Development of an on-line monitoring system of human keratinocyte growth by image analysis and its application to bioreactor culture

Human keratinocytes were cultured in serum-free medium for the purpose of on-line cell growth monitoring by image analysis. The validity of a process using a newly developed video microscopy system with image analysis for growth-rate monitoring in real time was verified by the measurement of the degree of confluence of keratinocytes in T-flasks and Petriperm dishes. The growth rate of keratinocytes was calculated subsequently from the linear relationship between average degree of confluence and cell concentration. This technique was applied to the culture in the bioreactor "KERATOR" in which a special video microscopy system using a CCD camera was built. The cell concentration evaluated by image analysis agreed well with that evaluated by conventional direct cell counting after enzymatic digestion, and the on-line monitoring of the specific growth rate allowed identification of both lag- and exponential-growth phases of the culture.     

Morphometric analysis of LPS-stimulated human monocytes by computer-assisted image analysis.

This report describes the results of applying the computer-assisted image analysis system for the measurement of some cytological parameters of LPS-stimulated and nonstimulated human monocytes. The experiments were carried out by means of the digital cell image analysis of haematoxilyn stained monocytes. Five different parameters describing the morphology of monocytes and their nuclei were selected to quantitate the differences between control and activated cells area, perimeter, elongation, dispersion, and extension of images of cell projections. The results suggest that all of the analysed parameters can be used to discriminate stimulated from nonstimulated monocytes which permits detailed monitoring of the changes in cell morphology during monocyte activation.     

Differentiation of allergenic fungal spores by image analysis, with application to aerobiological counts

The ability of an image analysis routine to differentiate between spores of eleven allergenic fungal genera was tested using analysis based on seven basic and up to 17 more complex features, extracted from digitised images. Fungal spores of Alternaria, Cladosporium, Fusarium, Aspergillus, Penicillium, Botrytis, Epicoccum, Exserohilum, Ustilago, Coprinus and Psilocybe were examined in a series of experiments designed to differentiate between spores at the genus and species level. Linear and Quadratic Discriminant Analysis of feature measurements, recorded for 100 to 1600 spores per taxon, differentiated between genera and species with a high level of accuracy. Genus comparisons using only seven basic features resulted in 98% accuracy for the recognition of conidia belonging to Cladosporium, Fusarium and Epicoccum. Differentiation between conidia of Aspergillus and Penicillium was the least reliable, with 56% of Aspergillus conidia correctly identified and 41% misidentified as Penicillium. At the species level, conidia of Cladosporium macrocarpum, Fusarium moniliforme (microconidia), F. oxysporum (microconidia), F. solani (macroconidia), Alternaria helianthi and A. brassicae were consistently identified with 86--100% accuracy. Reduced levels of accuracy in the identification of spores by image analysis reflected similarities between species in their spore morphology. The application of image analysis to aerobiological counting methods is discussed in relation to the results obtained.     

A chemogenomic analysis of the human proteome: application to enzyme families

Sequence-based phylogenies (SBP) are well-established tools for describing relationships between proteins. They have been used extensively to predict the behavior and sensitivity toward inhibitors of enzymes within a family. The utility of this approach diminishes when comparing proteins with little sequence homology. Even within an enzyme family, SBPs must be complemented by an orthogonal method that is independent of sequence to better predict enzymatic behavior. A chemogenomic approach is demonstrated here that uses the inhibition profile of a 130,000 diverse molecule library to uncover relationships within a set of enzymes. The profile is used to construct a semimetric additive distance matrix. This matrix, in turn, defines a sequence-independent phylogeny (SIP). The method was applied to 97 enzymes (kinases, proteases, and phosphatases). SIP does not use structural information from the molecules used for establishing the profile, thus providing a more heuristic method than the current approaches, which require knowledge of the specific inhibitor's structure. Within enzyme families, SIP shows a good overall correlation with SBP. More interestingly, SIP uncovers distances within families that are not recognizable by sequence-based methods. In addition, SIP allows the determination of distance between enzymes with no sequence homology, thus uncovering novel relationships not predicted by SBP. This chemogenomic approach, used in conjunction with SBP, should prove to be a powerful tool for choosing target combinations for drug discovery programs as well as for guiding the selection of profiling and liability targets.     

Clinical applications of image cytometry to human tumour analysis

Image cytometry (ICM) is widely applied to the automated screening, the detection, the diagnosis, the classification, the prognosis and the therapeutic follow-up of different types of cancers (breast, bladder, cervix,...). This review describes the analysis methods and the applications of nuclear image analysis, the determination of DNA content and the analysis of morphometry and of nuclear texture. DNA content analysis can contribute to a prognostic information in addition to other prognostic factors for breast, renal and prostate cancers. For ovarian cancer, aneuploidy seems to be related to prognosis. Bladder tumours with DNA aneuploidy were frequently of high malignancy while ploidy was significantly correlated to relapse risk. For digestive cancers, patients presenting DNA diploid tumours show a better survival than patients with aneuploid ones. Morphometry seems to be a more important criterion than other conventional prognostic factors of invasive breast and digestive carcinomas. A differential diagnosis between normal and neoplastic thyroids is more precise when based on a quantitative evaluation of texture associated to morphometry. Textural parameters permit the discrimination of two populations of patients having a different prognosis and could thus be an aid for prognosis in prostatic cancers. Morphonuclear parameters contribute to separate low and high grade bladder carcinomas. Although ICM was frequently reported, results from the reported examples were not always obvious. In conclusion, the measurements obtained with ICM could be helpful for a decision in several cancers but could not be a substitute for the classical approach of the pathologist.     

Proteomic analysis of human vessels: application to atherosclerotic plaques

Atherosclerosis is a chronic disease that affects medium and large arteries. This process originates from the interaction between cells of the arterial wall, lipoproteins and inflammatory cells, leading to the development of complex lesions or plaques that protrude into the arterial lumen. Plaque rupture and thrombosis result in acute clinical complications such as myocardial infarction and stroke. Owing to the heterogeneous cellular composition of the plaques, a proteomic analysis of the whole lesion is not appropriate. Therefore, we have studied the proteins secreted by human carotid atherosclerotic plaques, obtained by endarterectomy. Normal artery segments and different regions of the surgical pieces (noncomplicated plaque, complicated plaque with thrombus) were cultured in protein-free medium and the secreted proteins (supernatants) analyzed by two-dimensional gel electrophoresis. Normal artery segments secreted a moderate number of proteins (42 spots). However in the two-dimensional (2-D) gels (pH 3-10) of segments bearing a plaque, the number of spots increased markedly (154). The number of spots also increased (202) in the 2-D gels of artery segments with a ruptured plaque and thrombus. Thus, the more complicated the lesion, the higher the number of secreted proteins, suggesting the production of specific proteins relating to the complexity of the atherosclerotic lesion.     

Localization of the nucleolar organizer by computer-aided analysis of a variant no. 21 in a human isolate

Summary A variant chromosome no. 21 consisting of two stalks and two satellites in tandem was detected during a survey of a human isolate. The variant segregated in three generations of a large kindred. One male had the variant no. 21, a metacentric Y, and a 47, XXY complement; however, no other evidence of chromosomal nondisjunction was found. Computer-aided analysis of sequentially stained variant no. 21 chromosomes indicated that silver-stained material corresponded to the proximal stalk region (as defined defined by Giemsa). These data support the hypothesis that human nucleolar organizers are localized to the stalks of acrocentric chromosomes.     

A framework for the automated analysis of subcellular patterns in human protein atlas images

The systematic study of subcellular location patterns is required to fully characterize the human proteome, as subcellular location provides critical context necessary for understanding a protein's function. The analysis of tens of thousands of expressed proteins for the many cell types and cellular conditions under which they may be found creates a need for automated subcellular pattern analysis. We therefore describe the application of automated methods, previously developed and validated by our laboratory on fluorescence micrographs of cultured cell lines, to analyze subcellular patterns in tissue images from the Human Protein Atlas. The Atlas currently contains images of over 3000 protein patterns in various human tissues obtained using immunohistochemistry. We chose a 16 protein subset from the Atlas that reflects the major classes of subcellular location. We then separated DNA and protein staining in the images, extracted various features from each image, and trained a support vector machine classifier to recognize the protein patterns. Our results show that our system can distinguish the patterns with 83% accuracy in 45 different tissues, and when only the most confident classifications are considered, this rises to 97%. These results are encouraging given that the tissues contain many different cell types organized in different manners, and that the Atlas images are of moderate resolution. The approach described is an important starting point for automatically assigning subcellular locations on a proteome-wide basis for collections of tissue images such as the Atlas.     

An image analysis method for the precise selection and quantitation of fluorescently labeled cellular constituents: application to the measurement of human muscle cells in culture

The accurate measurement of the morphological characteristics of cells with nonuniform conformations presents difficulties. We report here a straightforward method using immunofluorescent staining and the commercially available imaging program Adobe Photoshop, which allows objective and precise information to be gathered on irregularly shaped cells. We have applied this measurement technique to the analysis of human muscle cells and their immunologically marked intracellular constituents, as these cells are prone to adopting a highly branched phenotype in culture. Use of this method can be used to overcome many of the long-standing limitations of conventional approaches for quantifying muscle cell size in vitro. In addition, wider applications of Photoshop as a quantitative and semiquantitative tool in immunocytochemistry are explored.     

Metabolomic analysis of human disease and its application to the eye

Metabolomics, the analysis of the metabolite profile in body fluids or tissues, is being applied to the analysis of a number of different diseases as well as being used in following responses to therapy. While genomics involves the study of gene expression and proteomics the expression of proteins, metabolomics investigates the consequences of the activity of these genes and proteins. There is good reason to think that metabolomics will find particular utility in the investigation of inflammation, given the multi-layered responses to infection and damage that are seen. This may be particularly relevant to eye disease, which may have tissue specific and systemic components. Metabolomic analysis can inform us about ocular or other body fluids and can therefore provide new information on pathways and processes involved in these responses. In this review, we explore the metabolic consequences of disease, in particular ocular conditions, and why the data may be usefully and uniquely assessed using the multiplexed analysis inherent in the metabolomic approach.     

Quantitative DNA determination by image analysis. II. Application to human and canine pulmonary cytology

Comparative DNA measurements in human and canine preneoplastic and neoplastic tracheobronchial cells were made with the application of computerized image analysis. Canine studies demonstrated that the sequence of cellular events that precede epidermoid lung cancer simulates precisely the progression observed in humans. DNA studies concomitantly confirmed that there is a stepwise increase in DNA content with advancing nuclear atypia in metaplastic respiratory cells in both species. All carcinomas, regardless of histologic type, were significantly hyperploid to aneuploid (4c to 6c). Small-cell carcinoma exhibited a narrow modal distribution in the 4c region. The uniformity of the cytologic and quantitative DNA changes among these disparate species tends to confirm that humans and canines share biologic characteristics in bronchogenic carcinogenesis. The quantitative DNA measurements provide an objective cellular marker and may be used clinically for diagnostic purposes.     

Gene geography of human population: computer-generated regional genogeographic atlas

Gene geography is considered in this work as the instrument for analysis of population's gene pool. To be effective in this analysis, gene geography should move from mapping of gene frequencies for each gene (and phenes) to construction of genogeographical atlas, as a collection of maps generated by computer, following some strongly defined principles and methods, and joined together, according to general task and the programme of investigation. Brief version of regional genogeographical atlas of Mongolians and the other peoples of Central Asia is presented in the article. This atlas includes computer-generated maps of AB0, Hp, Gc, G'3, Tf, GLO, EstD and PGM1 gene frequencies as well as computer-generalized maps of Mongolian gene pool.     

Proteomic analysis of human lysosomes: application to monocytic and breast cancer cells

To date, about fifty lysosomal hydrolases have been identified, and most of them are targeted towards the lysosomes through a specific mannose-6-phosphate (M-6-P) tag. As more lysosomal hydrolases were expected to be discovered, we performed a proteomic study of soluble lysosomal proteins. Human cells were induced to secrete M-6-P proteins which were affinity purified on immobilized M-6-P receptor. The purified proteins were resolved by two-dimensional electrophoresis and analyzed by mass spectrometry. Twenty-two proteins were identified, among which 16 were well-known lysosomal hydrolases. The remaining species distributed as follows: epididymis-specific alpha-mannosidase is a new mannosidase homolog, cystatin F and CREG (cellular repressor of E1A-stimulated genes) were previously identified as M-6-P proteins (Journet et al., Electrophoresis 2000, 21, 3411-3419), and the last three, which are not hydrolases, were up to now considered as nonlysosomal. This two-dimensional reference map of human U937 M-6-P proteins was afterwards used for comparison with M-6-P proteins purified either from U937 differentiated into macrophage-like cells, or from human breast cancer MCF7 cells. Phorbol ester induced differentiation of U937 cells led to limited proteolytic cleavage or maturation of a discrete number of hydrolases. Five additional lysosomal hydrolases were identified from MCF7 samples. These results prove the usefulness of such a procedure to analyze the lysosomal content of various cell lines, to discover new M-6-P proteins, as well as to point towards unknown biological processes.     

Osteocyte-based image analysis for quantitation of histologically apparent femoral head osteonecrosis: application to an emu model

Femoral head osteonecrosis is often characterized histologically by the presence of empty lacunae in the affected bony regions. The shape, size and location of a necrotic lesion influences prognosis, and can, in principle, be quantified by mapping the distribution of empty lacunae within a femoral head. An algorithm is here described that automatically identifies the locations of osteocyte-filled vs. empty lacunae. The algorithm is applied to necrotic lesions surgically induced in the emu, a large bipedal animal model in which osteonecrosis progresses to collapse, as occurs in humans. The animals' femoral heads were harvested at sacrifice, and hematoxylin and eosin-stained histological preparations of the coronal midsections were digitized and image-analyzed. The algorithm's performance in detecting empty lacunae was validated by comparing its results to corresponding assessments by six trained histologists. The percentage of osteocyte-filled lacunae identified by the algorithm vs. by the human readers was statistically indistinguishable.     

Toward a confocal subcellular atlas of the human proteome

Information on protein localization on the subcellular level is important to map and characterize the proteome and to better understand cellular functions of proteins. Here we report on a pilot study of 466 proteins in three human cell lines aimed to allow large scale confocal microscopy analysis using protein-specific antibodies. Approximately 3000 high resolution images were generated, and more than 80% of the analyzed proteins could be classified in one or multiple subcellular compartment(s). The localizations of the proteins showed, in many cases, good agreement with the Gene Ontology localization prediction model. This is the first large scale antibody-based study to localize proteins into subcellular compartments using antibodies and confocal microscopy. The results suggest that this approach might be a valuable tool in conjunction with predictive models for protein localization.     

Pepper seed germination assessed by combined X-radiography and computer-aided imaging analysis

A lot of pepper seeds having 87 % germination were subjected to X-ray inspection using a non lethal dose of radiation. Seeds with less than 2.7 % (on the basis of total seed area) of free space area, i.e. the spaces between embryo and endosperm, were classified as highly viable seeds (97–100 % germination) with the lowest level of abnormal seedlings. Seeds X-ray classified as good were subjected to a computerised image analysis to study seed imbibition and radicle elongation. The patterns of seed area increase, chosen as the most accurate indicator of seed swelling, resembled the triphasic curve of water uptake. The first phase was completed at 9 h followed by a second phase that varied widely in time until completion of germination between 52 and 96 h. The proportion of seeds with radicle protrusion between 52–56 h and 64–72 h assessed with the image analysis was significantly higher than that recorded using a conventional germination test. In addition, the rate of increase of seed area during the third phase of imbibition, mostly due to protrusion of the radicle tip and its growth, was highly correlated with the corresponding radicle elongation rate.