Comparison of human hepatoma HepaRG cells with human and rat hepatocytes in uptake transport assays in order to predict drug induced hepatotoxicity

Human hepatocytes are the gold standard for toxicological studies but they have several drawbacks, like scarce availability, high inter-individual variability, a short lifetime, which limits their applicability. The aim of our investigations was to determine, whether HepaRG cells could replace human hepatocytes in uptake experiments for toxicity studies. HepaRG is a hepatoma cell line with most hepatic functions, including a considerable expression of uptake transporters in contrast to other hepatic immortalized cell lines. We compared the effect of cholestatic drugs (bosentan, cyclosporinA, troglitazone,) and bromosulfophthalein on the uptake of taurocholate and estrone-3-sulfate in human and rat hepatocytes and HepaRG cells. The substrate uptake was significantly slower in HepaRG cells than in human hepatocytes, still, in the presence of drugs we observed a concentration dependent decrease in uptake. In all cell types, the culture time had a significant impact not only on the uptake process but on the inhibitory effect of drugs too. The most significant drug effect was measured at 4 h after seeding. Our report is among the first concerning interactions of the uptake transporters in the HepaRG, at the functional level. Results of the present study clearly show that concerning the inhibition of taurocholate uptake by cholestatic drugs, HepaRG cells are closer to human hepatocytes than rat hepatocytes. In conclusion, we demonstrated that HepaRG cells provide a suitable tool for hepatic uptake studies

Non-traumatic chest pain in patients presenting to an urban emergency Department in sub Saharan Africa: a prospective cohort study in Tanzania.

BACKGROUND:Non-traumatic chest pain (NTCP) is a common reason for emergency department (ED) attendance in high-income countries, with the primary concern focused on life threatening cardiovascular diseases. There is general lack of data on aetiologies, diagnosis and management of NTPC in Sub Sahara African (SSA) countries. We aimed to describe evaluation, diagnosis and outcomes of adult patients presenting with NTCP to an urban ED in Tanzania. METHOD:This was a prospective observational cohort study of consecutive adult (≥18 years) patients presenting with non-traumatic chest pain to the Emergency Medicine Department (EMD) of Muhimbili National Hospital (MNH) in Dar es salaam from September 2017 to April 2018. Structured case report form was used to collected demographics, clinical presentation, investigations, diagnosis, and EMD disposition and in hospital mortality. We determined frequency of NTCP among our patients, aetiologies, 24-h and 7-day in-hospital mortality, and predictors for mortality. RESULTS:We screened 29,495 adults attending EMD-MNH during the study and 389 (1.3%) presented with NTCP of these, 349 (90%) were enrolled. The median age was 45 (IQR 29-60) years and 177 (50.7%) were female. Overall, 69.1% patients received electrocardiography (ECG) in the EMD and 34.1% had a troponin test. Heart failure and pulmonary tuberculosis (PTB) were the leading hospital diagnoses (12.6% each), followed by chronic kidney disease (10%) and acute coronary syndrome (ACS) (9.6%). Total of 167 (48%) patients were admitted, and the 24-h and 7-day in-hospital mortality were 5 (3%) and 16 (9.6%) respectively. Univariate risk factors for mortality were a Glasgow Coma Scale of < 15 [RR = 3.4 (95%CI 3.2-23)], Acute Coronary Syndrome [RR = 5.7 (95% CI 1.7-11.8) and Troponin > 0.04 ng/ml [RR 2.9 (95%CI 1.2-7.3)]. Features distinguishing cardiovascular from other causes were: bradycardia [RR = 2.6 (95%CI 2.1-3.2)], heart beat awareness [RR = 2.3 (95%CI 1.7-3.2)] and history of diabetic mellitus [RR = 2.2 (95% CI 1.6-3.0)]. CONCLUSION:In this ED of SSA country, heart failure and pulmonary tuberculosis were the leading causes of NCTP, and ACS was present in 9.6%. NTCP in this setting carries high mortality, and ACS was the leading risk factor for death. ED providers in SSA must increasingly consider cardiovascular causes of NTCP

Viability of Non-Coplanar VMAT for Liver SBRT as Compared to Coplanar VMAT and Beam Orientation Optimized 4π IMRT.

PurposeThe 4π static non-coplanar radiotherapy delivery technique has demonstrated better normal tissue sparing and dose conformity than the clinically used volumetric modulated arc therapy (VMAT). It is unclear whether this is a fundamental limitation of VMAT delivery or the coplanar nature of its typical clinical plans. The dosimetry and the limits of normal tissue toxicity constrained dose escalation of coplanar VMAT, non-coplanar VMAT and 4π radiotherapy are quantified in this study.Methods and materialsClinical stereotactic body radiation therapy plans for 20 liver patients receiving 30-60 Gy using coplanar VMAT (cVMAT) were re-planned using 3-4 partial non-coplanar arcs (nVMAT) and 4π with 20 intensity-modulated non-coplanar fields. The conformity number (CN), homogeneity index (HI), 50% dose spillage volume (R50), normal liver volume receiving >15 Gy (VL>15), dose to organs at risk (OARs), and tumor control probability (TCP) were compared for all three treatment plans. The maximum tolerable dose (MTD) yielding a normal liver normal tissue control probability (NTCP) below 1%, 5%, and 10% was calculated with the Lyman-Kutcher-Burman model for each plan, as well as the resulting survival fractions at one, two, three, and four years.ResultsCompared to cVMAT, the nVMAT and 4π plans reduced VL>15 by an average of 5 cm3 and 80 cm3, respectively. 4π reduced the 50% dose spillage volume by ~23% compared to both VMAT plans, and either significantly decreased or maintained OAR doses. The 4π MTDs and survival fractions were significantly higher than both cVMAT and nVMAT (p<0.05) for all normal liver NTCP limits used in this study.ConclusionsThe 4π technique provides significantly better OAR sparing than both cVMAT and vMAT and enables more clinically relevant dose escalation for tumor local control. Therefore, despite the current accessibility of nVMAT, it is not a viable alternative to 4π for liver SBRT

Synthesis, in vitro and in vivo evaluation of 3β-[18F]fluorocholic acid for the detection of drug-induced cholestasis in mice

Introduction : Drug-induced cholestasis is a liver disorder that might be caused by interference of drugs with the hepatobiliary bile acid transporters. It is important to identify this interference early on in drug development. In this work, Positron Emission Tomography (PET)-imaging with a F-18 labeled bile acid analogue was introduced to detect disturbed hepatobiliary transport of bile acids. Methods : 3 beta-[F-18]fluorocholic acid ([F-18]FCA) was prepared by nucleophilic substitution of a mesylated precursor with [F-18]fluoride, followed by deprotection with sodium hydroxide. Transport of [F-18]FCA was assessed in vitro using CHO-NTCP, HEK-OATP1B1, HEK-OATP1B3 transfected cells and BSEP & MRP2 membrane vesicles. Investigation of [F-18]FCA metabolites was performed with primary mouse hepatocytes. Hepatobiliary transport of [F-18]FCA was evaluated in vivo in wild-type, rifampicin and bosentan pretreated FVB-mice by dynamic mu PET scanning. Results : Radiosynthesis of [F-18] FCA was achieved in a moderate radiochemical yield (8.11-1.94%; non-decay corrected; n = 10) and high radiochemical purity (>99%). FCA was transported by the basolateral bile acid uptake transporters NTCP, OATP1B1 and OATP1B3. For canalicular efflux, BSEP and MRP2 are the relevant bile acid transporters. [F-18]FCA was found to be metabolically stable. In vivo, [F-18]FCA showed fast hepatic uptake (4.5-0.5 min to reach 71.8-1.2% maximum % ID) and subsequent efflux to the gallbladder and intestines (93.3-6.0% ID after 1 hour). Hepatobiliary transport of [F-18]FCA was significantly inhibited by both rifampicin and bosentan. Conclusion : A F-18 labeled bile acid analogue, [F-18]FCA, has been developed that shows transport by NTCP, OATP, MRP2 and BSEP. [F-18]FCA can be used as a probe to monitor disturbed hepatobiliary transport in vivo and accumulation of bile acids in blood and liver during drug development

Influence of respiratory motion management technique on radiation pneumonitis risk with robotic stereotactic body radiation therapy.

Purpose/objectivesFor lung stereotactic body radiation therapy (SBRT), real-time tumor tracking (RTT) allows for less radiation to normal lung compared to the internal target volume (ITV) method of respiratory motion management. To quantify the advantage of RTT, we examined the difference in radiation pneumonitis risk between these two techniques using a normal tissue complication probability (NTCP) model.Materials/method20 lung SBRT treatment plans using RTT were replanned with the ITV method using respiratory motion information from a 4D-CT image acquired at the original simulation. Risk of symptomatic radiation pneumonitis was calculated for both plans using a previously derived NTCP model. Features available before treatment planning that identified significant increase in NTCP with ITV versus RTT plans were identified.ResultsPrescription dose to the planning target volume (PTV) ranged from 22 to 60 Gy in 1-5 fractions. The median tumor diameter was 3.5 cm (range 2.1-5.5 cm) with a median volume of 14.5 mL (range 3.6-59.9 mL). The median increase in PTV volume from RTT to ITV plans was 17.1 mL (range 3.5-72.4 mL), and the median increase in PTV/lung volume ratio was 0.46% (range 0.13-1.98%). Mean lung dose and percentage dose-volumes were significantly higher in ITV plans at all levels tested. The median NTCP was 5.1% for RTT plans and 8.9% for ITV plans, with a median difference of 1.9% (range 0.4-25.5%, pairwise P < 0.001). Increases in NTCP between plans were best predicted by increases in PTV volume and PTV/lung volume ratio.ConclusionsThe use of RTT decreased the risk of radiation pneumonitis in all plans. However, for most patients the risk reduction was minimal. Differences in plan PTV volume and PTV/lung volume ratio may identify patients who would benefit from RTT technique before completing treatment planning

Cochlea-sparing acoustic neuroma treatment with 4π radiation therapy.

PurposeThis study investigates whether 4π noncoplanar radiation therapy can spare the cochleae and consequently potentially improve hearing preservation in patients with acoustic neuroma who are treated with radiation therapy.Methods and materialsClinical radiation therapy plans for 30 patients with acoustic neuroma were included (14 stereotactic radiation surgery [SRS], 6 stereotactic radiation therapy [SRT], and 10 intensity modulated radiation therapy [IMRT]). The 4π plans were created for each patient with 20 optimal beams selected using a greedy column generation method and subsequently recalculated in Eclipse for comparison. Organ-at-risk (OAR) doses, homogeneity index, conformity, and tumor control probability (TCP) were compared. Normal tissue complication probability (NTCP) was calculated for sensorineural hearing loss (SNHL) at 3 and 5 years posttreatment. The dose for each plan was then escalated to achieve 99.5% TCP.Results4π significantly reduced the mean dose to both cochleae by 2.0 Gy (32%) for SRS, 3.2 Gy (29%) for SRT, and 10.0 Gy (32%) for IMRT. The maximum dose to both cochleae was also reduced with 4π by 1.6 Gy (20%), 2.2 Gy (15%), and 7.1 Gy (18%) for SRS, SRT, and IMRT plans, respectively. The reductions in mean/maximum brainstem dose with 4π were also statistically significant. Mean doses to other OARs were reduced by 19% to 56% on average. 4π plans had a similar CN and TCP, with a significantly higher average homogeneity index (0.93 vs 0.92) and significantly lower average NTCP for SNHL at both 3 years (30.8% vs 40.8%) and 5 years (43.3% vs 61.7%). An average dose escalation of approximately 116% of the prescription dose achieved 99.5% TCP, which resulted in 32.6% and 43.4% NTCP for SNHL at 3 years and 46.4% and 64.7% at 5 years for 4π and clinical plans, respectively.ConclusionsCompared with clinical planning methods, optimized 4π radiation therapy enables statistically significant sparing of the cochleae in acoustic neuroma treatment as well as lowering of other OAR doses, potentially reducing the risk of hearing loss

Modelling the impact of treatment uncertainties in radiotherapy

Uncertainties are inevitably part of the radiotherapy process. Uncertainty in the dose deposited in the tumour exists due to organ motion, patient positioning errors, fluctuations in machine output, delineation of regions of interest, the modality of imaging used, and treatment planning algorithm assumptions among others; there is uncertainty in the dose required to eradicate a tumour due to interpatient variations in patient-specific variables such as their sensitivity to radiation; and there is uncertainty in the dose-volume restraints that limit dose to normal tissue. This thesis involves three major streams of research including investigation of the actual dose delivered to target and normal tissue, the effect of dose uncertainty on radiobiological indices, and techniques to display the dose uncertainty in a treatment planning system. All of the analyses are performed with the dose distribution from a four-field box treatment using 6 MV photons. The treatment fields include uniform margins between the clinical target volume and planning target volume of 0.5 cm, 1.0 cm, and 1.5 cm. The major work is preceded by a thorough literature review on the size of setup and organ motion errors for various organs and setup techniques used in radiotherapy. A Monte Carlo (MC) code was written to simulate both the treatment planning and delivery phases of the radiotherapy treatment. Using MC, the mean and the variation in treatment dose are calculated for both an individual patient and across a population of patients. In particular, the possible discrepancy in tumour position located from a single CT scan and the magnitude of reduction in dose variation following multiple CT scans is investigated. A novel convolution kernel to include multiple pretreatment CT scans in the calculation of mean treatment dose is derived. Variations in dose deposited to prostate and rectal wall are assessed for each of the margins and for various magnitudes of systematic and random error, and penumbra gradients. The linear quadratic model is used to calculate prostate Tumour Control Probability (TCP) incorporating an actual (modelled) delivered prostate dose. The Kallman s-model is used to calculate the normal tissue complication probability (NTCP), incorporating actual (modelled) fraction dose in the deforming rectal wall. The impact of each treatment uncertainty on the variation in the radiobiological index is calculated for the margin sizes.Thesis (Ph.D.)--Department of Physics and Mathematical Physics, 2002

Blocking entry of hepatitis B and D viruses to hepatocytes as a novel immunotherapy for treating chronic infections

Background. Chronic hepatitis B and D virus (HBV/HDV) infections can cause cancer. Current HBV therapy using nucleoside analogues (NAs) is life-long and reduces but does not eliminate the risk of cancer. A hallmark of chronic hepatitis B is a dysfunctional HBV-specific T-cell response. We therefore designed an immunotherapy driven by naive healthy T cells specific for the HDV antigen (HDAg) to bypass the need for HBV-specific T cells in order to prime PreS1-specific T cells and PreS1 antibodies blocking HBV entry. Methods. Ten combinations of PreS1 and/or HDAg sequences were evaluated for induction of PreS1 antibodies and HBV- and HDV-specific T cells in vitro and in vivo. Neutralization of HBV by PreS1-specific murine and rabbit antibodies was evaluated in cell culture, and rabbit anti-PreS1 were tested for neutralization of HBV in mice repopulated with human hepatocytes. Results. The best vaccine candidate induced T cells to PreS1 and HDAg, and PreS1 antibodies blocking HBV entry in vitro. Importantly, adoptive transfer of PreS1 antibodies prevented, or modulated, HBV infection after a subsequent challenge in humanized mice. Conclusions. We here describe a novel immunotherapy for chronic HBV/HDV that targets viral entry to complement NAs and coming therapies inhibiting viral maturation