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Biomedical subjects

E F Potworowski

Publications and source records attributed to E F Potworowski.

At least 19 recordsLinked to original sources
PubMed1995

Cloning of a thymic stromal cell capable of protecting thymocytes from apoptosis.

A clone of thymic stromal cells, namely 2BH4, was established by primary culture, cellular transfection and limiting dilution. Morphological analysis by transmission electron microscopy revealed that these cells grow as multilayers, producing a well-defined basement membrane to which they attach and frequently form structures similar to hemidesmosomes. The adjoining cells are connected by intercellular junctions, as tight junctions, intermediate junctions, and desmosome-like junctions, as well as interdigitations. Their cytoplasm contains microtubules, strands of actin filaments, and scarce intermediate filaments. Fluorescence microscopy revealed that 2BH4 cells stain with anti-cytokeratin antibodies, the majority of them giving a faint reaction. In addition, they express Thy-1.1, LFA-1, ICAM-1, and the gp23 epithelial antigen, and synthesize laminin. They have a doubling time of 16 hr and are able to bind thymocytes. Thymocytes cultured in the presence of 2BH4 cells are partially protected from both spontaneous and PMA- or dexamethasone-induced apoptosis. This protection is conferred neither by soluble factors normally produced by the 2BH4 cells nor by the sole contact with fixed 2BH4 cells. Rather, thymocytes must interact with metabolically active 2BH4 cells in order to receive the protective signal(s).

Animals↗
PubMed1994

Protein tyrosine kinases transcribed in a murine thymic medullary epithelial cell line.

Medullary epithelial cells of the thymus can be activated by contact with thymocytes. We identified a number of protein tyrosine kinases (PTKs) that could be responsible for the tyrosine phosphorylation observed in a murine thymic medullary epithelial cell line (E-5) following complex formation with thymocytes. Degenerate oligodeoxyribonucleotides (oligos) derived from the amino acid (aa) sequence motifs of PTK catalytic domains were used as oligo primers for PCR amplification to determine the PTK genes which are normally transcribed in the E-5 cell line. Amplicons were cloned, sequenced and the deduced aa sequences were compared to known PTK sequences. Among the 13 distinct PTK catalytic domains identified in E-5 cells, two were novel: they were encoded by eteck, a member of the eph sub-family of PTKs, and thy, a member of the src sub-family of PTKs.

Amino Acid Sequence↗
PubMed1994

Up-regulation of a thymic epithelial ligand after contact with thymocytes.

Thymic medullary epithelial cells of the E-5 line were shown to form in vitro complexes with thymocytes resulting in no apparent modification to the thymocytes participating in the complex, but in tyrosine phosphorylation on a glycoprotein associated with the epithelial adhesion molecule. Because signal transduction from lymphocytes to stromal cells is poorly documented, we examine in this work events which follow epithelial cell activation. Our findings indicate that one chain of the epithelial adhesion molecule (gp23), after complex formation with thymocytes, undergoes a rapid and transient tyrosine kinase-dependent up-regulation.

Cell Adhesion↗
PubMed1993

Increased PCR sensitivity by using paraffin wax as a reaction mix overlay.

The use of mineral oil as a reaction mix overlay in conventional PCR may lead to problems. In addition to more difficult handling, traces of mineral oil in amplicon suspensions have been shown to decrease the efficiency of post-PCR manipulations. Commercial alternatives aimed at resolving the problem more than double the cost of an amplification. This is an important drawback when dealing with a large number of clinical samples. The use of inexpensive paraffin wax as a reaction mix overlay eliminated problems associated with the presence of mineral oil while being more practical and safer in handling potentially contaminated clinical samples. Moreover, when used in conjunction with a modified hot start technique, the use of paraffin wax increased the specificity and sensitivity of PCR amplifications over mineral oil in similar hot start conditions. Using the aforementioned method, the increase in specificity and sensitivity has enabled specific detection of viral DNA in clinical samples which the conventional PCR method failed to detect.

Base Sequence↗
PubMed1993

Identification of a 16-kDa thymocyte membrane glycoprotein involved in the thymocyte/thymic medullary epithelial cell interaction.

We have previously described a type of lymphoepithelial interaction involving CD4+ CD8+ thymocytes and a medullary epithelial cell line (E-5). This interaction is mediated by the recently described gp23/45 epithelial adhesion molecule and an as yet unknown thymocyte receptor. The present work describes a thymocyte surface glycoprotein of 16 kDa which binds both to E-5 cells and to the purified gp23/45 adhesion molecule. In addition, a thymic lymphoma cell line (Ti-6), which interacts with the E-5 cells via the gp23/45 receptor, also present a 16-kDa glycoprotein on its surface. Taken together, the data suggest that the 16-kDa thymocyte surface glycoprotein participates in the binding between these cells and the thymic epithelium.

Animals↗
PubMed1992

Tyrosine kinase activation in thymic epithelial cells: necessity of thymocyte contact through the gp23/45/90 adhesion complex.

Interactions between thymocytes and thymic stromal cells are necessary for T cell differentiation, maturation and proliferation. The signals required for these events to occur often necessitate close contact, and indeed adhesion, between the cell types involved. While the transmission of signals from stromal cells to thymocytes has been well documented, there is little evidence that binding of thymocytes to stromal cells can result in stromal cell activation. We have recently identified a novel thymic epithelial adhesion complex composed of three non-covalently associated glycoproteins (gp23, gp45 and gp90). While gp23 and gp45 are jointly required for adhesion to thymocytes, the function of gp90 is unknown. In the present work, we show that gp23/45-mediated contact with thymocytes induces de novo tyrosine phosphorylation of gp90. Furthermore, the protein tyrosine kinase responsible for gp90 neophosphorylation is itself an integral part of the adhesion complex.

Animals↗
PubMed1990

A novel thymic epithelial adhesion molecule.

Interactions between thymocytes and thymic stromal cells are responsible for positive and negative selection of T cell, their differentiation, maturation and proliferation. The signals required for these events to occur often necessitate close contact, and indeed adhesion, between the cell types involved. The identification of specific adhesion molecules in this context, is, therefore, a vital first step in determining the nature of the signal they mediate or facilitate at a given stage of differentiation. In the present work we identify, isolate and partially characterize a ligand present on thymic medullary epithelial cells which selectively binds CD4+ CD8+ thymocytes found primarily in the thymic cortex. This adhesion molecule is composed of two non-covalently associated glycoproteins of 23 kDa and 45 kDa, respectively, both of which are needed to bind to thymocytes. The importance of the finding is that the ligand, in isolated immobilized form, binds the same thymocyte subset as the original epithelial cell line from which it was isolated. The CD4+ CD8+ thymocyte subset is the precursor of single-positive mature T cells; hence the putative biological activity of the ligand in question takes place at a pivotal stage of T cell differentiation.

Animals↗
PubMed1990

Selection of CD4+CD8+ thymocytes by complex formation with medulla-derived epithelial cells.

The role of lymphostromal complexes in T-cell differentiation is far from elucidated, mainly because a clear association of a particular stromal cell type with a distinct thymocyte subset has never been identified. Using an in vitro system, detecting the adherence of thymocytes to a thymic medullary epithelial cell line (E-5), we showed that the phenotype of these thymocytes was that of cortical type: Thy-1hi, LFA-1+, PNAhi, CD4+CD8+, MEL-14-/lo, IL-2R-, CD3-/lo, and TcR V beta 8-/lo. They were enriched in cells in G2/M at the time of complex formation, showed a higher basal proliferation in culture, and did not respond to PHA, IL-2 and only marginally to Con A. These data show that complex formation with mouse thymic medullary epithelium selects for CD4+CD8+ thymocytes, as shown by the marked decrease in CD4+CD8-/CD4-CD8+ thymocytes, and the incapacity of CD4-CD8- thymocytes to adhere.

Animals↗
PubMed1989

Thymic lymphoma cells as a model for complex-formation between thymocytes and thymic medullary epithelial cells.

The formation of complexes between thymocytes and thymic stromal elements is known to be involved in T cell differentiation. We have previously described one type of lympho-stromal interaction involving CD4+ CD8+ thymocytes and a medullary epithelial cell line (E-5). In this study we report the potential for complex formation of two different thymic lymphoma cell lines (Ti-6 and RDM-4). Ti-6 cells were shown to adhere to the E-5 cells, while RDM-4 cells were totally incompetent. Phenotypic characterization of these cell lines suggests that immature thymocytes are not capable of forming complexes with medullary epithelium and that a certain level of differentiation is required to do so. Comparison of their phenotypes showed that the possibility of some classical T cell surface markers being the receptor for the E-5 ligand can be dismissed.

Animals↗
PubMed1989

Mouse thymic virus-mediated immunosuppression: association with decreased helper T cells and increased suppressor T cells.

Mouse thymic virus (MTV) is a herpesvirus which, when administered to newborn mice, induces an extensive but temporary thymic necrosis associated with immunosuppression. In the present study, the T cell subsets in the thymus of MTV infected newborn C57Bl/6 mice were evaluated at 4, 7, 14, 28, 56, and 84 days after infection, using labeled monoclonal anti-CD4 and anti-CD8 antibodies with two-color flow cytometry. At 7 and 14 days, the percentages of CD4+8- and CD4+8+ cells were significantly decreased whereas the percentage of CD4-8+ cell was increased. At days 28 and 56 percentages had returned to normal. These results indicate that the virus has an affinity for CD4+ T cells (helper cells and their precursors). Increased percentage of CD4-8+ T cells (suppressor cells) is also associated with depressed immune functions in MTV infected newborn mice.

Animals↗
PubMed1989

Dynamics of complex formation between thymocytes and thymic medullary epithelial cells.

Direct cell contact is an intrinsic part of several differentiation processes. A case in point is the formation of complexes between thymic lymphocytes and stromal cells, which are essential for T-cell maturation. The objective of the present work was to gain an insight into the mechanisms underlying the formation and dissociation of such lymphostromal complexes. Using an in vitro system, we show that the number of thymocytes adhering to a thymic medullary epithelial cell line (E-5) increases with time and reaches a plateau, after which some thymocytes spontaneously detach, while the rest remain attached to epithelium. The detached and forcibly removed thymocytes were analysed for their expression of L3T4 and Ly-2 antigens. The detached thymocytes showed a markedly lowered expression of both antigens. Both of these subtypes were shown to be totally refractory to form further complexes after a first encounter with E-5 cells. We also show that, once a mean adherence level of one thymocyte per E-5 cell was reached, (a) the level of further adherence increased exponentially, and (b) during this phase, binding of thymocytes to E-5 cells occurred in clusters, indicating pre-existing polarity or contact-induced polarization of the receptors at the E-5 cell surface.

Animals↗
PubMed1989

Binding of cortical thymocytes to a medullary epithelial cell line: a brief review.

In this review, we bring together data regarding complexes formed between thymocytes and a line of thymic epithelial cells, E-5. We show that E-5 has all the markers which identify it as of epithelial and medullary origin. We show that thymocytes which form complexes with it belong to the double positive, CD4 + CD8 + population, and that the receptor responsible for binding to epithelial cells is likely to be an as yet unidentified glycoprotein. The ligand on E-5 cells is also a glycosylated protein. Adhering thymocytes are in a state of mitotic activity, and their incubation with E-5 cells does not induce increased responsiveness to polyclonal mitogens. Non-adhered thymocytes, on the other hand coincubated With E-5 cells increase their responsiveness to ConA and initiate responsiveness to PHA. Differentiation of adhering thymocytes is evidenced by their acquisition of refractoriness to a secondary adherence, and by the modification of their surface phenotype.

Animals↗
PubMed1988

Interactions between lymphoid cells and a thymic stromal cell line in vitro.

In the present study, we have optimalized the adherence assay to allow monitoring of the level of contact between thymocytes and a thymic epithelial cell line, E-5, in vitro. This type of interaction is not MHC-restricted, thus is unlikely to participate in the education of thymocytes to self. It was also shown that adherence does not vary from strain to strain, except for B6lpr/lpr immunodeficient mice which showed a markedly decreased adherence. This might be caused by the high level of L3T4-, Lyt-2- thymocytes in these mice (Davignon et al., 1985), since enriched double negative cells were shown not to adhere to E-5 cells. Preliminary characterization of adhering thymocytes suggests an heterogeneous mature phenotype. These cells appear around day 16 of fetal life and increase gradually until birth to remain constant throughout life. On the basis of contact duration, two populations of adhering thymocytes exist: one spontaneously detached after 1.5 hr, refractive to further adherence and the other which adheres for up to 14 hr. Contact between lymphoid and E-5 cells was shown to induce PHA responsiveness.

Animals↗
PubMed1988

Abrogation of graft-versus-host reaction by dieldrin in mice.

Sublethal exposure to the organochlorine pesticide, dieldrin, decreased the T-cell immune response in mice. Indeed, a transient inhibition of the mixed lymphocyte reactivity (MLR) was noted at 7 days after intraperitoneal exposure to 0.6 LD50 dieldrin. The present study was undertaken to further investigate the effects of dieldrin on the T-cell immune response, using the graft-versus-host reaction (GVHR) as a model, in order to assess T-cell subset efficiency. Lymphoid cells of A/J mice injected intraperitoneally 7 days earlier with 36 mg/kg body weight dieldrin were transferred into H-2-incompatible F1 hybrids. With this model, known to induce a marked GVHR, we have observed that dieldrin inhibited the potential of parental cells to induce a GVHR in hybrid mice. This effect could not be attributed to a direct cell cytotoxicity, nor to the modulation of major T-cell subsets as shown by thymic and peripheral T-cell subpopulation analysis. Collaboration processes between these cellular subsets seem to represent a potential site for the dieldrin-induced suppression.

Animals↗
PubMed1988

Resistance to RadLV-induced leukemia: non-participation of splenic natural killer cells.

The phenotypic expression of genetically determined resistance to radiation leukemia virus (RadLV)-induced leukemia in mice has been shown to reside in the bone marrow. Because the bone marrow contains precursors of natural killer (NK) cells, known to play a role in retrovirally induced infections, and because these cells have been suggested as participating in resistance to radiation-induced leukemia, it was pertinent to establish whether their levels differed in strains of mice susceptible and resistant to leukemia. We therefore tested splenic NK cell levels in C57BL/Ka (susceptible) and B10.A(5R) (resistant) mice before viral inoculation, immediately after viral inoculation, and throughout the preleukemic period and showed that they were not different. This indicates that splenic NK cell levels have no bearing on the resistance to RadLV-induced leukemia and that other immune or non-immune mechanisms must be sought.

Animals↗
PubMed1988

Characterization of the in vitro interaction between thymocytes and a medullary thymic epithelial cell line.

T-cell differentiation is known to be mediated by lympho-stromal interactions. However, the precise role of cellular complexes formed during this process is far from clear. We have previously established a thymic medullary epithelial cell-line, E-5, and have shown the adherence of thymocytes by a receptor-mediated-mechanism. We report here that the thymocytes able of complex-formation with E-5 cells appear at day 16 of gestation. Moreover, while the proportion of such thymocytes is constant throughout post-natal life, their absolute number decreases with thymic involution. We have also investigated the influence of the genetic background of thymocytes on adherence and found that polymorphic regions of H-2 genes were not involved in contact recognition. Therefore, this type of lympho-stromal interactions is unlikely to participate in the education to self MHC-restriction. However, thymocytes from B6/lpr mice, which spontaneously develop systemic lupus erythematosus (SLE) and have impaired T-cell differentiation, were shown incapable of adhering to E-5 cells. These results are interpreted as showing that interaction between thymocytes and medullary epithelial cells reflect a discrete stage of T-cell differentiation.

Animals↗
PubMed1987

Bone marrow phenotype determines genetic resistance to RadLV-induced leukemia in radiation chimeric mice.

The development of RadLV-induced T-cell leukemia is a multistep process which evolves along the bone marrow-thymus axis. This process has been shown to be under the control of resistance and susceptibility genes. The relative importance of bone marrow and thymic phenotypes in this genetic control have not been established. We have constructed radiation chimeras with bone marrow from susceptible C57BL/Ka and thymus from resistant B10.A(5R) mice (and vice versa). The rate of leukemia development in the various groups indicates that the phenotype of the bone marrow and not that of the thymus determines the expression of resistance or susceptibility.

Animals↗