Targeted disruption of mouse fibroblast activation protein.

Human fibroblast activation protein (FAP), a member of the serine prolyl oligopeptidase family, is a type II cell surface glycoprotein selectively expressed by fibroblastic cells in areas of active tissue remodeling, such as the embryonic mesenchyme, areas of wound healing, the gravid uterus, and the reactive stroma of epithelial cancers. Homologues of FAP have been identified in the mouse and Xenopus laevis. FAP is a dual-specificity enzyme that acts as a dipeptidyl peptidase and collagenase in vitro. To explore the role of FAP in vivo, Fap(-/-) mice were generated by homologous recombination. RNase protection analysis and reverse transcription-PCR confirmed the absence of full-length Fap transcripts in mouse embryonic tissues. No FAP protein was detected in Fap(-/-) animals by immunohistochemistry, and no FAP-specific dipeptidyl peptidase activity was found. We report that Fap(-/-) mice are fertile, show no overt developmental defects, and have no general change in cancer susceptibility.

BIBW 22, a dipyridamole analogue, acts as a bifunctional modulator on tumor cells by influencing both P-glycoprotein and nucleoside transport.

BIBW 22, a phenylpteridine analogue of dipyridamole (DPM), enhanced vincristine cytotoxicity approximately 10 times more than DPM in a multidrug-resistant (MDR) KB V20C cell line. Using rhodamine 123 accumulation in KB V20C cells as an indicator of MDR phenotype, BIBW 22 was shown to be about 100 times more potent than DPM in inhibiting the MDR-associated efflux of rhodamine 123. Photolabeling of P-glycoprotein in KB V20C plasma membranes with 0.2 microM [3H]azidopine was strongly inhibited by 1 microM BIBW 22, indicating that this compound reverses the MDR phenotype by interfering with MDR-associated P-glycoprotein. In addition, BIBW 22 at 1 microM could also enhance the cytotoxicity of 5-fluorouracil in KB cells about 20-fold. Its potency in inhibiting nucleoside transport is 7-fold more potent than that of DPM. These results suggest that BIBW 22 is a potent bifunctional modulator which influences both P-glycoprotein and nucleoside transport in tumor cells. Potential use of this compound as a modulator of combination chemotherapy involving antimetabolites and drugs affected by MDR should be explored.

Effect of platelet-activating factor on human polymorphonuclear leukocyte enhancement of chemiluminescence and antibody-dependent cellular cytotoxicity.

The effect of platelet-activating factor on human granulocytes was determined by luminol-dependent chemiluminescence (CL), antibody-dependent cellular cytotoxicity (ADCC), and rosette assay. An activation of the respiratory burst could be measured by CL and be shown to depend on the presence of extracellular calcium. This direct stimulation of PMN was proved to be inhibited by oxygen radical scavengers as well as by the calcium channel blocker diltiazem. Furthermore, the presence of PAF enhanced the activation of PMN via Fc- or C3b-receptors, as demonstrated by CL and ADCC. On the other hand, no influence on the rosette-forming capacity of PMN could be detected. The results support the concept of the import role of PAF in inflammatory processes.

Expression of the fibroblast activation protein during mouse embryo development.

Human Fibroblast Activation Protein (FAP), a member of the serine prolyl oligopeptidase family, is a type II cell surface glycoprotein that acts as a dual-specificity dipeptidyl-peptidase (DPP) and collagenase in vitro. Its restricted expression pattern in embryonic mesenchyme, in wound healing and in reactive stromal fibroblasts of epithelial cancers, has suggested a role for the FAP protease in extracellular matrix degradation or growth factor activation in sites of tissue remodeling. The FAP homologue in Xenopus laevis has been reported to be induced in the thyroid hormone-induced tail resorption program during tadpole metamorphosis supporting a role for FAP in tissue remodeling processes during embryonic development. However, Fap-deficient mice show no overt developmental defects and are viable. To study the expression of FAP during mouse embryogenesis, a second Fap-deficient mouse strain expressing beta-Galactosidase under the control of the Fap promoter was generated by homologous recombination (Fap-/- lacZ mice). FAP deficiency was confirmed by the absence of FAP-specific dipeptidyl-peptidase activity in detergent-soluble extracts isolated from 17.5 d.p.c. Fap-/- lacZ embryos. We report that Fap-/- lacZ mice express beta-Galactosidase at regions of active tissue remodeling during embryogenesis including somites and perichondrial mesenchyme from cartilage primordia.

The influence of BIBW22BS, a dipyridamole derivative, on the antiproliferative effects of 5-fluorouracil, methotrexate and gemcitabine in vitro and in human tumour xenografts.

Dipyridamole is known as a potent inhibitor of facilitated diffusion-mediated nucleoside transport as well as a modulator of 'classical' multidrug resistance. BIBW22BS, a derivative of dipyridamole, has been found to be 20- to 100-fold more potent in the reversal of multidrug resistance when compared to the parent compound. In parallel, we studied the efficacy of BIBW22BS in the modulation of the antiproliferative effects of 5-fluorouracil, methotrexate and gemcitabine in human cancer cell lines. BIBW22BS, at non-toxic concentrations up to 1.0 microM, increased the antiproliferative effects of 5-fluorouracil 2- to 6-fold in seven of the eight colon cancer cell lines tested in a dose-dependent manner. The addition of 1.0 microM BIBW22BS to methotrexate resulted in a slight increase in the antiproliferative effects, but inhibited the activity of gemcitabine 30- to 100-fold in various cancer cell lines. In vitro, no notable difference was found between BIBW22BS and dipyridamole in their capacity to modulate the activity of the antimetabolites studied. BIBW22BS did not affect the growth inhibition induced by 5-fluorouracil or gemcitabine in human tumour xenografts grown subcutaneously in nude mice. We confirmed the higher potency of BIBW22BS when compared to dipyridamole in the reversal of drug resistance in the Pgp-positive COLO 320 cell line.

Localisation of human peripheral blood leukocytes after transfer to C.B-17 scid/scid mice.

Severe combined immunodeficient (scid) mice of the inbred strain C.B-17 lack functional T and B cells and, because of this, they tolerate xenografts. We reconstituted scid mice with human peripheral blood leukocytes (PBL) by i.p. injection. In order to determine the human PBL in lymphoid organs of these reconstituted scid mice, we labelled the human PBL prior to transfer with the fluorescent dye PKH 26-GL. With this experimental approach it was possible to detect the human cells in lymphoid organs and peritoneal exudate of the reconstituted scid mice by cytofluorimetrical and histological methods. This method is thus helpful for the determination of xenografts transplanted upon scid mice as well as in other experimental settings including adoptive transfer.

Chloroquine resistance in Plasmodium falciparum is not reversed by BIBW-22, a compound reversing the multidrug resistance phenotype in mammalian cancer cells.

The pteridine derivative BIBW-22 (4-[N-(2-hydroxy-2-methyl-propyl)-ethanolamino]-2,7-bis(cis-2,6-di methyl-morpholino)-6-phenylpteridine), which had been developed for the treatment of multidrug-resistant cancer and binds to P-glycoprotein, was tested against chloroquine resistant Plasmodium falciparum strains in culture. Based on the result that BIBW-22 enhanced rather than lowered chloroquine resistance in vitro, it is concluded that chloroquine resistance in malaria parasites may not be mechanistically linked to the multidrug-resistant phenotype of chloroquine resistant P. falciparum.

Cross-binding activity and protective capacity of monoclonal antibodies to lipid A.

Six hybridoma clones (1M, 4M, 9M, 11M, 18M and 31G), secreting monoclonal antibodies (mAbs) against lipid A were obtained after fusion between cells of mouse myeloma line X63-Ag8.653 and spleen cells from BALB/c mice immunized with acid treated Salmonella minnesota bacteria coated with additional free lipid A. The specificity and cross-binding activity of the mAbs were characterized in ELISA by using synthetic lipid A analogs as well as different lipid A and lipopolysaccharides (LPS) extracted from R- and S-form bacteria. It was found that the antibodies recognize epitopes in which phosphate groups, especially those at the C4' position of the glucosamine backbone of lipid A, were present. These epitopes were accessible also for the antibodies in purified intact LPS. By using a set of core glycolipids with increasing completion of the core region of the molecule and S-LPSs it was shown that the mAbs cross-reacted with a variety of R- and S-form LPS. The binding activity decreased with increasing length of the polysaccharide chain. The mAb did not prevent ultimate lethality of mice challenged with Klebsiella pneumoniae B and Salmonella typhimurium C5. However a delay of mortality rate of mice pretreated with antibodies 18M and 31G and infected with K. pneumoniae was seen.

Killer-cell lines derived from mouse thymus, resembling large granular lymphocytes and expressing natural killer-like cytotoxicity.

Murine thymus cells were maintained in vitro with supernatant-factors derived from Concanavalin-A-stimulated spleen cells. After an initial phase of vigorous proliferation, large granular cells (GC), which were not observed in fresh thymus cell preparations, appeared in these cultures. GC, derived from C3Hf/Tif-, BALB/c-, and C57BL/10-thymus cultures, could be slowly expanded and have been maintained as increasingly homogeneous (oligoclonal) lines for up to six months. During this time, other types of thymus cells died or were diluted out. Thymic GC differ functionally and histochemically from macrophages and mast cells. They do not phagocytize zymosan particles, bind opsonized SRBC, express nonspecific esterases or contain detectable amounts of histamine. GC share many features with natural killer (NK) cells and large granular lymphocytes (LGL). One morphologically representative line (C3Hf/Tif) had the following surface phenotype: Thy-1+, Lyt-1-, Lyt-2-, H-2K+, I-A-, asialo Gm1+. GC bind peanut agglutinin (PNA) on their surface and contain azurophilic granules. These cytoplasmic granules are considerably larger than those in LGL. Cells of a GC line derived from mouse strain C3Hf/Tif (H-2k) lysed the NK-sensitive YAC-1 (H-2a) and EL-4 (H-2b), but not the NK-insensitive P815 cells.

Modulation of multidrug resistance by BIBW22BS in blasts of de novo or relapsed or persistent acute myeloid leukemia ex vivo.

The phenylpteridine derivative BIBW22BS (BIBW22) is a potent modulator of multidrug resistance (MDR). We investigated BIBW22 in comparison to dexniguldipine and verapamil as modifier of MDR in blasts of de novo, relapsed or persistent acute myeloid leukemia (AML) in vitro. All patients with relapsed or persistent AML had been pretreated with idarubicin and cytosine arabinoside. The degree of MDR was determined by efflux kinetics of rhodamine 123 (R123), daunorubicin, and idarubicin measured by flow cytometry (FACS). A total of 51 patients with AML, 25 de novo and 26 relapsed or persistent, were investigated. While only 6 out of 25 de novo AML blast populations showed moderate efflux of R123 and daunorubicin, 17 out of 26 blast populations of relapsed or persistent AML had an efflux between 20% and 44% within 15 min ex vivo. This efflux could be significantly inhibited by 1 microM BIBW22, 1 microM dexniguldipine, or 10 microM verapamil. For idarubicin we found an effusion of 40+/-9% within 15 min in all blast populations that could not be inhibited by the modulators. Clinically achievable drug concentrations causing only moderate side-effects are in the range of 0.5 microM dexniguldipine and 3 microM verapamil. Up to now, BIBW22 has not been investigated clinically. Thus the potential toxicity of concentrations of 0.5-1 microM BIBW22, sufficient for an optimal efflux inhibition ex vivo, is not known yet. We conclude from our ex vivo investigations in blast populations of de novo, relapsed or persistent AML that BIBW22 is a potent modulator of MDR.

[Histological and cytofluorometric investigations of immunodeficient CB17 scid/scid mice]. / Histologische und zytofluorometrische Untersuchungen von immundefekten Mäusen des Stammes CB17 scid/scid.

Blood cell differentiation and development is under the control of a number of genes, and mutations in one of these genes could result in an immunodefective phenotype. Mice of the CB17 scid/scid ("severe combined imunodeficient") strain are characterized by a mutation which affects early lymphoid differentiation and lack functional T and B lymphocytes. We investigated the serum level of murine immunoglobulins of CB17 scid/scid mice in comparison with the wild type CB17. Furthermore we performed FACS-analysis of the peripheral blood cells as well as cells from the spleen and thymus of these animals. Histological methods were used to study the morphology of the lymphoid tissues. In the sera of CB17 scid/scid mice murine immunoglobuline levels below 50 ng/ml were found. Mice with a level between this and the normal level (5-10 mg/ml) were called "leakys". Cytofluorometric as well as histological investigations revealed no differences between scid and leaky mice, which both appeared quite different than immunocompetent CB17 (wildtype). On the other hand animals with lymphomas show mononuclear infiltrations in the lymphoid tissues. Inflammatory reactions in scid are characterized by leukocytic infiltrations in the draining lymphnodes.

Immunogenicity and immunomodulatory activity of bovine surfactant (SF-RI 1).

Respiratory distress syndrome in preterm infants can be treated successfully by endotracheal administration of a bovine surfactant preparation (SF-RI 1). Before the routine use of xenogenic surfactant preparations can be recommended, their immunogenicity as well as their in-vivo and in-vitro immunomodulatory activity have to be investigated. High titers of anti-surfactant antibodies were detected by a sensitive ELISA after immunizing rats, rabbits and mice with SF-RI 1. Repeated endotracheal administration of SF-RI 1 resulted in a humoral antibody response in three out of eight rabbits. After treatment of 34 preterm infants with SF-RI 1 (50-200 mg/kg), a humoral immune response to SF-RI 1 could not be detected. In-vitro restimulation of peripheral blood lymphocytes with SF-RI 1 after primary in-vivo administration did not result in cell proliferation as measured by 3H-thymidine incorporation. SF-RI 1 did not stimulate peripheral blood lymphocytes of neonates in vitro. The mitogenic response of these cells to stimulation with PHA, ConA or PWM was heavily impaired in the presence of SF-RI 1 concentrations increasing from 0.04 to 4 mg/ml. These data indicate that SF-RI 1 is immunogenic and that it may have an influence on lymphocyte proliferation in vivo.

BIBW22 BS, potent multidrug resistance-reversing agent, binds directly to P-glycoprotein and accumulates in drug-resistant cells.

The expression of P-glycoprotein (P-gp) in tumor cells causes a multidrug resistance (MDR) phenotype. P-gp has been shown to mediate the transport of structurally dissimilar drugs across the cell membrane in an energy-dependent manner. In this report, we show that BIBW22 BS, a phenylpteridine analog, reverses the MDR phenotype of CEM human lymphoma cells in a dose-dependent fashion. Using a photoactive analog of BIBW22 BS {[3H]azido-4-[N-(2-hydroxy-2-methylpropyl)-ethanolamino]-2, 7-bis(cis-2,6-dimethyl-morpholino)-6-phenylpteridine}, we show the photoaffinity labeling of a 170-kDa protein in drug-resistant cells immunoprecipitated with P-gp-specific monoclonal antibodies. The photolabeling of P-gp by [3H]azido-BIBW22 BS was specific and saturable. Furthermore, BIBW22 BS, vinblastine, and verapamil, but not colchicine, inhibited the photolabeling of P-gp by [3H]azido-BIBW22 BS. Drug binding studies showed that membranes from MDR cells bound more BIBW22 BS than parental drug-sensitive cells, and this binding was inhibited with vinblastine and, to a lesser extent, with uridine. However, drug transport studies demonstrated that BIBW22 BS is not a substrate for P-gp efflux pump. Interestingly, BIBW22 BS was shown to accumulate more in resistant cells. Also, BIBW22 BS accumulation in drug-sensitive and -resistant cells was not energy dependent. These results are in contrast with the observed decrease in accumulation or enhanced efflux of [3H]vinblastine seen in the same MDR cells. A comparison of [3H]azido-BIBW22 BS or [3H]azidopine photolabeled P-gp by Cleveland mapping with Staphylococcus aureus V8 protease showed differences in the photolabeled peptides. Taken together, the results of this study show that BIBW22 BS is a potent MDR-reversing agent that binds directly to P-gp but is not effluxed from drug-resistant cells.

Anti-idiotypic antibodies as staphylococcal enterotoxin receptor probes on monkey mast cells.

The immediate-type skin reaction in unsensitized monkeys upon challenge with staphylococcal enterotoxin B (SEB) was studied to define the role of mast cell receptors in the action of the toxin. For this purpose anti-idiotypic antibodies (anti-Id) were raised in BALB/c mice against monoclonal anti-SEB antibodies and purified by idiotype affinity chromatography. Anti-Id completely abolished skin reactions upon challenge with SEB without having biological functions itself. The data are compatible with the view that receptors for staphylococcal enterotoxin actually exist on the mast cell membrane of primates and anti-Id may be of potential value to influence the course of staphylococcal enterotoxin-mediated effects.

Human peripheral blood leukocytes transplanted on CB17 scid-scid mice are transferred to their offspring.

Severe combined immunodeficient (scid) mice are deficient in functional T cells and B cells. Hence, scid mice reconstituted with human peripheral blood leukocytes (scid-huPBL) provide an excellent model for analysis of the human immune response under in vivo conditions. We have investigated this model further by analyzing human immune responses in the progeny of scid-huPBL (termed scid-humo). We find markedly elevated levels of human immunoglobulins (Ig) in the serum of scid-humo for more than 12 weeks indicating materno-fetal transfer of human B lymphocytes. Consistent with this finding we obtained evidence for the existence of human lymphocytes in scid-humo. Murine Ig levels in scid-humo were also elevated and surface Ig-expressing cells (probably B cells) were demonstrable. In this respect scid-humo resembled "leaky" scid. In contrast to "leaky" scid, scid-humo accepted transfer of human blood leukocytes. Not only leukocytes from autologous but also those from heterologous donors were accepted. Human Ig levels in scid-humo increased more rapidly as compared to normal scid mice. Thus, despite these increased B cell activities in scid-humo, transferred human leukocytes were not affected indicating that materno-fetal transfer of human cells had caused tolerization or conditioning. This is in contrast to scid mice in which elevated Ig levels correlate with increased failure rates of reconstitution with human blood leukocytes. We propose that scid-humo provide an improved model for studying the human immune responses in an in vivo setting.

Biochemical modulation of 'classical' multidrug resistance by BIBW22BS, a potent derivative of dipyridamole.

BACKGROUND: Modulators of the 'classical' multidrug resistance (mdr) phenotype have low efficacy in patients with solid tumors. We analyzed BIBW22BS, 4-[N-(2-hydroxy-2-met- hyl-propyl)-ethanolamino]-2,7-bis(cis-2,6-dimethyl-morpho- lino)-6-phenylpteridine, a derivative of dipyridamole, for its higher potential to modulate mdr. MATERIALS AND METHODS: Four human malignant cell lines: BRO, A2780, GLC4, SW1573, the Pgp-positive sublines: BRO/mdr1.1, 2780AD and the non-Pgp sublines: GLC4/ADR, SW1573/2R120 were used in vitro to investigate BIBW22BS as a modulator of the antiproliferative effects of vincristine and doxorubicin and to compare the potency of BIBW22BS with that of dipyridamole, verapamil, bepridil and flunarizine. BRO/mdr1.1 s.c. well-established xenografts in nude mice were used to study the modulating properties of BIBW22BS 50 mg/kg i.v. followed after one h by vincristine 1 mg/kg i.p. or doxorubicin 8 mg/kg i.p. weekly x 2. RESULTS: BIBW22BS was 20- to 100-fold more potent than dipyridamole in the reversal of resistance in the Pgp-positive sublines. Reversal of resistance was obtained in a dose-dependent manner and was complete at concentrations of 0.5-2.5 microM. At non-toxic, equimolar concentrations of 1.0 microM BIBW22BS showed higher modulating potency than the calcium-channel blockers. BIBW22BS did not affect resistance in the non-Pgp sublines. BRO/mdr1.1 s.c. xenografts have stable multidrug-resistance characteristics upon serial transplantation. BIBW22BS, vincristine, or doxorubicin as single agents were not effective in vivo, while the addition of BIBW22BS could significantly reduce the tumor growth expressed as the T/C% of vincristine from 109% to 48% and that of doxorubicin from 55% to 32%. However, reversal of vincristine resistance in BRO/mdr1.1 xenografts was not complete when compared to the efficacy of vincristine in BRO xenografts. CONCLUSION: The results encourage the further preclinical development of BIBW22BS as a modulator of 'classical' multidrug resistance in cancer patients.

Anti-idiotypic antibodies that inhibit immediate-type skin reactions in unsensitized monkeys on challenge with staphylococcal enterotoxin.

The staphylococcal enterotoxin B (SEB)-induced immediate-type skin reaction in unsensitized monkeys was used as a nonimmunological mast cell stimulus to examine whether the toxin exerts its effect via specific receptors on the target cell membrane. Anti-idiotypic antibodies (anti-Id) were raised in BALB/c mice against monoclonal anti-SEB antibodies (anti-SEB) and purified by idiotype affinity chromatography. The anti-Id nature of the antibody was demonstrated by its ability to inhibit the binding of 125I-labeled anti-SEB to the ligand in a concentration-dependent manner. Moreover, binding of anti-SEB to anti-Id was antagonized by the SEB ligand in a competitive way. These antibodies completely abolished skin reactions in unsensitized monkeys on challenge with SEB and impeded those provoked by staphylococcal enterotoxins A and C1 but did not have the biological activity of the toxin. These data are compatible with the view that receptors for staphylococcal enterotoxins may exist on the membrane of mast cells in the skin of unsensitized monkeys. The data suggest an experimental approach for producing anti-cell receptor antibodies that are of potential value to influence the course of staphylococcal enterotoxin-mediated effects.