Normal Human Populations Research Articles

Field size effects on DNA damage and proliferation in normal human cell populations irradiated with X-ray microbeams

To clarify the health risks of internal radiation exposure, it is important to investigate the radiological effects of local exposure at cell levels from radioactive materials taken up by organs. Focusing on the response of cell populations post-irradiation, X-ray microbeams are very effective at reproducing the effects of local exposure within an internal exposure in vitro. The present study aims to clarify the effects of local exposure by investigating the response of normal human cell (MRC-5) populations irradiated with X-ray microbeams of different beam sizes to DNA damage. The populations of MRC-5 were locally irradiated with X-ray microbeams of 1 Gy at 0.02–1.89 mm2 field sizes, and analyzed whether the number of 53BP1 foci as DSB (DNA double strand break) per cell changed with the field size. We found that even at the same dose, the number of DSB per cell increased depending on the X-irradiated field size on the cell population. This result indicated that DNA damage repair of X-irradiated cells might be enhanced in small size fields surrounded by non-irradiated cells. This study suggests that X-irradiated cells received some signal (a rescue signal) from surrounding non-irradiated cells may be involved in the response of cell populations post-irradiation.

Ki-67 index as a predictor of vestibular schwannoma regrowth or recurrence.

Ki-67 is a monoclonal antibody that provides a means of evaluating the growth fraction of normal and neoplastic human cell populations. A Ki-67 index of less than 3 per cent is expected for a typical schwannoma. Vestibular schwannomas with an index of greater than 3 per cent are presumed to be actively proliferating and pose a theoretically higher risk for regrowth or recurrence. A retrospective chart review was conducted. Ki-67 staining was performed and specimens were divided into two groups according to Ki-67 activity: less than 3 per cent (low index), and 3 per cent or greater (elevated index). Eight patients (53.3 per cent) with elevated Ki-67 had recurrence or regrowth, versus five (8.5 per cent) in the low Ki-67 group. Among the 13 patients with recurrence or regrowth, the average Ki-67 value was 4.3 per cent. Among the 61 patients without recurrence or regrowth, the average Ki-67 value was 1.0 per cent. The Ki-67 labelling index reliably identifies vestibular schwannomas with an elevated potential for recurrence or regrowth in subtotal or total resection cases. In patients with a Ki-67 index greater than 3 per cent, more frequent clinical examination and radiological follow up are recommended.

A Human Adult Stem Cell Signature Marks Aggressive Variants across Epithelial Cancers

Cancer progression to an aggressive phenotype often co-opts aspects of stem cell biology. Here, we developed gene signatures for normal human stem cell populations to understand the relationship between epithelial cancers and stem cell transcriptional programs. Using a pan-cancer approach, we reveal that aggressive epithelial cancers are enriched for a transcriptional signature shared by epithelial adult stem cells. The adult stem cell signature selected for epithelial cancers with worse overall survival and alterations of oncogenic drivers. Lethal small cell neuroendocrine lung, prostate, and bladder cancers transcriptionally converged onto the adult stem cell signature and not other stem cell signatures tested. We found that DNA methyltransferase expression correlated with adult stem cell signature status and was enriched in small cell neuroendocrine cancers. DNA methylation analysis uncovered a shared epigenomic profile between small cell neuroendocrine cancers. These pan-cancerfindings establish a molecular link between human adult stem cells and aggressive epithelial cancers.

A high-sensitivity electrochemiluminescence-based ELISA for the measurement of the oxidative stress biomarker, 3-nitrotyrosine, in human blood serum and cells.

The generation of 3-nitrotyrosine, within proteins, is a post-translational modification resulting from oxidative or nitrative stress. It has been suggested that this modification could be used as a biomarker for inflammatory diseases. Despite the superiority of mass spectrometry-based determinations of nitrotyrosine, in a high-throughput clinical setting the measurement of nitrotyrosine by an enzyme-linked immunosorbent assay (ELISA) is likely to be more cost-effective. ELISAs offer an alternative means to detect nitrotyrosine, but many commercially available ELISAs are insufficiently sensitive to detect nitrotyrosine in healthy human serum. Here, we report the development, validation and clinical application of a novel electrochemiluminescence-based ELISA for nitrotyrosine which provides superior sensitivity (e.g. a 50-fold increase in sensitivity compared with one of the tested commercial colorimetric ELISAs). This nitrotyrosine ELISA has the following characteristics: a lower limit of quantitation of 0.04 nM nitrated albumin equivalents; intra- and inter-assay coefficients of variation of 6.5% and 11.3%, respectively; a mean recovery of 106 ± 3% and a mean linearity of 0.998 ± 0.001. Far higher nitration levels were measured in normal human blood cell populations when compared to plasma. Mass spectrometry was used to validate the new ELISA method. The analysis of the same set of chemically modified albumin samples using the ELISA method and mass spectrometry showed good agreement for the relative levels of nitration present in each sample. The assay was applied to serum samples from patients undergoing elective surgery which induces the human inflammatory response. Matched samples were collected before and one day after surgery. An increase in nitration was detected following surgery (median (IQR): 0.59 (0.00-1.34) and 0.97 (0.00-1.70) nitrotyrosine (fmol of nitrated albumin equivalents/mg protein) for pre- and post-surgery respectively. The reported assay is suitable for nitrotyrosine determination in patient serum samples, and may also be applicable as a means to determine oxidative stress in primary and cultured cell populations.

Axon guidance pathways served as common targets for human speech/language evolution and related disorders

Human and several nonhuman species share the rare ability of modifying acoustic and/or syntactic features of sounds produced, i.e. vocal learning, which is the important neurobiological and behavioral substrate of human speech/language. This convergent trait was suggested to be associated with significant genomic convergence and best manifested at the ROBO-SLIT axon guidance pathway. Here we verified the significance of such genomic convergence and assessed its functional relevance to human speech/language using human genetic variation data. In normal human populations, we found the affected amino acid sites were well fixed and accompanied with significantly more associated protein-coding SNPs in the same genes than the rest genes. Diseased individuals with speech/language disorders have significant more low frequency protein coding SNPs but they preferentially occurred outside the affected genes. Such patients’ SNPs were enriched in several functional categories including two axon guidance pathways (mediated by netrin and semaphorin) that interact with ROBO-SLITs. Four of the six patients have homozygous missense SNPs on PRAME gene family, one youngest gene family in human lineage, which possibly acts upon retinoic acid receptor signaling, similarly as FOXP2, to modulate axon guidance. Taken together, we suggest the axon guidance pathways (e.g. ROBO-SLIT, PRAME gene family) served as common targets for human speech/language evolution and related disorders.

Reduced Intestinal Motility, Mucosal Barrier Function, and Inflammation in Aged Monkeys.

We aimed to examine the general health and intestinal physiology of young and old non-human primates with comparable life histories and dietary environments. Vervet monkeys (Chlorcebus aethiops sabaeus) in stable and comparable social and nutritional environments were selected for evaluation. Health phenotype, circulating cytokines and biomarkers of microbial translocation (MT) were measured (n=26-44). Subsets of monkeys additionally had their intestinal motility, intestinal permeability, and fecal microbiomes characterized. These outcomes document age-related intestinal changes present in the absence of nutritional stressors, which are all known to affect gastrointestinal motility, microbiome, and MT. We found that old monkeys have greater systemic inflammation and poor intestinal barrier function as compared to young monkeys. Old monkeys have dramatically reduced intestinal motility, and all changes in motility and MT are present without large differences in fecal microbiomes. We conclude that deteriorating intestinal function is a feature of normal aging and could represent the source of inflammatory burden yet to be explained by disease or diet in normal aging human primate populations. Intestinal changes were seen independent of dietary influences and aging within a consistent environment appears to avoid major microbiome shifts. Our data suggests interventions to promote intestinal motility and mucosal barrier function have the potential to support better health with aging.

Behavioral modeling of human choices reveals dissociable effects of physical effort and temporal delay on reward devaluation.

There has been considerable interest from the fields of biology, economics, psychology, and ecology about how decision costs decrease the value of rewarding outcomes. For example, formal descriptions of how reward value changes with increasing temporal delays allow for quantifying individual decision preferences, as in animal species populating different habitats, or normal and clinical human populations. Strikingly, it remains largely unclear how humans evaluate rewards when these are tied to energetic costs, despite the surge of interest in the neural basis of effort-guided decision-making and the prevalence of disorders showing a diminished willingness to exert effort (e.g., depression). One common assumption is that effort discounts reward in a similar way to delay. Here we challenge this assumption by formally comparing competing hypotheses about effort and delay discounting. We used a design specifically optimized to compare discounting behavior for both effort and delay over a wide range of decision costs (Experiment 1). We then additionally characterized the profile of effort discounting free of model assumptions (Experiment 2). Contrary to previous reports, in both experiments effort costs devalued reward in a manner opposite to delay, with small devaluations for lower efforts, and progressively larger devaluations for higher effort-levels (concave shape). Bayesian model comparison confirmed that delay-choices were best predicted by a hyperbolic model, with the largest reward devaluations occurring at shorter delays. In contrast, an altogether different relationship was observed for effort-choices, which were best described by a model of inverse sigmoidal shape that is initially concave. Our results provide a novel characterization of human effort discounting behavior and its first dissociation from delay discounting. This enables accurate modelling of cost-benefit decisions, a prerequisite for the investigation of the neural underpinnings of effort-guided choice and for understanding the deficits in clinical disorders characterized by behavioral inactivity.

Quantitative Analysis of Cell Migration Using Optical Flow

Neural crest cells exhibit dramatic migration behaviors as they populate their distant targets. Using a line of zebrafish expressing green fluorescent protein (sox10:EGFP) in neural crest cells we developed an assay to analyze and quantify cell migration as a population, and use it here to characterize in detail the subtle defects in cell migration caused by ethanol exposure during early development. The challenge was to quantify changes in the in vivo migration of all Sox10:EGFP expressing cells in the visual field of time-lapse movies. To perform this analysis we used an Optical Flow algorithm for motion detection and combined the analysis with a fit to an affine transformation. Through this analysis we detected and quantified significant differences in the cell migrations of Sox10:EGFP positive cranial neural crest populations in ethanol treated versus untreated embryos. Specifically, treatment affected migration by increasing the left-right asymmetry of the migrating cells and by altering the direction of cell movements. Thus, by applying this novel computational analysis, we were able to quantify the movements of populations of cells, allowing us to detect subtle changes in cell behaviors. Because cranial neural crest cells contribute to the formation of the frontal mass these subtle differences may underlie commonly observed facial asymmetries in normal human populations.

Defining antibody responses in normal and non-malignant human aging populations

Event Abstract Back to Event Defining antibody responses in normal and non-malignant human aging populations Sarah M. Tete1, Kasper Wilting2, Gerda Horst1, Michiel Klijn3, Johanna Westra1, Aalzen De Haan4, Anke Huckriede4, Hanneke Kluin-Nelemans5, Surinder Sahota6, Marc Bijl7 and Nico Bos1* 1 University Medical Centre Groningen, Reumatology and Clinical Immunology, Netherlands 2 University Medical Centre Groningen, Department of Medical Microbiology, Netherlands 3 General Practitioners Practice, Huisarten Boterdiep, Netherlands 4 University Medical Centre Groningen, Department of Medical Microbiology, Molecular Virology section, Netherlands 5 University Medical Centre Groningen, Department of Hematology, Netherlands 6 University of Southampton, Cancer Sciences Division, School of Medicine, United Kingdom 7 Martini Ziekenhuis, Department of Internal Medicine and Rheumatology, Netherlands Understanding immunosenescence in ageing is a pressing need to counter the increased susceptibility to infections, as apparent in the inadequate response to influenza virus that underlies>90% of deaths associated with infection in the elderly. The emergence of non-malignant conditions like Monoclonal Gammopathy of Undetermined Significance (MGUS) in the elderly may further alter this susceptibility, and remain poorly defined. To investigate this, we examined linked B- and T-cell responses to influenza vaccination in a healthy elderly cohort (n=19) and contrasted these with aged individuals with MGUS (n=19), a benign plasma cell monoclonal expansion in the bone marrow that secretes elevated levels of monoclonal protein. Influenza H1N1- and H3N2-specific IgG levels were measured and the frequencies of H1N1- and H3N2-specific IFN-γ-secreting cells assayed by enzyme-linked immunospot. Comparable influenza-specific IgG titers were seen in HCs and MGUS at a cohort level, and MGUS patients showed a significant increase in influenza-specific IgG responses post-vaccination. However, significant differences emerged in MGUS in relation to M-protein levels. MGUS with low M-protein showed increased influenza-specific IgG titers post-vaccination but MGUS with high M-protein had lower influenza-specific IgG titers which they failed to expand post-vaccination. Contrary to HCs, MGUS patients revealed impaired T-cell responses, as the number of IFN-γ-secreting cells didnot increase post-vaccination. M-protein concentration inversely correlated with influenza-specific IgG response at d7 (p=0.020) and d28 (p=0.012) as well as with the number of IFN-γ-secreting cells (p=0.018) at d28 post-vaccination. MGUS, as a non-malignant clonal expansion, depresses immune responses to influenza vaccination that varies strikingly with M-protein levels.

Simulation of the pharmacokinetics of bisoprolol in healthy adults and patients with impaired renal function using whole-body physiologically based pharmacokinetic modeling

To develop and evaluate a whole-body physiologically based pharmacokinetic (WB-PBPK) model of bisoprolol and to simulate its exposure and disposition in healthy adults and patients with renal function impairment. Bisoprolol dispositions in 14 tissue compartments were described by perfusion-limited compartments. Based the tissue composition equations and drug-specific properties such as log P, permeability, and plasma protein binding published in literatures, the absorption and whole-body distribution of bisoprolol was predicted using the 'Advanced Compartmental Absorption Transit' (ACAT) model and the whole-body disposition model, respectively. Renal and hepatic clearances were simulated using empirical scaling methods followed by incorporation into the WB-PBPK model. Model refinements were conducted after a comparison of the simulated concentration-time profiles and pharmacokinetic parameters with the observed data in healthy adults following intravenous and oral administration. Finally, the WB-PBPK model coupled with a Monte Carlo simulation was employed to predict the mean and variability of bisoprolol pharmacokinetics in virtual healthy subjects and patients. The simulated and observed data after both intravenous and oral dosing showed good agreement for all of the dose levels in the reported normal adult population groups. The predicted pharmacokinetic parameters (AUC, C(max), and T(max)) were reasonably consistent (<1.3-fold error) with the observed values after single oral administration of doses ranging from of 5 to 20 mg using the refined WB-PBPK model. The simulated plasma profiles after multiple oral administration of bisoprolol in healthy adults and patient with renal impairment matched well with the observed profiles. The WB-PBPK model successfully predicts the intravenous and oral pharmacokinetics of bisoprolol across multiple dose levels in diverse normal adult human populations and patients with renal insufficiency.

Identification of pluripotent and adult stem cell genes unrelated to cell cycle and associated with poor prognosis in multiple myeloma.

Gene expression-based scores used to predict risk in cancer frequently include genes coding for DNA replication, repair or recombination. Using two independent cohorts of 206 and 345 previously-untreated patients with Multiple Myeloma (MM), we identified 50 cell cycle-unrelated genes overexpressed in multiple myeloma cells (MMCs) compared to normal human proliferating plasmablasts and non-proliferating bone marrow plasma cells and which have prognostic value for overall survival. Thirty-seven of these 50 myeloma genes (74%) were enriched in genes overexpressed in one of 3 normal human stem cell populations – pluripotent (18), hematopoietic (10) or mesenchymal stem cells (9) - and only three genes were enriched in one of 5 populations of differentiated cells (memory B lymphocytes, T lymphocytes, polymorphonuclear cells, monocytes, osteoclasts). These 37 genes shared by MMCs and adult or pluripotent stem cells were used to build a stem cell score (SCscore), which proved to be strongly prognostic in the 2 independent cohorts of patients compared to other gene expression-based risk scores or usual clinical scores using multivariate Cox analysis. This finding highlights cell cycle-unrelated prognostic genes shared by myeloma cells and normal stem cells, whose products might be important for normal and malignant stem cell biology.

Genetic, morphometric, and behavioral factors linked to the midsagittal area of the corpus callosum.

The corpus callosum is the main commissure connecting left and right cerebral hemispheres, and varies widely in size. Differences in the midsagittal area of the corpus callosum (MSACC) have been associated with a number of cognitive and behavioral phenotypes, including obsessive-compulsive disorders, psychopathy, suicidal tendencies, bipolar disorder, schizophrenia, autism, and attention deficit hyperactivity disorder. Although there is evidence to suggest that MSACC is heritable in normal human populations, there is surprisingly little evidence concerning the genetic modulation of this variation. Mice provide a potentially ideal tool to dissect the genetic modulation of MSACC. Here, we use a large genetic reference panel – the BXD recombinant inbred line – to dissect the natural variation of the MSACC. We estimated the MSACC in over 300 individuals from nearly 80 strains. We found a 4-fold difference in MSACC between individual mice, and a 2.5-fold difference among strains. MSACC is a highly heritable trait (h2 = 0.60), and we mapped a suggestive QTL to the distal portion of Chr 14. Using sequence data and neocortical expression databases, we were able to identify eight positional and plausible biological candidate genes within this interval. Finally, we found that MSACC correlated with behavioral traits associated with anxiety and attention.

A role for the CISD2 gene in lifespan control and human disease

CISD2, the causative gene for Wolfram syndrome 2 (WFS2), is an evolutionarily conserved novel gene. Recently, we have demonstrated that CISD2 is involved in mammalian lifespan control; this work also establishes WFS2 as a mitochondria-mediated disorder and effectively links CISD2 gene function, mitochondrial integrity, and aging in mammals. In wild-type mice, the expression levels of CISD2 decrease in an age-dependent manner during the naturally aging process; this correlates with mitochondrial breakdown and parallels the development of an aged phenotype. Future work will examine how the CISD2 knockout mouse helps us to understand WFS2 pathogenesis, as well as exploring the potential effects of increased CISD2 expression. In addition, it will be of great interest to compare gene activity and/or protein function between normal human populations and long-lived centenarian groups. Together, human and mouse genetic studies should provide evidence as to whether CISD2 is a "master gene" for extreme old age.

Markers Distinguishing Cancer Stem Cells from Normal Human Neuronal Stem Cell Populations in Malignant Glioma Patients

Markers Distinguishing Cancer Stem Cells from Normal Human Neuronal Stem Cell Populations in Malignant Glioma Patients

Lactate dehydrogenase (LDH) isoenzymes and proliferative activity of lymphoid cells—an immunocytochemical study

In a recent immunohistochemical study, we suggested that elevation of LDH isoenzymes is generally related to cell proliferation. To explore this relationship further, we have now examined the expression of H- and M-type LDH isoenzymes immunocytochemically in human resting and mitogen-activated B and T lymphocytes. In the resting state, T lymphocytes showed strong staining for H-type LDH but showed little or no staining for M-type LDH, while B lymphocytes showed only weak staining for M-type LDH. During activation of T cells, M-type LDH started to increase when cells entered the early stages of the cell cycle. The staining intensity increased to a maximum when the percentage of the T cells at the S/G2/M phases of the cell cycle reached its peak. M-type LDH expression declined when the activated T cells returned to their resting state. Staining for H-type LDH remained strong in T cells during activation. In B lymphocytes, both H- and M-type LDH isoenzymes increased concomitantly following activation and the staining intensity also correlated well with the percentage of the S/G2/M fraction. The expression of H- and M-type LDH was also determined in fresh leukaemia and a variety of lymphoid cell lines. It was noted that cells of chronic lymphocytic leukaemia (CLL) and pro-lymphocytic leukaemia (PLL), morphologically similar to normal lymphocytes, showed a LDH staining pattern resembling that of resting B lymphocytes, while lymphoblasts in T cell acute lymphoblastic leukaemia (T-ALL), high grade B cell lymphoma and Epstein-Barr virus (EBV) transformed cell lines showed a LDH staining pattern similar to that in activated T or B lymphocytes. Taken together, our results have demonstrated a significant correlation between expression of LDH and proliferative activity of cells. Immunostaining with the MoAbs to H- and M-type LDH can, therefore, provide a useful means not only for identification of T and B lymphocytes but also for rapid evaluation of the proliferating fraction of normal and neoplastic human cell populations.

C. albicans Colonization of Human Mucosal Surfaces

Background Candida albicans is a low level commensal organism in normal human populations with the continuous potential to expand and cause a spectrum of clinical conditions.Methodology/Principal FindingsUsing ex vivo human organ cultures and populations of primary human cells, we have developed several related experimental systems to examine early-stage interactions between C. albicans and mucosal surfaces. Experiments have been conducted both with exogenously added C. albicans and with overtly normal human mucosal surfaces supporting pre-existing infections with natural isolates of Candida. Under different culture conditions, we have demonstrated the formation of C. albicans colonies on human target cells and filament formation, equivalent to tissue invasion.Conclusions/SignificanceThese organ culture systems provide a valuable new resource to examine the molecular and cellular basis for Candida colonization of human mucosal surfaces.

Variation of metabolites in normal human urine

Urine is often sampled from patients participating in clinical and metabolomic studies. Biological homeostasis occurs in humans, but little is known about the variability of metabolites found in urine. It is important to define the inter- and intra-individual metabolite variance within a normal population before scientific or clinical conclusions are made regarding different pathophysiologies. This study investigates the variability of selected urine metabolites in a group of 60 healthy men and women over a period of 30 days. To monitor individual variation, 6 women from the normal population were randomly selected and followed for 30 days. To determine the influence of extraneous environmental factors urine was collected from 25 guinea pigs with similar genetics, diet, and living environment. For both studies, 24 metabolites were identified and quantified using high-resolution 1H nuclear magnetic resonance spectroscopy (NMR). The data demonstrated large inter and intra-individual variation in metabolite concentrations in both normal human and control animal populations. A defined normal baseline is essential before any conclusions may be drawn regarding changes in urine metabolite concentrations.

Transformation of Different Human Breast Epithelial Cell Types Leads to Distinct Tumor Phenotypes

We investigated the influence of normal cell phenotype on the neoplastic phenotype by comparing tumors derived from two different normal human mammary epithelial cell populations, one of which was isolated using a new culture medium. Transformation of these two cell populations with the same set of genetic elements yielded cells that formed tumor xenografts exhibiting major differences in histopathology, tumorigenicity, and metastatic behavior. While one cell type (HMECs) yielded squamous cell carcinomas, the other cell type (BPECs) yielded tumors closely resembling human breast adenocarcinomas. Transformed BPECs gave rise to lung metastases and were up to 10(4)-fold more tumorigenic than transformed HMECs, which are nonmetastatic. Hence, the pre-existing differences between BPECs and HMECs strongly influence the phenotypes of their transformed derivatives.

Effects of maternal vitamin A status on kidney development: a pilot study

Nephron endowment ranges widely in normal human populations. Recent autopsy studies have drawn attention to the possibility that subtle congenital nephron deficits may be associated with increased risk of developing hypertension later in life. Since modest maternal vitamin A deficiency reduces nephron number in rats, we designed a pilot study to determine the prevalence of maternal vitamin A deficiency in Montreal (Canada) and Bangalore (India) and the usefulness of newborn renal volume as a surrogate for nephron endowment. Among 48 pregnant Montreal women, two (4%) had one isolated mid-gestation retinol level slightly below the accepted limit of normal (0.9 mumol/L), whereas 25 (55%) of 46 pregnant women in Bangalore had at least one sample below this limit. Average estimated retinoid intake was correlated with mean serum retinol in pregnant women from Bangalore. In Montreal where maternal vitamin A deficiency was negligible, we found that newborn renal volume (estimated by renal ultrasonography at 2-6 weeks of age) was correlated with surface area at birth and was inversely correlated with serum creatinine at 1 month. Interestingly, renal volume adjusted for body surface area in Montreal (184+/-44 ml/m(2)) was significantly greater than in Bangalore (114+/-33 ml/m(2)) (p<0.01). Definitive studies are needed to establish whether maternal vitamin A deficiency accounts for subtle renal hypoplasia in Indian newborns. If so, there may be important public health implications for regions of the world where maternal vitamin A deficiency is prevalent.

Human endogenous retrovirus-W envelope (syncytin) is expressed in both villous and extravillous trophoblast populations

The placenta is unique amongst normal tissues in transcribing numerous different human endogenous retroviruses at high levels. In this study, RT-PCR and immunohistochemistry were used to investigate the expression of syncytin in human trophoblast. Syncytin transcripts were found in first-trimester trophoblast cells with both villous and extravillous phenotypes and also in the JAR and JEG-3 choriocarcinoma cell lines. Syncytin protein was detected in villous trophoblast and in all extravillous trophoblast subpopulations of first- and second-trimester placental tissues. It was also present in ectopic trophoblast from tubal implantations. This study confirms that syncytin is expressed widely by a variety of normal human trophoblast populations, as well as choriocarcinoma cell lines.