Diverse potential of secretome from natural killer cells and monocyte-derived macrophages in activating stellate cells.

Chronic liver diseases, such as non-alcoholic steatohepatitis (NASH)-induced cirrhosis, are characterized by an increasing accumulation of stressed, damaged, or dying hepatocytes. Hepatocyte damage triggers the activation of resident immune cells, such as Kupffer cells (KC), as well as the recruitment of immune cells from the circulation toward areas of inflammation. After infiltration, monocytes differentiate into monocyte-derived macrophages (MoMF) which are functionally distinct from resident KC. We herein aim to compare the in vitro signatures of polarized macrophages and activated hepatic stellate cells (HSC) with ex vivo-derived disease signatures from human NASH. Furthermore, to shed more light on HSC activation and liver fibrosis progression, we investigate the effects of the secretome from primary human monocytes, macrophages, and NK cells on HSC activation. Interleukin (IL)-4 and IL-13 treatment induced transforming growth factor beta 1 (TGF-ß1) secretion by macrophages. However, the supernatant transfer did not induce HSC activation. Interestingly, PMA-activated macrophages showed strong induction of the fibrosis response genes COL10A1 and CTGF, while the supernatant of IL-4/IL-13-treated monocytes induced the upregulation of COL3A1 in HSC. The supernatant of PMA-activated NK cells had the strongest effect on COL10A1 induction in HSC, while IL-15-stimulated NK cells reduced the expression of COL1A1 and CTGF. These data indicate that other factors, aside from the well-known cytokines and chemokines, might potentially be stronger contributors to the activation of HSCs and induction of a fibrotic response, indicating a more diverse and complex role of monocytes, macrophages, and NK cells in liver fibrosis progression.

Automated characterization of patient-ventilator interaction using surface electromyography.

BACKGROUND: Characterizing patient-ventilator interaction in critically ill patients is time-consuming and requires trained staff to evaluate the behavior of the ventilated patient. METHODS: In this study, we recorded surface electromyography ([Formula: see text]) signals from the diaphragm and intercostal muscles and esophageal pressure ([Formula: see text]) in mechanically ventilated patients with ARDS. The sEMG recordings were preprocessed, and two different algorithms (triangle algorithm and adaptive thresholding algorithm) were used to automatically detect inspiratory patient effort. Based on the detected inspirations, major asynchronies (ineffective, auto-, and double triggers and double efforts), delayed and synchronous triggers were computationally classified. Reverse triggers were not considered in this study. Subsequently, asynchrony indices were calculated. For the validation of detected efforts, two experts manually annotated inspiratory patient activity in [Formula: see text], blinded toward each other, the [Formula: see text] signals, and the algorithmic results. We also classified patient-ventilator interaction and calculated asynchrony indices with manually detected inspirations in [Formula: see text] as a reference for automated asynchrony classification and asynchrony index calculation. RESULTS: Spontaneous breathing activity was recognized in 22 out of the 36 patients included in the study. Evaluation of the accuracy of the algorithms using 3057 inspiratory efforts in [Formula: see text] demonstrated reliable detection performance for both methods. Across all datasets, we found a high sensitivity (triangle algorithm/adaptive thresholding algorithm: 0.93/0.97) and a high positive predictive value (0.94/0.89) against expert annotations in [Formula: see text]. The average delay of automatically detected inspiratory onset to the [Formula: see text] reference was [Formula: see text]79 ms/29 ms for the two algorithms. Our findings also indicate that automatic asynchrony index prediction is reliable. For both algorithms, we found the same deviation of [Formula: see text] to the [Formula: see text]-based reference. CONCLUSIONS: Our study demonstrates the feasibility of automating the quantification of patient-ventilator asynchrony in critically ill patients using noninvasive sEMG. This may facilitate more frequent diagnosis of asynchrony and support improving patient-ventilator interaction.

Application of human iPSC-derived macrophages in a miniaturized high-content-imaging-based efferocytosis assay.

Macrophages play a pivotal role in drug discovery due to their key regulatory functions in health and disease. Overcoming the limited availability and donor variability of human monocyte-derived macrophages (MDMs), human induced pluripotent stem cell (iPSC)-derived macrophages (IDMs) could provide a promising tool for both disease modeling and drug discovery. To access large numbers of model cells for medium- to high-throughput application purposes, an upscaled protocol was established for differentiation of iPSCs into progenitor cells and subsequent maturation into functional macrophages. These IDM cells resembled MDMs both with respect to surface marker expression and phago- as well as efferocytotic function. A statistically robust high-content-imaging assay was developed to quantify the efferocytosis rate of IDMs and MDMs allowing for measurements both in the 384- and 1536-well microplate format. Validating the applicability of the assay, inhibitors of spleen tyrosine kinase (Syk) were shown to modulate efferocytosis in IDMs and MDMs with comparable pharmacology. The miniaturized cellular assay with the upscaled provision of macrophages opens new routes to pharmaceutical drug discovery in the context of efferocytosis-modulating substances.

Senescent cells suppress macrophage-mediated corpse removal via upregulation of the CD47-QPCT/L axis.

Progressive accrual of senescent cells in aging and chronic diseases is associated with detrimental effects in tissue homeostasis. We found that senescent fibroblasts and epithelia were not only refractory to macrophage-mediated engulfment and removal, but they also paralyzed the ability of macrophages to remove bystander apoptotic corpses. Senescent cell-mediated efferocytosis suppression (SCES) was independent of the senescence-associated secretory phenotype (SASP) but instead required direct contact between macrophages and senescent cells. SCES involved augmented senescent cell expression of CD47 coinciding with increased CD47-modifying enzymes QPCT/L. SCES was reversible by interfering with the SIRPα-CD47-SHP-1 axis or QPCT/L activity. While CD47 expression increased in human and mouse senescent cells in vitro and in vivo, another ITIM-containing protein, CD24, contributed to SCES specifically in human epithelial senescent cells where it compensated for genetic deficiency in CD47. Thus, CD47 and CD24 link the pathogenic effects of senescent cells to homeostatic macrophage functions, such as efferocytosis, which we hypothesize must occur efficiently to maintain tissue homeostasis.

Over- and under-prescribing, and their association with functional disability in older patients at risk of further decline in Germany - a cross-sectional survey conducted as part of a randomised comparative effectiveness trial.

BACKGROUND: Older patients at risk of functional decline are frequently affected by polypharmacy. This is associated with a further loss of independence. However, a relationship between functional disability and medications, such as 'Potentially Inappropriate Medications' (PIMs) and 'Potential Prescribing Omissions' (PPOs), as itemised for (de) prescribing in practice-orientated medication lists, has yet to be established. METHODS: As part of a randomised comparative effectiveness trial, LoChro, we conducted a cross-sectional analysis of the association between PIMs and PPOs measured using the 'Screening Tool of Older Persons' Prescription Criteria / Screening Tool To Alert to Right Treatment' (STOPP/START) Version 2, with functional disability assessed using the 'World Health Organization Disability Assessment Schedule 2.0' (WHODAS). Individuals aged 65 and older at risk of loss of independence were recruited from the inpatient and outpatient departments of the local university hospital. Multiple linear regression analysis was used to model the potential prediction of functional disability using the numbers of PIMs and PPOs, adjusted for confounders including multimorbidity. RESULTS: Out of 461 patients, both the number of PIMs and the number of PPOs were significantly associated with an increase in WHODAS-score (Regression coefficients B 2.7 [95% confidence interval: 1.5-3.8] and 1.5 [95% confidence interval: 0.2-2.7], respectively). In WHODAS-score prediction modelling the contribution of the number of PIMs exceeded the one of multimorbidity (standardised coefficients beta: PIM 0.20; multimorbidity 0.13; PPO 0.10), whereas no significant association between the WHODAS-score and the number of medications was seen. 73.5 % (339) of the participants presented with at least one PIM, and 95.2% (439) with at least one PPO. The most common PIMs were proton pump inhibitors and analgesic medication, with frequent PPOs being pneumococcal and influenza vaccinations, as well as osteoporosis prophylaxis. CONCLUSIONS: The results indicate a relationship between inappropriate prescribing, both PIMs and PPOs, and functional disability, in older patients at risk of further decline. Long-term analysis may help clarify whether these patients benefit from interventions to reduce PIMs and PPOs.

Blind source separation of inspiration and expiration in respiratory sEMG signals.

Objective.Surface electromyography (sEMG) is a noninvasive option for monitoring respiratory effort in ventilated patients. However, respiratory sEMG signals are affected by crosstalk and cardiac activity. This work addresses the blind source separation (BSS) of inspiratory and expiratory electrical activity in single- or two-channel recordings. The main contribution of the presented methodology is its applicability to the addressed muscles and the number of available channels.Approach.We propose a two-step procedure consisting of a single-channel cardiac artifact removal algorithm, followed by a single- or multi-channel BSS stage. First, cardiac components are removed in the wavelet domain. Subsequently, a nonnegative matrix factorization (NMF) algorithm is applied to the envelopes of the resulting wavelet bands. The NMF is initialized based on simultaneous standard pneumatic measurements of the ventilated patient.Main results.The proposed estimation scheme is applied to twelve clinical datasets and simulated sEMG signals of the respiratory system. The results on the clinical datasets are validated based on expert annotations using invasive pneumatic measurements. In the simulation, three measures evaluate the separation success: The distortion and the correlation to the known ground truth and the inspiratory-to-expiratory signal power ratio. We find an improvement across all SNRs, recruitment patterns, and channel configurations. Moreover, our results indicate that the initialization strategy replaces the manual matching of sources after the BSS.Significance.The proposed separation algorithm facilitates the interpretation of respiratory sEMG signals. In crosstalk affected measurements, the developed method may help clinicians distinguish between inspiratory effort and other muscle activities using only noninvasive measurements.

Cross-lagged relations between mentoring received from supervisors and employee OCBs: Disentangling causal direction and identifying boundary conditions.

Although mentoring has documented relationships with employee attitudes and outcomes of interest to organizations, neither the causal direction nor boundary conditions of the relationship between mentoring and organizational citizenship behaviors (OCBs) has been fully explored. On the basis of Social Learning Theory (SLT; Bandura, 1977, 1986), we predicted that mentoring received by supervisors would causally precede OCBs, rather than employee OCBs resulting in the receipt of more mentoring from supervisors. Results from cross-lagged data collected at 2 points in time from 190 intact supervisor-employee dyads supported our predictions; however, only for OCBs directed at individuals (OCB-Is) and not for OCBs directed at the organization (OCB-Os). Further supporting our theoretical rationale for expecting mentoring to precede OCBs, we found that coworker support operates as a substitute for mentoring in predicting OCB-Is. By contrast, no moderating effects were found for perceived organizational support. The results are discussed in terms of theoretical implications for mentoring and OCB research, as well as practical suggestions for enhancing employee citizenship behaviors.

Aging and alcohol interact to alter hepatic DNA hydroxymethylation.

BACKGROUND: Aging and chronic alcohol consumption are both modifiers of DNA methylation, but it is not yet known whether chronic alcohol consumption also alters DNA hydroxymethylation, a newly discovered epigenetic mark produced by oxidation of methylcytosine. Furthermore, it has not been tested whether aging and alcohol interact to modify this epigenetic phenomenon, thereby having an independent effect on gene expression. METHODS: Old (18 months) and young (4 months) male C57BL/6 mice were pair-fed either a Lieber-DeCarli liquid diet with alcohol (18% of energy) or an isocaloric Lieber-DeCarli control diet for 5 weeks. Global DNA hydroxymethylation and DNA methylation were analyzed from hepatic DNA using a new liquid chromatography-tandem mass spectrometry method. Hepatic mRNA expression of the Tet enzymes were measured via quantitative real-time polymerase chain reaction. RESULTS: In young mice, mild chronic alcohol exposure significantly reduced global DNA hydroxymethylation compared with control mice (0.22 ± 0.01 vs. 0.29 ± 0.06%, p = 0.004). Alcohol did not significantly alter hydroxymethylcytosine levels in old mice. Old mice fed the control diet showed decreased global DNA hydroxymethylation compared with young mice fed the control diet (0.24 ± 0.02 vs. 0.29 ± 0.06%, p = 0.04). This model suggests an interaction between aging and alcohol in determining DNA hydroxymethylation (pinteraction  = 0.009). Expression of Tet2 and Tet3 was decreased in the old mice relative to the young (p < 0.005). CONCLUSIONS: The observation that alcohol alters DNA hydroxymethylation indicates a new epigenetic effect of alcohol. This is the first study demonstrating the interactive effects of chronic alcohol consumption and aging on DNA hydroxymethylation.

Quality of life in Parkinson's disease patients with motor fluctuations and dyskinesias in five European countries.

BACKGROUND: Little is known about the relationship between specific subtypes of treatment-associated motor complications and different domains of health-related Quality of Life (QoL) in patients with Parkinson's disease (PD). Larger studies that investigate these aspects within a cross-cultural setting are scarce. OBJECTIVE: To assess QoL and its association with on-off fluctuations, peak-dose dyskinesias, biphasic dyskinesias, and off-dystonias in PD patients from five European countries. METHODS: Data from 817 PD patients were collected cross-sectionally in France, Germany, Italy, Spain, and the UK. QoL was measured with the generic EuroQoL 5-Dimension questionnaire (EQ-5D) and the disease-specific Parkinson's Disease Questionnaire-39 (PDQ-39). Multivariable linear regression analyses were performed to test the associations of motor complication subtypes with QoL. RESULTS: Thirty-three percent of the patients (varying from 23% in Italy to 58% in France) suffered from motor complications, either a single subtype or a combination of different subtypes. On-off fluctuations were associated with a 7.1 percentage point decrease in the EQ-5D (p < 0.001) and a 3.6 percentage point deterioration in the PDQ-39 (p = 0.01). Dyskinesias were not seen to affect global QoL scores, but had detrimental effects on the PDQ-39 dimensions activities of daily living, cognitions, stigma, and bodily discomfort. Patients from Spain, Italy, and France had lower global QoL scores in the multivariable analyses than patients from Germany and the UK. CONCLUSION: Motor complications, primarily on-off fluctuations, may impact QoL in PD patients. This substantiates the importance of clinical strategies targeting the prevention, delay of onset, and management of motor complications in PD patients.

Effective Clinical Supervision in Substance Use Disorder Treatment Programs and Counselor Job Performance.

When mental health counselors have limited and/or inadequate training in substance use disorders (SUDs), effective clinical supervision (ECS) may advance their professional development. The purpose of the current study was to investigate whether ECS is related to the job performance of SUD counselors. Data were obtained in person via paper-and-pencil surveys from 392 matched SUD counselor-clinical supervisor dyads working in 27 SUD treatment organizations across the United States. ECS was rated by counselors and measured with five multi-item scales (i.e., sponsoring counselors' careers, providing challenging assignments, role modeling, accepting/confirming counselors' competence, overall supervisor task proficiency). Clinical supervisors rated counselors' job performance, which was measured with two multi-item scales (i.e., task performance, performance within supervisory relationship). Using mixed-effects models, we found that most aspects of ECS are related to SUD counselor job performance. Thus, ECS may indeed enhance counselors' task performance and performance within the supervisory relationship, and, as a consequence, offset limited formal SUD training.

An interdisciplinary meta-analysis of the potential antecedents, correlates, and consequences of protégé perceptions of mentoring.

This meta-analysis summarized youth, academic, and workplace research on the potential antecedents (demographics, human capital, and relationship attributes), correlates (interaction frequency, relationship length, performance, motivation, and social capital), and consequences (attitudinal, behavioral, career-related, and health-related outcomes) of protégé perceptions of instrumental support, psychosocial support, and relationship quality to the mentor or to the relationship. A total of 173 meta-analytic correlations were computed based on data from 173 samples and a combined N of 40,737. Among antecedents, positive protégé perceptions were most strongly associated with greater similarity in attitudes, values, beliefs, and personality with their mentors (ρ ranged from .38 to .59). Among correlates, protégé perceptions of greater instrumental support (ρ = .35) and relationship quality (ρ = .54) were most strongly associated with social capital while protégé perceptions of greater psychosocial support were most strongly associated with interaction frequency (ρ = .25). Among consequences, protégé perceptions of greater instrumental support (ρ = .36) and relationship quality (ρ = .38) were most strongly associated with situational satisfaction while protégé perceptions of psychosocial support were most highly associated with sense of affiliation (ρ = .41). Comparisons between academic and workplace mentoring generally revealed differences in magnitude, rather than direction, of the obtained effects. The results should be interpreted in light of the methodological limitations (primarily cross-sectional designs and single-source data) and, in some instances, a small number of primary studies.

Clinical supervisor and counselor perceptions of clinical supervision in addiction treatment.

Little is empirically known about clinical supervision in addiction treatment. This study describes multiple domains of clinical supervision in addiction treatment from the perspectives of clinical supervisors and their counselors. Survey data were obtained from 484 matched clinical supervisor-counselor dyads working in diverse addiction treatment programs across the United States. Supervisors report wide-ranging experiences and training in supervision. Counselors generally perceive their supervisors' job performance as effective. Supervisors and their counselors largely differ in their perceptions of supervision practices, with supervisors reporting greater supervision given and their counselors reporting less supervision received. The implications are discussed.

Folate supplementation differently affects uracil content in DNA in the mouse colon and liver.

High folate intake may increase the risk of cancer, especially in the elderly. The present study examined the effects of ageing and dietary folate on uracil misincorporation into DNA, which has a mutagenic effect, in the mouse colon and liver. Old (18 months; n 42) and young (4 months; n 42) male C57BL/6 mice were pair-fed with four different amino acid-defined diets for 20 weeks: folate deplete (0 mg/kg diet); folate replete (2 mg/kg diet); folate supplemented (8 mg/kg diet); folate deplete (0 mg/kg diet) with thymidine supplementation (1·8 g/kg diet). Thymidylate synthesis from uracil requires folate, but synthesis from thymidine is folate independent. Liver folate concentrations were determined by the Lactobacillus casei assay. Uracil misincorporation into DNA was measured by a GC/MS method. Liver folate concentrations demonstrated a stepwise increase across the spectrum of dietary folate levels in both old (P = 0·003) and young (P < 0·001) mice. Uracil content in colonic DNA was paradoxically increased in parallel with increasing dietary folate among the young mice (P trend = 0·033), but differences were not observed in the old mice. The mean values of uracil in liver DNA, in contrast, decreased with increasing dietary folate among the old mice, but it did not reach a statistically significant level (P < 0·1). Compared with the folate-deplete group, thymidine supplementation reduced uracil misincorporation into the liver DNA of aged mice (P = 0·026). The present study suggests that the effects of folate and thymidine supplementation on uracil misincorporation into DNA differ depending on age and tissue. Further studies are needed to clarify the significance of increased uracil misincorporation into colonic DNA of folate-supplemented young mice.

Ageing, chronic alcohol consumption and folate are determinants of genomic DNA methylation, p16 promoter methylation and the expression of p16 in the mouse colon.

Older age, dietary folate and chronic alcohol consumption are important risk factors for the development of colon cancer. The present study examined the effects of ageing, folate and alcohol on genomic and p16-specific DNA methylation, and p16 expression in the murine colon. Old (aged 18 months; n 70) and young (aged 4 months; n 70) male C57BL/6 mice were pair-fed either a Lieber-DeCarli liquid diet with alcohol (18 % of energy), a Lieber-DeCarli diet with alcohol (18 %) and reduced folate (0.25 mg folate/l) or an isoenergetic control diet (0.5 mg folate/l) for 5 or 10 weeks. Genomic DNA methylation, p16 promoter methylation and p16 gene expression were analysed by liquid chromatography-MS, methylation-specific PCR and real-time RT-PCR, respectively. Genomic DNA methylation was lower in the colon of old mice compared with young mice (P < 0.02) at 10 weeks. Alcohol consumption did not alter genomic DNA methylation in the old mouse colon, whereas it tended to decrease genomic DNA methylation in young mice (P = 0.08). p16 Promoter methylation and expression were higher in the old mouse colon compared with the corresponding young groups. There was a positive correlation between p16 promoter methylation and p16 expression in the old mouse colon (P < 0.02). In young mice the combination of alcohol and reduced dietary folate led to significantly decreased p16 expression compared with the control group (P < 0.02). In conclusion, ageing and chronic alcohol consumption alter genomic DNA methylation, p16 promoter methylation and p16 gene expression in the mouse colon, and dietary folate availability can further modify the relationship with alcohol in the young mouse.

Too much folate: a risk factor for cancer and cardiovascular disease?

PURPOSE OF REVIEW: The intent of this evidence-based review is to analyze the role of folate in chronic diseases, focusing on cancer and cardiovascular disease. RECENT FINDINGS: Low folate status has been shown to be a risk factor for cancer and cardiovascular disease. Although epidemiological data suggest an inverse association between folate status and disease risk, intervention studies give equivocal results, suggesting the response to folate intake does not follow a linear continuum. Moreover, recent folate intervention trials raise concern about possible adverse effects of folate supplementation and suggest that too much folate in inopportune settings may be potentially harmful in individuals at higher risk for cardiovascular disease and cancer. SUMMARY: Although folate intake at sufficient levels appears to be an effective cancer chemopreventive strategy, high-dose supplementation of folate has generally not been effective in reducing recurrence of cardiovascular events or colorectal adenomas in clinical intervention trials. Although controversial, high folate status achieved through folate fortification or supplementation may increase the risk of certain chronic diseases among certain individuals, possibly by interfering with the homeostasis of one-carbon metabolism. Further research is urgently needed to accurately define the relationship between supraphysiological intake of folate and chronic diseases.

Apple polyphenols modulate expression of selected genes related to toxicological defence and stress response in human colon adenoma cells.

Apples contain significant amounts of flavonoids that are potentially cancer risk reducing by acting antioxidative or antiproliferative and by favorably modulating gene expression. The purpose of this study was to investigate whether polyphenols from apples modulate expression of genes related to colon cancer prevention in preneoplastic cells derived from colon adenoma (LT97). For this, LT97 cells were treated with effective concentrations of apple extracts (AEs). RNA was isolated and used for synthesis and labeling of cDNA that was hybridized to cDNA-arrays. Gene expression studies were performed using a commercial cDNA-array from Superarray that contains a limited number of genes (96 genes) related to drug metabolism, and a custom-made cDNA microarray that contains a higher number of genes (300 genes, including some genes from Superarray) related to mechanisms of carcinogenesis or chemoprevention. Real-time PCR and enzyme activity assays were additionally performed to confirm selected array results. Treatment of cells with AE resulted in 30 and 46 genes expressed over cut-off values (>or=1.5- or

Overview of experimental data on reduction of colorectal cancer risk by inulin-type fructans.

Colorectal cancer is related to diet, lifestyle, physical inactivity, and obesity. The responsible carcinogens cause mutations or enhance cell growth. Inulin-type fructans may counteract the effects via their gut flora-mediated fermentation products in vitro and in vivo. Important products formed by fermentation of inulin-type fructans with human gut flora are short-chain fatty acids. Of these, butyrate and propionate inhibit growth of colon tumor cells and histone deacetylases. Butyrate also causes apoptosis, reduces metastasis in colon cell lines, and protects from genotoxic carcinogens by enhancing expression of enzymes involved in detoxification. Fermentation supernatants of inulin have similar growth-inhibitory effects on colon adenoma and carcinoma cells and induce histone hyperacetylation by inhibiting histone deacetylases. In animal models inulin-type fructans prevent and retard colorectal carcinogenesis. Several studies reported the reduction of chemically induced preneoplastic lesions or tumors in the colon of rodents treated with inulin-type fructans. The human intervention study (SYNCAN project) sought to provide the experimental evidence for risk reduction by inulin-type fructans in humans. One group of polypectomized people at high risk for colon cancer and another of colon cancer volunteers after curative resection were given a synbiotic preparation. There were clear functional effects of the synbiotic because numerous different cancer risk markers were favorably altered. In conclusion, there is considerable experimental evidence that inulin modulates parameters of colon cancer risks in human colon cells, in animals, and in a human intervention trial. The involved mechanisms possibly include reduction of exposure to risk factors and suppression of tumor cell survival.

Physiological concentrations of butyrate favorably modulate genes of oxidative and metabolic stress in primary human colon cells.

Butyrate, a metabolite of gut flora-mediated fermentation of dietary fibre, was analysed for effects on expression of genes related to oxidative stress in primary human colon cells. An induction of detoxifying, antioxidative genes is expected to contribute to dietary chemoprevention. Cells were treated with butyrate (3.125-50 mM; 0.5-8 h), and kinetics of uptake and survival were measured. Gene expression was determined with a pathway-specific cDNA array after treating colon epithelium stripes with nontoxic doses of butyrate (10 mM, 12 h). Changes of hCOX-2, hSOD2 and hCAT expression were confirmed with real-time polymerase chain reaction (PCR) and by measuring catalase-enzyme activity. Primary colon cells consumed 1.5 and 0.5 mM butyrate after 4- and 12-h treatment, respectively. Cell viability was not changed by butyrate during 0.5-2-h treatment, whereas cell yields decreased after 1 h. Metabolic activity of remaining cells was either increased (4 h, 50 mM) or retained at 97% (8 h, 50 mM). Expression of hCAT was enhanced, whereas hCOX-2 and hSOD2 were lowered according to both array and real-time PCR analysis. An enhanced catalase-enzyme activity was detected after 2 h butyrate treatment. Healthy nontransformed colon cells well tolerated butyrate (50 mM, 2 h), and lower concentrations (10 mM, 12 h) modulated cyclooxygenase 2 (COX-2) and catalase genes. This points to a dual role of chemoprotection, since less COX-2 could reduce inflammatory processes, whereas more catalase improves detoxification of hydrogen peroxide (H(2)O(2)), a compound of oxidative stress. Changes of this type could reduce damaging effects by oxidants and protect cells from initiation.

Products formed during fermentation of the prebiotic inulin with human gut flora enhance expression of biotransformation genes in human primary colon cells.

Inulin-type fructans are fermented by gut bacteria to yield SCFA, including butyrate which is trophic for colonocytes and induces glutathione S-transferases (GST) that detoxify carcinogens. Since little is known on similar effects by complex fermentation samples, we studied related products in non-transformed human colonocytes. Inulin enriched with oligofructose (1:1, Synergy1) was fermented with human gut flora. SCFA were quantified and a SCFA mixture was prepared accordingly. Colonocytes were incubated (4-12 h) with the Synergy1 fermentation supernatant (SFS), faeces control, a mixture of the three major SCFA (each 0-15 %, v/v) or butyrate (0-50 mM). Metabolic activity was determined to assess trophic effects and cytotoxicity. Expression of ninety-six genes related to biotransformation was studied using cDNA macroarrays. Results on modulated GST were reassessed with real-time PCR and GST activity was measured. Fermentation of inulin resulted in 2-3-fold increases of SCFA. The samples were non-cytotoxic. SFS increased metabolic activity, pointing to trophic effects. The samples modulated gene expression with different response patterns. Key results were that GSTM2 (2.0-fold) and GSTM5 (2.2-fold) were enhanced by SFS, whereas the SCFA mixture reduced expression. The faeces control enhanced GSTA4 (2.0-fold), but reduced GSTM2 (0.2-fold) and GSTM5 (0.2-fold). Realtime qPCR confirmed the induction of GSTM2 and GSTM5 by SFS and of GSTA4 and GSTT2 by butyrate. Activity of GST was not modulated. High concentrations of fermentation products were well tolerated by primary colonocytes, pointing to trophic effects. The induction of GST by the SFS may protect the cells from carcinogenic compounds.

Apple flavonoids inhibit growth of HT29 human colon cancer cells and modulate expression of genes involved in the biotransformation of xenobiotics.

Flavonoids from fruits and vegetables probably reduce risks of diseases associated with oxidative stress, including cancer. Apples contain significant amounts of flavonoids with antioxidative potential. The objectives of this study were to investigate such compounds for properties associated with reduction of cancer risks. We report herein that apple flavonoids from an apple extract (AE) inhibit colon cancer cell growth and significantly modulate expression of genes related to xenobiotic metabolism. HT29 cells were treated with AE at concentrations delivering 5-50 microM of one of the major ingredients, phloridzin ("phloridzin-equivalents," Ph.E), to the cell culture medium, with a synthetic flavonoid mixture mimicking the composition of the AE or with 5-100 microM individual flavonoids. HT29 cell growth was inhibited by the complex extract and by the mixture. HT29 cells were treated with nontoxic doses of the AE (30 microM, Ph.E) and after 24 h total RNA was isolated to elucidate patterns of gene expression using a human cDNA-microarray (SuperArray) spotted with 96 genes of drug metabolism. Treatment with AE resulted in an upregulation of several genes (GSTP1, GSSTT2, MGST2, CYCP4F3, CHST5, CHST6, and CHST7) and downregulation of EPHX1, in comparison to the medium controls. The enhanced transcriptional activity of GSTP1 and GSTT2 genes was confirmed with real-time qRT-PCR. On the basis of the pattern of differential gene expression found here, we conclude that apple flavonoids modulate toxicological defense against colon cancer risk factors. In addition to the inhibition of tumor cell proliferation, this could be a mechanism of cancer risk reduction.