Characterization of monoclonal anti-DNA antibodies and visualization of binding to chromosomes and cell nuclei

Monoclonal antibodies against DNA from two hybridoma cell lines were produced and characterized. One had specificity for single stranded (ss) DNA with some cross-reactivity to RNA, while the other was specific for both single (ss) and double stranded (ds) DNA. The latter ds and ss DNA-binding antibody was used as a model for analysing the distribution of the epitope in chromosomes and cell nuclei. A linear correlation between antibody binding and propidium iodide counterstaining was found on flow cytometric analysis of suspended chromosomes. Immunofluorescence of rat myoblast cells showed a speckled distribution of the antibody in the nucleus with a variability between the cells. Using electron microscopy to visualize antibody binding with gold particles, codistribution with uranyl acetate staining of leucocytes was found. These results suggested that the antibody preferentially binds to condensed chromatin in cells and chromosomes.     

Seeing right through you: applications of optical imaging to the study of the human brain

A new set of techniques allows for the study of brain function by near-infrared light, exploiting two optical phenomena: Changes in light absorption are determined by changes in the concentration of substances like oxy- and deoxyhemoglobin, and changes in light scattering occur as a consequence of variations of properties of membranes and corpuscles in the neural tissue. Methods based on light absorption can be used to study hemodynamic changes in the brain, whereas those based on light scattering can be used to study neuronal activity and to provide anatomical information at a cellular and subcellular level. Three optical imaging approaches can be used to study living tissue: reflection, optical coherence tomography (OCT), and photon migration. These three approaches vary in their penetration (from less than a millimeter for reflection to up to 3-5 cm for photon migration) and spatial resolution (from a micron level for reflection and OCT to a millimeter and centimeter level for photon migration). This issue includes a collection of articles reviewing applications of these technologies to the study of brain and other bodily functions in humans.     

GABAergic neurons comprise a major cell type in rodent visual relay nuclei: an immunocytochemical study of pretectal and accessory optic nuclei

Summary The enzyme glutamic acid decarboxylase (GAD) has been localized in sections of rodent brains (gerbil, rat) using conventional immunocytochemical techniques. Our findings demonstrate that large numbers of GAD-positive neurons and axon terminals (puncta) are present in the visual relay nuclei of the pretectum and the accessory optic system. The areas of highest density of these neurons are in the nucleus of the optic tract (NOT) of the pretectum, the dorsal and lateral terminal accessory optic nuclei (DTN, LTN), the ventral and dorsal subdivisions of the medial terminal accessory optic nucleus (MTNv, MTNd), and the interstitial nucleus of the posterior fibers of the superior fasciculus (inSFp). The findings indicate that 27% of the NOT neurons are GAD-positive and that these neurons are distributed over all of the NOT except the most superficial portion of the NOT caudally. The GAD-positive neurons of the NOT are statistically smaller (65.9 m²) than the total population of neurons of the NOT (84.3 [j,m2) but are otherwise indistinguishable in shape from the total neuron population. The other visual relay nuclei that have been analyzed (DTN, LTN, MTNv, MTNd, inSFp) are similar in that from 21% to 31% of their neurons are GAD-positive; these neurons are smaller in diameter and are more spherical than the total populations of neurons. The data further show that a large proportion of the neurons in these visual relay nuclei are contacted by GAD-positive axon terminals. It is estimated that approximately one-half of the neurons of the NOT and the terminal accessory optic nuclei receive a strong GABAergic input and have been called GAD-recipient neurons. Further, the morphology of the GAD-positive neurons combined with their similar distribution to the GAD-recipient neurons suggest that many of these neurons are acting as GABAergic, local circuit neurons. On the other hand, the large number of GAD-positive neurons in the NOT and MTN (20–30%) in relation to estimates of projection neurons (75%) presents the possibility that some may in fact be projection neurons. The overall findings provide morphological evidence which supports the general conclusion that GABAergic neurons play a significant role in modulating the output of the visually related NOT and terminal accessory optic nuclei.Abbreviations to Figures A Cerebral aqueduct - CP Posterior commissure - DK Nucleus of Darkschewitsch - DMN Deep mesencephalic nucleus - DTN Dorsal terminal nucleus, accessory optic system - HITr Habenulointerpeduncular tract - IGL Intergeniculate leaflet - INC Interstitial nucleus of Cajal - inSFp Interstitial nucleus, superior fasciculus, posterior fibers - LGNd Dorsal lateral geniculate nucleus - LGNv Ventral posterior nucleus - LP Lateral posterior nucleus - LTN Lateral terminal nucleus, accessory optic system - MB Mammillary body - MGN Medial geniculate nucleus - ML Medial lemniscus - MTNd Medial terminal nucleus, dorsal subdivision, accessory optic system - MTNv Medial terminal nucleus, ventral subdivision, accessory optic system - NOT Nucleus of the optic tract - NPC Nucleus of posterior commissure - OT Optic tract - PA Anterior pretectal nucleus - PAG Periaqueductal gray - pbp Nucleus parabrachialis pigmentosus - pC Cerebral peduncle - PM Medial pretectal nucleus - pn Nucleus paranigralis - PO Pretectal olivary nucleus - pp Posterior pretectal nucleus - PPN Peripeduncular nucleus - RNm Magnocellular division, red nucleus - RNp Parvocellular division, red nucleus - SC Superior colliculus - SGP Stratum griseum profundus, superior colliculus - SGS Stratum griseum superficiale, superior colliculus - SGM Stratum griseum medium, superior colliculus - SNc Substantia nigra, pars compacta - SNr Substantia nigra, pars reticulata - SO Stratum opticum, superior colliculus - VB Ventrobasal complex - ZI Zona incerta - 3N Oculomotor nerve, root fibers - 3V Third ventricle Supported by USPHS grants EY03642, NS15669, NS20228, EY03018, and NS15321. C.E.R. is the recipient of a Klingenstein Fellowship in the Neurosciences; R.H.I.B. is a Research Career Development Fellow of the National Eye Institute; and J.H.F. is a Research Career Development Fellow of the National Institutes of Health     

Technology of building an expert system based on a set of quantitative features of tumor cell nuclei for diagnosing breast cancer

The technology of building an expert system for diagnosing malignant nature of invasive tumors of the mammary gland based on a set of quantitative features of the cell nuclei has been developed. Its peculiarity was the presence of weighting coefficients in all the features. Quantitative features were obtained by transforming the initial morphometric data with the help of simple (evaluation of mean values and building of histograms) and complex (regression analysis) mathematical operations. The expert system consisted of one‐dimensional X‐matrix used for investigations and two‐dimensional standard S‐matrix. The X‐matrix elements were assigned for filling with the quantitative features of the studied sample with a nonestablished diagnosis. The S‐matrix elements contained threshold values of quantitative features from the system of diagnostic decision criteria for malignant forms of diseases and their weighting coefficients. Threshold values of nuclear features (larger or smaller) were determined taking into account the range of their values in the groups of malignant and benign pathology. Significance of quantitative features in diagnosing diseases has been assessed. The presence of weighting coefficients allowed diagnosing malignant and benign pathology in a quantitative form by the diagnostic index value. Diagnostic index was calculated by the sum of weighting coefficients of features of the studied sample, which fell within the range of system of the S‐matrix diagnostic decision criteria. Clinical trials revealed high efficiency of the developed approach while diagnosis of breast cancer invasive forms at a preoperative stage. Diagn. Cytopathol. 2013. © 2012 Wiley Periodicals, Inc.     

JPEG2000 for automated quantification of immunohistochemically stained cell nuclei: a comparative study with standard JPEG format

The Joint Photographic Experts Group (JPEG) standard format is one of the most widely used in image compression technologies. More recently, JPEG2000 format has emerged as a state-of-the-art technology that provides substantial improvements in picture quality at higher compression ratios. However, there has been no attempt to date to determine which of the two compression formats produces less variability in the automated evaluation of immunohistochemically stained digital images in agreement with their compression rates and complexity degrees. The evaluation of Ki67 and FOXP3 immunohistochemical nuclear markers was performed in a total of 329 digital images: 47 were captured in uncompressed Tagged Image File Format (TIFF), 141 were converted to three JPEG compressed formats (47 each with 1:3, 1:23 and 1:46 compression) and 141 were converted to three JPEG2000 compressed formats (47 each with 1:3, 1:23 and 1:46 compression). The count differences between images in TIFF versus JPEG formats were compared with those obtained between images in TIFF versus JPEG2000 formats at the three levels of compression. It was found that, using JPEG2000 compression, the results of the stained nuclei count are close enough to the results obtained with uncompressed images, especially in highly complex images at minimum and medium compression. Otherwise, in images of low complexity, JPEG and JPEG2000 had similar count efficiency to that of the original TIFF images at all compression levels. These data suggest that JPEG2000 could give rise to an efficient means of storage, reducing file size and storage capacity, without compromise on the immunohistochemical analytical quality.     

Emerging parasitosis ans mycosis: risk and threats for the new millenium

For multiple reasons, the emergent infectious risks do not stop increasing these last twenty years. The climatic modifications and the human interventions modifying the biotope as well as the rapid spreading of resistant strains to treatments, generate re-emergence or emergence, all the more dramatic as the means of fight are reduced. These emergent or re-emergent diseases are extremely worrisome as their diagnosis and their prevention are often difficult. The important infesting power of parasites and the particularly effective capacities of adaptation of these eucaryotes contributed to the public health problems. Anthropozoonoses and zoonoses constitute a permanent risk the control of which is imaginary. The new pathogenic agents, the unusual clinical demonstrations in the context of deficiency of the host immune functions imply attentiveness and a permanent up to date of the knowledge of the biologist and of the different professionals of health. The risks with which are confronted the humanity during this century underline the necessity of determining mechanisms involved in the pathogenesis. The determination of the specific and vital biologic processes for the microorganism, could allow to define the most appropriated targets and the most effective and original means of fight.     

Acute leukemias: a paradigm for the integration of new technologies in diagnosis and classification.

Acute leukemias (involving the myeloid and lymphoid lineages) and the related myelodysplastic and myeloproliferative syndromes are an extremely heterogeneous group of clonal neoplastic disorders. Traditionally, these diseases were classified solely by morphologic and cytochemical criteria, as originally championed in the 1976 French-American-British Group proposal. Since 1976, however, phenomenal scientific advances have provided new insights into the biology and genetics of the acute leukemias and myeloproliferative/myelodysplastic diseases. Immunophenotyping approaches have begun to supplant more traditional cytochemical assays for lineage determination, and cytogenetic and molecular genetic studies led to the identification and cloning of the genes involved in a large number of the recurrent genetic abnormalities in these diseases. New automated molecular technologies, reviewed in this article, now allow rapid and sensitive detection of these genetic abnormalities in leukemic cells. Such immunophenotyping and molecular genetic information is now considered crucial for both diagnostic and therapeutic decision-making. As our scientific knowledge and diagnostic sophistication increases, classification schemes for these disorders based solely on morphologic features are increasingly seen as unsatisfactory. This review highlights progress in the development of new morphologic classification schemes for the acute leukemias that integrate critical clinical, biologic, and genetic features.     

Public health and valorization of genome-based technologies: a new model

Background

The success rate of timely translation of genome-based technologies to commercially feasible products/services with applicability in health care systems is significantly low. We identified both industry and scientists neglect health policy aspects when commercializing their technology, more specifically, Public Health Assessment Tools (PHAT) and early on involvement of decision makers through which market authorization and reimbursements are dependent. While Technology Transfer (TT) aims to facilitate translation of ideas into products, Health Technology Assessment, one component of PHAT, for example, facilitates translation of products/processes into healthcare services and eventually comes up with recommendations for decision makers. We aim to propose a new model of valorization to optimize integration of genome-based technologies into the healthcare system.

Methods

The method used to develop our model is an adapted version of the Fish Trap Model and the Basic Design Cycle.

Results

We found although different, similarities exist between TT and PHAT. Realizing the potential of being mutually beneficial justified our proposal of their relative parallel initiation. We observed that the Public Health Genomics Wheel should be included in this relative parallel activity to ensure all societal/policy aspects are dealt with preemptively by both stakeholders. On further analysis, we found out this whole process is dependent on the Value of Information. As a result, we present our LAL (Learning Adapting Leveling) model which proposes, based on market demand; TT and PHAT by consultation/bi-lateral communication should advocate for relevant technologies. This can be achieved by public-private partnerships (PPPs). These widely defined PPPs create the innovation network which is a developing, consultative/collaborative-networking platform between TT and PHAT. This network has iterations and requires learning, assimilating and using knowledge developed and is called absorption capacity. We hypothesize that the higher absorption capacity, higher success possibility. Our model however does not address the phasing out of technology although we believe the same model can be used to simultaneously phase out a technology.

Conclusions

This model proposes to facilitate optimization/decrease the timeframe of integration in healthcare. It also helps industry and researchers to come to a strategic decision at an early stage, about technology being developed thus, saving on resources, hence minimizing failures.

     

Phosphatases in concert with kinases set the gain for signal transduction through the T cell receptor

The 'tunable activation thresholds' model for signal transduction through the T cell receptor (TCR)/CD3 signaling complex proposes that rapid cycles of phosphorylation and dephosphorylation are integral to regulating the frequency of protein-protein interaction, thus having considerable influence over the activation of downstream signaling pathways. Co-temporal activation of kinases and phosphatases could serve to modulate the ongoing signaling response, depending on the relative balance of their opposing enzymatic activities. Although recent reports have addressed the mechanisms by which specific kinase/phosphatase pairs contribute to the initiation and termination of signaling, we sought a more global understanding of the ability of the kinase/phosphatase balance to regulate, or "tune", the very proximal steps of TCR signaling in primary human T cells. Herein, we provide biochemical evidence that phosphotyrosine induction via the TCR is subject to fine-tuning based on the overall activity of kinases and phosphatases relative to one another, leading to cycles of phosphorylation and dephosphorylation, with implications for developing the next generation of immunotherapeutic agents.     

Prevention of mitochondrial diseases: a hope through assisted reproductive technologies

Mitochondrial diseases are a heterogenic and poorly studied group of diseases, considered serious in most cases and currently without treatment. Although assisted reproduction proposed strategies to prevent them, such as pre-implantation genetic diagnosis, these techniques are not sufficiently successful. However, the recent publication of two assistedreproduction techniques – meiotic spindle transfer in nonhuman primates and pronuclear transfer in humans – generate a clear ray of hope for the prevention of these diseases. This review analyzes the characteristics and meaning of these new findings and their future clinical implications.     

Emerging insights into human health and NK cell biology from the study of NK cell deficiencies

Human NK cells are innate immune effectors that play a critical roles in the control of viral infection and malignancy. The importance of their homeostasis and function can be demonstrated by the study of patients with primary immunodeficiencies (PIDs), which are part of the family of diseases known as inborn defects of immunity. While NK cells are affected in many PIDs in ways that may contribute to a patient's clinical phenotype, a small number of PIDs have an NK cell abnormality as their major immunological defect. These PIDs can be collectively referred to as NK cell deficiency (NKD) disorders and include effects upon NK cell numbers, subsets, and/or functions. The clinical impact of NKD can be severe including fatal viral infection, with particular susceptibility to herpesviral infections, such as cytomegalovirus, varicella zoster virus, and Epstein‐Barr virus. While NKD is rare, studies of these diseases are important for defining specific requirements for human NK cell development and homeostasis. New themes in NK cell biology are emerging through the study of both known and novel NKD, particularly those affecting cell cycle and DNA damage repair, as well as broader PIDs having substantive impact upon NK cells. In addition, the discovery of NKD that affects other innate lymphoid cell (ILC) subsets opens new doors for better understanding the relationship between conventional NK cells and other ILC subsets. Here, we describe the biology underlying human NKD, particularly in the context of new insights into innate immune cell function, including a discussion of recently described NKD with accompanying effects on ILC subsets. Given the impact of these disorders upon human immunity with a common focus upon NK cells, the unifying message of a critical role for NK cells in human host defense singularly emerges.     

Experimental assessment of new stent technologies: validation of a comparative paired rabbit iliac artery study model

Preventing coronary in-stent restenosis is a major challenge for physicians and industry. To assess new stent technologies, a comparative paired iliac artery model in rabbits is proposed. One tubular stent was implanted in each external iliac artery in 12 rabbits (i.e., 24 stents). An artery overdilatation level of 20% was strictly observed. Restenosis was examined at 30 days by angiography, intravascular ultrasound (IVUS) examination, and histomorphometry. On quantitative angiography, the mean loss of angiographic diameter was 9.8 +/- 4.4% in the right as compared to 9.3 +/- 55% in the left artery (p = 0.75). On IVUS, the volume of intrastent neointimal proliferation was 26.6 +/- 4.9 mm(3) in the right and 25.8 +/- 3.5 mm(3) in the left artery (p = 0.58). In histomorphometry, the neointimal proliferation area was 0.78 +/- 17 mm(2) in the right and 0.76 +/- 0.17 mm(2) in the left artery (p = 0.87). Intrastent neointimal proliferation was comparable between the left and right arteries of all rabbits. The model has three main advantages: (1) arterial dilatation and thus arterial wall aggression are controlled, (2) pairing makes each animal its own control subject, and (3) the statistical power for comparative testing is maximized. The model enables the effect of a new drug-delivery device to be assessed.