Single molecule microscopy in 3D cell cultures and tissues

From the onset of the first microscopic visualization of single fluorescent molecules in living cells at the beginning of this century, to the present, almost routine application of single molecule microscopy, the method has well-proven its ability to contribute unmatched detailed insight into the heterogeneous and dynamic molecular world life is composed of. Except for investigations on bacteria and yeast, almost the entire story of success is based on studies on adherent mammalian 2D cell cultures. However, despite this continuous progress, the technique was not able to keep pace with the move of the cell biology community to adapt 3D cell culture models for basic research, regenerative medicine, or drug development and screening. In this review, we will summarize the progress, which only recently allowed for the application of single molecule microscopy to 3D cell systems and give an overview of the technical advances that led to it. While initially posing a challenge, we finally conclude that relevant 3D cell models will become an integral part of the on-going success of single molecule microscopy.

Stochastic dynamics of interacting haematopoietic stem cell niche lineages

Since we still know very little about stem cells in their natural environment, it is useful to explore their dynamics through modelling and simulation, as well as experimentally. Most models of stem cell systems are based on deterministic differential equations that ignore the natural heterogeneity of stem cell populations. This is not appropriate at the level of individual cells and niches, when randomness is more likely to affect dynamics. In this paper, we introduce a fast stochastic method for simulating a metapopulation of stem cell niche lineages, that is, many sub-populations that together form a heterogeneous metapopulation, over time. By selecting the common limiting timestep, our method ensures that the entire metapopulation is simulated synchronously. This is important, as it allows us to introduce interactions between separate niche lineages, which would otherwise be impossible. We expand our method to enable the coupling of many lineages into niche groups, where differentiated cells are pooled within each niche group. Using this method, we explore the dynamics of the haematopoietic system from a demand control system perspective. We find that coupling together niche lineages allows the organism to regulate blood cell numbers as closely as possible to the homeostatic optimum. Furthermore, coupled lineages respond better than uncoupled ones to random perturbations, here the loss of some myeloid cells. This could imply that it is advantageous for an organism to connect together its niche lineages into groups. Our results suggest that a potential fruitful empirical direction will be to understand how stem cell descendants communicate with the niche and how cancer may arise as a result of a failure of such communication.

Atmospheric pressure gas plasma-induced colorectal cancer cell death is mediated by Nox2-ASK1 apoptosis pathways and oxidative stress is mitigated by Srx-Nrf2 anti-oxidant system

Atmospheric pressure gas plasma (AGP) generates reactive oxygen species (ROS) that induce apoptosis in cultured cancer cells. The majority of cancer cells develop a ROS-scavenging anti-oxidant system regulated by Nrf2, which confers resistance to ROS-mediated cancer cell death. Generation of ROS is involved in the AGP-induced cancer cell death of several colorectal cancer cells (Caco2, HCT116 and SW480) by activation of ASK1-mediated apoptosis signaling pathway without affecting control cells (human colonic sub-epithelial myofibroblasts; CO18, human fetal lung fibroblast; MRC5 and fetal human colon; FHC). However, the identity of an oxidase participating in AGP-induced cancer cell death is unknown. Here, we report that AGP up-regulates the expression of Nox2 (NADPH oxidase) to produce ROS. RNA interference designed to target Nox2 effectively inhibits the AGP-induced ROS production and cancer cell death. In some cases both colorectal cancer HT29 and control cells showed resistance to AGP treatment. Compared to AGP-sensitive Caco2 cells, HT29 cells show a higher basal level of the anti-oxidant system transcriptional regulator Nrf2 and its target protein sulfiredoxin (Srx) which are involved in cellular redox homeostasis. Silencing of both Nrf2 and Srx sensitized HT29 cells, leads to ROS overproduction and decreased cell viability. This indicates that in HT29 cells, Nrf2/Srx axis is a protective factor against AGP-induced oxidative stress. The inhibition of Nrf2/Srx signaling should be considered as a central target in drug-resistant colorectal cancer treatments.

Tissue requirements for establishing long-term CD4+ T cell-mediated immunity following Leishmania donovani infection

Organ-specific immunity is a feature of many infectious diseases, including visceral leishmaniasis caused by Leishmania donovani. Experimental visceral leishmaniasis in genetically susceptible mice is characterized by an acute, resolving infection in the liver and chronic infection in the spleen. CD4+ T cell responses are critical for the establishment and maintenance of hepatic immunity in this disease model, but their role in chronically infected spleens remains unclear. In this study, we show that dendritic cells are critical for CD4+ T cell activation and expansion in all tissue sites examined. We found that FTY720-mediated blockade of T cell trafficking early in infection prevented Ag-specific CD4+ T cells from appearing in lymph nodes, but not the spleen and liver, suggesting that early CD4+ T cell priming does not occur in liver-draining lymph nodes. Extended treatment with FTY720 over the first month of infection increased parasite burdens, although this associated with blockade of lymphocyte egress from secondary lymphoid tissue, as well as with more generalized splenic lymphopenia. Importantly, we demonstrate that CD4+ T cells are required for the establishment and maintenance of antiparasitic immunity in the liver, as well as for immune surveillance and suppression of parasite outgrowth in chronically infected spleens. Finally, although early CD4+ T cell priming appeared to occur most effectively in the spleen, we unexpectedly revealed that protective CD4+ T cell-mediated hepatic immunity could be generated in the complete absence of all secondary lymphoid tissues.

Comparison of oropharyngeal and oral cavity squamous cell cancer incidence and trends in New Zealand and Queensland, Australia

Background Increases in the incidence of squamous cell oropharyngeal cancer (OPC) have been reported from some countries, but have not been assessed in Australia or New Zealand. This study examines trends for squamous cell OPC and squamous cell oral cavity cancer (OCC) in two similarly sized populations, New Zealand and Queensland, Australia. Methods Incidence data for 1982–2010 were obtained from the respective population-based cancer registries for squamous cell OPC and OCC, by subsite, sex, and age. Time trends and annual percentage changes (APCs) were assessed by joinpoint regression. Results The incidence rates of squamous cell OPC in males in New Zealand since 2005 and Queensland since 2006 have increased rapidly, with APCs of 11.9% and 10.6% respectively. The trends were greatest at ages 50–69 and followed more gradual increases previously. In females, rates increased by 2.1% per year in New Zealand from 1982, but by only 0.9% (not significant) in Queensland. In contrast, incidence rates for OCC decreased by 1.2% per year in males in Queensland since 1982, but remained stable for females in Queensland and for both sexes in New Zealand. Overall, incidence rates for both OCC and OPC were substantially higher in Queensland than in New Zealand. In males in both areas, OPC incidence is now higher than that of OCC. Conclusions Incidence rates of squamous cell OPC have increased rapidly in men, while rates of OCC have been stable or reducing, showing distinct etiologies. This has both clinical and public health importance, including implications for the extension of human papilloma virus (HPV) vaccination to males.

Analysis of focal adhesion turnover: A quantitative live-cell imaging example

Recent advances in optical and fluorescent protein technology have rapidly raised expectations in cell biology, allowing quantitative insights into dynamic intracellular processes like never before. However, quantitative live-cell imaging comes with many challenges including how best to translate dynamic microscopy data into numerical outputs that can be used to make meaningful comparisons rather than relying on representative data sets. Here, we use analysis of focal adhesion turnover dynamics as a straightforward specific example on how to image, measure, and analyze intracellular protein dynamics, but we believe this outlines a thought process and can provide guidance on how to understand dynamic microcopy data of other intracellular structures.

High incidence of contaminating maternal cell overgrowth in human placental mesenchymal stem/stromal cell cultures: A systematic review

Placenta is a readily accessible translationally advantageous source of mesenchymal stem/stromal cells (MSCs) currently used in cryobanking and clinical trials. MSCs cultured from human chorion have been widely assumed to be fetal in origin, despite evidence that placental MSCs may be contaminated with maternal cells, resulting in entirely maternally derived MSC cultures. To document the frequency and determinants of maternal cell contamination in chorionic MSCs, we undertook a PRISMA-compliant systematic review of publications in the PubMed, Medline, and Embase databases (January 2000 to July 2013) on placental and/or chorionic MSCs from uncomplicated pregnancies. Of 147 studies, only 26 (18%) investigated fetal and/or maternal cell origin. After excluding studies that did not satisfy minimal MSC criteria, 7 of 15 informative studies documented MSC cultures as entirely fetal, a further 7 studies reported cultured human chorionic MSC populations to be either maternal (n=6) or mixed (n=1), whereas 1 study separately cultured pure fetal and pure maternal MSC from the same placenta. Maternal cell contamination was associated with term and chorionic membrane samples and greater passage number but was still present in 30% of studies of chorionic villous MSCs. Although most studies assume fetal origin for MSCs sourced from chorion, this systematic review documents a high incidence of maternal-origin MSC populations in placental MSC cultures. Given that fetal MSCs have more primitive properties than adult MSCs, our findings have implications for clinical trials in which knowledge of donor and tissue source is pivotal. We recommend sensitive methods to quantitate the source and purity of placental MSCs.

Dissecting the prostate cancer stem cell niche inside the bone marrow

Prostate cancer frequently metastasizes to bone, which becomes incurable; yet how cancer cells manage to migrate and grow inside the bone remains unknown. In this study I have discovered that both bone and fat cells within the bone marrow actively promote the survival and expansion of prostate cancer cells, and have subsequently developed approaches that can effectively inhibit these processes. Therefore, my work offers opportunities for the development of new prognostic and therapeutic approaches against metastatic prostate cancer and have the potential for improving the treatment outcome of the patients.

Utility of temporal artery biopsy samples for genome-wide analysis of giant cell arteritis

Giant Cell Arteritis (GCA) is the most common vasculitis affecting the elderly. Archived formalin-fixed paraffin-embedded (FFPE) temporal artery biopsy (TAB) specimens potentially represent a valuable resource for large-scale genetic analysis of this disease. FFPE TAB samples were obtained from 12 patients with GCA. Extracted TAB DNA was assessed by real time PCR before restoration using the Illumina HD FFPE Restore Kit. Paired FFPE-blood samples were genotyped on the Illumina OmniExpress FFPE microarray. The FFPE samples that passed stringent quality control measures had a mean genotyping success of >97%. When compared with their matching peripheral blood DNA, the mean discordant heterozygote and homozygote single nucleotide polymorphisms calls were 0.0028 and 0.0003, respectively, which is within the accepted tolerance of reproducibility. This work demonstrates that it is possible to successfully obtain high-quality microarray-based genotypes FFPE TAB samples and that this data is similar to that obtained from peripheral blood.

Long-term effect of childhood liver transplantation on body cell mass

Malnutrition is common in end-stage liver disease, but a correction after transplantation is expected. Body cell mass (BCM) assessment using total body potassium (TBK) measurements is considered the gold standard for assessing nutritional status. The aim of this study was to examine the BCM and, therefore, nutritional status of long-term survivors after childhood liver transplantation. © 2014 American Association for the Study of Liver Diseases.

Genetic risk and a primary role for cell-mediated immune mechanisms in multiple sclerosis

Multiple sclerosis is a common disease of the central nervous system in which the interplay between inflammatory and neurodegenerative processes typically results in intermittent neurological disturbance followed by progressive accumulation of disability. Epidemiological studies have shown that genetic factors are primarily responsible for the substantially increased frequency of the disease seen in the relatives of affected individuals, and systematic attempts to identify linkage in multiplex families have confirmed that variation within the major histocompatibility complex (MHC) exerts the greatest individual effect on risk. Modestly powered genome-wide association studies (GWAS) have enabled more than 20 additional risk loci to be identified and have shown that multiple variants exerting modest individual effects have a key role in disease susceptibility. Most of the genetic architecture underlying susceptibility to the disease remains to be defined and is anticipated to require the analysis of sample sizes that are beyond the numbers currently available to individual research groups. In a collaborative GWAS involving 9,772 cases of European descent collected by 23 research groups working in 15 different countries, we have replicated almost all of the previously suggested associations and identified at least a further 29 novel susceptibility loci. Within the MHC we have refined the identity of the HLA-DRB1 risk alleles and confirmed that variation in the HLA-A gene underlies the independent protective effect attributable to the class I region. Immunologically relevant genes are significantly overrepresented among those mapping close to the identified loci and particularly implicate T-helper-cell differentiation in the pathogenesis of multiple sclerosis. © 2011 Macmillan Publishers Limited. All rights reserved.

The multiple sclerosis whole blood mRNA transcriptome and genetic associations indicate dysregulation of specific T cell pathways in pathogenesis

Multiple sclerosis (MS) is an autoimmune disease with a genetic component, caused at least in part by aberrant lymphocyte activity. The whole blood mRNA transcriptome was measured for 99 untreated MS patients: 43 primary progressive MS, 20 secondary progressive MS, 36 relapsing remitting MS and 45 age-matched healthy controls. The ANZgene Multiple Sclerosis Genetics Consortium genotyped more than 300 000 SNPs for 115 of these samples. Transcription from genes on translational regulation, oxidative phosphorylation, immune synapse and antigen presentation pathways was markedly increased in all forms of MS. Expression of genes tagging T cells was also upregulated (P < 10-12) in MS. A T cell gene signature predicts disease state with a concordance index of 0.79 with age and gender as co-variables, but the signature is not associated with clinical course or disability. The ANZgene genome wide association screen identified two novel regions with genome wide significance: one encoding the T cell co-stimulatory molecule, CD40; the other a region on chromosome 12q13-14. The CD40 haplotype associated with increased MS susceptibility has decreased gene expression in MS (P < 0.0007). The second MS susceptibility region includes 17 genes on 12q13-14 in tight linkage disequilibrium. Of these, only 13 are expressed in leukocytes, and of these the expression of one, FAM119B, is much lower in the susceptibility haplotype (P tdthomlt; 10-14). Overall, these data indicate dysregulation of T cells can be detected in the whole blood of untreated MS patients, and supports targeting of activated T cells in therapy for all forms of MS.

Adjusting body cell mass for size in women of differing nutritional status

Background: Body cell mass (BCM) may be estimated in clinical practice to assess functional nutritional status, eg, in patients with anorexia nervosa. Interpretation of the data, especially in younger patients who are still growing, requires appropriate adjustment for size. Previous investigations of this general issue have addressed chemical rather than functional components of body composition and have not considered patients at the extremes of nutritional status, in whom the ability to make longitudinal comparisons is of particular importance. Objective: Our objective was to determine the power by which height should be raised to adjust BCM for height in women of differing nutritional status. Design: BCM was estimated by K-40 counting in 58 healthy women, 33 healthy female adolescents, and 75 female adolescents with anorexia nervosa. The relation between BCM and height was explored in each group by using log-log regression analysis. Results: The powers by which height should be raised to adjust BCM,A,ere 1.73. 1.73, and 2.07 in the women, healthy female adolescents, and anorexic female adolescents, respectively. A simplified version of the index, BCM/height(2), was appropriate for all 3 categories and was negligibly correlated with height. Conclusions: In normal-weight women, the relation between height and BCM is consistent with that reported previously between height and fat-free mass. Although the consistency of the relation between BCM and fat-free mass decreases with increasing weight loss, the relation between height and BCM is not significantly different between normal-weight and underweight women. The index BCM/height(2) is easy to calculate and applicable to both healthy and underweight women. This information may be helpful in interpreting body-composition data in clinical practice.

Energy expenditure and the body cell mass in cystic fibrosis

Poor nutritional status in patients with cystic fibrosis (CF) is associated with severe lung disease, and possible causative factors include inadequate intake, malabsorption, and increased energy requirements. Body cell mass (which can be quantified by measurement of total body potassium) provides an ideal standard for measurements of energy expenditure. The aim of this study was to compare resting energy expenditure (REE) in patients with CF with both predicted values and age-matched healthy children and to determine whether REE was related to either nutritional status or pulmonary function. REE was measured by indirect calorimetry and body cell mass by scanning with total body potassium in 30 patients with CF (12 male, mean age = 13.07 ± 0.55 y) and 18 healthy children (six male, mean age = 12.56 ± 1.25 y). Nutritional status was expressed as a percentage of predicted total body potassium. Lung function was measured in the CF group by spirometry and expressed as the percentage of predicted forced expiratory volume in 1 s. Mean REE was significantly increased in the patients with CF compared with healthy children (119.3 ± 3.1% predicted versus 103.6 ± 5% predicted, P < 0.001) and, using multiple regression techniques, REE for total body potassium was significantly increased in patients with CF (P = 0.0001). There was no relation between REE and nutritional status or pulmonary disease status in the CF group. In conclusion, REE is increased in children and adolescents with CF but is not directly related to nutritional status or pulmonary disease.

Nutritional status of children with cystic fibrosis measured by total body potassium as a marker of body cell mass: Lack of sensitivity of anthropometric measures

Objective: To investigate measures aimed at defining the nutritional status of cystic fibrosis (CF) populations, this study compared standard anthropometric measurements and total body potassium (TBK) as indicators of malnutrition. Methods: Height, weight, and TBK measurements of 226 children with CF from Royal Children's Hospital, Brisbane, Australia, were analyzed. Z scores for height for age, weight for age, and weight for height were analyzed by means of the National Centre for Health Statistics reference. TBK was measured by means of whole body counting and compared with predicted TBK for age. Two criteria were evaluated with respect to malnutrition: (1) a z score < -2.0 and (2) a TBK for age <80% of predicted. Results: Males and females with CF had lower mean height-for-age and weight-for-age z scores than the National Centre for Health Statistics reference (P < .01), but mean weight-for-height z score was not significantly different. There were no significant gender differences. According to anthropometry, only 7.5% of this population were underweight and 7.6% were stunted. However, with TBK as an indicator of nutritional status, 29.9% of males and 22.0% of females were malnourished. Conclusion: There are large differences in the percentage of patients with CF identified as malnourished depending on whether anthropometry or body composition data are used as the nutritional indicator. At an individual level, weight-based indicators are not sensitive indicators of suboptimal nutritional status in CF, significantly underestimating the extent of malnutrition. Current recommendations in which anthropometry is used as the indicator of malnutrition in CF should be revised.