PubMed Health⌕ Search

Biomedical subjects

P Koolwijk

Publications and source records attributed to P Koolwijk.

36 records · Page 2Linked to original sources
PubMed1998

Effect of steroid hormones and retinoids on the formation of capillary-like tubular structures of human microvascular endothelial cells in fibrin matrices is related to urokinase expression.

Angiogenesis, the formation of new capillary blood vessels, is a feature of a variety of pathological processes. To study the effects of a specific group of hormones (all ligands of the steroid/retinoid/thyroid hormone receptor superfamily) on the angiogenic process in humans, we have used a model system in which human microvascular endothelial cells from foreskin (hMVEC) are cultured on top of a human fibrin matrix in the presence of basic fibroblast growth factor and tumor necrosis factor-alpha. This model mimics the in vivo situation where fibrin appears to be a common component of the matrix present at sites of chronic inflammation and tumor stroma. Our results show that testosterone and dexamethasone are strong inhibitors and all-trans retinoic acid (at-RA) and 9-cis retinoic acid (9-cis RA) are potent stimulators of the formation of capillary-like tubular structures. These effects are mediated by their respective nuclear hormone receptors as demonstrated by the use of specific synthetic receptor agonists and antagonists. 17beta-estradiol, progesterone, and 1,25-dihydroxyvitamin D3 did not affect or only weakly affected in vitro angiogenesis, which may be related to the lack of significant nuclear receptor expression. Although hMVEC express both thyroid hormone receptors alpha and beta, no effect of thyroid hormone on tube formation was found. The effects of testosterone, dexamethasone, at-RA, and 9-cis RA on tube formation were accompanied by parallel changes in urokinase-type plasminogen activator (u-PA) expression, at both mRNA and antigen levels. Exogenous suppletion of the medium with single chain u-PA enhances tube formation in our in vitro model, whereas quenching of u-PA activity (but not of tissue-type plasminogen activator activity) or of u-PA binding to u-PA receptor by specific antibodies suppressed basal and retinoid-stimulated tube formation. Moreover, addition of scu-PA to testosterone- or dexamethasone-treated hMVEC restored the suppressed angiogenic activity for a substantial part. Aprotinin, an inhibitor of plasmin activity, completely inhibited tube formation, indicating that the proteolytic properties of the u-PA/u-PA receptor complex are crucial in this process. Our results show that steroid hormones (testosterone and dexamethasone) and retinoids have strong, but opposite effects on tube formation in a human in vitro model reflecting pathological angiogenesis in the presence of fibrin and inflammatory mediators. These effects can be explained by hormone-receptor-mediated changes in u-PA expression, resulting in enhanced local proteolytic capacity of the u-PA/u-PA receptor complex.

Aprotinin↗
PubMed1998

A novel and simple immunocapture assay for determination of gelatinase-B (MMP-9) activities in biological fluids: saliva from patients with Sjögren's syndrome contain increased latent and active gelatinase-B levels.

Here we describe a new principle for accessing the activity of the different members of the human matrix-metalloproteinases (MMPs) by a colorimetric assay. Using protein engineering, a modified pro-urokinase was made in which the activation sequence, normally recognized by plasmin (ProArgPheLys/IleIleGlyGly), was replaced by a sequence that is specifically recognized by MMPs (ArgProLeuGly/IleIleGlyGly). The active urokinase resulting from the activation of this modified pro-urokinase by MMPs can be measured directly using a chromogenic peptide substrate for urokinase. The assay has been made specific for MMP-9 using an MMP-9 specific monoclonal antibody. Using this antibody MMP-9 is captured from biological fluids or tissue culture media, and MMP-activity of both active and latent MMP-9 can be analysed. We determined the gelatinase-B (MMP-9) activity present in saliva from patients with Sjögren's syndrome. Using a general gelatinase assay with radioactively-labeled gelatinated collagen it was observed that gelatinase activity was slightly, though not significantly, increased in patients: general gelatinase activity in patients versus healthy controls: 17.0 +/- 4.9 vs 12.2 +/- 2.5 x 10(4) cpm/ml (p > 0.05, and 44.0 (4.0 vs 36.1 +/- 1.9 x 10(4) cpm/ml (p > 0.05), for active and latent gelatinase, respectively. However, using the immunocapture activity assay (using modified urokinase) specifically MMP-9 activity was measured, which was significantly increased in saliva from patients compared to healthy controls: MMP-9 (already active): patients 8.9 +/- 2.5 U/mg, controls 1.0 +/- 0.5 U/mg (p = 0.002); latent plus active MMP-9: patients 53.1 +/- 9.8 U/mg, controls 16.5 +/- 2.6 U/mg (p = 0.01). This assay, measuring MMP-9 activity using modified pro-urokinase as a substrate can easily be adapted for the specific detection of the various members of the MMP-family or other difficult to measure proteases, in a format that can be used for high throughput screening of compounds or samples.

Animals↗
PubMed1998

Influence of fibrin structure on the formation and maintenance of capillary-like tubules by human microvascular endothelial cells.

Fibrin is a temporary matrix which not only covers a wound, but also provides a structure for invading cells during healing. Changes in the polymerization conditions before gelation of the clot affect the structure of fibrin and thus might influence the interaction with invading cells. Therefore we tested whether changes in the fibrin structure influence the formation of capillary-like tubular structures by human microvascular endothelial cells (hMVEC) in an in vitro angiogenesis model. Opaque [125I]fibrin structures prepared at pH 7.0, fibrin matrices at pH 7.4 and transparent [125I]fibrin structures prepared at pH 7.8 were neutralized (pH 7.4) before seeding hMVEC on top of them in confluent density. Endothelial cells were stimulated with a growth factor [basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF)165] and a cytokine [tumor necrosis factor (TNF)-alpha] to induce the u-PA/u-PA receptor-dependent formation of capillary-like tubular structures. The formation of these structures was quantified by determining the length of the invasive structures by image analysis and by measuring the accompanying [125I]fibrin degradation. Ingrowth of tubular structures proceeded at a faster rate in opaque matrices consisting of thick fibrin fibers as compared to transparent gels with fine fibrin fibers. The more rapid ingrowth of tubular structures in opaque fibrin gels induced by bFGF/TNF-alpha or VEGF165/TNF-alpha was accompanied by a larger extent of fibrin degradation. Both processes were inhibited by aprotinin and epsilon-aminocaproic acid indicating the involvement of plasmin. They were also inhibited by anti-u-PA or anti-u-PA receptor IgG, but not by anti-t-PA IgG, suggesting the involvement of cell-bound u-PA activity. However, in the opaque fibrin gels, the tubular structures dissolved upon prolonged incubation due to excessive fibrin degradation. Simulation of hMVEC with bFGF alone did not induce tubular structures, but ca used a high degree of t-PA- and plasmin-dependent fibrin lysis, and, after several days, a partial detachment of sheets of cells. Gradual inhibition of the excessive fibrin degradation by a series of aprotinin concentrations did not lead to tube formation in bFGF-treated cells. These data indicate that the formation and stability of tubular structures by hMVEC in fibrin is accompanied by controlled fibrinolysis and depends critically not only on cell-bound u-PA-dependent plasminogen activation, but also on the fibrin structure. Because the fibrin structure is largely influenced by the conditions in which fibrin has been polymerized, these conditions may have considerable impact on angiogenesis during wound healing and vascularization of tumour stroma.

Journal Article↗
PubMed1998

Inhibition of MMP synthesis by doxycycline and chemically modified tetracyclines (CMTs) in human endothelial cells.

Doxycycline is a commonly used broad-spectrum antibiotic. Recently, it has been shown that it also inhibits the activity of mammalian collagenases and gelatinases, an activity unrelated to its antimicrobial efficacy. In this study, we show that doxycycline not only inhibits MMP-8 and MMP-9 (gelatinase B) activity, but also the synthesis of MMPs in human endothelial cells. Doxycycline (50 microM) completely inhibited the phorbol-12-myristate-13-acetate (PMA)-mediated induction of MMP-8 and MMP-9, as measured by Western blotting and gelatin zymography, respectively. The inhibition was also observed at the mRNA level. No effect was observed on the expression of MMP-2 and of the MMP inhibitors TIMP-1 and TIMP-2. Chemically modified tetracyclines (CMTs) showed an inhibition similar to that of doxycycline, albeit less efficient. These observations demonstrate that endothelial cells display a specific regulation of MMPs, which may have implications for the pharmaceutical interaction in angiogenesis and angiogenesis-related diseases.

Anti-Bacterial Agents↗
PubMed1997

Role of fibrin and plasminogen activators in repair-associated angiogenesis: in vitro studies with human endothelial cells.

Angiogenesis, the formation of new blood vessels from existing ones, plays a central role in development and in a number of pathological conditions. Tissue repair-associated angiogenesis usually involves cell invasion into a fibrin structure and the presence of inflammatory cells. In this chapter the role of plasminogen activators in the dissolution of fibrin and the invasion of endothelial cells into a fibrin matrix is described. Tissue-type plasminogen activator is stored in endothelial cells and can be released acutely into the vessel lumen upon stimulation of the endothelium to activate fibrinolysis and to prevent fibrin deposition. At the basolateral side of the cell, urokinase-type plasminogen activator (uPA) bound to a specific cellular receptor is involved in the proteolytic modulation of matrix proteins and cell-matrix interaction. The cytokine tumor necrosis factor-alpha (TNF-alpha) cooperates with the angiogenic factors basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF) in inducing human microvascular endothelial cells in vitro to invade a three dimensional fibrin matrix and to form capillary-like tubular structures. The formation of these capillary-like tubules requires cell-bound uPA activity.

Animals↗
PubMed1996

Cooperative effect of TNFalpha, bFGF, and VEGF on the formation of tubular structures of human microvascular endothelial cells in a fibrin matrix. Role of urokinase activity.

In angiogenesis associated with tissue repair and disease, fibrin and inflammatory mediators are often involved. We have used three-dimensional fibrin matrices to investigate the humoral requirements of human microvascular endothelial cells (hMVEC) to form capillary-like tubular structures. bFGF and VEGF165 were unable to induce tubular structures by themselves. Simultaneous addition of one or both of these factors with TNFalpha induced outgrowth of tubules, the effect being the strongest when bFGF, VEGF165, and TNFalpha were added simultaneously. Exogenously added u-PA, but not its nonproteolytic amino-terminal fragment, could replace TNFalpha, suggesting that TNFalpha-induced u-PA synthesis was involved. Soluble u-PA receptor (u-PAR) or antibodies that inhibited u-PA activity prevented the formation of tubular structures by 59-99%. epsilon-ACA and trasylol which inhibit the formation and activity of plasmin reduced the extent of tube formation by 71-95%. TNFalpha or u-PA did not induce tubular structures without additional growth factors. bFGF and VEGF165 enhanced of the u-PAR by 72 and 46%, but TNFalpha itself also increased u-PAR in hMVEC by 30%. Induction of mitogenesis was not the major contribution of bFGF and VEGF165 because the cell number did not change significantly in the presence of TNFalpha, and tyrphostin A47, which inhibited mitosis completely, reduced the formation of tubular structures only by 28-36%. These data show that induction of cell-bound u-PA activity by the cytokine TNFalpha is required in addition to the angiogenic factors VEGF165 and/or bFGF to induce in vitro formation of capillary-like structures by hMVEC in fibrin matrices. These data may provide insight in the mechanism of angiogenesis as occurs in pathological conditions.

Capillaries↗
PubMed1995

Activated gelatinase-B (MMP-9) and urokinase-type plasminogen activator in synovial fluids of patients with arthritis. Correlation with clinical and experimental variables of inflammation.

OBJECTIVE: To evaluate the occurrence of gelatinase-B (matrix metalloproteinase 9, MMP-9) in synovial fluids (SF) of patients with arthritis to investigate the possible role of this neutral MMP in joint destruction. METHODS: In paired (series I) and unpaired SF (series II) we examined the occurrence of gelatinase-B, prostromelysin-1, and urokinase-type plasminogen activator (u-PA). RESULTS: In the paired SF a parallelism between the presence of activated gelatinase-B and the local arthritis activity scores of the knees was observed. Activated gelatinase-B correlated well with the presence of stromelysin-1 and u-PA, 2 enzymes probably involved in the activation process of gelatinase-B. In the 2nd series, activated gelatinase-B was found in 56 SF samples, whereas 82 samples did not exhibit activated gelatinase-B. The SF samples with the activated form of gelatinase-B showed a significantly higher ability to induce permeability changes in cultured monolayers of human endothelial cells, had more myeloperoxidase activity--secreted by infiltrated leukocytes--and had higher u-PA antigen concentrations, compared to SF samples without the activated form of gelatinase-B. CONCLUSION: Our data suggest that the presence of gelatinase-B is a reflection of the inflammatory condition of the joints of patients with arthritis, and that the activation of gelatinase-B in the joints, which may occur in a u-PA/plasmin dependent and/or a stromelysin dependent way, contributes to the progression of arthritis.

Adult↗
PubMed1994

Genistein reduces tumor necrosis factor alpha-induced plasminogen activator inhibitor-1 transcription but not urokinase expression in human endothelial cells.

The plasminogen activator inhibitor PAI-1 is markedly elevated in vivo and in vitro upon exposure to the inflammatory mediators tumor necrosis factor alpha (TNF alpha), interleukin-1 (IL-1), and bacterial lipopolysaccharide. Here we report that the isoflavone compound genistein prevents the increase in synthesis of PAI-1 induced by these inflammatory mediators in human endothelial cells in vitro, and partially reduces the basal PAI-1 production by these cells. These effects of genistein were accompanied by a decrease in PAI-1 mRNA and in a suppression of the PAI-1 transcription rate as shown by run-on assay. A specific action of genistein, probably by inhibiting a tyrosine protein kinase, is likely, because the structural genistein analogue daidzein, which has a low tyrosine protein kinase inhibitor activity, did not inhibit PAI-1 synthesis. Vanadate, a tyrosine protein phosphatase inhibitor, increased PAI-1 production. The effect of genistein on PAI-1 synthesis was rather selective. Herbimycin A also reduced PAI-1 synthesis, but several other tyrosine protein kinase inhibitors, namely tyrphostin A47, methyl-2,5-dihydroxy-cinnamate, and compound 5, were unable to do so. All these tyrosine protein kinase inhibitors reduced basic fibroblast growth factor (b-FGF)-induced [3H]thymidine incorporation in endothelial cells. This indicates that the effect of genistein on PAI-1 transcription proceeds independently of its effect on mitogenesis. In contrast to TNF-alpha-induced PAI-1 production, the transcription and synthesis of urokinase-type plasminogen activator (u-PA) was not inhibited by genistein. A TNF-alpha-mutant (Trp32Thr86TNF alpha) that specifically recognizes the 55-kD TNF-receptor, mimicked the effects of TNF alpha on both PAI-1 and u-PA. Because genistein affected PAI-1, but not u-PA induced by this mutant, involvement of different TNF-receptors cannot underlie the difference in the effects of genistein on PAI-1 and u-PA synthesis. Because genistein also inhibited PAI-1 induction by thrombin and IL-4, it is likely that genistein does not act on a TNF alpha-receptor-coupled protein kinase but on the signal transduction pathway enhancing PAI-1 transcription. Our results suggest that the TNF alpha-induced signal transduction pathway of PAI-1 transcription involves a genistein-sensitive step that is not involved in the induction of u-PA by TNF alpha. Given the limited sensitivity to several other tyrosine protein kinase inhibitors, this genistein-sensitive step may be a potential target for pharmacologic intervention to reduce elevated plasma PAI-1 levels.

Cells, Cultured↗
PubMed1993

Regulation of matrix metalloproteinase expression in human vein and microvascular endothelial cells. Effects of tumour necrosis factor alpha, interleukin 1 and phorbol ester.

Matrix metalloproteinases (MMPs) play a role in tissue remodelling and angiogenesis. We have investigated the expression and regulation of MMP-1 (interstitial collagenase), MMP-2 (gelatinase A), MMP-3 (stromelysin 1), MMP-7 (matrilysin), MMP-9 (gelatinase B) and their inhibitors TIMP-1 and TIMP-2 in human umbilical vein, femoral vein and microvascular endothelial cells, and compared these data with those obtained with human synovial fibroblasts. Non-stimulated vein endothelial cells expressed the mRNAs for MMP-1, MMP-2, TIMP-1 and TIMP-2. MMP-3 mRNA and protein were undetectable or only weakly expressed, but could be stimulated by the inflammatory mediator tumour necrosis factor alpha (TNF alpha). The expression of MMP-3 and MMP-1 was further enhanced by phorbol 12-myristate 13-acetate (PMA). Phorbol ester also induced TIMP-1 and MMP-9, the expression of the latter being further enhanced by TNF alpha or interleukin 1 alpha (IL-1 alpha). Similar stimulatory effects were observed in microvascular endothelial cells. Hence the inflammatory mediator TNF alpha induces/enhances the production of several matrix metalloproteinases in human endothelial cells. On the other hand, MMP-2 and TIMP-2 were not affected or were affected in a variable way by TNF alpha and/or phorbol ester, suggesting a dissimilar regulation of these proteins. The cyclic AMP-enhancing agent forskolin affected the production of MMPs in a cell-type-specific way. In human vein endothelial cells it enhanced the PMA-mediated induction of MMP-9, whereas it suppressed this induction in human microvascular endothelial cells and in synovial fibroblasts. On the other hand, forskolin suppressed the PMA-mediated induction of MMP-1 and MMP-3 in synovial fibroblasts, while it enhanced or did not affect this induction in various types of human endothelial cells. These observations may have implications for future pharmacological intervention in angiogenesis.

Capillaries↗
PubMed1992

Human monocyte-mediated cytotoxicity towards erythrocytes induced by hybrid mouse monoclonal antibodies: effect of antibody binding valency on IgG-Fc gamma R interaction.

In this study, we describe the ability of hybrid mouse monoclonal antibody (mAb) to induce monocyte-mediated cytotoxicity towards human IgA1-coated E (HuIgA1-E), and the effect of mAb binding valency on Fc gamma RI-mediated ADCC. All hybrid monospecific (ms) anti-HuIgA1 and bispecific (bs) anti-HuIgA1/HRP mAb were capable of inducing monocyte-mediated lysis of HuIgA1-E, in spite of differences in mAb densities essential for optimal lysis. The cytotoxicity induced by hybrid mAb which consist of one or more mIgG2a H chains was predominantly mediated via Fc gamma RI, as shown by inhibition studies on monocytes with Fc gamma RI-blocking mAb TB-3 (approximately 80% inhibition). However, partial inhibition of mIgG1-2a and mIgG2a-2b-induced cytotoxicity (20-50%) was observed by using Fc gamma RII-blocking mAb IV.3 or CIKM5. For hybrid mIgG1-1 mAb the opposite was true; the cytotoxicity was predominantly mediated via Fc gamma RII (70-80%) and less via Fc gamma RI (20-30%). Comparing the hybrid ms anti-HuIgA1 mAb-induced cytotoxicity with the cytotoxicity induced by hybrid bs anti-HuIgA1/HRP mAb of the same isotype, we observed a decrease in cytotoxicity towards HuIgA1-E sensitized with univalently bound bs anti-HuIgA1/HRP mAb. This decrease was only found for Fc gamma RI-mediated ADCC (mIgG2a-2a, mIgG1-2a and mIgG2a-2b). This diminished recognition of univalently bound IgG relative to bivalently bound IgG by Fc gamma RI was also observed with U937 effector cells. In conclusion, this work shows that hybrid mAb are able to induce monocyte-mediated cytotoxicity towards E-HuIgA1 and that there appears to be an effect of Ag-IgG binding valency on Fc gamma RI-mediated cytotoxicity.

Antibodies, Monoclonal↗
PubMed1991

An isotype-specific spot-ELISA for the enumeration of antibody-secreting hybridomas and the determination of isotype switch variants.

A solid-phase spot enzyme-linked immunosorbent assay (spot-ELISA) using rat monoclonal antibodies (MAbs) and an image-processing system is described. This isotype-specific spot-ELISA permits the enumeration of antibody-secreting cells irrespective of the specificity of the secreted antibodies. When used in combination with an ELISA, the antibody production per cell can also be evaluated. In addition, isotype switch variants, which arise spontaneously in antibody-producing cell lines, can be determined. This study compared four assays: three antigen-specific spot-ELISAs, using enzyme-conjugated polyclonal antibodies as well as rat MAbs; and an isotype-specific spot-ELISA using rat MAbs. There were no significant differences between these four spot-ELISA systems. For one tested cell line (alpha huIgA1/gamma 1), the number of antibody-secreting cells fluctuated between 60% and 95% during several passages. For the other tested cell line (alpha huIgA1/gamma 2b), the number of antibody-secreting cells decreased from 90% to 70% after several passages. The results of the spot-ELISA were in agreement with flow cytometric (FC) analysis of cytoplasmic IgG. This indicates that for these two cell lines, the synthesized IgG was also secreted into the culture fluid. Using the isotype-specific spot-ELISA, the switch frequency of five murine hybridomas (alpha huIgA1/gamma 1, alpha huIgA1/gamma 2b, alpha HRP, RIV6, MN12) was determined. The switch frequencies varied from 1/82,000 for the alpha HRP cell line to 1/660,000 for the alpha huIgA1/gamma 2b cell line.

Animals↗
PubMed1991

Induction of intracellular Ca2+ mobilization and cytotoxicity by hybrid mouse monoclonal antibodies. Fc gamma RII regulation of Fc gamma RI-triggered functions or signaling?

We studied the interaction of bispecific mouse mAb with human IgG Fc receptors, and assessed their ability to activate the monocytic cell line U937. Binding of monomeric hybrid anti-HuIgA1/HRP mAb to the high-affinity IgG receptor, Fc gamma RI, on U937 cells was only observed when mAb with one or more mIgG2a H chains (hybrid mIgG1-2a, mIgG2a-2b, and mIgG2a-2a) were used. These Fc gamma RI-bound hybrid mAb were capable of enhancing the internal free cytosolic Ca2+ concentration ([Ca2+]i) in U937 cells only when bound mIgG were cross-linked using F(ab')2 fragments of goat anti-mIg antibody. A hybrid mIgG1-2a mAb were cross-linked using goat anti-mIgG1 antibody, showing that the hybrid mAb themselves mediate the induction of Ca2+ increase. Remarkably, anti-Fc gamma RII mAb IV.3 was able to inhibit the Ca2+ increase induced via mIgG2a-1 or mIgG1-2a hybrid mAb completely, despite the fact that we could not detect any effect of IV.3 on binding of monomeric hybrid mIgG1-2a or mIgG2a-1 mAb to U937. The hybrid mAb were also able to induce lysis of HuIgA1-coated E using U937 effector cells. This lysis was completely inhibited by preincubation of U937 cells with mIgG2a mAb TB-3, which blocks Fc gamma RI via its Fc-part ("Kurlander phenomenon"). In contrast, Fc gamma RII-blocking mAb IV.3 and CIKM5 caused a significant enhancement of the antibody-dependent cellular cytotoxicity (ADCC) activity mediated by hybrid mIgG1-2a and mIgG2a-2b mAb. This enhancement did not occur when the parental anti-HuIgA1/2a or the hybrid anti-HuIgA1/HRP/2a-2a mAb were evaluated for ADCC activity. These findings suggest that hybrid mAb not only can bind to Fc gamma RI, but can mediate functional activation of myeloid cells. Given the effect of mAb IV.3 on [Ca2+]i changes and ADCC triggered through IgG1-2a mAb, we suggest that Fc gamma RII may have a role in the regulation of Fc gamma RI-triggered functions or signaling.

Antibodies, Monoclonal↗
PubMed1991

Binding of the human complement subcomponent C1q to hybrid mouse monoclonal antibodies.

Activation of the classical pathway of the complement system is initiated by the binding of C1q to antibody complexes. Here we evaluated the C1q binding capacity of series of monospecific and bispecific hybrid mouse monoclonal antibodies (mAb) and compared them with parental (conventional) mAb. The hierarchy in C1q binding capacity of the bispecific anti-HuIgA1/HRP mAb with homologous H-H chain combinations (IgG2a-2a, IgG2b-2b and IgG1-1) and the parental anti-HuIgA1 or anti-HRP mAb was identical; IgG2a greater than IgG2b much greater than IgG1. Hybrid IgG1-2a mAb bind intermediate amounts of C1q when compared with the IgG1 and IgG2a parental antibodies. IgG1-2b and IgG1-1 hybrid mAb did not bind any C1q, like the IgG1 mAb. We could not observe any difference in C1q binding efficiency between monovalently bound IgG1-2a, IgG2a-2a and IgG2b-2b anti-HuIgA1 HRP mAb and the bivalently bound IgG1-2a, IgG2a-2a and IgG2b-2b anti-HuIgA1 mAb, respectively. Furthermore, these hybrid ms anti-HuIgA1 and bs anti-HRP/HuIgA1 mAb were able to lyse HuIgA1-coated erythrocytes, in the presence of 50% human serum, as efficiently as their parental counterparts. These data indicate that a simultaneous binding of both F(ab') fragment to antigen is not a necessary prerequisite for binding and activation of C1q.

Antibodies, Monoclonal↗
PubMed1989

Interaction between hybrid mouse monoclonal antibodies and the human high-affinity IgG FcR, huFc gamma RI, on U937. Involvement of only one of the mIgG heavy chains in receptor binding.

Here we have used hybrid mouse IgG1-2a and IgG2a-2b mAb to demonstrate that the interaction between the human high-affinity IgG FcR (huFc gamma RI) and monomeric mouse IgG2a mAb requires only one of the mIgG2a H chains. Recently, we reported a method for the generation and isolation of hybrid hybridomas, producing hybrid mouse mAb. Using this method we have obtained hybrid mouse (m)IgG1-2a and mIgG2a-2b mAb reacting with either horseradish peroxidase or human IgA1 (monospecific mAb) or with both Ag (bispecific mAb). Using protein A- or Ag-affinity chromatography purified hybrid mAb, we demonstrate here the binding of monomeric hybrid mIgG1-2a and mIgG2a-2b mAb to huFc gamma R on U937 cells, whereas no binding could be observed to the K562 cell line. Monomeric mouse IgG2a mAb and human IgG1 were found to be capable of inhibiting the binding of these hybrid mIgG1-2a and mIgG2a-2b mAb in a manner similar to the way they inhibited binding of monomeric mIgG2a mAb to U937 cells; this is in contrast to our findings for mIgG1 and mIgG2b mAb which did not inhibit the binding of both hybrid mAb. In addition, the binding of the hybrid mIgG1-2a and mIgG2a-2b mAb could be blocked by mAb TB-3, which is known to block huFc gamma RI-mediated binding by the "Kurlander phenomenon" and not by the anti-Fc gamma RII mAb CIKM5 and IV.3. These results indicate that both types of monomeric hybrid mAb are bound by the huFc gamma RI. Scatchard plots of mIgG2a, hybrid mIgG1-2a, and mIgG2a-2b mAb binding revealed similar numbers of binding sites and similar affinity constants of huFc gamma RI for these mAb (0.9 to 3.6 x 10(8) M-1). These results suggest that huFc gamma RI, present on the U937 cell line, are capable of binding monomeric hybrid mIgG1-2a and mIgG2a-2b mAb, and that this interaction requires only one of the mIgG2a H chains.

Animals↗
PubMed1988

Enrichment and selection of hybrid hybridomas by Percoll density gradient centrifugation and fluorescent-activated cell sorting.

Hybrid hybridomas, producing bi-specific monoclonal antibodies that react with horseradish peroxidase and human IgA1 were isolated by sorting the double-fluorescent cells on single-cell basis after fusion of two hybridomas, previously labelled green or red by octadecylamine-FITC or -TRITC, respectively. The double-fluorescent fused cells were significantly different in AXL (size) and RAS (internal structure) distribution compared with the (non-fused) mono-fluorescent cells. The percentage of double-fluorescent cells and the viability of these cells could be increased by Percoll density gradient centrifugation. As a result, there was an 8-fold increase of total isolated hybrid hybridomas (up to 30% of all tested clones) compared to isolations without Percoll density gradient centrifugation. All the isolated hybrid hybridoma clones had similar amounts of DNA, equal to the sum of the DNA of both parental hybridomas.

Antibodies, Monoclonal↗
PubMed1986

Transport of trehalose in Salmonella typhimurium.

We have studied trehalose uptake in Salmonella typhimurium and the possible involvement of the phosphoenolpyruvate:carbohydrate phosphotransferase system (PTS) in this process. Two transport systems could recognize and transport trehalose, the mannose PTS and the galactose permease. Uptake of trehalose via the latter system required that it be expressed constitutively (due to a galR or galC mutation). Introduction of a ptsM mutation, resulting in a defective IIMan/IIIMan system, in S. typhimurium strains that grew on trehalose abolished growth on trehalose. A ptsG mutation, eliminating IIGlc of the glucose PTS, had no effect. In contrast, a crr mutation that resulted in the absence of IIIGlc of the glucose PTS prevented growth on trehalose. The inability of crr and also cya mutants to grow on trehalose was due to lowered intracellular cyclic AMP synthesis, since addition of extracellular cyclic AMP restored growth. Subsequent trehalose metabolism could be via a trehalose phosphate hydrolase, if trehalose phosphate was formed via the PTS, or trehalase. Trehalose-grown cells contained trehalase activity, but we could not detect phosphoenolpyruvate-dependent phosphorylation of trehalose in toluenized cells.

Bacterial Proteins↗
PubMedDate not supplied

Steroids and cytokines in endometrial angiogenesis.

Angiogenesis, the formation of new blood vessels from pre-existing ones, occurs physiologically in the endometrium and pathologically e.g. during tumour growth. Sex-steroid hormones affect angiogenesis in the endometrium during the menstrual cycle and might be implicated in cancer angiogenesis. Little information is presently available regarding the exact mechanisms by which these steroids exert their function on the process of both physiological and pathological angiogenesis. In this overview a survey is given of factors important for angiogenesis and of the effects of steroids on the expression of these factors. We have focused on endometrial angiogenesis, because the endometrium is unique in its cyclic growth pattern for which angiogenesis is indispensable. Stimulators and inhibitors of endometrial angiogenesis, that have been found (or suggested) to respond to ovarian steroids, are discussed These factors include vascular endothelial growth factor (VEGF), fibroblast growth factors (FGFs), tumour necrosis factor a (TNF-alpha), erythropoietin (Epo) and trombospondin-1 (TSP-1). Also, the influence of steroids on the expression of matrixdegrading proteases, in particular the plasminogen activator/plasmin system and matrixdegrading metalloproteinases (MMPs) are reviewed, because these proteases play an important role in the migration and invasion of endothelial cells during the process of angiogenesis. An insight into the effects of steroids on endometrial angiogenesis may be helpful to understand and anticipate the potential stimulatory and inhibitory effect of various steroids on angiogenesis in other tissues, in particular tumours.

Animals↗