Survey of ex vivo drug combination effects in chronic lymphocytic leukemia reveals synergistic drug effects and genetic dependencies.

Drug combinations that target critical pathways are a mainstay of cancer care. To improve current approaches to combination treatment of chronic lymphocytic leukemia (CLL) and gain insights into the underlying biology, we studied the effect of 352 drug combination pairs in multiple concentrations by analysing ex vivo drug response of 52 primary CLL samples, which were characterized by "omics" profiling. Known synergistic interactions were confirmed for B-cell receptor (BCR) inhibitors with Bcl-2 inhibitors and with chemotherapeutic drugs, suggesting that this approach can identify clinically useful combinations. Moreover, we uncovered synergistic interactions between BCR inhibitors and afatinib, which we attribute to BCR activation by afatinib through BLK upstream of BTK and PI3K. Combinations of multiple inhibitors of BCR components (e.g., BTK, PI3K, SYK) had effects similar to the single agents. While PI3K and BTK inhibitors produced overall similar effects in combinations with other drugs, we uncovered a larger response heterogeneity of combinations including PI3K inhibitors, predominantly in CLL with mutated IGHV, which we attribute to the target's position within the BCR-signaling pathway. Taken together, our study shows that drug combination effects can be effectively queried in primary cancer cells, which could aid discovery, triage and clinical development of drug combinations.

Vitamin D Deficiency Is Highly Prevalent in Critically Ill Patients and a Risk Factor for Mortality: A Prospective Observational Study Comparing Noncirrhotic Patients and Patients With Cirrhosis.

INTRODUCTION: A 25-hydroxyvitamin D, 25(OH)D, deficiency is common among critically ill patients and correlated with increased mortality. Furthermore, deficiency is associated with advanced liver disease. However, there are no studies available comparing the dimensions and consequences of a 25(OH)D deficiency between patients with and without liver cirrhosis in the setting of intensive care units (ICUs). This study focuses on differences in 25(OH)D status between critically ill noncirrhosis patients and patients with cirrhosis (primary end point), hypothesizing that deficiency and its impact on mortality risk are even more pronounced in patients with cirrhosis. METHODS: We performed a prospective observational study of 176 patients (noncirrhosis patients, N = 114; patients with cirrhosis, N = 62) with a laboratory assessment of 25(OH)D on ICU admission and survival analyses after 180 days. RESULTS: On admission, 55% of patients showed a severe deficiency, 25(OH)D <10 ng/mL, and a further 23% moderate deficiency (10-19 ng/mL). The overall median level of 25(OH)D was 8.0 (5.0-18.0) ng/mL (10.5 [6.0-21.3] in noncirrhosis patients vs 7.0 [4.8-10.0] in patients with cirrhosis; P < .001). We found extremely low levels particularly in patients without prior vitamin D supplementation (6.0 [4.0-7.5] in patients with cirrhosis vs 8.0 [5.0-12.0] ng/mL in noncirrhosis patients; P = .004). Vitamin D status correlated inversely with the sequential organ failure assessment, acute and physiology chronic health evaluation, model of end-stage liver disease, and Child-Pugh scores. Survival analyses categorized 25(OH)D levels <10 ng/mL as a high-risk factor for mortality 180 days after admission (hazard ratio [HR]: 2.45, 95% confidence interval [CI] = 1.60-3.70; P < .001). In patients with cirrhosis, a severe deficiency (<10 ng/mL) involved a significantly higher mortality risk than in noncirrhosis patients (HR: 2.30, 95% CI = 1.39-3.82; P = .001). In cases of admission levels ≥10 ng/mL, however, mortality risk was similar between patients with cirrhosis and noncirrhosis patients (HR: 1.08, 95% CI = 0.43-2.73; P = .873). CONCLUSIONS: Hypovitaminosis D is a highly frequent disorder in critically ill patients admitted to ICU. A severe deficiency with levels <10 ng/mL is a high risk factor for increased mortality, especially in patients with cirrhosis.

Influence of Sustained Low-Efficiency Dialysis Treatment on Isavuconazole Plasma Levels in Critically Ill Patients.

Isavuconazole plasma concentrations were measured before and after sustained low-efficiency dialysis (SLED) treatment in 22 critically ill adult patients with probable invasive aspergillosis and underlying hematological malignancies. Isavuconazole levels were significantly lower after SLED treatment (5.73 versus 3.36 µg/ml; P < 0.001). However, even after SLED treatment, isavuconazole concentrations exceeded the in vivo MICs for several relevant Aspergillus species.

Changes in pathogen spectrum and antimicrobial resistance development in the time-course of acute necrotizing pancreatitis.

BACKGROUND AND AIM: In contrast to the first peak of multi-organ failure in acute pancreatitis, the second peak is mostly triggered by septic complications. Our aim was to analyze the spectrum of pathogens and antimicrobial resistance development in relation to the time-course of the disease and its clinical outcome. METHODS: One hundred twenty-two patients with acute necrotizing pancreatitis undergoing pancreas puncture at two tertiary academic medical centers in Germany were retrospectively analyzed. RESULTS: At species level, there was a change in spectrum from Enterococcus faecalis (∆d150 - d1 = 14.6% - 16.7% = -2.1%) to Enterococcus faecium (∆d150 - d1 = 93.1% - 16.3% = 76.8%) (P < 0.001) and from Candida albicans (∆d150 - d1 = 39.7% - 23.6% = 16.1%) to non-albicans Candida spp. (∆d150 - d1 = 43.5% - 6.4% = 37.1%) (P = 0.005). Time-to-event analysis of acquired antimicrobial resistance showed that the overall number of patients with Enterobacteriaceae presented an antimicrobial susceptibility decrease by 59.7% (∆d1 - d100 = 87.0% - 27.3% = 59.7%). The cumulative incidence of multi-resistant bacteria increased with length of hospital stay (∆d150 - d1 = 49.1% - 3.1% = 46.0%) (P = 0.004). Multivariable logistic regression analysis in relation to the pathogen spectrum and antimicrobial resistance development showed a significantly higher mortality for non-albicans Candida spp. (P = 0.039, odds ratio [OR] = 3.32 [95% confidence interval [CI]: 1.07-10.35]), E. faecium (P = 0.009, OR = 3.73 [95% CI: 1.38-10.05]), and multi-resistant bacteria (P = 0.007, OR = 5.08 [95% CI: 1.55-16.66]). CONCLUSIONS: Antimicrobial treatment of infected pancreatic necrosis becomes more challenging over time, owing to a change in spectrum favoring difficult-to-treat pathogens and an increase in multi-resistant bacteria associated with worse clinical outcomes (World Health Organization trial registration number: DRKS00014785).

Protective effects of coffee against induction of DNA damage and pre-neoplastic foci by aflatoxin B1.

SCOPE: Aim of the study was to investigate the protective properties of coffee towards aflatoxin B1 (AFB1) induced formation of pre-neoplastic hepatic foci and the identification of the constituents and molecular mechanisms that account for these effects. MATERIALS AND METHODS: Rats consumed three different brews and were subsequently treated with AFB1 (0.75 mg/kg b.w. intraperitoneally). Ten weeks later, the numbers and areas of hepatic foci were determined. Furthermore, the impact of the brews on AFB1-induced DNA damage was quantified in single cell gel electrophoresis assays and the activities of drug metabolising enzymes and glutathione-related parameters were monitored. Additionally, single cell gel electrophoresis assay experiments were conducted with pure caffeine. CONCLUSION: All brews reduced the frequencies of the hepatic foci. The most pronounced protection (reduction 82%) was seen with the caffeine containing metal and paper filtered brews. DNA migration was reduced between 65 and 75% with the caffeine containing brews. In additional experiments, clear protective effects were found with caffeine at dose levels that corresponded to those contained in the coffee. This observation indicates that the alkaloid accounts partly for the protective effects of coffee. Furthermore, our findings indicate that induction of UDP-glucuronosyltransferase contributes to the chemopreventive effects of coffee since all brews increased the activity of this detoxifying enzyme.

Food-derived peroxidized fatty acids may trigger hepatic inflammation: a novel hypothesis to explain steatohepatitis.

BACKGROUND & AIMS: Obesity and hepatic steatosis are frequently associated with the development of a non-alcoholic steatohepatitis (NASH). The mechanisms driving progression of a non-inflamed steatosis to NASH are largely unknown. Here, we investigated whether ingestion of peroxidized lipids, as being present in Western style diet, triggers the development of hepatic inflammation. METHODS: Corn oil containing peroxidized fatty acids was administered to rats by gavage for 6 days. In a separate approach, hepatocytes (HC), endothelial (EC) and Kupffer cells (KC) were isolated from untreated livers, cultured, and incubated with peroxidized linoleic acid (LOOH; linoleic acid (LH) being the main fatty acid in corn oil). Samples obtained from in vivo and in vitro studies were mainly investigated by qRT-PCR and biochemical determinations of lipid peroxidation products. RESULTS: Rat treatment with peroxidized corn oil resulted in increased hepatic lipid peroxidation, upregulation of nitric oxide synthetase-2 (NOS-2), cyclooxygenase-2 (COX-2), interleukin-1ß (IL-1ß), and tumor necrosis factor-α (TNFα), elevation of total nitric oxides, and increase in cd68-, cd163-, TNFα-, and/or COX-2 positive immune cells in the liver. When investigating liver cell types, LOOH elevated the secretion of TNFα, p38MAPK phosphorylation, and mRNA levels of NOS-2, COX-2, and TNFα, mainly in KC. The elevation of gene expression could be abrogated by inhibiting p38MAPK, which indicates that p38MAPK activation is involved in the pro-inflammatory effects of LOOH. CONCLUSIONS: These data show for the first time that ingestion of peroxidized fatty acids carries a considerable pro-inflammatory stimulus into the body which reaches the liver and may trigger the development of hepatic inflammation.

Instant coffee with high chlorogenic acid levels protects humans against oxidative damage of macromolecules.

SCOPE: Coffee is among the most frequently consumed beverages. Its consumption is inversely associated to the incidence of diseases related to reactive oxygen species; the phenomenon may be due to its antioxidant properties. Our primary objective was to investigate the impact of consumption of a coffee containing high levels of chlorogenic acids on the oxidation of proteins, DNA and membrane lipids; additionally, other redox biomarkers were monitored in an intervention trial. METHODS AND RESULTS: The treatment group (n=36) consumed instant coffee co-extracted from green and roasted beans, whereas the control consumed water (800 mL/P/day, 5 days). A global statistical analysis of four main biomarkers selected as primary outcomes showed that the overall changes are significant. 8-Isoprostaglandin F2α in urine declined by 15.3%, 3-nitrotyrosine was decreased by 16.1%, DNA migration due to oxidized purines and pyrimidines was (not significantly) reduced in lymphocytes by 12.5 and 14.1%. Other markers such as the total antioxidant capacity were moderately increased; e.g. LDL and malondialdehyde were shifted towards a non-significant reduction. CONCLUSION: The oxidation of DNA, lipids and proteins associated with the incidence of various diseases and the protection against their oxidative damage may be indicative for beneficial health effects of coffee.

Effects of coffee and its chemopreventive components kahweol and cafestol on cytochrome P450 and sulfotransferase in rat liver.

Coffee drinking appears to reduce cancer risk in liver and colon. Such chemoprevention may be caused by the diterpenes kahweol and cafestol (K/C) contained in unfiltered beverage. In animals, K/C treatment inhibited the mutagenicity/tumorigenicity of several carcinogens, likely explicable by beneficial modifications of xenobiotic metabolism, particularly by stimulation of carcinogen-detoxifying phase II mechanisms. In the present study, we investigated the influence of K/C on potentially carcinogen-activating hepatic cytochrome P450 (CYP450) and sulfotransferase (SULT). Male F344 rats received 0.2% K/C (1:1) in the diet for 10 days or unfiltered and/or filtered coffee as drinking fluid. Consequently, K/C decreased the metabolism of four resorufin derivatives representing CYP1A1, CYP1A2, CYP2B1, and CYP2B2 activities by approximately 50%. For CYP1A2, inhibition was confirmed at the mRNA level, accompanied by decreased CYP3A9. In contrast to K/C, coffee increased the metabolism of the resorufin derivatives up to 7-fold which was only marginally influenced by filtering. CYP2E1 activity and mRNA remained unchanged by K/C and coffee. K/C but not coffee decreased SULT by approximately 25%. In summary, K/C inhibited CYP450s by tendency but not universally. Inhibition of CYP450 and SULT may contribute to chemoprevention with K/C but involvement in the protection of coffee drinkers is unlikely. The data confirm that the effects of complex mixtures may deviate from those of their putatively active components.

Modification of N-acetyltransferases and glutathione S-transferases by coffee components: possible relevance for cancer risk.

Enzymes of xenobiotic metabolism are involved in the activation and detoxification of carcinogens and can play a pivotal role in the susceptibility of individuals toward chemically induced cancer. Differences in such susceptibility are often related to genetically predetermined enzyme polymorphisms but may also be caused by enzyme induction or inhibition through environmental factors or in the frame of chemopreventive intervention. In this context, coffee consumption, as an important lifestyle factor, has been under thorough investigation. Whereas the data on a potential procarcinogenic effect in some organs remained inconclusive, epidemiology has clearly revealed coffee drinkers to be at a lower risk of developing cancers of the colon and the liver and possibly of several other organs. The underlying mechanisms of such chemoprotection, modifications of xenobiotic metabolism in particular, were further investigated in rodent and in vitro models, as a result of which several individual chemoprotectants out of the >1000 constituents of coffee were identified as well as some strongly metabolized individual carcinogens against which they specifically protected. This chapter discusses the chemoprotective effects of several coffee components and whole coffee in association with modifications of the usually protective glutathione-S-transferase (GST) and the more ambivalent N-acetyltransferase (NAT). A key role is played by kahweol and cafestol (K/C), two diterpenic constituents of the unfiltered beverage that were found to reduce mutagenesis/tumorigenesis by strongly metabolized compounds, such as 2-amino-1-methyl-6-phenylimidazo-[4,5-b]pyridine, 7,12-dimethylbenz[a]anthracene, and aflatoxin B(1), and to cause various modifications of xenobiotic metabolism that were overwhelmingly beneficial, including induction of GST and inhibition of NAT. Other coffee components such as polyphenols and K/C-free coffee are also capable of increasing GST and partially of inhibiting NAT, although to a somewhat lesser extent.

Potential chemoprotective effects of the coffee components kahweol and cafestol palmitates via modification of hepatic N-acetyltransferase and glutathione S-transferase activities.

Coffee drinking has been associated with reduced incidence of colorectal cancer, possibly via chemoprotection/modification of the metabolism of dietary heterocyclic amine carcinogens such as 2-amino-1-methyl-6-phenylimidazo-[4,5-b]pyridine (PhIP) by kahweol and cafestol palmitates (K/C), two components of unfiltered coffee. Using the PhIP-exposed male Fisher F344 rat as a model, K/C have been shown to reduce colonic PhIP-DNA adducts by > 50%. We have used the male F344 rat to investigate the effects of dietary K/C (0.02-0.2% as a 1:1 mixture) on the metabolism of PhIP by N-acetyltransferase- (NAT), sulfotransferase- (SULT), and glutathione-dependent pathways. K/C decreased hepatic NAT-dependent PhIP activation by up to 80% in a dose-dependent manner. Conversely, hepatic glutathione S-transferase (GST) activity/expression increased, e.g., 3-4 fold toward 1-chloro-2,4-dinitrobenzene (total activity), up to 23-fold toward 4-vinylpyridine (rGSTP1), and approximately 7-fold for rGSTA2 protein. These effects had fully developed after 5 days of the test diet and persisted for at least 5 days after withdrawal of K/C. Hepatic glutathione increased two- to threefold and this increase was more short-lived than other changes. K/C did not modify hepatic SULT activity or colon NAT and GST activities. Benzylisothiocyanate and black tea, which have also been shown to reduce the formation of PhIP-DNA adducts in this model, had little effect on hepatic NAT, SULT, GST, or GSH. In primary culture of rat hepatocytes, both kahweol and cafestol palmitates reduced NAT activity by 80%. In summary, the unique potential of K/C to convert rapid acetylators to a slow acetylator phenotype, accompanied by GST induction, might contribute to chemoprevention against cancers associated with heterocyclic amines.

Coffee and its chemopreventive components Kahweol and Cafestol increase the activity of O6-methylguanine-DNA methyltransferase in rat liver--comparison with phase II xenobiotic metabolism.

A lower rate of colon cancer was observed in consumers of coffee with a high content of the diterpenes Kahweol and Cafestol (K/C). In animal models, K/C have been found to protect against the mutagenic/carcinogenic effects of compounds such as 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), aflatoxin B1, and 7,12-dimethylbenz[a]anthracene. Thus far, such chemoprotection by K/C has been attributed to modifications of xenobiotic metabolism, e.g. enhanced detoxification by UDP-glucuronosyltransferase (UDPGT) and/or glutathione transferase (GST). In the present study, we investigated the potential of several coffee-related treatments (K/C [1:1], Cafestol-alone, Turkish coffee) to modify the expression level of the DNA repair protein O(6)-methylguanine-DNA methyltransferase (MGMT) which is involved in the reversal of the precarcinogenic DNA damage O(6)-alkylguanine induced by alkylating agents. The results show that, in the male F344 rat, K/C and Cafestol increase hepatic MGMT in a dose-dependent manner up to a maximum of 2.6-fold at 0.122% K/C in the feed. Turkish coffee led to enhancements of up to 16%, the more moderate increase being associated with the lower estimated K/C intake through the beverage. In the livers of the rats receiving Turkish coffee, we also found 10-30% increases in several GST-related parameters (overall GST, GST-pi, glutathione, gamma-glutamylcysteine-synthetase) and a two-fold increase in UDPGT activity. Dose-response studies with K/C revealed that MGMT increased in parallel with three of the four GST-related parameters whereas the dose-response curves of UDPGT and of GST-pi activity displayed a steeper slope. Increased expression level of MGMT may extend the antimutagenic/anticarcinogenic potential of coffee components to protection against DNA alkylating agents.

Chemoprotective effects of garden cress (Lepidium sativum) and its constituents towards 2-amino-3-methyl-imidazo[4,5-f]quinoline (IQ)-induced genotoxic effects and colonic preneoplastic lesions.

The chemoprotective effect of garden cress (GC, Lepidium sativum) and its constituents, glucotropaeolin (GT) and benzylisothiocyanate (BITC), a breakdown product of GT, towards 2-amino-3-methyl-imidazo [4,5-f] quinoline (IQ)-induced genotoxic effects and colonic preneoplastic lesions was investigated in single cell gel electrophoresis (SCGE) assays and in aberrant crypt foci (ACF) experiments, respectively. Pretreatment of F344 rats with either fresh GC juice (0.8 ml), GT (150 mg/kg) or BITC (70 mg/kg) for three consecutive days caused a significant (P < 0.05) reduction in IQ (90 mg/kg, 0.2 ml corn oil/animal)-induced DNA damage in colon and liver cells in the range of 75-92%. Chemical analysis of GC juice showed that BITC does not account for the effects of the juice as its concentration in the juice was found to be 1000-fold lower than the dose required to cause a chemoprotective effect. Parallel to the chemoprotection experiments, the modulation of the activities of cytochrome P4501A2, glutathione-S-transferase (GST) and UDP glucuronosyltransferase (UDPGT) by GC juice, GT and BITC was studied. Whereas GT and BITC did not affect the activity of any of the enzymes significantly, GC juice caused a significant (P < 0.05) increase in the activity of hepatic UDPGT-2. In the ACF assay, IQ was administered by gavage on 10 alternating days in corn oil (dose 100 mg/kg). Five days before and during IQ treatment, subgroups received drinking water which contained 5% cress juice. The total number of IQ-induced aberrant crypts and ACF as well as ACF with crypt multiplicity of > or =4 were reduced significantly (P < 0.05) in the group that received IQ plus GC juice compared with the group that was fed with IQ only. However, crypt multiplicity was not significantly different in these two groups when all ACF with all classes of crypt multiplicity were considered in the analysis. This is the first report on the inhibition of HA-induced DNA damage and preneoplastic lesions by a cruciferous plant. Our findings suggest that the chemoprotective effect of GC is mediated through enhancement of detoxification of IQ by UDPGT.

Enhancement of the chemoprotective enzymes glucuronosyl transferase and glutathione transferase in specific organs of the rat by the coffee components kahweol and cafestol.

The coffee components kahweol and cafestol (K/C) have been reported to protect the colon and other organs of the rat against the formation of DNA adducts by 2-amino-1-methyl-6-phenylimidazo[4,5- b]pyridine (PhIP) and aflatoxin B1. PhIP is a cooked-food mutagen to which significant human exposure and a role in colon cancer etiology are attributed, and, interestingly, such cancers appear to develop at a lower rate in consumers of coffees with high amounts of K/C. Earlier studies in rodent liver have shown that a key role in the chemopreventive effect of K/C is likely to be due to the potential of these compounds to induce the detoxification of xenobiotics by glutathione transferase (GST) and to enhance the synthesis of the corresponding co-factor glutathione. However, mutagens like PhIP may also be detoxified by UDP-glucuronosyl transferase (UDPGT) for which data are lacking regarding a potential effect of K/C. Therefore, in the present study, we investigated the effect of K/C on UDPGT and, concomitantly, we studied overall GST and the pattern of individual GST classes, particularly GST-theta;, which was not included in earlier experiments. In addition, we analyzed the organ-dependence of these potentially chemopreventive effects. K/C was fed to male F344 rats at 0.122% in the chow for 10 days. Enzyme activities in liver, kidney, lung, colon, salivary gland, pancreas, testis, heart and spleen were quantified using five characteristic substrates and the hepatic protein pattern of GST classes alpha, mu, and pi was studied with affinity chromatography/HPLC. Our study showed that K/C is not only capable of increasing overall GST and GST classes alpha, mu, and pi but also of enhancing UDGPT and GST-theta. All investigated K/C effects were strongest in liver and kidney, and some response was seen in lung and colon but none in the other organs. In summary, our results show that K/C treatment leads to a wide spectrum of increases in phase II detoxification enzymes. Notably, these effects occurred preferentially in the well perfused organs liver and kidney, which may thus not only contribute to local protection but also to anti-carcinogenesis in distant, less stimulated organs such as the colon.