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M D Kramer

Publications and source records attributed to M D Kramer.

At least 55 records · Page 3Linked to original sources
PubMed1995

Complement component C7 is a plasminogen-binding protein.

Ab deposition, whether by reaction with the specific Ag or by preformed immune complexes, is followed by activation and deposition of complement components. Tissue destruction is observed in the Ab- and complement-induced lesions. The proteolytic enzyme plasmin is thought to participate in the Ab- and complement-mediated organ pathology. Plasmin is generated from plasma-derived plasminogen by cell-derived plasminogen activators (PAs). Two types of PAs are known, urokinase-type PA (uPA) and tissue-type PA (tPA). We investigated whether the PA system and the complement system can interact to promote local plasmin generation. Among the terminal complement components C5b6, C7, C8, and C9, the nonenzymatic component C7 is a plasminogen-binding protein. Radioligand binding studies revealed that the isolated component, as well as C7 after its incorporation into the terminal complement complex C5b-9, can bind plasminogen. Binding was inhibited by the lysine analogues 6-aminohexanoic acid and tranexamic acid, implicating the lysine binding sites of plasminogen into the binding interaction. tPA-mediated plasminogen activation was enhanced in the presence of C7. Based on these findings, an interaction is proposed between the complement system and the plasminogen activator system; a mechanism that may focus plasmin activity to structures that have been tagged by Ab and complement deposition.

Autoradiography↗
PubMed1995

Plasmin abrogates alpha v beta 5-mediated adhesion of a human keratinocyte cell line (HaCaT) to vitronectin.

At cellular surfaces, urokinase-type plasminogen activator (uPA) is bound to a specific receptor (uPA-R). When bound to this receptor, uPA activates plasminogen, which is derived from plasma or the interstitial fluids. Thus, plasmin is provided for proteolysis of pericellular proteinaceous substrates. Here we demonstrate by immunocytology and laser scan microscopy that in the human keratinocyte cell line HaCaT uPA-R and uPA are localized together with the integrin alpha v beta 5 in focal contacts. Via the integrin alpha v beta 5, HaCaT cells adhere to vitronectin in a RGD-dependent manner. Plasmin interfered with the alpha v beta 5-mediated keratinocyte adhesion to vitronectin, most likely via cleavage of vitronectin and destruction of its cell binding function. Our findings demonstrate that plasmin, when generated by the uPA-dependent cell surface-associated pathway of plasminogen activation, can abrogate the cell-binding function of vitronectin and can thus disturb the adhesive interaction with this matrix molecule. In focal contacts molecules are assembled that are crucial for adhesion to vitronectin (i.e., the integrin alpha v beta 5), as well as for the generation of plasmin (i.e., uPA-R and uPA), which can negatively influence the binding interaction. We suggest that the plasmin-mediated abrogation of the interaction between the integrin alpha v beta 5 and vitronectin is a pathway of negative regulation; the codistribution of uPA-R/uPA and alpha v beta 5 in focal contacts may restrict this process to areas of cell/matrix contact.

Amino Acid Sequence↗
PubMed1995

Differential expression of urokinase-type plasminogen activator (uPA), its receptor (uPA-R), and inhibitor type-2 (PAI-2) during differentiation of keratinocytes in an organotypic coculture system.

Cultured keratinocytes resemble migrating keratinocytes under conditions of reepithelialization during wound healing. Such keratinocytes express urokinase-type plasminogen activator (uPA) and its specific receptor (uPA receptor). Receptor-bound uPA activates plasminogen, thus providing plasmin for pericellular proteolysis. uPA is regulated by the plasminogen activator inhibitors PAI-1 and PAI-2. As indicated by immunohistology, neither uPA nor uPA receptor is expressed in normal epidermis. Thus, the down-regulation of uPA and uPA-receptor expression in keratinocytes appears to be an important event in epidermal healing and restoration of a normal epidermal tissue architecture. We have addressed this matter by using a culture and differentiation system for keratinocytes in vitro. Keratinocytes were grown in organotypic cocultures for 4, 7, and 14 days. Frozen sections were analyzed with indirect immunofluorescence staining and overlay zymography, the latter detecting activity of plasminogen activators. While tPA and PAI-1 stainings were consistently negative over the entire observation period, uPA and uPA receptor were expressed by basal keratinocytes at Days 4 and 7, but not at Day 14. Accordingly, overlay zymography revealed uPA activity at Days 4 and 7. PAI-2 was found throughout the entire observation period, but with varying distribution: at Days 4 and 7 all suprabasal keratinocytes stained positive for PAI-2. At Day 14, PAI-2-specific stainings were confined to the uppermost cells of the stratum spinosum. Our data demonstrate that uPA and uPA receptor, which are up-regulated in cultured keratinocytes, are down-regulated upon restoration of an epidermis-like structure. The distribution of PAI-2 varied over the observation period and at Day 14 resembled the distribution of PAI-2 in normal epidermis. Taken together, keratinocytes in organotypic coculture behave like keratinocytes in healing wounds in vivo with respect to the expression of the plasminogen activator system.

Animals↗
PubMed1995

Increased risk of placenta previa among women of Asian origin.

OBJECTIVE: To investigate the frequency of placenta previa among Asian women. METHODS: We conducted a population-based case-control study using Washington state birth certificate data from 1984-1987. Our study population included 810 women with pregnancies complicated by placenta previa and 2917 randomly selected controls. Unconditional logistic regression was used to estimate odds ratios (OR) and their 95% confidence intervals (CI), and interaction terms were used to examine effect modification. Potential confounding by maternal age, gravidity and parity, maternal smoking during pregnancy, and a history of abortion or cesarean delivery was adjusted for in the analysis. RESULTS: The frequency of placenta previa during the study period was 3.3 per 1000 live births. Women of Asian origin were 86% more likely (OR 1.86, 95% CI 1.38-2.51) to have a delivery complicated by placenta previa than were white women. This association was stronger among women without a previous live birth (OR 2.51, 95% CI 1.57-4.01) than those who previously had experienced a live birth (OR 1.50, 95% CI 1.01-2.25). CONCLUSION: Asian women residing in the United States are at increased risk of placenta previa. If confirmed by others, our results suggest that obstetricians should consider meticulous ultrasound evaluations during pregnancy to rule out the presence of placenta previa in Asian-American women.

Abortion, Induced↗
PubMed1995

Cell surface-bound urokinase-type plasminogen activator facilitates infiltration of freshly isolated granulocytes into fibrin matrix.

Human cell lines of myelo/monocytic origin express the cellular receptor for urokinase-type plasminogen activator (uPA-R). The receptor localizes urokinase-type plasminogen activator (uPA) to the surface of the cell, where it can convert plasminogen to the active serine proteinase plasmin. Plasmin may subsequently account for proteolysis of pericellular proteins. We demonstrated the expression of the uPA-R by freshly isolated neutrophilic granulocytes by using a specific mAb. In freshly isolated granulocytes we detected only a weak occupation of the uPA-R; further uPA binding by granulocytes was saturable and proceeded in a dose-dependent manner. Receptor-bound uPA retained its enzymatic activity. Saturation of isolated granulocytes with exogenous uPA enhanced cellular infiltration into a fibrin matrix in vitro. uPA-dependent infiltration was inhibited by an anti-catalytic monoclonal anti-uPA antibody. The findings show that circulating neutrophilic granulocytes express the cell surface uPA-R and suggest that surface-binding of uPA may facilitate the infiltration of granulocytes into a fibrin clot, a process that might add to thrombolysis in vivo.

Cell Movement↗
PubMed1995

tPA of human keratinocytes: contribution to cell surface-associated plasminogen activation and upregulation by retinoic acid.

We tested distinct variants of a human keratinocyte line (HaCaT) for the expression of tissue-type plasminogen activator (tPA)-specific mRNA, as well as cell surface-associated and secreted tPA. Cells of early passages (passage no. 22) only expressed urokinase plasminogen activator (uPA)- but not tPA-specific mRNA. Cells after prolonged culture (passage no. 44) expressed uPA- and tPA-specific mRNA, but did not release tPA in the extracellular space and did not display surface-associated tPA. HaCaT cells transformed with the c-Ha-ras oncogene (HaCaTras) showed both secreted and surface-associated tPA antigen. The secreted and the surface-associated plasminogen activator (PA)-activity of HaCaTras cells were in part inhibitable by anticatalytic anti-tPA antibodies, thus indicating that tPA contributes to extracellular and surface-associated plasminogen activation. Finally, we demonstrate that tPA secretion of HaCaT 44 cells can be induced by retinoic acid, most likely via interaction of retinoic acid with nuclear-associated retinoic acid-receptor(s).

Base Sequence↗
PubMed1995

Molecular cloning and immunological characterization of a novel linear-plasmid-encoded gene, pG, of Borrelia burgdorferi expressed only in vivo.

Previously we have found that sera from immunocompetent mice infected either naturally by ticks or experimentally with low numbers of Borrelia burgdorferi ZS7 bacteria lack OspA- and OspB-specific antibodies but confer optimal protection on severe combined immunodeficiency mice against challenge with spirochetes (U.E. Schaible, L. Gern, R. Wallich, M. D. Kramer, M. Prester, and M. M. Simon, Immunol. Lett. 36:219-226, 1993). We have now used the latter immune sera to identify new spirochetal structures with relevance for protection from an expression library of the virulent European strain B. burgdorferi ZS7. Here we report the cloning and characterization of a novel lipoprotein, designated pG, the gene for which is located on a 48-kb linear plasmid. Sequence analysis of the pG gene revealed an open reading frame encoding a putative lipoprotein of 196 amino acids with a calculated molecular mass of 22 kDa and a consensus cleavage sequence (Leu-X-Y-Z-Cys) recognized by signal peptidase II. Restriction fragment length polymorphism analyses of pG derived from independent B. burgdorferi isolates from different geographic areas revealed that the gene is species specific, with, however, extensive genotypic heterogeneity. Comparison of the protein sequence of pG with those of other known B. burgdorferi outer surface lipoproteins (OspA to OspF and P27) demonstrated that pG is most related to OspF. Furthermore, the upstream region of pG exhibited extensive sequence homology (> 94%) with the ospEF promoter region. Mouse immune sera to recombinant pG did not recognize a corresponding molecule in lysates of in vitro-propagated ZS7 spirochetes. However, experimental or natural infection of mice with ZS7 resulted in the induction of antibodies with reactivity for pG and the potential to delay the development of clinical arthritis. Together with the finding that sera from Lyme disease patients also contain antibodies to pG, our data suggest that the pG gene is preferentially expressed in the mammal environment.

Amino Acid Sequence↗
PubMed1995

A competitive chromogenic assay to study the functional interaction of urokinase-type plasminogen activator with its receptor.

Urokinase-type plasminogen activator (uPA) converts plasminogen to plasmin which degrades various extracellular matrix components. uPA is focused to the cell surface via binding to a specific receptor (uPAR, also termed CD87). uPAR-bound uPA mediates pericellular proteolysis in a variety of biological processes, e.g. cell migration, tissue remodeling and tumor invasion. We have developed a competitive microtiter plate-based chromogenic assay which allows the analysis of uPA/uPAR interaction. The plates are coated with recombinant uPAR expressed in Chinese hamster ovary (CHO) cells. Proteolytically active uPA (HMW-uPA) is added to the microtiter plate-attached uPAR. The amount of receptor-bound uPA is then determined indirectly via addition of plasminogen, which is activated to plasmin, followed by cleavage of a plasmin-specific chromogenic substrate. Substances interfering with binding of HMW-uPA to uPAR diminish the generation of plasmin, as indicated by a reduction of cleaved chromogenic substrate. This assay was used to analyze the inhibitory capacity of a variety of proteins and peptides, respectively, on the uPA/uPAR interaction: i) uPAR and uPAR-variants expressed in CHO cells, yeast or E. coli, ii) the aminoterminal fragment (ATF) of human uPA or yeast recombinant pro-uPA, iii) synthetic peptides derived from the sequence of the uPAR-binding region of uPA, and iv) antibodies directed against uPAR. This assay may be helpful in identifying uPA and uPAR analogues or antagonists which efficiently block uPA/uPAR interaction.

Amino Acid Sequence↗
PubMed1995

Plasminogen activation by human keratinocytes: molecular pathways and cell-biological consequences.

Keratinocytes are the major cellular constituent of stratified epithelia. Defects in these epithelia are re-epithelialized by keratinocytes migrating from the edge of the defect into the wound. The cells form a monolayer with subsequent differentiation into a multilayered epithelium. It is thought that plasminogen activation by migrating keratinocytes is an important event during re-epithelialization. In the present report we summarize the studies on plasminogen activation by human keratinocytes in vitro and in vivo. Under the aspect of pericellular proteolysis the discussion is focused on the molecular mechanisms of plasminogen activation at the keratinocyte surface and on the cell-biological consequences of pericellular plasmin formation. We describe a cell surface-associated pathway of plasminogen activation which crucially depends on cell surface receptors for (pro)-uPA and plasmin(ogen). uPA bound to its receptor converts cell-bound plasminogen into the active protease plasmin. Compared to plasminogen activation in solution, activation at the keratinocyte cell surface is accelerated by a factor of approx. 7-10, and the plasmin generated and bound at the cell surface is protected against its specific inhibitor alpha 2-antiplasmin. Plasmin thus provided in the pericellular space leads to detachment of cultured keratinocytes from the growth substratum. Plasmin interferes with the adhesion of keratinocytes to fibrin, but not with the adhesion to collagen type I. By demonstrating that keratinocytes of the epithelial outgrowth in healing skin wounds express uPA and the uPA-R and that plasmin(ogen) is colocalized with uPA and/or uPA-R, indirect evidence is provided that this pathway may be operative in vivo. In view of previous findings that plasminogen activation is also observed under certain pathologic conditions in the epidermis, we conclude that plasminogen activation by keratinocytes is rather related to tissue damage and subsequent repair mechanisms than to a specific pathologic situation.

Fibrinolysin↗
PubMed1995

The outer surface lipoprotein A of Borrelia burgdorferi provides direct and indirect augmenting/co-stimulatory signals for the activation of CD4+ and CD8+ T cells.

Naive CD4+ and CD8+ T cells require two distinct signals to proliferate and to express effector functions [1]. One is provided by the antigen receptor on the T cell (TCR) after its encounter with antigenic peptides associated with class I or II major histocompatibility complex (MHC) molecules on antigen-presenting cells (APC) [2]. The second signal, which is not antigen-specific but essential for proliferation and differentiation of naive T cells, is provided by co-stimulatory structures. The major co-stimulatory molecules for CD4+ T cells seem to be B7 [3], B7.2 [4,5], and heat-stable antigen (HSA) [6]. These molecules are expressed on a variety of naive and/or activated APC and bind to CD28 and CTLA-4 and possibly other, as yet undefined, TCRs [3,7]. Optimal T cell activation only occurs when co-stimulatory molecules and ligands for the TCR are expressed on the same APC [8,9]. However, co-stimulation for T cells may also be provided via bystander cells [8,9] or by glycoproteins of the extracellular matrix, like fibronectin [10] and laminin [11]. In this case, T-cell VLA integrins function as signaling molecules [10,11]. This indicates that antigen-specific T-cell activation may also occur in areas where antigens are presented in association with extracellular matrix proteins. The recent finding that the invasion protein of Yersinia spp. delivers co-stimulatory signals to anti-CD3-activated human T cells, most probably through the b1 integrins, suggests that bacterial products can also bind to contribute to the activation of T cells [12].(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1994

The outer surface protein A of the spirochete Borrelia burgdorferi is a plasmin(ogen) receptor.

The spirochete Borrelia burgdorferi is the causative agent of Lyme borreliosis (Lyme disease) and is transmitted to mammalian hosts by tick vectors. In humans, the bacteria induce a complex disease, which involves the skin, joints, heart, and nervous system. However, the pathogenic principles of this multisystem illness are far from being understood. To disseminate from the site of the tick bite and invade multiple organ sites, spirochetes have to penetrate normal tissue barriers, such as vascular basement membranes and other organized extracellular matrices. Substantial evidence from other invasive bacterial infections suggest that spirochetes may use endogenous or host-derived enzymes--in particular, proteinases--for this purpose. Here we show that B. burgdorferi binds human plasmin(ogen)--mainly via its outer cell surface lipoprotein A. Binding of plasminogen to spirochetal receptor leads to an accelerated formation of active plasmin in the presence of host-derived plasminogen activator. The cell-surface-associated plasmin cannot be regulated by the serum inhibitor alpha 2-antiplasmin and degrades high-molecular-weight glycoproteins, such as fibronectin. It is suggested that the acquisition of host-derived proteinase plasmin(ogen) contributes to the pathogenicity of B. burgdorferi.

Antigens, Surface↗
PubMed1994

Urokinase-type plasminogen activator enhances invasion of human T cells (Jurkat) into a fibrin matrix.

The receptor for urokinase-type plasminogen activator (uPA-R) localizes uPA to the cell surface. The receptor-bound uPA converts plasminogen to the trypsin-like endopeptidase plasmin. Thus uPA is involved in the initiation of pericellular proteolysis. Pericellular proteolysis is assumed to facilitate the cellular infiltration into surrounding tissue. The uPA-R has recently been identified as a surface antigen of activated human T lymphocytes. We have characterized the uPA-R of the human CD4 T cell line Jurkat by immunological (flow cytometry), biochemical (ligand blotting), and physico-chemical (Scatchard blotting) methods. The collective data suggest that the human CD4+ T cell line Jurkat expresses a cell surface receptor for uPA similar to that of myelo/monocytes and normal T cells with regard to size, affinity, ligand specificity, and antigenicity. Binding studies using exogenous uPA and subsequent functional assays revealed that receptor-bound uPA retains its enzymatic activity. Saturation of the Jurkat cell uPA-R with exogenous uPA facilitated cellular invasion into fibrin matrices in vitro. uPA-dependent invasion was inhibited in the presence of an anti-catalytic monoclonal anti-uPA antibody. We propose that uPA-R-bound uPA may facilitate the invasiveness of uPA-R-positive T lymphocytes.

CD4-Positive T-Lymphocytes↗
PubMed1994

The receptor for urokinase-type plasminogen activator of a human keratinocyte line (HaCaT).

It is assumed that plasmin participates in pericellular proteolysis in the epidermis. Plasmin is generated by keratinocyte-associated plasminogen activators from the proenzyme plasminogen; plasminogen activation can proceed at the keratinocyte surface. The resultant plasmin interferes with cell to matrix adhesion and does possibly contribute to keratinocyte migration during reepithelialization. Here we describe the receptor for urokinase-type plasminogen activator (uPA-R) in the human keratinocyte cell line HaCaT, which serves to direct plasminogen activation to the cell surface; we relate the receptor to the uPA-R previously described in human myelo-/monocytes. Binding of uPA to the receptor accelerated plasminogen activation by a factor of approximately 10, compared to uPA in solution. Receptor-bound uPA was susceptible to inhibition by the plasminogen activator inhibitors 1 and 2. uPA and uPA-R antigen, as well as uPA activity, were localized to the leading front of expanding sheets of HaCaT cells. Exposure of HaCaT cells to plasminogen was followed by detachment of the cells. Detachment was prevented by an anticatalytic anti-uPA antibody, by the plasmin-specific inhibitor aprotinin, and by the lysine analogue tranexamic acid, the latter of which prevents plasmin(ogen) binding to the cell surface. Our findings support the hypothesis that uPA-mediated plasminogen activation is characteristic of mobile rather than sessile keratinocytes. Moreover, the uPA-R seems to focalize plasminogen activation to the surface of cells at the site of keratinocyte migration.

Base Sequence↗
PubMed1994

[Sclerodermiform skin changes in Borrelia burgdorferi infection. Diagnostic use of polymerase chain reaction].

We report on a 61-year-old patient with sclerodermiform skin lesions of the extremities and polyneuropathy. Borrelia (B.) burgdorferi DNA was detected in lesional skin by the polymerase chain reaction. Serological testing revealed IgG antibodies to B. burgdorferi. Histology revealed an inflammatory stage of a sclerotic reaction in the lesional skin. The admixture of plasma cells and the perineural distribution of the cellular infiltrate was suggestive for a borrelia infection. Immunohistochemical staining for the B. burgdorferi flagellin (41 kDa) revealed a positive staining reaction in the epidermis of lesional skin. The improvement of both the dermatological and the neurological symptoms upon antibiotic treatment with ceftriaxone was taken as further support for the diagnosis of a. B. burgdorferi infection.

Antibodies, Bacterial↗
PubMed1994

Granzyme A.

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Animals↗
PubMed1994

Characterization of Borrelia burgdorferi isolated from different organs of Ixodes ricinus ticks collected in nature.

Borrelia burgdorferi was isolated from 22 out of 133 adult Ixodes ricinus ticks collected from vegetation at two sites in Switzerland. From 17 ticks, spirochetes could be isolated from more than one organ. When the different isolates obtained from one tick were compared by SDS-PAGE analysis, differences in the protein profiles were observed in 8 cases. The isolates were further compared by immunological methods using mono- and polyclonal antibodies. Differences were observed in the proteins of 31-35 kDa and 18-25 kDa. Genetic divergence among isolates was evaluated by use of a B. burgdorferi specific gene probe for ospA. Correlation could be observed between immunological differences in OspA defined by monoclonal antibody LA31 and genetic variation of ospA as judged by restriction fragment length polymorphism (RFLP). Our findings indicate that systemic infection in unfed I. ricinus adults, as reflected by isolation of B. burgdorferi from multiple organs of one tick, is more frequent (8/22, 36%) than previously described (5%). Moreover, the presence of different B. burgdorferi phenotypes/genotypes in one tick is described for the first time. The findings may have bearings (i) on the time of tick attachment required for spirochete transmission since borreliae are already present in the salivary glands of systemically infected ticks at the beginning of the blood meal and (ii) perhaps also on the diversity of B. burgdorferi phenotypes inoculated by these ticks.

Animals↗
PubMed1994

Comparison in the immunological properties of Borrelia burgdorferi isolates from Ixodes ricinus derived from three endemic areas in Switzerland.

Borrelia burgdorferi isolates were obtained from Ixodes ricinus from three sites in Switzerland. They were examined by SDS-PAGE and immunoblotting. The phenotypes, in respect of three outer surface proteins (Osp), differed between the sites of collection. In site 1, most isolates had an OspA of 31 kDa and an OspB of 34 kDa; in site 2, isolates presenting an OspA of 33 kDa dominated and in site 3, the isolates with an OspA of 32 kDa and an OspB of 35 kDa were most frequent. This distribution differed significantly. About half of the isolates from sites 1 and 3 reacted with anti-OspA monoclonal antibody H5332 compared to 29% from site 2. Site 1 isolates reacted significantly more frequently (81%) with another anti-OspA monoclonal antibody LA-31 than isolates from site 3 (P < 0.0001). These findings have implications for the epidemiology of Lyme borreliosis, for the further development of serodiagnostic reagents and for the development of a vaccine.

Animals↗