Ghost Cells for Mechanical Circulatory Support In Vitro Testing: A Novel Large Volume Production

The aim of this work is to establish a large volume batch production system to produce sufficient volumes of ghost cells to facilitate hemolysis testing of mechanical circulatory support devices. A volume of more than 405 mL with a hematocrit of at least 28% is required to perform in vitro hemolysis testing of mechanical circulatory support devices according to international standards. The established ghost cell production method performed at the institute is limited to 3.1 mL of concentrated cells, that is, cells with 100% hematocrit, due to predominantly manual process steps. Through semi‐automation of the existing method by using the large volume batch production system, productivity is increased 60‐fold to 188 mL while almost doubling process efficiency to 23.5%. Time‐consuming manual work such as pipetting is now supported by sensor‐based process engineering. With the help of the large volume batch production system, the objective of producing large quantities of ghost cells is successfully achieved. Thus, this work lays the foundation for spatially resolved hemolysis evaluation of mechanical circulatory support devices in combination with the small‐scale fluorescent hemolysis detection method.     

Target cells for avian myeloblastosis virus in embryonic yolk sac and relationship of cell differentiation to cell transformation

The yolk sac of the 12-day chicken embryo retains the blast stage progenitors to cells of the myeloid lineages with a very low level of contamination by more mature myeloid cells which have begun to express the characteristic myeloid cell markers. Both in vivo and in vitro experiments have supported the hypothesis that target cells for the BAI-A strain of avian myeloblastosis virus are contained within the myeloid lineages. An assay system for avian myeloblastosis virus was developed which utilizes this yolk sac cell system and which appears to be more sensitive than previous published assays. In addition, the kinetics of a liquid culture transformation system is presented in which at least 4% of the yolk sac cell population was transformed in a relatively synchronous fashion at 2 days after infection. The morphological transformation preceded an increased rate of cell proliferation. Cell separation procedures provided a 10- to 20-fold enrichment of target cells and demonstrated that the target cell population copurifies with macrophage colony-forming cells which are the committed progenitors to the macrophage lineage. In combination with earlier work, this work demonstrated that cells committed to the macrophage lineage at all stages of differentiation may serve as target cells for infection by avian myeloblastosis virus.     

HEC-1 cells

HEC-1 cell line was the first in vitro cell line of a human endometrial adenocarcinoma which enabled us to perform research work on the endometrium and endometrial carcinoma at a simplified cellular system, contributing cell and molecular biological studies on endometrial carcinoma. Once a cell line is established, it provides a stable experimental system that facilitates and progresses in the study of the tissues and/or neoplasias from which they are derived. In this article we report how HEC-1 cells have been established and cleared the proposed requirements to characterize the established cell line. Also to show the usefulness of the cell line for research work, once it was established, we illustrate these concepts by recalling results obtained with HEC-1 cells and reviewing the literature on what has been achieved by using these cells.     

YopK regulates the Yersinia pestis type III secretion system from within host cells

The pathogenic Yersinia species share a conserved type III secretion system, which delivers cytotoxic effectors known as Yops into target mammalian cells. In all three species, YopK (also called YopQ) plays an important role in regulating this process. In cell culture infections, yopK mutants inject higher levels of Yops, leading to increase cytotoxicity; however, in vivo the same mutants are highly attenuated. In this work, we investigate the mechanism behind this paradox. Using a β-lactamase reporter assay to directly measure the effect of YopK on translocation, we demonstrated that YopK controls the rate of Yop injection. Furthermore, we find that YopK cannot regulate effector Yop translocation from within the bacterial cytosol. YopE is also injected into host cells and was previously shown to contribute to regulation of the injectisome. In this work we show that YopK and YopE work at different steps to regulate Yop injection, with YopK functioning independently of YopE. Finally, by expressing YopK within tissue culture cells, we confirm that YopK regulates translocation from inside the host cell, and we show that cells pre-loaded with YopK are resistant to Yop injection. These results suggest a novel role for YopK in controlling the Yersinia type III secretion system.     

Ca2+/H+ exchange through plasma membrane in the system of transport of calcium and hydrogen ions

The survey is aimed to review the data from literature, concerning possible mechanisms of Ca2+ and H+ transport through the plasma membrane of a cells, and also possibility of existence of Ca2+/H(+)-exchange in the plasma membrane of the muscle cells. It is known that the modification of pHl (delta pH) also can influence the work of the contractile system of muscle cells, and the transition of Ca2+ through the plasma membrane of the cells. Thus, one can suppose a direct relation between Ca2+ and H+ transport, through Ca2+/H+ exchange, and indirect relation through connection with other systems of transport of both Ca2+ (Ca(2+)-ATPase, Na+/Ca2+ exchange), and H+ (Na+/H(+)-exchange, H(+)-ATPase). For example it is shown, that the activator (inhibitor) of the Na+/H(+)-exchange through the plasma membrane of muscle cells, influence the work of the retractive system. And as is known, Ca2+ takes main part in involvement in the system excitation--contraction, and, thus, influencing the work of the Na+/H(+)-exchange, it is possible to regulate transport of Ca2+ through the plasma membrane of a muscle cell. The problem about a possibility of existence of Ca2+/H+ exchange, or functioning of Ca2+/H(+)-exchanger, is still far from the solution. Therefore, in the given review the attempt is made to analyze available information about possible connection between Ca2+ and H+ transport through the plasma cell membrane.     

Modelling and simulation of vertebrate retina: Extended network

In a recent work (Oguztöreli, 1980) a mathematical model for studying the neutral activities in a vertebrate retina has been investigated, where the basic retinal network involves interconnected five neurons of different kind. This model is general enough to cover a great variety of neurons in the same retina as well as in the retinas of different species. In the present work we deal with an extension of the basic network considered in (Oguztöreli, 1980). This extended model contains interconnected twelve neurons: three receptor cells, two horizontal cells, two bipolar cells, two amacrine cells and three ganglion cells. The performance of the model under different conditions, and, the experimental determination of the system parameters are discussed. The background of the modelling and simulations can be found in (Oguztöreli, 1979, 1980).This work was supported by grants from the Natural Sciences and Engineering Research Council Canada under Grant NSERC-A-4345 and Grant NSERC G0377 to MNO through the University of Alberta     

Dendritic cells bask in the limelight

Ralph Steinman is to receive the 2007 Albert Lasker Award for Basic Medical Research for his discovery of dendritic cells and his path-breaking work demonstrating their central role as the principal antigen-presenting cells of the immune system and key activators of T cell responses.     

The scoop on the fly brain: glial engulfment functions in Drosophila

Glial cells provide support and protection for neurons in the embryonic and adult brain, mediated in part through the phagocytic activity of glia. Glial cells engulf apoptotic cells and pruned neurites from the developing nervous system, and also clear degenerating neuronal debris from the adult brain after neural trauma. Studies indicate that Drosophila melanogaster is an ideal model system to elucidate the mechanisms of engulfment by glia. The recent studies reviewed here show that many features of glial engulfment are conserved across species and argue that work in Drosophila will provide valuable cellular and molecular insight into glial engulfment activity in mammals.     

Secondary sensory cells in the gravity receptor system of the statocyst of Octopus vulgaris

Summary The presence of secondary sensory cells in the Octopus gravity receptor system has been demonstrated. In serial thin sections of the receptor cells (hair cells) no axons were found leaving the cells. Instead, synapses were observed with synaptic vesicles lying inside the receptor cells. Both data clearly indicate that the receptor hair cells represent secondary sensory cells. In addition, efferent contacts to the receptor cells could be confirmed.This work was supported in part by grant Wo 160/5 of the Deutsche Forschungsgemeinschaft to Prof. Dr. H.G. WolffThe experimental work was done in part at the Zoological Station in Naples and at the Sechenov Institute of Evolutionary Physiology and Biochemistry of the USSR Academy of Sciences (Laboratory of Prof. Dr. Ya.A. Vinnikov), Leningrad, USSR. The authors thank Prof. Vinnikov and Dr. Tsirulis for stimulating discussions     

A new self: MHC-class-I-independent natural-killer-cell self-tolerance

A fundamental tenet of the immune system is the requirement for lymphocytes to respond to transformed or infected cells while remaining tolerant of normal cells. Natural killer (NK) cells discriminate between self and non-self by monitoring the expression of MHC class I molecules. According to the 'missing-self' hypothesis, cells that express self-MHC class I molecules are protected from NK cells, but those that lack this self-marker are eliminated by NK cells. Recent work has revealed that there is another system of NK-cell inhibition, which is independent of MHC class I molecules. Newly discovered NK-cell inhibitory receptors that have non-MHC-molecule ligands broaden the definition of self as seen by NK cells.     

Some aspects of the regeneration process carried out by means of pluripotent stem cells

This work is devoted to the vital topic of regeneration by stem cells. Cells-predecessors and differentiated cells can divide a limited number of times (Alberts et al., 1994) and are not capable of providing tissue regeneration throughout the ontogenesis. The tissue renewal during such a long period is impossible without participation of a specialized system responsible for regeneration. The given system is submitted by stem cells which are capable of being differentiated in all types of somatic cells and in a line of germ cells, and also have ability to self-renew during the whole life of an organism. Results of our research suggest that stem cells make up a universal mechanism of regeneration which has been formed during evolution.     

A formal model of an artificial immune system

The paper presents a mathematical model based on the features of antigen-antibody bindings in the immune system. In the natural immune system, local binding of immune cells and molecules to antigenic peptides is based generally on the behavior of surface proteins. In particular, immune cells contain proteins on their receptors, and apparently, these proteins play the key role both in immune response and recognition processes. In this work, we consider the immune cells in the form of formal B-cell and formal T-cell and develop a mathematical model of their interactions. We refer this model as the formal immune system (FIS). The paper provides an analysis of a network of bindings (or interactions) among the formal proteins of the FIS.     

Sorting out the multiple roles of Fas ligand

Fas ligand can both be used by the immune system to initiate cell death, and be used by non-lymphoid cells to evade death. Recent work has shown that Fas ligand is differentially sorted in different cell types. Here we present the viewpoint that the differential sorting plays an important part in determining the role of Fas ligand in different cells.     

Tubulin folding cofactors: half a dozen for a dimer

Tubulin folding cofactors control the availability of tubulin subunits and microtubule stability in eukaryotic cells. Recent work on Arabidopsis mutants has provided a new experimental system for understanding the cellular functions of tubulin folding cofactors.     

Surface morphology of immunocompetent cells isolated from spleen of Bufo himalayanus (Günther)

Immunocompetent cells were isolated from spleen of B. himalayanus and studied surface morphology of the three different cell types--(i) plastic adherent; (ii) nylon wool adherent; and (iii) nylon wool non-adherent cells. As revealed by scanning electron microscopy, they resembled the macrophages, B and T cells, respectively. Presence of such cell types indicated that Bufo himalayanus possessed a well-organized immune system. Further work is needed to characterize the functional efficacy of these immunocompetent cells found in B. himalayanus.     

Cellular distribution of growth hormone-releasing hormone receptor in human reproductive system and breast and prostate cancers

Growth hormone releasing hormone receptor (GHRH-R) mRNA and protein was first localized to the anterior pituitary gland, consequent with the action of its ligand on GH synthesis and release. Subsequent studies found GHRH-R also expressed in the hypothalamus and in systemic tissues including those of the reproductive system. In the present work, we studied the distribution of GHRH-R in human reproductive system of males and females by immunohistochemical method. GHRH-R immunostaining was localized in male reproductive system: Leydig cells, Sertoli and basal germ cells of the seminiferous tubules and prostate secretory cells. GHRH-R immunostaining was also demonstrated in the ovary: oocytes, follicular cells, granulosa, thecal and corpus luteum cells. Endometrial glands, placenta and normal mammary glands also showed GHRH-R immunostaining. Our results demonstrate the localization of GHRH-R in the reproductive system, which may mediate the direct action of GHRH in these tissues. Moreover, GHRH-R was demonstrated in prostate and breast carcinomas, opening a variety of possibilities for the use of GHRH antagonists in the treatment of prostatic and mammary tumors.     

Selection of intracellularly active ribozymes in mammalian cells.

Ribozymes are expected to be useful as antiviral agents and powerful tools of functional analysis of unknown gene products in vivo. For use of ribozymes in vivo, they must be fully functional in the intracellular environment. Not all ribozymes selected in vitro would be expected to work in vivo, whereas ribozymes selected in the intracellular environment should retain their function in vivo. With the eventual aim of using ribozymes as antiviral agents or biological tools in mammalian cells, we then devised a novel selection system in mammalian cells of active ribozymes by targeting at a gene for the cyclin dependent kinase inhibitor (CDKI), p16INK4a. In this system, we found that p16INK4a-knockdown cells became malignant and they formed foci. In the mammalian system, we confirmed that the selected cells harbored the active ribozyme, indicating that our positive selection systems in vivo were operational.     

Hearing mechanics: a fly in your ear

Recent work suggests that the auditory organ of Drosophila may serve as an excellent model system for understanding the complex mechanical signal processing that takes place in sensory hair cells of the vertebrate inner ear.     

Both p53-PUMA/NOXA-Bax-mitochondrion and p53-p21cip1 pathways are involved in the CDglyTK-mediated tumor cell suppression

CDglyTK fusion suicide gene has been well characterized to effectively kill tumor cells. However, the exact mechanism and downstream target genes are not fully understood. In our study, we found that CDglyTK/prodrug treatment works more efficiently in p53 wild-type (HONE1) cells than in p53 mutant (CNE1) cells. We then used adenovirus-mediated gene delivery system to either knockdown or overexpress p53 and its target genes in these cells. Consistent results showed that both p53-PUMA/NOXA/Bcl2-Bax and p53-p21 pathways contribute to the CDglyTK induced tumor cell suppression. Our work for the first time addressed the role of p53 related genes in the CDglyTK/prodrug system.     

The reversible effect of colchicine on the taste bud cells of the central circumvallate papilla in the rat

Summary It is believed that differentiation and maintenance of taste buds in vertebrates is dependent on the trophic function of their sensory nerve supply. In the present work colchicine was injected into the circumvallate papilla of the rat. This produced a reversible blockade of neuroplasmic transport and disappearance of taste buds. Colchicine inhibited the further differentiation of bud cells, but apparently did not change the life cycle of the cells present already at the time of injection. It is speculated that the neurotrophic factors in this particular cell system are effective to induce cell differentiation only.This work was supported by CAIT Grant No 1776