Isolation of Primary Human Hepatocytes from Normal and Diseased Liver Tissue: A One Hundred Liver Experience

Successful and consistent isolation of primary human hepatocytes remains a challenge for both cell-based therapeutics/transplantation and laboratory research. Several centres around the world have extensive experience in the isolation of human hepatocytes from non-diseased livers obtained from donor liver surplus to surgical requirement or at hepatic resection for tumours. These livers are an important but limited source of cells for therapy or research. The capacity to isolate cells from diseased liver tissue removed at transplantation would substantially increase availability of cells for research. However no studies comparing the outcome of human hepatocytes isolation from diseased and non-diseased livers presently exist. Here we report our experience isolating human hepatocytes from organ donors, non-diseased resected liver and cirrhotic tissue. We report the cell yields and functional qualities of cells isolated from the different types of liver and demonstrate that a single rigorous protocol allows the routine harvest of good quality primary hepatocytes from the most commonly accessible human liver tissue samples

Need for weight management in Switzerland: findings from National Blood Pressure Week 2009

<p>Abstract</p> <p>Background</p> <p>The Swiss Health Survey (SHS) provides the only source of data for monitoring overweight and obesity in the general population in Switzerland. However, this survey reports body mass index (BMI) based on self-reported height and weight, and is therefore subject to measurement errors. Moreover, it is not possible to differentiate between overall and abdominal overweight. In this study, we aimed to gain a better understanding of the need for weight management in the general population of Switzerland by exploring and comparing prevalence rates of BMI and waist circumference (WC) based on physical measurements by trained observers, based on data from the 2009 National Blood Pressure Week (NBPW).</p> <p>Methods</p> <p>Sample selection was based on a one-stage cluster design. A total of 385 pharmacies representing 3,600 subjects were randomly selected from pharmacies participating in NBPW. BMI measures based on physical weight and height (NBPW) were compared with self-reported BMI measures from the SHS. BMI and WC measurements from NBPW were then used to produce population estimates of overweight and obesity.</p> <p>Results</p> <p>BMI-based overall prevalence of overweight and obesity was 43.6%, which was 4.7% higher than the value based on the respective SHS data. Overweight and obesity were more common in men (54.3%) than in women (33.5%). However, the overall prevalence of increased WC in the general population was estimated to be 64.4%, with more women (68.4%) than men (60.1%) exhibiting a WC above the threshold. The prevalence of subjects requiring weight management in the Swiss population remained high, even after adjusting WC for false positive and negative cases.</p> <p>Conclusions</p> <p>Firstly, it may be more appropriate for health promotion programs to address the wider group identified by WC, which includes subjects who need to reduce their weight, or gain no further weight. Secondly, the gender differences are reversed depending on the use of WC or BMI to identify subjects suitable for health promotion programs; more women than men are identified by WC, and more men than women using BMI. These differences should be accounted for in gender-specific health promotion programs.</p

Monitoring integrity and localization of modified single-stranded RNA oligonucleotides using ultrasensitive fluorescence methods

Short single-stranded oligonucleotides represent a class of promising therapeutics with diverse application areas. Antisense oligonucleotides, for example, can interfere with various processes involved in mRNA processing through complementary base pairing. Also RNA interference can be regulated by antagomirs, single-stranded siRNA and single-stranded microRNA mimics. The increased susceptibility to nucleolytic degradation of unpaired RNAs can be counteracted by chemical modification of the sugar phosphate backbone. In order to understand the dynamics of such single-stranded RNAs, we investigated their fate after exposure to cellular environment by several fluorescence spectroscopy techniques. First, we elucidated the degradation of four differently modified, dual-dye labeled short RNA oligonucleotides in HeLa cell extracts by fluorescence correlation spectroscopy, fluorescence cross-correlation spectroscopy and Forster resonance energy transfer. We observed that the integrity of the oligonucleotide sequence correlates with the extent of chemical modifications. Furthermore, the data showed that nucleolytic degradation can only be distinguished from unspecific effects like aggregation, association with cellular proteins, or intramolecular dynamics when considering multiple measurement and analysis approaches. We also investigated the localization and integrity of the four modified oligonucleotides in cultured HeLa cells using fluorescence lifetime imaging microscopy. No intracellular accumulation could be observed for unmodified oligonucleotides, while completely stabilized oligonucleotides showed strong accumulation within HeLa cells with no changes in fluorescence lifetime over 24 h. The integrity and accumulation of partly modified oligonucleotides was in accordance with their extent of modification. In highly fluorescent cells, the oligonucleotides were transported to the nucleus. The lifetime of the RNA in the cells could be explained by a balance between release of the oligonucleotides from endosomes, degradation by RNases and subsequent depletion from the cells

Changes in gut bacterial populations and their translocation into liver and ascites in alcoholic liver cirrhotics

Background The liver is the first line of defence against continuously occurring influx of microbial-derived products and bacteria from the gut. Intestinal bacteria have been implicated in the pathogenesis of alcoholic liver cirrhosis. Escape of intestinal bacteria into the ascites is involved in the pathogenesis of spontaneous bacterial peritonitis, which is a common complication of liver cirrhosis. The association between faecal bacterial populations and alcoholic liver cirrhosis has not been resolved. Methods Relative ratios of major commensal bacterial communities (Bacteroides spp., Bifidobacterium spp., Clostridium leptum group, Enterobactericaea and Lactobacillus spp.) were determined in faecal samples from post mortem examinations performed on 42 males, including cirrhotic alcoholics (n = 13), non-cirrhotic alcoholics (n = 15), non-alcoholic controls (n = 14) and in 7 healthy male volunteers using real-time quantitative PCR (RT-qPCR). Translocation of bacteria into liver in the autopsy cases and into the ascites of 12 volunteers with liver cirrhosis was also studied with RT-qPCR. CD14 immunostaining was performed for the autopsy liver samples. Results Relative ratios of faecal bacteria in autopsy controls were comparable to those of healthy volunteers. Cirrhotics had in median 27 times more bacterial DNA of Enterobactericaea in faeces compared to the healthy volunteers (p = 0.011). Enterobactericaea were also the most common bacteria translocated into cirrhotic liver, although there were no statistically significant differences between the study groups. Of the ascites samples from the volunteers with liver cirrhosis, 50% contained bacterial DNA from Enterobactericaea, Clostridium leptum group or Lactobacillus spp.. The total bacterial DNA in autopsy liver was associated with the percentage of CD14 expression (p = 0.045). CD14 expression percentage in cirrhotics was significantly higher than in the autopsy controls (p = 0.004). Conclusions Our results suggest that translocation of intestinal bacteria into liver may be involved as a one factor in the pathogenesis of alcoholic liver cirrhosis.BioMed Central open acces