bronchiolar epithelial cells. Interestingly, MATE1 was strongly expressed in alveolar macrophages as demonstrated both in lung tissue and in BAL cells, and in inflammatory cells including CD3 positive T cells. P-gp, OCTN1 and OCTN2 were also expressed in the alveolar epithelial cells and in inflammatory cells including alveolar macrophages. In BAL cells from smokers, MATE1 and P-gp mRNA expression was significantly lower compared to cells from non-smokers whereas no difference was observed between COPD patients and healthy subjects. THP-1 cells were evaluated as a model for alveolar macrophages but did not reflect the transporter expression observed in BAL cells. Conclusions We conclude that MATE1, P-gp, OCTN1 and OCTN2 are expressed in pulmonary lung epithelium, in alveolar macrophages and in other inflammatory cells. This is important to consider in the development of drugs treating pulmonary disease as the transporters may impact drug disposition in the lung and consequently affect pharmacological efficacy and toxicity.

Abstract Background In a prospective cohort study, we wanted to detect thresholds distinguishing between patients with a satisfactory and an unsatisfactory outcome after total knee replacement (TKR) based on Patient-Reported Outcome Measures (PROMs), namely the Oxford Knee Score (OKS), using patient satisfaction and patient-perceived function as global transition items. Methods Seventy-three TKR patients completed the OKS questionnaire before surgery and were invited to complete the same questionnaire again 6 (4 to 9) months after surgery. Correlations between outcome measures and anchors were calculated using Pearson’s correlation coefficient. Thresholds were established by receiver operating characteristics (ROC) analysis, using multiple anchor-based approaches. Results Patients showed a mean increase of 16.5 (SD 9.5) in OKS following TKR. Significant positive correlations were found between outcome measures and anchors. Six different thresholds were determined for outcome measures coupled with satisfaction, patient-perceived function and a combination thereof using a cut-off of 50 and 70. Conclusions This study has established a set of clinically meaningful thresholds for Oxford Knee scores that may help to detect TKR patients who might be in need of post-operative evaluation.