Investigating the role of ultrasound-based shear wave elastography in kidney transplanted patients: correlation between non-invasive fibrosis detection, kidney dysfunction and biopsy results-a systematic review and meta-analysis.

INTRODUCTION: Interstitial fibrosis and tubular atrophy are leading causes of renal allograft failure. Shear wave elastography could be a promising noninvasive method for providing information on the state of the kidney, with specific regard to fibrosis but currently available data in the literature are controversial. Our study aimed to analyze the correlation between shear wave elastography and various kidney dysfunction measures. METHODS: This review was registered on PROSPERO (CRD42021283152). We systematically searched three major databases (MEDLINE, Embase, and CENTRAL) for articles concerning renal transplant recipients, shear wave elastography, fibrosis, and kidney dysfunction. Meta-analytical calculations for pooled Pearson and Spearman correlation coefficients (r) were interpreted with 95% confidence intervals (CIs). Heterogeneity was tested with Cochran's Q test. I2 statistic and 95% CI were reported as a measurement of between-study heterogeneity. Study quality was assessed with the QUADAS2 tool. RESULTS: In total, 16 studies were included in our meta-analysis. Results showed a moderate correlation between kidney stiffness and interstitial fibrosis and tubular atrophy, graded according to BANFF classification, on biopsy findings for pooled Pearson (r = 0.48; CI: 0.20, 0.69; I2 = 84%) and Spearman correlations (r = 0.57; CI: 0.35, 0.72; I2 = 74%). When compared to kidney dysfunction parameters, we found a moderate correlation between shear wave elastography and resistive index (r = 0.34 CI: 0.13, 0.51; I2 = 67%) and between shear wave elastography and estimated Glomerular Filtration Rate (eGFR) (r = -0.65; CI: - 0.81, - 0.40; I2 = 73%). All our outcomes had marked heterogeneity. CONCLUSION: Our results showed a moderate correlation between kidney stiffness measured by shear wave elastography and biopsy results. While noninvasive assessment of kidney fibrosis after transplantation is an important clinical goal, there is insufficient evidence to support the use of elastography over the performance of a kidney biopsy.

Intracellular Metabolomics Identifies Efflux Transporter Inhibitors in a Routine Caco-2 Cell Permeability Assay-Biological Implications.

Caco-2 screens are routinely used in laboratories to measure the permeability of compounds and can identify substrates of efflux transporters. In this study, we hypothesized that efflux transporter inhibition of a compound can be predicted by an intracellular metabolic signature in Caco-2 cells in the assay used to test intestinal permeability. Using selective inhibitors and transporter knock-out (KO) cells and a targeted Liquid Chromatography tandem Mass Spectrometry (LC-MS) method, we identified 11 metabolites increased in cells with depleted P-glycoprotein (Pgp) activity. Four metabolites were altered with Breast Cancer Resistance (BCRP) inhibition and nine metabolites were identified in the Multidrug Drug Resistance Protein 2 (MRP2) signature. A scoring system was created that could discriminate among the three transporters and validated with additional inhibitors. Pgp and MRP2 substrates did not score as inhibitors. In contrast, BCRP substrates and inhibitors showed a similar intracellular metabolomic signature. Network analysis of signature metabolites led us to investigate changes of enzymes in one-carbon metabolism (folate and methionine cycles). Our data shows that methylenetetrahydrofolate reductase (MTHFR) protein levels increased with Pgp inhibition and Thymidylate synthase (TS) protein levels were reduced with Pgp and MRP2 inhibition. In addition, the methionine cycle is also affected by both Pgp and MRP2 inhibition. In summary, we demonstrated that the routine Caco-2 assay has the potential to identify efflux transporter inhibitors in parallel with substrates in the assays currently used in many DMPK laboratories and that inhibition of efflux transporters has biological consequences.

Presence of human breast cancer xenograft changes the diurnal profile of amino acids in mice.

Human xenografts are extremely useful models to study the biology of human cancers and the effects of novel potential therapies. Deregulation of metabolism, including changes in amino acids (AAs), is a common characteristic of many human neoplasms. Plasma AAs undergo daily variations, driven by circadian endogenous and exogenous factors. We compared AAs concentration in triple negative breast cancer MDA-MB-231 cells and MCF10A non-tumorigenic immortalized breast epithelial cells. We also measured plasma AAs in mice bearing xenograft MDA-MB-231 and compared their levels with non-tumor-bearing control animals over 24 h. In vitro studies revealed that most of AAs were significantly different in MDA-MB-231 cells when compared with MCF10A. Plasma concentrations of 15 AAs were higher in cancer cells, two were lower and four were observed to shift across 24 h. In the in vivo setting, analysis showed that 12 out of 20 AAs varied significantly between tumor-bearing and non-tumor bearing mice. Noticeably, these metabolites peaked in the dark phase in non-tumor bearing mice, which corresponds to the active time of these animals. Conversely, in tumor-bearing mice, the peak time occurred during the light phase. In the early period of the light phase, these AAs were significantly higher in tumor-bearing animals, yet significantly lower in the middle of the light phase when compared with controls. This pilot study highlights the importance of well controlled experiments in studies involving plasma AAs in human breast cancer xenografts, in addition to emphasizing the need for more precise examination of exometabolomic changes using multiple time points.

Repurposing Vandetanib plus Everolimus for the Treatment of ACVR1-Mutant Diffuse Intrinsic Pontine Glioma.

Somatic mutations in ACVR1 are found in a quarter of children with diffuse intrinsic pontine glioma (DIPG), but there are no ACVR1 inhibitors licensed for the disease. Using an artificial intelligence-based platform to search for approved compounds for ACVR1-mutant DIPG, the combination of vandetanib and everolimus was identified as a possible therapeutic approach. Vandetanib, an inhibitor of VEGFR/RET/EGFR, was found to target ACVR1 (K d = 150 nmol/L) and reduce DIPG cell viability in vitro but has limited ability to cross the blood-brain barrier. In addition to mTOR, everolimus inhibited ABCG2 (BCRP) and ABCB1 (P-gp) transporters and was synergistic in DIPG cells when combined with vandetanib in vitro. This combination was well tolerated in vivo and significantly extended survival and reduced tumor burden in an orthotopic ACVR1-mutant patient-derived DIPG xenograft model. Four patients with ACVR1-mutant DIPG were treated with vandetanib plus an mTOR inhibitor, informing the dosing and toxicity profile of this combination for future clinical studies. SIGNIFICANCE: Twenty-five percent of patients with the incurable brainstem tumor DIPG harbor somatic activating mutations in ACVR1, but there are no approved drugs targeting the receptor. Using artificial intelligence, we identify and validate, both experimentally and clinically, the novel combination of vandetanib and everolimus in these children based on both signaling and pharmacokinetic synergies.This article is highlighted in the In This Issue feature, p. 275.

Treatment of benign thyroid nodules by radiofrequency thermal ablation / Jóindulatú pajzsmirigygöbök rádiófrekvenciás hoablatiós kezelése.

INTRODUCTION: Benign thyroid nodules are frequent findings in imaging studies, most of the time not requiring any intervention. Treatment is usually started when nodules increase in size, the patient becomes symptomatic or clinically relevant hyperthyroidism develops. Thermoablation is an effective alternative modality. In Hungary, our team has pioneered these interventions using radiofrequency ablation for decreasing the size of the nodules. AIM: We are presenting our results showing the effectiveness of this treatment after introducing the role, importance and technique of thermoablation in benign thyroid nodules. METHOD: Between June of 2016 and September of 2019, 186 nodules of 140 patients were treated with radiofrequency ablation and had at least 6 months of follow up. The volume and diameter of all the ablated nodules were measured and calculated, then the decreases of these parameters were followed using ultrasonography. The mean follow-up time was 12.5 ± 5.9 months. RESULTS: The size measurements at the follow-up ultrasonography examinations showed a decrease in size and vascularity. The mean volume reduction was 44.7 ± 17.6% at one-month post-treatment and 72.9 ± 17.9% at 6 months. There were 3 minor complications. CONCLUSIONS: Radiofrequency ablation represents a feasible, effective, well tolerated method for outpatient treatment of benign thyroid nodules. This method is a valuable alternative to surgical treatments in selected cases. Orv Hetil. 2020; 161(27): 1131-1136.

Metabolomic changes of the multi (-AGC-) kinase inhibitor AT13148 in cells, mice and patients are associated with NOS regulation.

INTRODUCTION: To generate biomarkers of target engagement or predictive response for multi-target drugs is challenging. One such compound is the multi-AGC kinase inhibitor AT13148. Metabolic signatures of selective signal transduction inhibitors identified in preclinical models have previously been confirmed in early clinical studies. This study explores whether metabolic signatures could be used as biomarkers for the multi-AGC kinase inhibitor AT13148. OBJECTIVES: To identify metabolomic changes of biomarkers of multi-AGC kinase inhibitor AT13148 in cells, xenograft / mouse models and in patients in a Phase I clinical study. METHODS: HILIC LC-MS/MS methods and Biocrates AbsoluteIDQ™ p180 kit were used for targeted metabolomics; followed by multivariate data analysis in SIMCA and statistical analysis in Graphpad. Metaboanalyst and String were used for network analysis. RESULTS: BT474 and PC3 cells treated with AT13148 affected metabolites which are in a gene protein metabolite network associated with Nitric oxide synthases (NOS). In mice bearing the human tumour xenografts BT474 and PC3, AT13148 treatment did not produce a common robust tumour specific metabolite change. However, AT13148 treatment of non-tumour bearing mice revealed 45 metabolites that were different from non-treated mice. These changes were also observed in patients at doses where biomarker modulation was observed. Further network analysis of these metabolites indicated enrichment for genes associated with the NOS pathway. The impact of AT13148 on the metabolite changes and the involvement of NOS-AT13148- Asymmetric dimethylarginine (ADMA) interaction were consistent with hypotension observed in patients in higher dose cohorts (160-300 mg). CONCLUSION: AT13148 affects metabolites associated with NOS in cells, mice and patients which is consistent with the clinical dose-limiting hypotension.

International Ring Trial of a High Resolution Targeted Metabolomics and Lipidomics Platform for Serum and Plasma Analysis.

A challenge facing metabolomics in the analysis of large human cohorts is the cross-laboratory comparability of quantitative metabolomics measurements. In this study, 14 laboratories analyzed various blood specimens using a common experimental protocol provided with the Biocrates AbsoluteIDQ p400HR kit, to quantify up to 408 metabolites. The specimens included human plasma and serum from male and female donors, mouse and rat plasma, as well as NIST SRM 1950 reference plasma. The metabolite classes covered range from polar (e.g., amino acids and biogenic amines) to nonpolar (e.g., diacyl- and triacyl-glycerols), and they span 11 common metabolite classes. The manuscript describes a strict system suitability testing (SST) criteria used to evaluate each laboratory's readiness to perform the assay, and provides the SST Skyline documents for public dissemination. The study found approximately 250 metabolites were routinely quantified in the sample types tested, using Orbitrap instruments. Interlaboratory variance for the NIST SRM-1950 has a median of 10% for amino acids, 24% for biogenic amines, 38% for acylcarnitines, 25% for glycerolipids, 23% for glycerophospholipids, 16% for cholesteryl esters, 15% for sphingolipids, and 9% for hexoses. Comparing to consensus values for NIST SRM-1950, nearly 80% of comparable analytes demonstrated bias of <50% from the reference value. The findings of this study result in recommendations of best practices for system suitability, quality control, and calibration. We demonstrate that with appropriate controls, high-resolution metabolomics can provide accurate results with good precision across laboratories, and the p400HR therefore is a reliable approach for generating consistent and comparable metabolomics data.

[Role of interventional radiology in the transplantation of abdominal solid organs]. / Az intervenciós radiológia szerepe a hasi szervek átültetésében.

The transplantation of the abdominal organs has a major role in the treatment of several diseases. All subspecialities affected with the transplantation showed a rapid development in the last decades. The cooperation of the specialists of different segments of medicine provides the success of organ transplantation. Teamwork is necessary throughout the whole process starting from securing the technical background and proper human workforce, followed by the lifelong management of organs and recipients as well. One of the key players of organ transplantation is radiology and interventional radiology - the role of the latter one is discussed in this review, including the minimally invasive treatment of pre- and post-transplantation situations and diseases. Besides vascular and non-vascular interventions, the options of interventional oncology will be mentioned based on international literature and Hungarian experience. Orv Hetil. 2018; 159(46): 1940-1947.

Modulation of Plasma Metabolite Biomarkers of the MAPK Pathway with MEK Inhibitor RO4987655: Pharmacodynamic and Predictive Potential in Metastatic Melanoma.

MAPK pathway activation is frequently observed in human malignancies, including melanoma, and is associated with sensitivity to MEK inhibition and changes in cellular metabolism. Using quantitative mass spectrometry-based metabolomics, we identified in preclinical models 21 plasma metabolites including amino acids, propionylcarnitine, phosphatidylcholines, and sphingomyelins that were significantly altered in two B-RAF-mutant melanoma xenografts and that were reversed following a single dose of the potent and selective MEK inhibitor RO4987655. Treatment of non-tumor-bearing animals and mice bearing the PTEN-null U87MG human glioblastoma xenograft elicited plasma changes only in amino acids and propionylcarnitine. In patients with advanced melanoma treated with RO4987655, on-treatment changes of amino acids were observed in patients with disease progression and not in responders. In contrast, changes in phosphatidylcholines and sphingomyelins were observed in responders. Furthermore, pretreatment levels of seven lipids identified in the preclinical screen were statistically significantly able to predict objective responses to RO4987655. The RO4987655 treatment-related changes were greater than baseline physiological variability in nontreated individuals. This study provides evidence of a translational exo-metabolomic plasma readout predictive of clinical efficacy together with pharmacodynamic utility following treatment with a signal transduction inhibitor. Mol Cancer Ther; 16(10); 2315-23. ©2017 AACR.

[Thermoablation therapy in the treatment of benign lesions]. / Thermoablatiós lehetoségek a benignus térfoglaló képletek gyógyításában.

INTRODUCTION: Depending on their size and location, some benign tumors can cause prolonged discomfort and even rupture and fatal bleeding in severe cases. Hitherto the therapeutic strategies for such lesions were observation, surgery and in selected cases transarterial embolization. AIM: Our aim was to present the possibilities of thermoablation for treating lesions. METHOD: Here we present interventions of four patients in Semmelweis University Department of Transplantation and Surgery. A thyroid adenoma and a kidney angiomyolipoma were treated with radiofrequency ablation. Two patients with a liver haemangioma were treated with microwave thermoablation technique. RESULTS: Complications were not observed in any of the cases. In most cases, the size of the treated lesions decreased. The mean decrease in volume was 32.7%. The contrast enhancement of the lesions also decreased, the mean reduction in contrast enhancing volume was 75.3%. CONCLUSIONS: Thermoablational procedures for the benign tumors presented above are safe. The therapy shows excellent cosmetic results, a shorter hospital stay and quicker recovery. Orv. Hetil., 2016, 157(51), 2040-2047.

8-Substituted Pyrido[3,4-d]pyrimidin-4(3H)-one Derivatives As Potent, Cell Permeable, KDM4 (JMJD2) and KDM5 (JARID1) Histone Lysine Demethylase Inhibitors.

We report the discovery of N-substituted 4-(pyridin-2-yl)thiazole-2-amine derivatives and their subsequent optimization, guided by structure-based design, to give 8-(1H-pyrazol-3-yl)pyrido[3,4-d]pyrimidin-4(3H)-ones, a series of potent JmjC histone N-methyl lysine demethylase (KDM) inhibitors which bind to Fe(II) in the active site. Substitution from C4 of the pyrazole moiety allows access to the histone peptide substrate binding site; incorporation of a conformationally constrained 4-phenylpiperidine linker gives derivatives such as 54j and 54k which demonstrate equipotent activity versus the KDM4 (JMJD2) and KDM5 (JARID1) subfamily demethylases, selectivity over representative exemplars of the KDM2, KDM3, and KDM6 subfamilies, cellular permeability in the Caco-2 assay, and, for 54k, inhibition of H3K9Me3 and H3K4Me3 demethylation in a cell-based assay.

A P-gp vesicular transport inhibition assay - optimization and validation for drug-drug interaction testing.

Accurate determination of potential drug-drug interaction mediated by efflux transporters (tDDI) is crucial to assess the risk of pharmacokinetic interaction and toxicity of drugs. Passive permeability and uptake transporter mediated transport are important covariates of cell-based inhibition assays that need to be taken into consideration when performing kinetic analysis of data. Vesicular uptake inhibition has been considered by regulatory agencies as a viable alternative for testing tDDI potential of low passive permeability drugs in particular. Membranes prepared from a P-gp overexpressing human cell line has superior transport properties over membranes prepared from Sf9 cells and cholesterol enriched Sf9 membranes. P-gp expressed in this membrane effluxes N-methyl-quinidine (NMQ) with high affinity (K(m) is 3.65 µM) and a high rate (V(max) is 656 pmol/mg protein/min). Digoxin, vinblastine and paclitaxel, established P-gp substrates inhibited transport of NMQ with estimated K(i) values of 250, 0.1 and 0.6 µM, respectively. A panel of 11 drugs that have been listed by regulatory agencies as reference inhibitors were used to validate the assay to predict clinical inhibition potential. All the drugs that have been implicated in P-gp mediated DDI were found to be inhibitors in a relevant concentration range.

Detection and management of renal cell carcinoma in the renal allograft.

PURPOSE: Tumours of the transplanted kidney represent a rare form of post-transplantation malignancies. An important aspect of the treatment option is whether the transplanted kidney can be saved or not. Aim of our study was the analysis of our allograft tumours. METHODS: In the Budapest Centre, 3,530 kidney transplantations were performed between 1973 and 2012. Retrospective analysis of 9 patients who developed renal cell carcinoma (RCC) in the transplanted kidney was done. RESULTS: Mean age of recipients was 45.3 ± 13.4 years at the time of transplantation and 57.0 ± 11.6 years at the time of tumour detection. Mean age of their donors was 43.5 ± 11.5 years. Mean time from transplantation to tumour diagnosis was 134.6 ± 40.8 months. Seven RCC were stage pT1a, 1 was stage pT1b and 1 was pT3a. Eight patients had stage I. (pT1a-b, N0, M0) and 1 patient had stage IV. (pT3a, N1, M1) disease. Histological types were clear cell (n = 6), papillary (n = 2) and sarcomatoid (n = 1) carcinomas. The tumour growth rate of RCC was 16.7 ± 13.5 mm/year. In 4 cases, transplant nephrectomy was performed; 5 cases had percutaneous radiofrequency ablation (RFA). Ablative therapy had no influence on renal graft function. Six patients (including 5 patients who were treated with RFA) are still alive and tumour-free; 3 patients died. CONCLUSIONS: According to our observation, we can state that RCC of the kidney allograft diagnosed at an early stage can be successfully treated with RFA instead of graft removal. A longer follow-up is needed to assess the effectivity of the RFA treatment in these cases.

Fenretinide derivatives act as disrupters of interactions of serum retinol binding protein (sRBP) with transthyretin and the sRBP receptor.

Serum retinol binding protein (sRBP) is released from the liver as a complex with transthyretin (TTR), a process under the control of dietary retinol. Elevated levels of sRBP may be involved in inhibiting cellular responses to insulin and in generating first insulin resistance and then type 2 diabetes, offering a new target for therapeutic attack for these conditions. A series of retinoid analogues were synthesized and examined for their binding to sRBP and their ability to disrupt the sRBP-TTR and sRBP-sRBP receptor interactions. A number inhibit the sRBP-TTR and sRBP-sRBP receptor interactions as well as or better than Fenretinide (FEN), presenting a potential novel dual mechanism of action and perhaps offering a new therapeutic intervention against type 2 diabetes and its development. Shortening the chain length of the FEN derivative substantially abolished binding to sRBP, indicating that the strength of the interaction lies in the polyene chain region. Differences in potency against the sRBP-TTR and sRBP-sRBP receptor interactions suggest variant effects of the compounds on the two loops of sRBP guarding the entrance of the binding pocket that are responsible for these two protein-protein interactions.

Comparison of 3 assay systems using a common probe substrate, calcein AM, for studying P-gp using a selected set of compounds.

The multidrug resistance protein 1 (MDR1) transporter is the most abundantly investigated adenosine triphosphate (ATP)-Binding Cassette (ABC) transporter protein. Multiple assay systems were developed to study MDR1-mediated transport and possible drug-drug interactions. Yet, as different probe substrates are used in these assays, it is difficult to directly compare the results. In this study, a common probe substrate was applied in 3 assay systems developed to study MDR1: the cellular dye efflux assay, the ATPase assay, and the vesicular transport assay. This probe substrate is calcein acetoxymethyl ester (calcein AM), the acetoxymethyl ester derivative of the fluorescent dye, calcein. Using a common probe allows the investigation of the effect of passive permeability on the result obtained by testing various compounds. In this study, 22 compounds with different logP values were tested in the above-mentioned 3 assay systems. The vesicular transport assay proved most sensitive, detecting 18 of 22 interactions with the protein. The ATPase assay detected 15 interactions, whereas the cellular dye efflux assay was the least sensitive with only 10 hits. A correlation was found between the hydrophobicity of the compound and the ratio of cellular and vesicular transport IC(50) values, indicating the effect of passive permeability on the result. Based on hydrophobicity, the current study provides guidelines on applying the most correct tool for studying MDR1 interactions.

Expression and characterization of recombinant human retinol-binding protein in Pichia pastoris.

Plasma retinol-binding protein (RBP4) is the principal carrier of vitamin A in blood. Recent studies have suggested that RBP4 may have also a role in insulin resistance. To date the recombinant protein is usually produced by refolding inclusion bodies in Escherichia coli. Here we report the expression and characterization of recombinant human plasma RBP4 using the Pichia pastoris expression system. Simple and rapid purification allowed us to obtain 5mg/L of purified protein from the fermentation supernatant with no need to perform denaturing and refolding steps. The identity of the protein was verified by ion-trap MS and Western blotting. The functionality of recombinant RBP4, i.e., the binding to its physiologic ligand, retinol, and the interaction with transthyretin (TTR), was tested by fluorimetric and pull-down assays, respectively. The apparent dissociation constant for retinol to the recombinant protein of 2 x 10(-7)M was consistent with published data for native human protein. The recombinant protein interacted specifically with TTR. These results suggest that expression of recombinant human RBP4 in P. pastoris provides an efficient source of fully functional protein in soluble form for biochemical and biophysical studies.

ABCG2 (breast cancer resistance protein/mitoxantrone resistance-associated protein) ATPase assay: a useful tool to detect drug-transporter interactions.

The ATPase assay using membrane preparations from recombinant baculovirus-infected Spodoptera frugiperda ovarian (Sf9) cells is widely used to detect the interaction of compounds with different ATP-binding cassette transporters. However, Sf9 membrane preparations containing the wild-type ABCG2 transporter show an elevated baseline vanadate-sensitive ATPase activity, which cannot be further stimulated by substrates of ABCG2. Therefore, this assay system cannot be used for the detection of ABCG2 substrates. To overcome this difficulty we 1) purified membranes from a selected human cell line expressing wild-type ABCG2, and 2) inhibited the baseline ATPase activity with different inhibitors. In our modified assay, ABCG2 substrates were able to stimulate the baseline ATPase activity of ABCG2 expressed in membranes of human cells. Furthermore, using the specific ABCG2 inhibitors Ko143 or Ko134 allowed us to suppress the baseline vanadate-sensitive ATPase activity. Substrates of ABCG2 could stimulate this suppressed baseline ATPase, resulting in a better signal-to-background ratio and a robust assay to detect substrates of the ABCG2 transporter. The ATPase assay and the direct vesicular transport measurements for estrone-3-sulfate were in good accordance.

[In vitro methods suitable for the prediction of drug and ABC transporter, especially ABCG2 interactions]. / ABC transzporterek-különös tekintettel az ABCG2-re--és gyógyszerjelölt molekulák kölcsönhatásának predikciójára alkalmas in vitro módszerek.

ABCG2, a transporter of the ATP-binding cassette family, is known to play a prominent role in the absorption, distribution, metabolism, and excretion of xenobiotics. Drug-transporter interactions are commonly screened by in vitro systems using transfected insect and/or human cell lines.