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Biomedical subjects

P Groscurth

Publications and source records attributed to P Groscurth.

At least 91 records · Page 5Linked to original sources
PubMed1989

Use of fibrin glue as a substrate for in vitro endothelialization of PTFE vascular grafts.

The shear stress resistance of cultured human endothelium was investigated on 6 mm polytetrafluoroethylene vascular grafts. Endothelial cell attachment was promoted by precoating the grafts with fibrin glue, which contained human fibronectin and inhibitors of fibrinolysis (aprotinin and tranexam acid). To evaluate the possible effect of fibrinolysis on cell detachment, seven grafts were lined with adult human saphenous vein endothelial cells (AHSVEC) and 11 with fibrinolytically almost inactive human umbilical vein endothelial cells (HUVEC). Endothelial cell seeding was performed in a microprocessor-controlled rotation device, allowing a low inoculum of 12 X 10(4) endothelial cells/cm2. Grafts were then cultivated for 9 days to enable the maturation of the cytoskeleton, before they were exposed to pulsatile shear stress for 48 hours. A mock circulation simulated the flow patterns and the wall shear forces of the femoral artery. After a 3-hour seeding process, 45% of AHSVEC and 43% of HUVEC were attached to the fibrin matrix, forming a confluent monolayer. After 24 hours of perfusion, a cell loss of 23% in AHSVEC- and of 42% in HUVEC-lined grafts was encountered. In spite of a further cell loss during the following 24 hours of perfusion, the majority of the graft surface was still covered by endothelial cells. Therefore we conclude that fibrin glue is a suitable substrate for the formation of a shear stress-resistant endothelial cell monolayer on polytetrafluoroethylene vascular grafts.

Aprotinin↗
PubMed1989

Blood platelets in cardiopulmonary bypass operations. Recovery occurs after initial stimulation, rather than continual activation.

The ultrastructure of blood platelets was related to platelet function and secretion products before, during, and after cardiopulmonary bypass. Circulating platelets from 15 patients undergoing aorta-coronary bypass operations were investigated at ten predetermined points of time by scanning and transmission electron microscopy. Simultaneously, platelet adenosine triphosphate, diphosphate, and serotonin, as well as plasma levels of platelet factor 4, beta-thromboglobulin, serotonin, thromboxane B2, lactic dehydrogenase, and free hemoglobin were measured. Moreover, platelet responsiveness toward adenosine diphosphate and collagen was determined by optical aggregometry. By scanning electron microscopy, the number of unactivated platelets dropped from 96% +/- 4% to 54% +/- 19% (p less than 0.05) 8 minutes after the onset of bypass. Simultaneously, the percentage of "shape changed" platelets significantly increased. No major release reaction was detected at this time. After the initial activation, platelet morphology began to recover although the bypass continued. During the late period of bypass, a highly significant correlation between increasing plasma levels of alpha-granule compounds (platelet factor 4 and beta-thromboglobulin) and lysis parameters (lactic dehydrogenase and free hemoglobin) was found. However, transmission electron microscopic analysis of the arterial filter and scanning electron microscopic findings of circulating platelets indicated that the release products in plasma were due not only to platelet lysis but also to a limited extent to secondary aggregation. In an inverse and probably causative manner, platelet morphology recovered, whereas the sensitivity of platelets to adenosine diphosphate and collagen decreased toward the end of bypass.

Adenosine Triphosphate↗
PubMed1988

Characterization of granzymes A and B isolated from granules of cloned human cytotoxic T lymphocytes.

A human CD8+ CTL clone with cytolytic potential was shown to express two serine proteases, a 50-kDa homodimer and a 27-kDa monomer, which were purified from cytoplasmic granules. N-terminal sequencing of the purified proteins revealed that the 50-kDa homodimer is the gene product of the human Hanukah factor cDNA clone and that it represents the human homologue to granzyme A. Similarly, the 27-kDa protein was shown to be the serine esterase encoded by the human lymphocyte protease cDNA clone and corresponds to granzyme B. There was no evidence for the presence of other granzymes, in particular for the human homologues to murine granzymes C, D, E, and F. The substrate best cleaved by granzyme A was Gly-Pro-Arg-amido-4-methyl-coumarin after the Arg residue and the pH optimum was 8 to 8.5. Upon triggering of the TCR-CD3 complex with an anti-CD3 mAb, granzyme A was released into the culture medium. Furthermore, a granule-associated hemolytic activity was detected after salt extraction and partial purification of granule proteins. This suggests that hemolytically active human perforin can be obtained from inactive granules.

Amino Acid Sequence↗
PubMed1988

Distribution of polyhexylcyanoacrylate nanoparticles in nude mice over extended times and after repeated injection.

Carbon-14-labeled polyhexylcyanoacrylate nanoparticles, with diameters between 200 and 300 nm, were injected intravenously into nude mice. The distribution in liver, spleen, lung, heart, kidney, GI tract, gonads, brain, muscle, and serum was investigated by liquid scintillation counting. After a single injection, the radiolabel was cleared slowly with 45% remaining after 28 d and 8% remaining after 140 d. After repeated injection with an interval of 28 d (twice or thrice), relatively higher proportions of the dose were found in the spleen and lung as compared with those in other organs. No histological alterations were observed in the liver, spleen, or lung.

Animals↗
PubMed1988

Development of follicular dendritic cells in lymph nodes of B-cell-depleted mice.

We have studied follicular dendritic cells (FDC) in lymph nodes of normal and thymus dysgeneic nude mice depleted of B-cells by chronic treatment with anti-IgM antibodies. We found that B cell depletion was accompanied by the absence of mature FDC as defined morphologically at the ultrastructural level. Only precursor FDC (p-FDC) could be demonstrated. Upon release of B-cell suppression, the repopulation of lymph nodes with B-cells was associated with the reappearance of fully differentiated FDC in primary follicles of nude mice and in secondary follicles of T-cell competent mice. We conclude that mature B-cells and/or B-cell products are required for the development of mature follicular dendritic cells in the mouse lymph node.

Animals↗
PubMed1988

Identification and sequencing of cDNA clones encoding the granule-associated serine proteases granzymes D, E, and F of cytolytic T lymphocytes.

Cytoplasmic granules of cytolytic T lymphocytes contain at least six related serine esterases (granzymes) that are released together with perforin, a pore-forming protein related to complement component C9, during target-cell lysis. Polyclonal antibodies were used to isolate a large number of cDNA clones from an expression library derived from cytolytic-T-cell mRNA. Three distinct full-length cDNA clones coding for granzymes D, E, and F were identified by restriction site mapping and nucleotide sequencing. The three deduced amino acid sequences are highly similar to one another (between 72% and 90% amino acid identities) and to the sequences of granzymes B and C, cathepsin G, and rat mast-cell proteases I and II (between 43% and 57% amino acid identities). Cysteine residues capable of forming intramolecular disulfide bonds are conserved, as are the catalytic-site residues characteristic of serine proteases. Comparison of the cDNA-derived protein sequences with the amino termini of the isolated granzymes provides evidence that they are stored in a fully processed, activated form after removal of the signal peptide and two additional residues (propeptide) at the amino terminus. Immunoelectron microscopic studies demonstrated that granzymes D, E, and F are present in the same morphologically distinct cytoplasmic granules in which perforin has been found previously.

Amino Acid Sequence↗
PubMed1988

Low density lipoprotein causes general cellular activation with increased phosphatidylinositol turnover and lipoprotein catabolism.

Low density lipoprotein (LDL), at concentrations high enough for receptor binding but not high enough to saturate the receptor, induces activation of phosphatidylinositol (PtdIns) turnover in a variety of cell types with various biological functions. Using both biochemical and electron microscopic studies, we have shown that blood platelets take up and degrade LDL in a manner reminiscent of phagocytic cell types. The activation of both PtdIns turnover and LDL metabolism is inhibited by high density lipoprotein. Thus, LDL at hormonal concentrations causes general cellular activation. Since all cell types studied responded to LDL with increased PtdIns turnover and uptake of LDL cholesterol, the PtdIns cycle may also be involved in the cellular regulation of LDL cholesterol metabolism.

Animals↗
PubMed1988

Distinct patterns of virus-specific T cell-mediated cytolysis of transformed versus primary target cells.

Ideas about the mechanism(s) by which cytotoxic T lymphocytes (CTL) lyse appropriate target cells are still controversial. We studied the action of established murine CTL clones as well as of freshly prepared primarily induced CTL against two types of target cells. Transformed tumour cell lines (MC57G and L929) and untransformed cells such as peritoneal exudate cells (PEC) or fibroblasts were examined as target cells by scanning and transmission electron microscopy and with the 51Cr-release assay. We found independent of which CTL used, that in transformed cells the cell membrane seemed to be the first target of CTL attack, whereas in untransformed cells the first noticeable events appeared to take place in the nucleus of the target cells; the membrane of attacked untransformed PEC or fibroblasts was found to be intact at a time when the cellular organelles already were disintegrated. The morphological observations were paralleled by differences in the kinetics of 51Cr-release; untransformed target cells released their label only after a 2 h long period, whereas transformed cells released 51Cr considerably earlier.

Animals↗
PubMed1988

Effects of platelet activating factor (PAF) on human citrated whole blood.

Effects of PAF on citrated whole blood (C-WB) from 38 healthy donors have been studied by impedance aggregometry and by morphologic examination of blood cells using scanning and transmission electron microscopy. In the aggregometer, the C-WB samples showed distinct differences in PAF sensitivity. C-WB specimens from high responders (= 15 donors) displayed a dose-related response to PAF stimulation but those from low responders (= 23 donors) did not indicate an impedance alteration even after the addition of high PAF doses (greater than or equal to 10(-6) mol/l). Morphologic studies revealed shape-changed platelets and primary aggregates in all C-WB samples, whereas secondary aggregates occurred only in C-WB specimens from high responders. Monocytes and neutrophil PMNs showed typical morphologic alterations which were observed in PAF-stimulated C-WB samples from all donors. Both cell types appeared polarized in shape and exhibited large vacuoles in the cytoplasm after PAF activation. In addition, monocytes came into close contact with shape-changed platelets as well as primary and secondary aggregates, whereas PMNs had no special relationship to single or aggregated platelets. In summary, our study indicates that PAF acts on different cell types in C-WB including platelets, monocytes and PMNs. The sensitivity of platelets against PAF stimulation appears to vary between different donors and in certain cases seems to be limited to the formation of primary aggregates.

Blood↗
PubMed1988

Autonomous growth, but not autonomous function, in embryonic human thyroids: a clue to understanding autonomous goiter growth?

Thyroid glands from six 8- to 10-week-old fetuses obtained at the time of legal abortion were cryopreserved in liquid nitrogen and transplanted into nude nu/nu mice. Histological and autoradiographic studies of the grafts labeled with [3H]thymidine and [125I]iodine showed proliferation and functional differentiation of the fetal thyroid tissue. Despite T4-mediated suppression of host TSH secretion, up to 36% of the follicular cell nuclei incorporated the thymidine label, reflecting autonomous proliferation, while iodine organification was almost entirely obliterated. Methimazole-induced TSH hypersecretion readily stimulated both growth and function of the transplanted tissue. Thus, during early development, the human thyroid largely depends on TSH for function, but not for growth. Similar findings were obtained in newborn mice, in whom 58% of the thyroid follicular cells proliferated autonomously, i.e. in the absence of TSH. The number of autonomously proliferating cells gradually declined with increasing age to about 1% in 60-day-old animals and, as reported previously, in xenotransplanted normal human thyroid tissue, whereas the number of autonomously proliferating cells was previously found to be several times higher in xenotransplanted human multinodular goiters. We, therefore, hypothesize that the rapidly and autonomously replicating cells that initiate nodule formation in human multinodular goiters reflect the persistence in the adult gland of cells with fetal growth potential.

Age Factors↗
PubMed1987

Cellular localization of perforin 1 in murine cloned cytotoxic T lymphocytes.

The localization of perforin 1 (P1) in cytotoxic cells was studied by immuno-electron microscopy by using a monospecific rabbit antiserum against highly purified mouse P1 and protein A gold as a second ligand. P1 was found in specific granules of cloned cytotoxic T lymphocytes (CTL). Within the granules, P1 antigen was localized in the fine granular matrix, whereas the vesicular compartment remained free of gold particles. The amount of P1 antigen detectable by immuno-electron microscopy varied between different CTL clones. CTL with NK-like activity had the highest level of P1 antigen. A cytotoxicity loss CTL mutant had no detectable P1 antigen, suggesting an important role of P1 during cell-mediated cytolysis. P1 antigen was undetectable also in bone marrow macrophages, indicating a different cytolytic mechanism of these cells.

Animals↗
PubMed1987

Induction, maintenance, and reinduction of tumoricidal activity in bone marrow-derived mononuclear phagocytes by Corynebacterium parvum. Evidence for the involvement of a T cell- and interferon-gamma-independent pathway of macrophage activation.

Rat bone marrow-derived mononuclear phagocytes, virtually homogeneous with respect to the cell lineage, do not exhibit spontaneous tumoricidal activity in the resting state. When incubated with macrophage-activating lymphokines, rat recombinant interferon-gamma (IFN), or heat-killed Corynebacterium parvum, bone marrow-derived mononuclear phagocytes readily evolve tumoricidal activity. Whereas tumoricidal activity induced by lymphokines and/or rat recombinant IFN-gamma is short-lived, that elicited by C. parvum is maintained for at least 2 wk, provided that the C. parvum organisms are continuously present in the culture. After washing off extracellular organisms, C. parvum-induced tumoricidal activity decays rapidly, suggesting that sustained extracellular stimulation is required for its maintenance. Induction of tumoricidal activity by macrophage-activating lymphokines and/or rat recombinant IFN-gamma is fully prevented by polyclonal and monoclonal anti-IFN-gamma antibodies; in contrast, induction by C. parvum is not affected by anti-IFN-gamma. Since induction of tumoricidal activity by C. parvum takes place irrespective of the presence of anti-Thy-1 antisera or cyclosporin A, T cells and/or their products appear not to be involved in this type of macrophage activation. Accordingly, present findings provide evidence for the existence of lymphokine-independent pathways of macrophage activation.

Animals↗
PubMed1987

Antigen presentation and tumor cytotoxicity by interferon-gamma-treated microglial cells.

In this study microglial cells isolated from brain cell cultures of newborn mice were characterized and investigated for morphology, their responses to growth factors and their functional properties. The microglial cells were phagocytic, contained nonspecific esterase activity and expressed Fc (IgG1/2b) and type-3 complement receptors. Scanning electron microscopy revealed that in analogy to brain tissue two types of microglial cells are present in the cultures: the ameboid and the ramified type which both display similar appearance by transmission electron microscopy. Interleukin 3 and the granulocyte-macrophage colony-stimulating factor were potent growth factors for the cultured microglial cells. The cells were negative for class II antigens (Ia) of the major histocompatibility antigen complex. However, upon treatment with interferon-gamma (IFN-gamma) microglial cells became Ia+ and functioned as antigen-presenting cells when tested on ovalbumin-specific Ia-restricted helper T cells. Furthermore, microglial cells exposed to IFN-gamma and endotoxin developed tumor cell cytotoxicity and produced tumor necrosis factor alpha. Taken together, microglial cells share the characteristics of cells of the macrophage lineage.

Animals↗
PubMed1987

Effects of washing and gel filtration on the ultrastructure of human platelets.

The ultrastructure of washed and gel-filtered platelets has been examined by scanning- and transmission-electron microscopy. Three different washing procedures previously established for functional studies have been employed. Each washing method resulted in typical morphologic alterations of the platelets. Gel filtration of platelet-rich plasma appeared less harmful than washing. Specimens fixed immediately after elution contained numerous platelets with typical shape changes which were restored almost completely after incubation at 37 degrees C. Our study emphasizes the need for cautious interpretation of results obtained with separated platelets and mandates morphologic controls for such experiments.

Blood Platelets↗
PubMed1987

PGI2 and PGE1 induce morphological alterations in human platelets similar to those of the initial phase of activation.

The effects of PGI2 and PGE1 on the ultrastructure of human platelets were studied by scanning (SEM) and transmission (TEM) electron microscopy in relation to the record of an optical aggregometer. Addition of PGI2 or PGE1 to citrated platelet-rich plasma (C-PRP) resulted in a permanent slight decrease in percent light transmission (%T) recorded by the aggregometer. SEM investigation of the platelets showed marginal pseudopods and occasional large stomata after application of prostaglandins. These alterations occurred within the initial 30 s and remained constant during the subsequent 20 min of incubation. TEM studies revealed morphological changes of alpha granules and moderately electron-dense material in the dilated profiles of the surface-connected canalicular system (SCCS). Addition of 10 microM ADP to C-PRP preincubated for 30 s with either 2 ng/ml (5 nmol/liter) PGI2 or 30 ng/ml (85 nmol/liter) PGE1 resulted in a further decrease of %T followed by a slight increase. The alterations of the aggregometer tracing were characterized in SEM by platelet shape change and the generation of primary aggregates. C-PRP samples preincubated with 3 and 9 ng/ml (8 nmol/liter and 24 nmol/liter) PGI2 or 40 and 120 ng/ml (113 nmol/liter and 338 nmol/liter) PGE1 did not produce additional changes in the aggregometer curves or in the ultrastructure of platelets in response to ADP. Our morphological study indicates that antiaggregatory prostaglandins induce an early phase of platelet activation but inhibit "shape change" and the formation of aggregates.

Adenosine Diphosphate↗
PubMed1986

Firm persistent binding between activated macrophages and tumor cells is not a prerequisite for the mediation of cytolysis.

The present study seeks to reassess the roles of macrophage activation and persistent firm binding to tumor cells as a prerequisite for tumoricidal activity. To this end, macrophage effector populations from various tissues, expressing diverse functional activities, were made to interact in vitro with different suspension tumor cell lines. The resultant binding and cytolytic effector cell capacities were determined. Macrophage activation appears to be an absolute prerequisite for binding and for killing of tumor cells. Activated macrophages firmly bind and form clusters with D-12 rat fibrosarcoma cells, which are relatively resistant to macrophage-mediated tumoricidal action. In contrast, P-815 murine mastocytoma cells, highly susceptible to the lytic activity of macrophages, bind rather poorly with activated macrophages and do not form clusters. Since susceptible target cells are readily killed by activated rat macrophages in the absence of persistent firm contact, it appears that firm binding and killing are not causally related events.

Animals↗
PubMed1986

A monoclonal antibody against altered LFA-1 induces proliferation and lymphokine release of cloned T cells.

A murine monoclonal antibody (I-17, IgM) has the following functional effects on murine long-term T cell clones: inhibition of cell-mediated lysis, induction of proliferation, release of lymphokines and change of the cell morphology. The determinant detected by I-17 is expressed on long-term T lines but not on thymocytes, lymph node cells and spleen cells. I-17 precipitated proteins with apparent molecular mass of 220 kDa, 170 kDa, 150 kDa and 100 kDa. Biochemical studies indicate that the determinant recognized by I-17 is tunicamycin sensitive and that I-17 binds to the alpha chain of the lymphocyte function-associated antigen (LFA-1).

Animals↗