Information on human papillomavirus (HPV) type distribution is necessary to evaluate the potential impact of current and future HPV vaccines. We estimated the relative contribution (RC) to invasive cervical cancer (ICC) and precancerous cervical lesions of the nine HPV types (HPV 6/11/16/18/31/33/45/52/58) included in an HPV vaccine currently under development.
Estimations on ICC were based on an international study of 8,977 HPV positive cases and estimations on precancerous cervical lesions were extracted from a published meta-analysis including 115,789 HPV positive women. Globocan 2008 and 2010 World Population Prospects were used to estimate current and future projections of new ICC cases.
RC of the 9 HPV types in ICC was 89.4%, with 18.5% of cases positive for HPV 31/33/45/52/58. Regional variations were observed. RCs varied by histology, ranging between 89.1% in squamous cell carcinomas (SCC) and 95.5% in adenocarcinomas (ADC). HPV 16/18/45 were detected in 94.2% of ADC. RC of the 9 types altogether decreased with age (trend test p < 0.0001), driven by the decrease in older ages of HPV 16/18/45. In contrast, the RC of HPV 31/33/52/58 increased with age. Due to population growth alone, projected estimates of ICC cases attributable to the 9 types are expected to rise from 493,770 new cases in 2012 to 560,887 new cases in 2025.
The RCs of individual high risk HPV types varied by cytological and histological grades of HPV-positive precancerous cervical lesions, and there was an under representation of HPV 18 and 45 compared to ICC.
The addition of HPV 31/33/45/52/58 to HPV types included in current vaccines could prevent almost 90% of ICC cases worldwide. If the nine-valent vaccine achieves the same degree of efficacy than previous vaccines, world incidence rates could be substantially reduced.
Human papillomavirus; Cervical cancer; Genotype; Epidemiology; Human papillomavirus vaccines
Tumor necrosis factor alpha (TNF) is able to kill cancer cells via receptor-mediated cell death requiring adenosine triphosphate (ATP). Clinical usage of TNF so far is largely limited by its profound hepatotoxicity. Recently, it was found in the murine system that specific protection of hepatocytes against TNF's detrimental effects can be achieved by fructose-mediated ATP depletion therein. Before employing this quite attractive selection principle in a first clinical trial, we here comprehensively investigated the interdependence between ATP depletion and TNF hepatotoxicity in both in vitro and ex vivo experiments based on usage of primary patient tissue materials.
Primary human hepatocytes, and both non-tumorous and tumorous patient-derived primary liver tissue slices were used to elucidate fructose-induced ATP depletion and TNF-induced cytotoxicity.
PHH as well as tissue slices prepared from non-malignant human liver specimen undergoing a fructose-mediated ATP depletion were both demonstrated to be protected against TNF-induced cell death. In contrast, due to tumor-specific overexpression of hexokinase II, which imposes a profound bypass on hepatocytic-specific fructose catabolism, this was not the case for human tumorous liver tissues.
Normal human liver tissues can be protected transiently against TNF-induced cell death by systemic pretreatment with fructose used in non-toxic/physiologic concentrations. Selective TNF-targeting of primary and secondary tumors of the liver by transient and specific depletion of hepatocytic ATP opens up a new clinical avenue for the TNF-based treatment of liver cancers.
The lipidic cubic phase (LCP) has repeatedly proven to serve as a successful membrane-mimetic matrix for a variety of difficult-to-crystallize membrane proteins. While monoolein has been the predominant lipid of choice, there is a growing need for the characterization and use of other LCP host lipids, allowing exploration of a range of structural parameters such as bilayer thickness and curvature for optimal insertion, stability and crystallogenesis of membrane proteins. Here, we describe the development of a high-throughput (HT) pipeline to employ small angle X-ray scattering (SAXS) – the most direct technique to identify lipid mesophases and measure their structural parameters – to interrogate rapidly a large number of lipid samples under a variety of conditions, similar to those encountered during crystallization. Leveraging the identical setup format for LCP crystallization trials, this method allows the quickly assessment of lipid matrices for their utility in membrane protein crystallization, and could inform the tailoring of lipid and precipitant conditions to overcome specific crystallization challenges. As proof of concept, we present HT LCP-SAXS analysis of lipid samples made of monoolein with and without cholesterol, and of monovaccenin, equilibrated with solutions used for crystallization trials and LCP fluorescence recovery after photobleaching (FRAP) experiments.
lipidic cubic phase; monoolein; phase behavior; SAXS
Chronic low back pain (CLBP) has been shown to be associated with various pathophysiological changes at several level of the sensorimotor system, pointing to a general hypersensitivity in CLBP patients. The aim of the present study was to investigate signs of generalized mechanical pain hypersensitivity in CLBP patients on the hand and on the painful site of the back.
Pinprick stimulation according to a validated standardized quantitative sensory testing protocol was used in 14 female CLBP patients and 14 healthy controls (HC) matched for sex and age. Stimulus response functions to pinprick stimulation on the skin were examined at the affected back and reference sites (hand palmar and hand dorsum). Data from CLBP patients were compared with HC and with reference data from the German Research Network on Neuropathic Pain.
We found significant differences in the stimulus response functions between CLBP patients and HC. Pain ratings to the pinpricks were increased for low and moderate pinprick stimuli in CLBP patients. Importantly, this kind of specific pinprick hyperalgesia was found not only for the affected body site (back), but also for the remote reference sites (hand dorsum and hand palmar).
We interpret our results as pointing to changes in the nociceptive processing in CLBP at higher levels of the neuraxis, possibly thalamus and/or attentional control, rather than changes of spinal processing. Alternatively, there might be a higher vulnerability to noxious stimulation in CLBP patients.
Chronic Low Back Pain (CLBP); Mechanical pain thresholds; Pinprick hyperalgesia; Allodynia
The Fibromyalgia Survey Questionnaire (FSQ) assesses the key symptoms of fibromyalgia syndrome. The FSQ can be administrated in survey research and settings where the use of interviews to evaluate the number of pain sites and extent of somatic symptom intensity and tender point examination would be difficult. We validated the FSQ in a cross-sectional survey with FMS patients. In a cross-sectional survey, participants with physician diagnosis of FMS were recruited by FMS-self help organisations and nine clinical institutions of different levels of care. Participants answered the FSQ (composed by the Widespread Pain Index [WPI] and the Somatic Severity Score [SSS]) assessing the Fibromyalgia Survey Diagnostic Criteria (FSDC) and the Patient Health Questionnaire PHQ 4. American College of Rheumatology 1990 classification criteria were assessed in a subgroup of participants. 1,651 persons diagnosed with FMS were included into analysis. The acceptance of the FSQ-items ranged between 78.9 to 98.1% completed items. The internal consistency of the items of the SSS ranged between 0.75–0.82. 85.5% of the study participants met the FSDC. The concordance rate of the FSDC and ACR 1990 criteria was 72.7% in a subsample of 128 patients. The Pearson correlation of the SSS with the PHQ 4 depression score was 0.52 (p<0.0001) and with the PHQ anxiety score was 0.51 (p<0.0001) (convergent validity). 64/202 (31.7%) of the participants not meeting the FSDC criteria and 152/1283 (11.8%) of the participants meeting the FSDC criteria reported an improvement (slightly too very much better) in their health status since FMS-diagnosis (Chi2 = 55, p<0.0001) (discriminant validity). The study demonstrated the feasibility of the FSQ in a cross-sectional survey with FMS-patients. The reliability, convergent and discriminant validity of the FSQ were good. Further validation studies of the FSQ in clinical and general population settings are necessary.
Consumer surveys provide information on effectiveness and side effects of medical interventions in routine clinical care. A report of fibromyalgia syndrome (FMS) consumers has not been carried out in Europe.
The study was carried out from November 2010 to April 2011. Participants diagnosed with FMS rated the effectiveness and side effects of pharmacological and non-pharmacological FMS interventions on a 0 to 10 scale, with 10 being most efficacious (harmful). The questionnaire was distributed by the German League for people with Arthritis and Rheumatism and the German Fibromyalgia Association to their members and to all consecutive FMS patients of nine clinical centers of different levels of care.
1661 questionnaires (95% women, mean age 54 years, mean duration since FMS diagnosis 6.8 years) were analysed. The most frequently used therapies were self-management strategies, prescription pain medication and aerobic exercise. The highest average effectiveness was attributed to whole body and local warmth therapies, thermal bathes, FMS education and resting. The highest average side effects were attributed to strong opioids, local cold therapy, gamma-amino-butyric acid analogues (pregabalin and gabapentin), tramadol and opioid transdermal systems.
The German fibromyalgia consumer reports highlight the importance of non-pharmcological therapies in the long-term management of FMS, and challenges the strong recommendations for drug therapies given by FMS-guidelines.
Fibromyalgia syndrome; Consumer reports; Drugs; Non-pharmacological therapies
Network motifs, overrepresented small local connection patterns, are assumed to act
as functional meaningful building blocks of a network and, therefore, received considerable
attention for being useful for understanding design principles and functioning of networks.
We present an extension of the original approach to network motif detection in single,
directed networks without vertex labeling to the case of a sample of directed networks
with pairwise different vertex labels. A characteristic feature of this approach to network
motif detection is that subnetwork counts are derived from the whole sample and the
statistical tests are adjusted accordingly to assign significance to the counts. The associated
computations are efficient since no simulations of random networks are involved. The
motifs obtained by this approach also comprise the vertex labeling and its associated
information and are characteristic of the sample. Finally, we apply this approach to
describe the intricate topology of a sample of vertex-labeled networks which originate from
a previous EEG study, where the processing of painful intracutaneous electrical stimuli
and directed interactions within the neuromatrix of pain in patients with major depression
and healthy controls was investigated. We demonstrate that the presented approach yields
characteristic patterns of directed interactions while preserving their important topological
information and omitting less relevant interactions.
Glutamate is the main excitatory neurotransmitter of the central nervous system. Tissue-type plasminogen activator (tPA) is recognized as a modulator of glutamatergic neurotransmission. This attribute is exemplified by its ability to potentiate calcium signaling following activation of the glutamate-binding N-methyl-D-aspartate receptor (NMDAR). It has been hypothesized that tPA can directly cleave the NR1 subunit of the NMDAR and thereby potentiate NMDA-induced calcium influx. In contrast, here we show that this increase in NMDAR signaling requires tPA to be proteolytically active, but does not involve cleavage of the NR1 subunit or plasminogen. Rather, we demonstrate that enhancement of NMDAR function by tPA is mediated by a member of the Low-Density Lipoprotein Receptor (LDLR) family. Hence, this study proposes a novel functional relationship between tPA, the NMDAR, a LDLR and an unknown substrate which we suspect to be a serpin. Interestingly, whilst tPA alone failed to cleave NR1, cell-surface NMDARs did serve as an efficient and discrete proteolytic target for plasmin. Hence, plasmin and tPA can affect the NMDAR via distinct avenues. Altogether, we find that plasmin directly proteolyses the NMDAR whilst tPA functions as an indirect modulator of NMDA-induced events via LDLR engagement.
Tissue-type plasminogen activator; N-methyl-D-aspartate receptor; Low-density lipoprotein receptor family; Serine protease inhibitor; Plasmin
Hepatocellular lipid accumulation is a hallmark of non-alcoholic
fatty liver disease (NAFLD), which encompasses a spectrum ranging from simple steatosis to non-alcoholic steatohepatitis (NASH) and ultimately cirrhosis. Zinc finger protein 267 (ZNF267) belongs to the family of Kruppel-like transcription factors, which regulate diverse biological processes that include development, proliferation, and differentiation. We have previously demonstrated that ZNF267 expression is up-regulated in liver cirrhosis and is further increased in hepatocellular carcinoma (HCC). Here, we analyzed the expression of ZNF267 in tissue specimens of NAFLD patients and found a significant up-regulation compared to normal liver tissue. Noteworthy, ZNF267 mRNA was already significantly increased in steatotic liver tissue without inflammation. In line with this, incubation of primary human hepatocytes with palmitic acid induced a dose-dependent lipid accumulation and corresponding dose-dependent ZNF267 induction in vitro. Furthermore, hepatocellular lipid accumulation induced formation of reactive oxygen species (ROS), and also chemically induced ROS formation increased ZNF267 mRNA expression. In summary with previous findings, which revealed ZNF267 as pro-fibrogenic and pro-cancerogenic factor in chronic liver disease, the present study further suggests ZNF267 as promising therapeutic target particularly for NAFLD patients. In addition, it further indicates that hepatic steatosis per se has pathophysiological relevance and should not be considered as benign.
Non-alcoholic fatty liver disease; Kruppel-like factor; ZNF267
Signalling pathways are the cornerstone on understanding cell function and predicting cell behavior. Recently, logical models of canonical pathways have been optimised with high-throughput phosphoproteomic data to construct cell-type specific pathways. However, less is known on how signalling pathways can be linked to a cellular response such as cell growth, death, cytokine secretion, or transcriptional activity.
In this work, we measure the signalling activity (phosphorylation levels) and phenotypic behavior (cytokine secretion) of normal and cancer hepatocytes treated with a combination of cytokines and inhibitors. Using the two datasets, we construct "extended" pathways that integrate intracellular activity with cellular responses using a hybrid logical/data-driven computational approach. Boolean logic is used whenever a priori knowledge is accessible (i.e., construction of canonical pathways), whereas a data-driven approach is used for linking cellular behavior to signalling activity via non-canonical edges. The extended pathway is subsequently optimised to fit signalling and behavioural data using an Integer Linear Programming formulation. As a result, we are able to construct maps of primary and transformed hepatocytes downstream of 7 receptors that are capable of explaining the secretion of 22 cytokines.
We developed a method for constructing extended pathways that start at the receptor level and via a complex intracellular signalling pathway identify those mechanisms that drive cellular behaviour. Our results constitute a proof-of-principle for construction of "extended pathways" that are capable of linking pathway activity to diverse responses such as growth, death, differentiation, gene expression, or cytokine secretion.
The individual character of pharmacokinetics is of great importance in the risk assessment of new drug leads in pharmacological research. Amongst others, it is severely influenced by the properties and inter-individual variability of the enzymes and transporters of the drug detoxification system of the liver. Predicting individual drug biotransformation capacity requires quantitative and detailed models.
In this contribution we present the de novo deterministic modeling of atorvastatin biotransformation based on comprehensive published knowledge on involved metabolic and transport pathways as well as physicochemical properties. The model was evaluated on primary human hepatocytes and parameter identifiability analysis was performed under multiple experimental constraints. Dynamic simulations of atorvastatin biotransformation considering the inter-individual variability of the two major involved enzymes CYP3A4 and UGT1A3 based on quantitative protein expression data in a large human liver bank (n = 150) highlighted the variability in the individual biotransformation profiles and therefore also points to the individuality of pharmacokinetics.
A dynamic model for the biotransformation of atorvastatin has been developed using quantitative metabolite measurements in primary human hepatocytes. The model comprises kinetics for transport processes and metabolic enzymes as well as population liver expression data allowing us to assess the impact of inter-individual variability of concentrations of key proteins. Application of computational tools for parameter sensitivity analysis enabled us to considerably improve the validity of the model and to create a consistent framework for precise computer-aided simulations in toxicology.
The purpose of this study was to identify attitudes toward HPV vaccination among US women 27 to 45 years of age. A survey was mailed to 2,750 insured US women to assess perceptions of relevance or irrelevance of the HPV vaccine, the underlying reasons, and, for those reporting relevance, the likelihood of vaccination if it became available. Among the 451 eligible respondents, 304 (67.4%) reported that the HPV vaccine was relevant to them, whereas 143 (31.7%) stated that it was not at all relevant. The most common reasons for relevance were protection from cervical cancer (62.8%), vaginal cancer (58.2%), precancerous cells (55.9%), HPV (55.6%), and genital warts (46.4%). Reasons for irrelevance were most commonly being married (54.0%) or in a monogamous relationship (39.6%). Most respondents reporting relevance of the HPV vaccine were likely (33.4%) or extremely likely (37.7%) to receive the vaccine if approved for their age group.
Augmenter of liver regeneration (ALR), which is critically important in liver regeneration and hepatocyte proliferation, is highly expressed in cirrhotic livers and hepatocellular carcinomas (HCC). In the current study, the functional role of ALR in hepatocancerogenesis was analyzed in more detail. HepG2 cells, in which the cytosolic 15 kDa ALR isoform was reexpressed stably, (HepG2-ALR) were used in migration and invasion assays using modified Boyden chambers. Epithelial-mesenchymal transition (EMT) markers were determined in HepG2-ALR cells in vitro and in HepG2-ALR tumors grown in nude mice. ALR protein was quantified in HCC and nontumorous tissues by immunohistochemistry. HepG2-ALR, compared with HepG2 cells, demonstrated reduced cell motility and increased expression of the epithelial cell markers E-cadherin and Zona occludens-1 (ZO-1), whereas SNAIL, a negative regulator of E-cadherin, was diminished. Matrix metalloproteinase MMP1 and MMP3 mRNA expression and activity were reduced. HepG2-ALR cell-derived subcutaneously grown tumors displayed fewer necrotic areas, more epithelial-like cell growth and fewer polymorphisms and atypical mitotic figures than tumors derived from HepG2 cells. Analysis of tumor tissues of 53 patients with HCC demonstrated an inverse correlation of ALR protein with histological angioinvasion and grading. The 15 kDa ALR isoform was found mainly in HCC tissues without histological angioinvasion 0. In summary the present data indicate that cytosolic ALR reduces hepatoma cell migration, augments epithelial growth and, therefore, may act as an antimetastatic and EMT reversing protein.
Members of the interleukin-1 (IL-1) family of cytokines play major roles in host defense and immune system regulation in infectious and inflammatory diseases. IL-1 cytokines trigger a biological response in effector cells by assembling a heterotrimeric signaling complex with two IL-1 receptor chains, a high-affinity primary receptor and a low-affinity co-receptor. To gain insights into the signaling mechanism of the novel IL-1-like cytokine IL-33, we first solved its solution structure and then performed a detailed biochemical and structural characterization of the interaction between IL-33, its primary receptor ST2 and the co-receptor IL-1RAcP. Using NMR data, we obtained a model of the IL-33/ST2 complex in solution that is validated by small-angle X-ray scattering (SAXS) data and is similar to the IL-1β/IL-1R1 complex. We extended our SAXS analysis to the IL-33/ST2/IL-1RAcP and IL-1β/IL-1R1/IL-1RAcP complexes and propose a general model of the molecular architecture of IL-1 ternary signaling complexes.
Despite CDC recommendations regarding universal catch-up vaccination against human papillomavirus (HPV), only about ten percent of young adult women in the United States have been vaccinated. The purpose of this study was to better understand reasons for non-vaccination among insured 19-26 year-old women and to evaluate future vaccination intentions.
We used an administrative claims database from a large US managed care plan to identify women aged 19-26 for receipt of a mailed survey. From a sample of 1,375 women with no evidence of HPV vaccination from June 1, 2006 through April 30, 2007, 222 completed surveys were received, of which 185 were eligible for this analysis. The main outcome measures were unvaccinated women's attitudes and vaccine awareness, likelihood of future action regarding the vaccine, and reasons for inaction.
Among the 185 non-vaccinees, 25.4% were married, 83.2% were white, and 89.2% had a college or higher level education. The vaccine was described as very important by 32.4% of subjects, and 30.1% had discussed the vaccine with a doctor and received a doctor's recommendation. Half or fewer of respondents were "very" or "extremely" likely to discuss the vaccine with their doctor (50.0%), do additional research on the vaccine (42.6%), ask a doctor to get the vaccine (37.5%), or make an appointment to get the vaccine (27.8%), while 48.0% were "somewhat", "very", or "extremely" likely to do nothing to get the vaccine. Among the latter, reasons for taking no action included being married or in a monogamous relationship (54.9%), belief that the vaccine is too new (35.4%), not having enough information about the vaccine (31.7%), concerns about side effects (24.4%), and uncertainty about insurance coverage (24.4%).
Educational interventions may be needed to enhance HPV vaccination rates among 19-26 year-old women, particularly regarding information about vaccine safety, vaccine efficacy, insurance coverage, and the value of vaccination to women in monogamous relationships.
Nonalcoholic fatty liver disease (NAFLD) encompasses a spectrum ranging from simple steatosis to cirrhosis. Hepatocellular lipid accumulation is a hallmark of both nonalcoholic steatosis and steatohepatitis (NASH). The latter develops upon pro-inflammatory cell infiltration and is widely considered as the first relevant pathophysiological step in NAFLD-progression. The chemokine CCL5/RANTES plays an important role in the progression of hepatic inflammation and fibrosis. We here aimed to investigate its expression in NAFLD. Incubation of primary human hepatocytes with palmitic acid induced a dose-dependent lipid accumulation, and corresponding dose-dependent RANTES induction in vitro. Furthermore, we observed significantly elevated hepatic RANTES expression in a dietary model of NAFLD, in which mice were fed a high-fat diet for 12 weeks. This diet induced significant hepatic steatosis but only minimal inflammation. In contrast to the liver, RANTES expression was not induced in visceral adipose tissue of the group fed with high-fat diet. Finally, RANTES serum levels were elevated in patients with ultrasound-diagnosed NAFLD. In conclusion, our data indicate hepatocytes as cellular source of elevated hepatic as well as circulating RANTES levels in response to hepatic steatosis. Noteworthy, upregulation of RANTES in response to lipid accumulation occurs in the absence of relevant inflammation, which further indicates that hepatic steatosis per se has pathophysiological relevance and should not be considered as benign.
Hepatic steatosis; liver cirrhosis; nonalcoholic steatosis; steatohepatitis; chemokine CCL5; RANTES; inflammation; nonalcoholic fatty liver disease (NAFLD)
To examine whether adherence to osteoporosis medications can be improved by educational interventions targeted at primary care physicians (PCPs) and patients.
Post hoc analysis of data collected as part of a prospective randomized controlled trial to improve initiation of osteoporosis management such as bone mineral density testing or osteoporosis drug initiation.
The trial was conducted among patients at risk for osteoporosis enrolled in Horizon Blue Cross Blue Shield of New Jersey. For a 3-month period, randomly selected PCPs and their patients received education about osteoporosis diagnosis and treatment. The PCPs received face-to-face education by trained pharmacists, while patients received letters and automated telephone calls. The control group received no education. We assessed medication adherence during 10 months following the start of the intervention using the medication possession ratio (MPR), the ratio of available medication to the total number of days studied.
These analyses included 1867 patients (972 randomized to the intervention group and 875 to the control group) and their 436 PCPs. During 10 months following the intervention, the median MPRs were 74% (interquartile range [IQR], 19%–93%) for the intervention group and 73% (IQR, 0%–93%) for the control group (P =.18). The median times until medication discontinuation after the intervention were 85 days (IQR, 58–174 days) for the intervention group and 79 days (IQR, 31–158 days) for the control group.
The educational intervention did not significantly improve medication compliance or persistence with osteoporosis drugs.
Tissue factor (TF) and its inhibitor tissue factor pathway inhibitor (TFPI) are the main regulators of the initiation of the coagulation process, important in atherothrombosis. In this study we have investigated the frequency of six known TF and TFPI single nucleotide polymorphisms (SNPs) in CHD patients as compared to healthy individuals. These genotypes and the phenotypes (TF, TFPI free and total antigen) were evaluated with special reference to gender and diabetes in the CHD population.
Patients with angiographically verified CHD (n = 1001; 22% women, 20% diabetics), and 204 healthy controls (28% women), were included. The investigated SNPs were: TF -1812C/T and TF -603A/G in the 5'upstream region, TF 5466A/G in intron 2, TFPI -399C/T and TFPI -287T/C in the 5'upstream region and the TFPI -33T/C in intron 7.
No significant differences in frequencies between the CHD population and the controls of any polymorphisms were observed. In the CHD population, the TF 5466 A/G SNP were significantly more frequent in women as compared to men (p < 0.001). The TF-1812C/T and the TF-603A/G SNPs were significantly more frequent in women without type-2 diabetes compared to those with diabetes (p < 0.018, both), and the heterozygous genotypes were associated with significantly lower TF plasma levels compared to the homozygous genotypes (p < 0.02, both).
The TFPI-399C/T and the TFPI-33T/C SNPs were associated with lower and higher TFPI total antigen levels, respectively (p < 0.001, both).
Genetic variations in the TF and TFPI genes seem to be associated with gender and type-2 diabetes, partly affecting their respective phenotypes.
Nonalcoholic fatty liver disease (NAFLD) is characterized by hepatic lipid accumulation which starts with simple hepatic steatosis and may progress toward inflammation (nonalcoholic steatohepatitis [NASH]). Fatty acid synthase (FASN) catalyzes the last step in fatty acid biosynthesis, and thus, it is believed to be a major determinant of the maximal hepatic capacity to generate fatty acids by de novo lipogenesis. The aim of this study was to analyze the correlation between hepatic steatosis and inflammation with FASN expression. In vitro incubation of primary human hepatocytes with fatty acids dose-dependently induced cellular lipid-accumulation and FASN expression, while stimulation with TNF did not affect FASN levels. Further, hepatic FASN expression was significantly increased in vivo in a murine model of hepatic steatosis without significant inflammation but not in a murine NASH model as compared to control mice. Also, FASN expression was not increased in mice subjected to bile duct ligation, an experimental model characterized by severe hepatocellular damage and inflammation. Furthermore, FASN expression was analyzed in 102 human control or NAFLD livers applying tissue micro array technology and immunohistochemistry, and correlated significantly with the degree of hepatic steatosis, but not with inflammation or ballooning of hepatocytes. Quantification of FASN mRNA expression in human liver samples confirmed significantly higher FASN levels in hepatic steatosis but not in NASH, and expression of SREBP1, which is the main transcriptional regulator of FASN, paralleled FASN expression levels in human and experimental NAFLD. In conclusion, the transcriptional induction of FASN expression in hepatic steatosis is impaired in NASH, while hepatic inflammation in the absence of steatosis does not affect FASN expression, suggesting that FASN may serve as a new diagnostic marker or therapeutic target for the progression of NAFLD.
Nonalcoholic fatty liver disease (NAFLD); Fatty acid synthase (FASN); nonalcoholic steatohepatitis (NASH); SREBP1; expression; immunohistochemistry
The linker histone H1 has a key role in establishing and maintaining higher order chromatin structure and in regulating gene expression. Mammals express up to 11 different H1 variants, with H1.2 and H1.4 being the predominant ones in most somatic cells. Like core histones, H1 has high levels of covalent modifications; however, the full set of modifications and their biological role are largely unknown.
In this study, we used a candidate screen to identify enzymes that methylate H1 and to map their corresponding methylation sites. We found that the histone lysine methyltransferases G9a/KMT1C and Glp1/KMT1D methylate H1.2 in vitro and in vivo, and we mapped this novel site to lysine 187 (H1.2K187) in the C-terminus of H1. This H1.2K187 methylation is variant-specific. The main target for methylation by G9a in H1.2, H1.3, H1.5 and H1.0 is in the C-terminus, whereas H1.4 is preferentially methylated at K26 (H1.4K26me) in the N-terminus. We found that the readout of these marks is different; H1.4K26me can recruit HP1, but H1.2K187me cannot. Likewise, JMJD2D/KDM4 only reverses H1.4K26 methylation, clearly distinguishing these two methylation sites. Further, in contrast to C-terminal H1 phosphorylation, H1.2K187 methylation level is steady throughout the cell cycle.
We have characterised a novel methylation site in the C-terminus of H1 that is the target of G9a/Glp1 both in vitro and in vivo. To our knowledge, this is the first demonstration of variant-specific histone methylation by the same methyltransferases, but with differing downstream readers, thereby supporting the hypothesis of H1 variants having specific functions.
Glyoxalases (Glo1 and Glo2) are involved in the glycolytic pathway by detoxifying the reactive methylglyoxal (MGO) into D-lactate in a two-step reaction using glutathione (GSH) as cofactor. Inhibitors of glyoxalases are considered as anti-inflammatory and anti-carcinogenic agents. The recent finding that various polyphenols modulate Glo1 activity has prompted us to assess curcumin's potency as an Glo1 inhibitor.
Cultures of whole blood cells and tumor cell lines (PC-3, JIM-1, MDA-MD 231 and 1321N1) were set up to investigate the effect of selected polyphenols, including curcumin, on the LPS-induced cytokine production (cytometric bead-based array), cell proliferation (WST-1 assay), cytosolic Glo1 and Glo2 enzymatic activity, apoptosis/necrosis (annexin V-FITC/propidium iodide staining; flow cytometric analysis) as well as GSH and ATP content. Results of enzyme kinetics revealed that curcumin, compared to the polyphenols quercetin, myricetin, kaempferol, luteolin and rutin, elicited a stronger competitive inhibitory effect on Glo1 (Ki = 5.1±1.4 µM). Applying a whole blood assay, IC50 values of pro-inflammatory cytokine release (TNF-α, IL-6, IL-8, IL-1β) were found to be positively correlated with the Ki-values of the aforementioned polyphenols. Moreover, whereas curcumin was found to hamper the growth of breast cancer (JIMT-1, MDA-MB-231), prostate cancer PC-3 and brain astrocytoma 1321N1 cells, no effect on growth or vitality of human primary hepatocytes was elucidated. Curcumin decreased D-lactate release by tumor cells, another clue for inhibition of intracellular Glo1.
The results described herein provide new insights into curcumin's biological activities as they indicate that inhibition of Glo1 by curcumin may result in non-tolerable levels of MGO and GSH, which, in turn, modulate various metabolic cellular pathways including depletion of cellular ATP and GSH content. This may account for curcumin's potency as an anti-inflammatory and anti-tumor agent. The findings support the use of curcumin as a potential therapeutic agent.
Despite recommendations, osteoporosis screening rates among women aged 65 years and older remain low. We present results from a clustered, randomized trial evaluating patient mailed reminders, alone and in combination with physician prompts, to improve osteoporosis screening and treatment.
Primary care clinics (n = 15) were randomized to usual care, mailed reminders alone, or mailed reminders with physician prompts. Study patients were females aged 65–89 years (N = 10,354). Using automated clinical and pharmacy data, information was collected on bone mineral density testing, pharmacy dispensings, and other patient characteristics. Unadjusted/adjusted differences in testing and treatment were assessed using generalized estimating equation approaches.
Osteoporosis screening rates were 10.8% in usual care, 24.1% in mailed reminder, and 28.9% in mailed reminder with physician prompt. Results adjusted for differences at baseline indicated that mailed reminders significantly improved testing rates compared to usual care, and that the addition of prompts further improved testing. This effect increased with patient age. Treatment rates were 5.2% in usual care, 8.4% in mailed reminders, and 9.1% in mailed reminders with prompt. No significant differences were found in treatment rates between those receiving mailed reminders alone or in combination with physician prompts. However, women receiving usual care were significantly less likely to be treated.
The use of mailed reminders, either alone or with physician prompts, can significantly improve osteoporosis screening and treatment rates among insured primary care patients (Clinical Trials.gov number NCT00139425).
mailed reminders; osteoporosis; physician prompts; screening; bone mineral density testing
Despite accurate diagnostic tests and effective therapies, the management of osteoporosis has been observed to be suboptimal in many settings. We tested the effectiveness of an intervention to improve care in patients at-risk of osteoporosis.
Randomized controlled trial.
Primary care physicians and their patients at-risk of osteoporosis, including women 65 years and over, men and women 45 and over with a prior fracture, and men and women 45 and over who recently used ≥90 days of oral glucocorticoids.
A multifaceted program of education and reminders delivered to primary care physicians as well as mailings and automated telephone calls to patients. Outcome: Either undergoing a bone mineral density (BMD) testing or filling a prescription for a bone-active medication during the 10 months of follow-up.
After the intervention, 144 (14%) patients in the intervention group and 97 (10%) patients in the control group received either a BMD test or filled a prescription for an osteoporosis medication. This represents a 4% absolute increase and a 45% relative increase (95% confidence interval 9–93%, p = 0.01) in osteoporosis management between the intervention and control groups. No differences between groups were observed in the incidence of fracture.
An intervention targeting primary care physicians and their at-risk patients increased the frequency of BMD testing and/or filling prescriptions for osteoporosis medications. However, the absolute percentage of at-risk patients receiving osteoporosis management remained low.
osteoporosis; cluster randomized controlled trial; academic detailing; quality improvement
Hepatic homeostasis is essential for survival in critically ill and burned patients. Insulin administration improves survival and decreases infections in these patients. To determine the molecular mechanisms, the aim of the present study was to establish a stress model using primary human hepatocytes (PHHs) and to study the effects of insulin on the hepatic inflammatory signaling cascade. Liver tissue was obtained from general surgical patients, and PHHs were isolated and maintained in culture. Primary hepatocyte cultures were challenged with various doses of lipopolysaccharide (LPS), and the inflammatory signal transcription cascade was determined by real-time PCR. In subsequent experiments, primary hepatocyte cultures were challenged with LPS and insulin was added in various doses. Glucose was determined by colorimetric assays. PHHs treated with 100 μg/mL LPS showed a profound inflammatory reaction with increased expression of interleukin (IL)-6, IL-10, IL-1β, tumor necrosis factor (TNF), and signal transducer and activator of transcription 5 (STAT-5). Insulin at 10 IU/mL significantly decreased IL-6, TNF, and IL-1β at pretranslational levels, an effect associated with decreased STAT-5 mRNA expression (P < 0.05). Glucose concentration and cellular metabolic activity were not different between controls and insulin-treated cells. Based on our results, we suggest that primary hepatocyte cultures can be used to study the effect of LPS on the inflammatory cascade. Insulin decreases hepatic cytokine expression, which is associated with decreased STAT-5 expression.