An integrative computational model for intestinal tissue renewal.

OBJECTIVES: The luminal surface of the gut is lined with a monolayer of epithelial cells that acts as a nutrient absorptive engine and protective barrier. To maintain its integrity and functionality, the epithelium is renewed every few days. Theoretical models are powerful tools that can be used to test hypotheses concerning the regulation of this renewal process, to investigate how its dysfunction can lead to loss of homeostasis and neoplasia, and to identify potential therapeutic interventions. Here we propose a new multiscale model for crypt dynamics that links phenomena occurring at the subcellular, cellular and tissue levels of organisation. METHODS: At the subcellular level, deterministic models characterise molecular networks, such as cell-cycle control and Wnt signalling. The output of these models determines the behaviour of each epithelial cell in response to intra-, inter- and extracellular cues. The modular nature of the model enables us to easily modify individual assumptions and analyse their effects on the system as a whole. RESULTS: We perform virtual microdissection and labelling-index experiments, evaluate the impact of various model extensions, obtain new insight into clonal expansion in the crypt, and compare our predictions with recent mitochondrial DNA mutation data. CONCLUSIONS: We demonstrate that relaxing the assumption that stem-cell positions are fixed enables clonal expansion and niche succession to occur. We also predict that the presence of extracellular factors near the base of the crypt alone suffices to explain the observed spatial variation in nuclear beta-catenin levels along the crypt axis.

SysBioMed report: advancing systems biology for medical applications.

The following report selects and summarises some of the conclusions and recommendations generated throughout a series of workshops and discussions that have lead to the publication of the Science Policy Briefing (SPB) Nr. 35, published by the European Science Foundation. (Large parts of the present text are directly based on the ESF SPB. Detailed recommendations with regard to specific application areas are not given here but can be found in the SPB. Issues related to mathematical modelling, including training and the need for an infrastructure supporting modelling are discussed in greater detail in the present text.)The numerous reports and publications about the advances within the rapidly growing field of systems biology have led to a plethora of alternative definitions for key concepts. Here, with 'mathematical modelling' the authors refer to the modelling and simulation of subcellular, cellular and macro-scale phenomena, using primarily methods from dynamical systems theory. The aim of such models is encoding and testing hypotheses about mechanisms underlying the functioning of cells. Typical examples are models for molecular networks, where the behaviour of cells is expressed in terms of quantitative changes in the levels of transcripts and gene products. Bioinformatics provides essential complementary tools, including procedures for pattern recognition, machine learning, statistical modelling (testing for differences, searching for associations and correlations) and secondary data extracted from databases.Dynamical systems theory is the natural language to investigate complex biological systems demonstrating nonlinear spatio-temporal behaviour. However, the generation of experimental data suitable to parameterise, calibrate and validate such models is often time consuming and expensive or not even possible with the technology available today. In our report, we use the term 'computational model' when mathematical models are complemented with information generated from bioinformatics resources. Hence, 'the model' is, in reality, an integrated collection of data and models from various (possibly heterogeneous) sources. The present report focuses on a selection of topics, which were identified as appropriate case studies for medical systems biology, and adopts a particular perspective which the authors consider important. We strongly believe that mathematical modelling represents a natural language with which to integrate data at various levels and, in doing so, to provide insight into complex diseases: 1. Modelling necessitates the statement of explicit hypotheses, a process which often enhances comprehension of the biological system and can uncover critical points where understanding is lacking. 2. Simulations can reveal hidden patterns and/or counter-intuitive mechanisms in complex systems. 3. Theoretical thinking and mathematical modelling constitute powerful tools to integrate and make sense of the biological and clinical information being generated and, more importantly, to generate new hypotheses that can then be tested in the laboratory.Medical Systems Biology projects carried out recently across Europe have revealed a need for action: 4. While the need for mathematical modelling and interdisciplinary collaborations is becoming widely recognised in the biological sciences, with substantial implications for the training and research funding mechanisms within this area, the medical sciences have yet to follow this lead. 5. To achieve major breakthroughs in Medical Systems Biology, existing academic funding schemes for large-scale projects need to be reconsidered. 6. The hesitant stance of the pharmaceutical industry towards major investment in systems biology research has to be addressed. 7. Leading medical journals should be encouraged to promote mathematical modelling.

Tight junction composition is altered in the epithelium of polycystic kidneys.

Kidney cysts in autosomal dominant polycystic kidney disease (ADPKD) undergo progressive enlargement together with luminal fluid secretion. This involves active, uphill transcellular Cl(-) transport which drives passive Na(+) and water secretion. Implicit in this mechanism is the assumption that the paracellular permeability of the cyst epithelium to Cl(-) must be very low. Claudins are tight junction (TJ) transmembrane proteins that determine the ion selectivity of paracellular barriers. The aim of this study was to determine the expression and localization of claudins within renal cysts in a mouse hypomorphic model of ADPKD and in human patients. We found that the majority of cysts were of collecting duct origin. Claudins normally expressed in collecting duct (3, 4, 7, 8, and 10) were found in small cysts. However, only claudin-7 persisted at substantive levels in the dedifferentiated epithelium of large, presumably late-stage cysts, where it was localized both at the TJ and basolaterally. The constitutively expressed TJ proteins, ZO-1 and occludin, were also abundantly expressed and correctly localized, suggesting that the basic infrastructure of the TJ is preserved. A previous study suggested that claudin-7 may function as a paracellular Cl(-) barrier. We postulate that the role of claudin-7 in ADPKD is to seal the paracellular route in Cl(-)-secreting cyst epithelium, preventing backleak of Cl(-), and that it thereby plays a permissive role in fluid secretion and cyst growth.

Crypt dynamics and colorectal cancer: advances in mathematical modelling.

Mathematical modelling forms a key component of systems biology, offering insights that complement and stimulate experimental studies. In this review, we illustrate the role of theoretical models in elucidating the mechanisms involved in normal intestinal crypt dynamics and colorectal cancer. We discuss a range of modelling approaches, including models that describe cell proliferation, migration, differentiation, crypt fission, genetic instability, APC inactivation and tumour heterogeneity. We focus on the model assumptions, limitations and applications, rather than on the technical details. We also present a new stochastic model for stem-cell dynamics, which predicts that, on average, APC inactivation occurs more quickly in the stem-cell pool in the absence of symmetric cell division. This suggests that natural niche succession may protect stem cells against malignant transformation in the gut. Finally, we explain how we aim to gain further understanding of the crypt system and of colorectal carcinogenesis with the aid of multiscale models that cover all levels of organization from the molecular to the whole organ.

The embedded tumour: host physiology is important for the evaluation of tumour growth.

The growth potential of a tumour can significantly depend on host features such as age, cell proliferation rates and caloric intake. Although this is widely known, existing mathematical models for tumour growth do not account for it. We therefore developed a new model for tumour growth, starting from a mathematical framework that describes the host's physiology. The resulting tumour-in-host model allowed us to study the implications of various specific interactions between the energetics of tumour and host. The model accounts for the influence of both age and feeding regimen of the host organism on the behaviour of a tumour. Concerning the effects of a tumour on its host, it explains why tumour-mediated body-weight loss is often more dramatic than expected from the energy demands of the tumour. We also show how the model can be applied to study enhanced body-weight loss in presence of cachectic factors. Our tumour-in-host model thus appears a proper tool to unite a wide range of phenomena in tumour-host interactions.

A mathematical model that accounts for the effects of caloric restriction on body weight and longevity.

Several aspects of energy dynamics, such as energy expenditure and caloric intake, are known to affect the aging process. In this article we therefore model the aging process within a mathematical framework describing the energy dynamics of an organism. The resulting model comprises food intake, body growth and survival. The equation for the mortality rate accounts for food consumption and is suited to describe caloric restriction data. For non-growing animals, the expression for the mortality rate reduces to the well-known Gompertz equation. We successfully applied our model to growth and survival data on mice exposed to different food levels.

From exposure to effect: a comparison of modeling approaches to chemical carcinogenesis.

Standardized long-term carcinogenicity tests aim to reveal the relationship between exposure to a chemical and occurrence of a carcinogenic response. The analysis of such tests may be facilitated by the use of mathematical models. To what extent current models actually achieve this purpose is difficult to evaluate. Various aspects of chemically induced carcinogenesis are treated by different modeling approaches, which proceed very much in isolation of each other. With this paper we aim to provide for the non-mathematician a comprehensive and critical overview of models dealing with processes involved in chemical carcinogenesis. We cover the entire process of carcinogenesis, from exposure to effect. We succinctly summarize the biology underlying the models and emphasize the relationship between model assumptions and model formulations. The use of mathematics is restricted as far as possible with some additional information relegated to boxes.

Progestin-induced mammary growth hormone (GH) production.

Toxicity studies using beagle dogs revealed in the 1980s that synthetic progestins may induce a syndrome of growth hormone (GH) excess, known as acromegaly, and the development of predominantly benign mammary hyperplasia. In the early 1990s is was discovered that progestin-induced GH excess in the dog originates within the mammary gland. This mammary-derived GH may have endocrine, para/autocrine as well as exocrine effects. The expression of GH mRNA is also found in cats and humans indicating that mammary GH expression is not unique for the dog. The mammary gene is identical to the pituitary-expressed gene and uses the same promoter. Nevertheless a striking difference exists in the mammary gland. Pit-1, which is a prerequisite factor for pituitary GH mRNA expression, is likely not involved in the mammary gene expression. These studies shed new light on the mechanism of progesterone-induced mammary hyperplasia and urges for further research on potential adverse effects of synthetic progestins.

Growth hormone gene expression in normal lymph nodes and lymphomas of the dog.

Growth hormone (GH) is involved in the development, maturation, and function of the immune system. Recent studies have demonstrated that GH can be synthesized and secreted by lymphoid tissues, where it may act as an autocrine/paracrine growth factor. To determine whether GH may be involved in the development of hematological malignancies, GH gene expression in canine lymphomas was investigated. GH mRNA was detected in non-tumorous lymph nodes and in the majority of the lymphomas, by RT-PCR analysis. In situ and Northern blot hybridizations were negative. Analysis of the transcriptional start sites of the GH gene using 5'-RACE (rapid amplification of cDNA ends) showed that the canine lymphoid transcripts contained a 33-85 bp enlarged 5'-untranslated region compared to the pituitary and mammary GH transcripts. Part of the lymphoid GH transcripts contained intron 1, which would result in early termination of the translation due to an in-frame stopcodon. GH measurements in lymphoid tissues revealed a low content of immunoreactive GH. The results presented demonstrate that canine lymphoid tissue is an extrapituitary site of GH gene expression. However, GH production appeared to be low, indicating that lymphoid GH is probably not a major factor in the development or progression of canine lymphoma.

Cloning, characterization, and physical mapping of the canine Prop-1 gene (PROP1): exclusion as a candidate for combined pituitary hormone deficiency in German shepherd dogs.

Abnormalities in the genes encoding Pit-1 and Prop-1 have been reported to cause combined pituitary hormone deficiency (CPHD) in mice and humans. In dogs, a similar phenotype has been described in the German shepherd breed. We have previously reported that the Pit-1 gene (POU1F1) is not mutated in affected German shepherd dogs. In this study, we report the isolation and mapping of the canine Prop-1 gene (PROP1), and we assessed the involvement of PROP1 in German shepherd dog dwarfism. The canine PROP1 gene was found to contain three exons, encoding a 226 amino acid protein. The deduced amino acid sequence was 79% and 84% homologous with the mouse and human Prop-1 protein, respectively. Using fluorescence in situ hybridization, PROP1 was mapped to canine chromosome 11. Further mapping with a canine radiation hybrid panel showed co-localization with the polymorphic DNA marker AHT137. Sequence analysis of genomic DNA from dwarf German shepherd dogs revealed no alterations in the PROP1 gene. Moreover, linkage analysis of AHT137 revealed no co-segregation between the PROP1 locus and the CPHD phenotype, excluding this gene as candidate for canine CPHD and providing a new spontaneous model of hypopituitarism.

[Patients without referral treated in the emergency room: patient characteristics and motives]. / Patiënten zonder verwijzing op de afdeling Spoedeisende Hulp: patiëntkarakteristieken en motieven.

OBJECTIVES: To determine the motives of patients to directly visit the Emergency Department for medical treatment without seeing their family practitioner first and to establish the characteristics of this group. DESIGN: Prospective, descriptive. METHOD: Of the surgical patients attending the Emergency Room (ER) of Utrecht University Hospital, the Netherlands, between 1st April and 25th June 1997, the demographic and diagnostic data of the non-referred patients were compared with those of patients referred by the general practitioner (GP). The non-referred patients were asked about their reasons to attend without referral. RESULTS: A total of 1026 patients visited the ER at their own initiative: 603 males and 423 females. The most frequent reasons for the direct visits were 'had not thought of their family practitioner' (48%) and 'wants help that can only be provided in a hospital' (30%). The self-referring patients differed from the 962 referred patients in severity of the trauma: 76% versus 39% had a relatively minor trauma; sort of trauma: 19% versus 4% had a sport trauma; place of residence: 57% versus 45% lived in the near vicinity of the hospital. Of the self-referring patients 57% visited the hospital out of office hours, versus 39% of the referred patients. CONCLUSION: A large part of the non-referred patients visited the ER unnecessarily and should actually have consulted their GP. These were mainly young adults with minor injuries or sports injuries who lived close to the hospital. Important factors were unfamiliarity with or lack of understanding of the existing regulations.

Cloning and cellular localization of the canine progesterone receptor: co-localization with growth hormone in the mammary gland.

The mammary gland has been found to express the gene encoding growth hormone (GH) in several species. Within the mammary gland, it may act as an autocrine/paracrine growth factor for cyclic epithelial changes, and may be a determinant in mammary carcinogenesis. In the dog, progestins enhance mammary GH expression. To elucidate the mechanism of progestin-induced mammary GH expression, the canine progesterone receptor (PR) is characterized and the cellular localization of the PR in normal and tumorous mammary tissues is examined. Sequence analysis of the canine PR revealed two in-frame ATG codons, encoding a putative PR-B protein of 939 amino acids and a putative PR-A protein of 765 amino acids. Western blot analysis indicated that both isoforms occur in uterus and mammary gland issues. Immunohistochemical analysis of the PR revealed that the PR was differentially expressed in mammary tissue, with many PR-positive epithelial cells in the proliferation phase of the glandular tissue and a low number of PR-positive cells in differentiated mammary tissue. Stromal and myoepithelial cells had no specific PR staining. Mammary tumours had a variety of staining patterns, including no staining, normal nuclear staining, marked heterogeneous immunoreactivity and perinuclear staining of tumorous epithelial cells and cytoplasmic-staining of spindle cells. Double staining showed that all GH-producing cells were positive for PR, whereas not all PR containing cells stained for GH. It is concluded that the activated PR may transactivate GH expression in the mammary gland within the same cell and functions as a pre-requisite transcription factor. However, during malignant transformation this regulation may be lost.

Cloning of the canine gene encoding transcription factor Pit-1 and its exclusion as candidate gene in a canine model of pituitary dwarfism.

Combined pituitary hormone deficiency (CPHD) is an autosomal recessive inherited disease of German shepherd dogs characterized primarily by dwarfism. In mice and humans a similar genetic disorder has been described that results from an alteration in the gene encoding the transcription factor Pit-1. In this study we characterized the canine Pit-1 gene, determined the chromosomal localization of the Pit-1 gene, and screened dwarf German shepherd dogs for the presence of mutations in this gene. The full-length canine Pit-1 cDNA contained an open reading frame encoding 291 amino acids, 92 bp of 5'-untranslated region, and 1959 bp of 3'-untranslated region. The deduced amino acid sequence was highly homologous with Pit-1 of other mammalian species. Using a Pit-1 BAC clone as probe, the Pit-1 gene was mapped by FISH to canine Chromosome (Chr) 31. In dwarf German shepherd dogs a C to A transversion was detected, causing a Phe (TTC) to Leu (TTA) substitution at codon 81. This alteration was present neither in other canine breeds analyzed nor in other mammalian species. However, healthy German shepherd dogs were also homozygous for the mutant allele, indicating that it is not the primary disease-causing mutation. In addition, linkage analysis of polymorphic DNA markers flanking the Pit-1 gene, 41K19 and 52L05, revealed no co-segregation between the Pit-1 locus and the CPHD phenotype. These findings suggest that a gene other than Pit-1 is responsible for the pituitary anomaly in dwarf German shepherd dogs.

Mammary growth hormone and tumorigenesis--lessons from the dog.

The discovery in the early 1990s that progestin-induced growth hormone (GH) excess in the dog originates in the mammary gland can be seen as a hallmark in the research on the pathogenesis of mammary cancer in the dog. The local biosynthesis and release of GH may provide a highly proliferative environment in the mammary gland, which contributes to the development and/or progression of mammary tumours. Before final goals such as prevention of tumour formation or inhibition of tumour promotion can be achieved it is of eminent importance to elucidate the mechanism of progesterone-induced mammary GH production and the mechanism of local autocrine/paracrine action of GH. These local GH effects may be achieved through direct growth stimulating effects of GH as well as by indirect effects mediated by the stimulation of the biosynthesis of insulin-like growth factor-I (IGF-I). The biological effects of the IGFs largely depend on the presence of IGF binding proteins (IGFBPs) which may both enhance or inhibit the activity of the IGFs. This review concentrates on recent advances in the understanding of the local mammary GH-IGF axis and the lessons which can be drawn from the dog for mammary cancer research in other species.

Canine mammary growth hormone gene transcription initiates at the pituitary-specific start site in the absence of Pit-1.

Growth hormone (GH) gene expression has been reported in the mammary glands of various mammalian species. The mechanism by which the GH gene becomes activated in extrapituitary tissues is currently unclear. We have characterized the canine mammary and pituitary GH gene transcripts by Northern blot, 5'- and 3'-RACE (rapid amplification of cDNA ends), and DNA sequence analysis. Northern blot analysis detected GH gene transcripts in mammary glands of dogs which were exposed to high levels of progestins. The mammary and pituitary GH cDNAs were shown to be identical in both the coding region and untranslated regions. Pituitary GH gene expression is highly dependent upon the transcription factor Pit-1. Analysis of Pit-1 gene expression using RT-PCR followed by Southern hybridization revealed a strong pituitary signal but faint, weak or no hybridization signals in mammary gland samples. Among the negative samples were progestin-treated dogs with high mammary GH gene expression. These findings indicate that mammary and pituitary GH gene transcripts originate from the same transcription start site but are regulated differentially.

A high proportion of novel mutations in BRCA1 with strong founder effects among Dutch and Belgian hereditary breast and ovarian cancer families.

We have identified 79 mutations in BRCA1 in a set of 643 Dutch and 23 Belgian hereditary breast and ovarian cancer families collected either for research or for clinical diagnostic purposes. Twenty-eight distinct mutations have been observed, 18 of them not previously reported and 12 of them occurring more than once. Most conspicuously, a 2804delAA mutation has been found 19 times and has never been reported outside the Netherlands. A common haplotype spanning > or = 375 kb could be identified for each of the nine examined recurrent mutations, indicating the presence of multiple BRCA1 founder mutations in the Dutch population. The 2804delAA mutation has been estimated to have originated approximately 32 generations ago. No specific breast or ovarian cancer phenotype could be assigned to any of the common mutations, and the ovarian cancer incidence among 18 families with the 2804delAA mutation was heterogeneous.

Antiphospholipid antibodies other than lupus anticoagulant and anticardiolipin antibodies in women with recurrent pregnancy loss, fertile controls, and antiphospholipid syndrome.

OBJECTIVE: To determine whether antiphospholipid antibodies other than lupus anticoagulant and anticardiolipin are associated with recurrent pregnancy loss. METHODS: Sera from three groups of women were studied: 1) 147 women with recurrent pregnancy loss but no clinical signs or symptoms of autoimmune disease who tested negative for lupus anticoagulant and medium-to-high levels of immunoglobulin G anticardiolipin antibodies; 2) 104 healthy, fertile controls of similar age and gravidity; and 3) 43 women with well-characterized antiphospholipid syndrome. Serum antibody binding against six phospholipids (cardiolipin, phosphatidic acid, phosphatidylserine, phosphatidylcholine, phosphatidylethanolamine, and phosphatidylinositol) was determined using enzyme-linked immunoassays, and results were normalized using an anticardiolipin standard. RESULTS: Twenty-six (18%) women with recurrent pregnancy loss and nine (9%) controls tested positive (above the 99th percentile) for antiphospholipid antibodies. Sera from five (3.4%) women with recurrent pregnancy loss and four (3.8%) controls demonstrated binding to phospholipid antigens other than cardiolipin. In contrast, binding to phospholipid antigens was demonstrated in sera from more than 90% of women with antiphospholipid syndrome. Among women testing positive for antiphospholipid antibodies, the median positive value for women in the antiphospholipid syndrome group was significantly higher than for those with recurrent pregnancy loss or normal fertile controls. CONCLUSIONS: Women with recurrent pregnancy loss are no more likely than fertile controls to have elevated levels of antiphospholipid antibodies once lupus anticoagulant, anticardiolipin, and an obvious clinical history of autoimmune disease have been excluded. Testing for antiphospholipid antibodies other than lupus anticoagulant and anticardiolipin is not clinically useful in the evaluation of recurrent pregnancy loss.

P53 gene mutations in osteosarcomas in the dog.

Osteosarcomas in 18 dogs were examined for the presence of p53 mutations in exons 4-8 by single strand conformation polymorphism (SSCP) analysis, followed by sequence analysis in tumors demonstrating abnormal bands in the SSCP analysis. P53 mutations were found in four of the primary tumors in 17 dogs. Metastases studied in two of these dogs in which the primary tumor contained only wild type p53 did not contain mutations, nor those of one dog in which the primary tumor was not studied. The alterations that were found included three missense mutations and one 3 bp insertion.

Molecular screening for somatic mutations in corticotropic adenomas of dogs with pituitary-dependent hyperadrenocorticism.

Pituitary tumorigenesis is now generally regarded as a multistep process of genomic damage leading to uncoupling of interdependent systems that control cell proliferation and differentiation. The alterations include mutations in genes encoding for proteins involved in signal transduction pathways, such as G-proteins and the p21 protein encoded for by the ras genes. Apart from their excessive secretion of ACTH, corticotropic adenomas are characterized by decreased sensitivity to inhibition by glucocorticoids. Therefore, mutations in the glucocorticoid receptor leading to decreased sensitivity to glucocorticoids may contribute to corticotropic tumor formation. In this study, 16 corticotropic adenomas of dogs with pituitary-dependent hyperadrenocorticism were screened for mutations in the Gs alpha, H-, K-, N-ras genes and the coding region of the DNA-binding domain of the glucocorticoid receptor. The cDNA fragment of the Gs alpha gene encompassed codons 159-240. The K-, and N-ras fragments spanned codons 1-71. The H-ras gene was only screened for mutations in codons 12/13 by direct sequencing of the PCR product. The cDNA fragment of the DNA-binding domain of the glucocorticoid receptor encompassed codons 410-500. The Gs alpha, K-ras, N-ras genes and the DNA-binding domain of the glucocorticoid receptor were screened by single-strand conformation polymorphism analysis. No mutations were found in the Gs alpha gene, the ras genes and the DNA-binding domain of the glucocorticoid receptor. It is concluded that mutations in the Gs alpha gene (codons 159-240), the K- and N-ras genes (codons 1-71), the H-ras gene (codons 12/13) and mutations in the DNA-binding domain of the glucocorticoid receptor do not play a role in the tumorigenesis of canine corticotropic adenomas.