Biomedical subjects

D A Lappi

Publications and source records attributed to D A Lappi.

At least 37 records · Page 2Linked to original sources

PubMed1995

Tumor targeting through fibroblast growth factor receptors.

Fibroblast growth factor tyrosine kinase receptors are encoded by four genes, but alternate splicing can result in more than 100 possible protein sequences. The receptors have widespread expression in the developing embryo, but the expression becomes more restricted in the adult. The ligand-receptor relationship is complex, due to the diversity of the receptors and the large number of possible ligands: there are now nine (and probably more) members of the fibroblast growth factor family. This complicated ligand-receptor relationship creates many options to target cell types through the use of individual ligands or receptor-specific monoclonal antibodies. In-vivo data demonstrate that FGF receptors are expressed on tumor cells and can be used to target tumors for growth inhibition. Given the complexity, it is possible that a unique targetable FGF receptor isoform can be found in one or more tumor types. Examples of the targeting of growth inhibition agents to tumors through FGF receptors are discussed.

Amino Acid Sequence↗

PubMed1995

Destruction of the cholinergic basal forebrain using immunotoxin to rat NGF receptor: modeling the cholinergic degeneration of Alzheimer's disease.

Degeneration of cholinergic neurons in the basal forebrain (CBF) is a prominent neuropathological feature of Alzheimer's disease and is thought responsible for some cognitive deficits seen in patients. An animal model of pure CBF degeneration would be valuable for analysis of the function of these neurons and testing therapeutic strategies. CBF neurons express receptors for nerve growth factor. In order to selectively destroy these neurons, we developed an immunotoxin using monoclonal antibody (192 IgG) to rat NGF receptor (p75NGFr) armed with the ribosome inactivating protein, saporin. In vitro 192-saporin was highly toxic to neurons expressing p75NGFr. Intraventricular injections of 192-saporin destroyed the CBF and impaired passive avoidance learning. These results indicate that 192-saporin treated rats can be used to model a key feature of Alzheimer's disease and that anti-neuronal immunotoxins are a powerful approach to selective neural lesioning.

Alzheimer Disease↗

PubMed1995

192 immunoglobulin G-saporin produces graded behavioral and biochemical changes accompanying the loss of cholinergic neurons of the basal forebrain and cerebellar Purkinje cells.

Immunolesions of the cholinergic basal forebrain were produced in rats using various intraventricular doses of the immunotoxin 192 immunoglobulin G-saporin: 0.34, 1.34, 2.0, 2.7 and 4.0 micrograms/rat. A battery of behavioral tests, chosen on the basis of reported sensitivity to conventional medial septal or nucleus basalis lesions, was administered. Dose-dependent impairments were found in acquisition, spatial acuity and working memory in the water maze. Dose-dependent hyperactivity in the open field and in swimming speed was observed. The highest dose group (4.0 micrograms) exhibited motoric disturbances which were particularly apparent in swimming and in clinging to an inclined screen. Response and habituation to acoustic startle were diminished in the three higher dose groups. Histological results from acetylcholinesterase and low-affinity nerve growth factor receptor staining showed that the lesion was selective for cholinergic neurons bearing p75 nerve growth factor receptors in the basal forebrain nuclei. However, some Purkinje cells in the superficial layers of the cerebellum were also destroyed at the higher doses of immunotoxin. The activity of choline acetyltransferase, used as a marker of cholinergic deafferentation in regions innervated by the basal forebrain nuclei, was decreased with increasing doses to a plateau level of about 90% (average depletion) for the two highest dose groups. These two groups were the only ones to exhibit consistent and severe behavioral impairments on all behavioral tests performed. Thus, for a relatively selective cholinergic basal forebrain lesion, almost a 90% reduction in choline acetyltransferase activity is needed to produce substantial behavioral deficits. It appears that either a considerable safety factor exists or robust compensatory mechanisms can ameliorate behavioral deficits from a major, but incomplete loss of cholinergic basal forebrain innervation.

Animals↗

PubMed1995

Anti-dopamine beta-hydroxylase immunotoxin-induced sympathectomy in adult rats.

Anti-dopamine beta-hydroxylase immunotoxin (DHIT) is an antibody-targeted noradrenergic lesioning tool comprised of a monoclonal antibody against the noradrenergic enzyme, dopamine beta-hydroxylase, conjugated to saporin, a ribosome-inactivating protein. Noradrenergic-neuron specificity and completeness and functionality of sympathectomy were assessed. Adult, male Sprague-Dawley rats were given 28.5, 85.7, 142 or 285 micrograms/kg DHIT i.v. Three days after injection, a 6% to 73% decrease in the neurons was found in the superior cervical ganglia of the animals. No loss of sensory, nodose and dorsal root ganglia, neurons was observed at the highest dose of DHIT. In contrast, the immunotoxin, 192-saporin (142 micrograms/kg), lesioned all three ganglia. To assess the sympathectomy, 2 wk after treatment (285 micrograms/kg), rats were anesthetized with urethane (1 g/kg) and cannulated in the femoral artery and vein. DHIT-treated animals' basal systolic blood pressure and heart rate were significantly lower than controls. Basal plasma norepinephrine levels were 41% lower in DHIT-treated animals than controls. Tyramine-stimulated release of norepinephrine in DHIT-treated rats was 27% of controls. Plasma epinephrine levels of DHIT animals were not reduced. DHIT-treated animals exhibited a 2-fold hypersensitivity to the alpha-adrenergic agonist phenylephrine. We conclude that DHIT selectively delivered saporin to noradrenergic neurons resulting in destruction of these neurons. Anti-dopamine beta-hydroxylase immunotoxin administration produces a rapid, irreversible sympathectomy.

Adrenergic Agents↗

PubMed1994

Noradrenergic lesioning with an anti-dopamine beta-hydroxylase immunotoxin.

Sympathectomy has been achieved by a variety of methods but each has its limitations. These include lack of tissue specificity, incomplete lesioning, and the age range of susceptibility to the lesioning. To circumvent these drawbacks, an immunotoxin was constructed using a monoclonal antibody against the noradrenergic specific enzyme dopamine beta-hydroxylase (D beta H) coupled via a disulfide bond to saporin, a ribosomal inactivating protein. Three days after intravenous injection of the anti-D beta H immunotoxin (50 micrograms) into adult Sprague-Dawley rats, 66% of neurons in the superior cervical ganglia were chromatolytic. Superior cervical ganglia neurons were poisoned in 1 day old and 1 week old (86% of neurons) neonatal rats following subcutaneous injection of 3.75 and 15 micrograms, respectively. The anti-D beta H immunotoxin will be a useful tool in the study of the peripheral noradrenergic system in adult and neonatal animals.

Animals↗

PubMed1994

Cortical cholinergic deafferentation following the intracortical infusion of 192 IgG-saporin: a quantitative histochemical study.

The immunotoxin 192 IgG-saporin has been hypothesized to selectively lesion cholinergic neurons that bear the low-affinity p75 nerve growth factor (NGF) receptor. To evaluate the usefulness of this toxin in studies intended to determine the functions of cholinergic afferents of cortical areas, relatively small concentrations and volumes of the immunotoxin (0.01-0.05 micrograms/0.5-1.0 microliters) were infused into cortical areas of one hemisphere of rats, while the vehicle was infused into homologous areas of the contralateral hemisphere. The effects of these infusions on the density of cortical acetylcholinesterase (AChE)-positive fibers and of normal fibers (as revealed by a reduced silver stain) were quantified. The infusion of the immunotoxin did not produce local gliosis in excess of the gliosis resulting from the infusion of vehicle. When compared with the frontoparietal cortex of the intact hemisphere, the number of cortical AChE-positive fibers was reduced by 36-39% and the density of the silver-stained fibers was decreased by 20-25%. While the loss of AChE-positive fibers and silver-stained fibers correlated significantly in layers V/VI, a linear regression analysis suggested that the magnitude of the loss of AChE-positive fibers was greater than would be predicted on the basis of the residual density of normal fibers. Thus, the data suggest that infusions of 192 IgG-saporin into the cortex did not result in the loss of non-cholinergic afferents. Intracortical infusions of relatively small concentrations and volumes of 192 IgG-saporin appear to provide a useful approach for the examination of the functions of cholinergic inputs to specific cortical regions.

Acetylcholinesterase↗

PubMed1994

Anti-B16-F10 melanoma activity of a basic fibroblast growth factor-saporin mitotoxin.

BACKGROUND: The authors attached basic fibroblast growth factor (FGF-2), a growth factor for numerous tumors and normal cell types, to saporin (SAP), a ribosome-inactivating protein isolated from the plant Saponaria officinalis. The conjugate (FGF-SAP) then was tested for antitumor activity using B16-F10 melanoma cells. This rapidly growing murine melanoma cell line has been used classically as a model to screen antitumor agents. METHODS: B16-F10 cells in culture were used for in vitro experiments or introduced into C57BL/6 mice to demonstrate the in vivo antitumor activities of FGF-SAP. RESULTS: FGF-SAP was found to be an extremely effective cytocidal agent in vitro with an ED50 of 30-60 pM. The effects were specific for FGF-2 receptors, as shown by the ability of FGF-2 to block FGF-SAP action. In the in vivo models, FGF-SAP was found to increase survival time, inhibit tumor growth, and decrease metastases. CONCLUSIONS: The authors conclude that this mitotoxin has potent in vitro and in vivo effects on B16-F10 cells, supporting the hypothesis that ligand-mediated cytotoxicity can control tumor growth.

Animals↗

PubMed1994

Expression and activities of a recombinant basic fibroblast growth factor-saporin fusion protein.

A fusion protein containing the full-length sequences of the mitogen, basic fibroblast growth factor (FGF-2), and the ribosome-inactivating protein, saporin (SAP), has been expressed in E. coli. As expected, it binds with high affinity to heparin-Sepharose like FGF-2 and can displace the binding of radiolabeled FGF-2 to its high affinity receptor. In contrast, the fusion protein only has much lower ribosome-inactivating activity than free saporin, although full ribosome-inactivating protein activity can be generated by proteolytic removal of the FGF-2 moiety. Cytotoxicity experiments with B16-F10 mouse melanoma cells establish that the fusion protein is active as a chemical conjugate against these intact cells. Presumably these cells have the ability to activate the SAP component of the fusion protein through an intra-cellular metabolism of the fusion protein. Because we also show the fusion protein has tumor growth inhibition properties and antimetastatic activity in in vivo models of melanoma, the findings support the hypothesis that FGF-based ligand-mediated cytotoxicity can serve to target cytotoxic agents in vivo.

Animals↗

PubMed1994

Time course of cholinergic and monoaminergic changes in rat brain after immunolesioning with 192 IgG-saporin.

192 IgG-saporin, an immunotoxin targeted at the low affinity NGF receptor, was infused into the lateral ventricle of rat brain. Three days and one week post lesion, choline acetyltransferase activity was markedly decreased in cortex, hippocampus, olfactory bulbs, and septum (brain regions innervated by the cholinergic neurons of the basal forebrain) with no change in cerebellum, striatum or pons. Measurement of monoamine levels revealed increases in HVA, DOPAC and dopamine, primarily in the olfactory bulbs at the 28-day time point only, suggesting a compensation for cholinergic inactivity. High levels of basal forebrain cholinergic lesioning can be obtained with this immunotoxin with minimal or no effects on monoaminergic or other cholinergic systems.

Animals↗

PubMed1994

Behavioural, histochemical and biochemical consequences of selective immunolesions in discrete regions of the basal forebrain cholinergic system.

The effectiveness of a recently developed immunotoxin, 192 IgG-saporin, was evaluated for making selective lesions of subgroups of basal forebrain cholinergic neurons. Following a pilot series of injections into the nucleus basalis magnocellularis to establish the effective dose for intraparenchymal lesions, separate groups of rats received injections of the immunotoxin into the septum, into the diagonal band of Broca or into the nucleus basalis magnocellularis. The lesions produced extensive and effective loss of cholinergic neurons in the discrete areas of the basal forebrain, as identified by loss of cells staining for acetylcholinesterase and p75NGFr, with a parallel loss of acetylcholinesterase staining and choline acetyltransferase activity in the target areas associated with each injection site in the dorsolateral neocortex, cingulate cortex and hippocampus. The selectivity of the lesion for cholinergic neurons was supported by the lack of gliosis and sparing of small to medium-sized cells at the site of injection of the toxin, including the glutamate decarboxylase immunoreactive cells that contribute to the septohippocampal projection. In spite of the extensive disturbance in the cholinergic innervation of the neocortex and hippocampus, immunotoxin lesions produced no detectable deficit in the Morris water maze task in any of the lesion sites within the basal forebrain. By contrast small but significant deficits were seen on tests of nocturnal activity (septal and nucleus basalis magnocellularis lesions), open field activity (septal and diagonal band lesions), passive avoidance (nucleus basalis magnocellularis lesions) and delayed non-matching to position (septal lesions). The results indicate that the 192 IgG-saporin provides a powerful tool for making effective lesions of the basal forebrain cholinergic neurons, and that the behavioural sequelae of such lesions warrant further detailed investigation.

Acetylcholinesterase↗

PubMed1994

Fibroblast growth factor in the hypothalamic-pituitary axis: differential expression of fibroblast growth factor-2 and a high affinity receptor.

In situ hybridization and immunohistochemistry were used to map gene expression and protein distribution of basic fibroblast growth factor (FGF-2) in the hypothalamic-pituitary system. Although the expression of FGF-2 mRNA in the pituitary is low, the protein is widely distributed in both its neural and anterior lobes. In the anterior lobe, immunoreactive (ir-) FGF-2 localizes to basement membranes and select endocrine cells. In the neural lobe, ir-FGF-2 is detected in basement membranes, pituicytes, and Herring bodies. Analyses of FGF high affinity receptor (FGFR) immunoreactivity in the anterior pituitary establishes a distribution of FGFR similar to that of FGF-2. In the neural lobe, ir-FGFR is associated with nerve fibers, pituicytes, and Herring bodies. Unlike FGF-2, the distribution of FGFR1 mRNA correlates well with the presence of the immunoreactive receptor. In the hypothalamus, magnocellular neurons of paraventricular and supraoptic nuclei contain ir-FGF-2 and ir-FGFR. In the median eminence, ir-FGF-2 and ir-FGFR is associated with fibers, glial, and endothelial cells. Ependymal and subependymal cells lining the third ventricle also show high levels of ir-FGF-2 and ir-FGFR and mRNAs. Overall, there is a specific and selective distribution of FGF-2 and its high affinity receptor(s) in the hypothalamo-pituitary axis. This localization lead us to postulate a role in neurohypophyseal functions, possibly water balance.

Animals↗

PubMed1994

Differential effects on spatial navigation of immunotoxin-induced cholinergic lesions of the medial septal area and nucleus basalis magnocellularis.

The effects on anatomy and behavior of a ribosomal inactivating protein (saporin) coupled to a monoclonal antibody against the low-affinity NGF receptor (NGFr) were examined. In adult rats, NGFr is expressed predominantly in cholinergic neurons of the medial septal area (MSA), diagonal band nuclei, and nucleus basalis magnocellularis (nBM), but also in noncholinergic cerebellar Purkinje cells. Rats with immunotoxin injections to the MSA, nBM, and lateral ventricle were compared to controls on a spatial and cued reference memory task in the Morris maze. Toxin injections to the MSA slightly impaired the initial, but not asymptotic, phase of spatial navigation. Injections to the nBM impaired all phases of spatial navigation. Cued navigation, however, was not affected in either the MSA or nBM group. The ventricular injections severely affected spatial and cued navigation. Acetylcholinesterase (AChE) histochemistry and NGFr and choline acetyltransferase immunohistochemistry revealed a loss of (1) almost all NGFr-positive cholinergic neurons in the MSA and AChE fibers in hippocampus (MSA group); (2) almost all NGFr neurons in the nBM, some in the MSA, most AChE fibers in neocortex and some in the hippocampus (nBM group), and (3) almost all NGFr neurons in the MSA and nBM and their corresponding hippocampal and cortical AChE fibers (ventricular group). Cholinergic nBM projections to the amygdala were largely preserved in all groups. The amount of cholinergic fiber loss in the cortex correlated modestly, but significantly, with the severity of impairment of the asymptotic phase of performance of the spatial task. An unambiguous interpretation of the anatomical locus of behavioral deficits was not possible because of damage to cholinergic striatal interneurons (nBM group) and to noncholinergic cerebellar Purkinje cells (ventricular group). These data suggest that the cholinergic cortical system is critical to the performance of this spatial memory task. Cholinergic denervation of the hippocampus alone, however, is not sufficient to impair markedly performance of this task.

Animals↗

PubMed1994

Complete and selective cholinergic denervation of rat neocortex and hippocampus but not amygdala by an immunotoxin against the p75 NGF receptor.

The immunotoxin 192 IgG-saporin, produced by coupling the ribosome-inactivating protein saporin to the monoclonal 192 IgG antibody against the low-affinity p75 NGF receptor (NGFr), was injected into the cerebral ventricle, septal area, and substantia innominata of adult rats. Injections into the cerebral ventricle induced a complete loss of NGFr-positive basal forebrain neurons and their axons. Extensive loss of cholinergic neurons was found in the septum, diagonal band, and magnocellular preoptic nucleus but not in the nucleus basalis-substantia innominata complex, where many cholinergic, presumably NGFr-negative, neurons remained intact. Cholinergic fibers were completely lost in the neocortex and hippocampus, showed some preservation in allocortical areas, and showed only minor loss in the amygdala. The NGFr-positive cholinergic basal forebrain neurons progressively degenerated during the first 5 d and did not recover after 180 d. The effect of intraventricular 192 IgG-saporin injections on NGFr-positive basal forebrain neurons could be blocked by simultaneous intraventricular injection of colchicine. Intraparenchymal injections into the septal area or substantia innominata damaged cholinergic neurons mainly around the injection sites and reduced their respective cortical and hippocampal projections. Noncholinergic septal neurons containing parvalbumin and noncholinergic neurons containing calbindin-D28k or NADPHd, which were adjacent to cholinergic nucleus basalis-substantia innominata neurons, were not affected by 192 IgG-saporin. The ChAT immunoreactivity in cortical interneurons, habenula, and brainstem was unchanged. Dopaminergic and noradrenergic cortical afferents remained intact. 192 IgG-saporin damaged two neuronal groups outside the basal forebrain that express the p75 NGF receptor: NGFr-positive cerebellar Purkinje cells after intraventricular injection and cholinergic striatal interneurons after injections into the substantia innominata. These results indicate that the immunotoxin 192 IgG-saporin induces a complete and selective lesion of NGFr-positive cholinergic basal forebrain neurons projecting to hippocampus and neocortex.

Acetylcholinesterase↗

PubMed1993

A conjugate between human urokinase and saporin, a type-1 ribosome-inactivating protein, is selectively cytotoxic to urokinase receptor-expressing cells.

Urokinase-type plasminogen activator (uPA) confers invasive potential to transformed cells. Cancer cells express high numbers of uPA receptors (uPARs), which concentrate uPA activity at the invasive edge of cancer cells and the tumor mass. We synthesized a conjugate between human uPA and saporin (SAP), a ribosome-inactivating protein produced by Saponaria officinalis. Results of cell-killing assays showed that uPA is very effective at targeting saporin specifically to uPAR-expressing cells, whereas cell lines devoid of uPARs were not affected by the conjugate. Receptor-bound uPA is internalized only upon formation of a complex with one of its inhibitors (PAIs). However, our conjugate was highly cytotoxic even when the interaction between uPA and PAIs was prevented. Moreover, the alpha 2-macroglobulin receptor, which has been reported to mediate the internalization of uPA.PAI complexes, seems not to be involved in cell killing caused by the uPA.SAP conjugate. Thus, uPA.SAP might follow a mechanism of internalization different from that of unconjugated uPA complexed to PAIs, although still uPAR-mediated. Our results also suggest that alpha 2-macroglobulin and/or its receptor could mediate the internalization and cytotoxicity of unconjugated saporin, as it has been shown for other toxins.

Animals↗

PubMed1993

Reducing the heterogeneity of chemically conjugated targeted toxins: homogeneous basic FGF-saporin.

Basic fibroblast growth factor-saporin (FGF-SAP) is an extremely potent cytotoxic agent for cells bearing the FGF receptor. For its synthesis, the two free cysteines on the surface of basic FGF are available for linking by disulfide bridge to SAP which has been derivatized with a reactive sulfhydryl. Because of the heterogeneous nature of the synthesis, the resulting conjugate is heterogeneous as judged by gel electrophoresis. We have removed by site-directed mutagenesis one of the reactive cysteines of basic FGF and have purified monoderivatized SAP to use as the reactants. The resulting chemical conjugate shows a single band containing only 1 mol of basic FGF and 1 mol of SAP per molecule. This homogeneous conjugate is a potent cytocidal agent to cells bearing the FGF receptor.

3T3 Cells↗

PubMed1993

The expression of saporin, a ribosome-inactivating protein from the plant Saponaria officinalis, in Escherichia coli.

We have isolated and sequenced genomic clones from the DNA of Saponaria officinalis using a cDNA probe that encodes proteins with high homology to saporin-6, one of the most potent of the ribosome-inactivating proteins that is currently used for the construction of immunotoxins and mitotoxins. Sequence differences in the clones suggest a multigene family of proteins. These data agree with observations of several different proteins with ribosome-inactivating protein activity and similar structure. Two of the genomic clones encode proteins that have identical sequences to two of the four isoforms of saporin-6. We have inserted the DNA from one genomic clone into an Escherichia coli expression system that encodes a signal sequence for export to the bacterial periplasmic space. Exportation is observed and the isolated gene product has ribosome-inactivation activity similar to the native protein. Sequence analysis shows differential processing of the remaining plant signal sequence. The majority of the expressed protein remains intracellular and this material also shows high specific activity and differential processing. Saporin as an immunotoxin in clinical trial and as a mitotoxin in experimental models has been extremely efficacious. These data indicate the ability to produce fusion proteins with saporin and cell-binding ligands for production of new reagents for further clinical and experimental use.

Amino Acid Sequence↗

PubMed1993

Characterization of a saporin mitotoxin specifically cytotoxic to cells bearing the granulocyte-macrophage colony-stimulating factor receptor.

When granulocyte-macrophage colony-stimulating factor (GM-CSF) is chemically conjugated to the ribosome-inactivating protein saporin, the resulting protein conjugate is highly toxic for cells expressing the GM-CSF receptor. Structural and Western blot analyses of the purified conjugate establish that it contains equimolar amounts of the starting materials and is free of any contamination by the non-conjugated components. The resulting bifunctional reagent is specifically cytotoxic to cells expressing the GM-CSF receptor, but is ineffective to cells that do not express the receptor. The cytotoxic activity is inhibited in a dose-dependent manner by GM-CSF, but not by any one of five other peptide growth factors. This is the first report of a mitotoxin for cells that express the GM-CSF receptor and which promises to be a valuable tool to study the expression of the GM-CSF receptor in normal and pathological states.

Animals↗

PubMed1992

Elimination of smooth muscle cells in experimental restenosis: targeting of fibroblast growth factor receptors.

Factors in plasma and platelets do not fully account for the proliferation of smooth muscle cells in vascular injury, implying that additional factors are involved. Recently, we and others have observed that vascular injury regulates basic fibroblast growth factor, suggesting a further role for this pleiotropic factor. We report here that injury of rat arteries leads to an increase in fibroblast growth factor receptors in vascular smooth muscle cells. This up-regulation makes smooth muscle cells susceptible, in vitro and in vivo, to the lethal effects of a conjugate of basic fibroblast growth factor with the ribosome inactivator saporin. Saporin alone has no effect, whereas the conjugate kills proliferating, but not quiescent, smooth muscle cells in vitro. In vivo, one to three doses inhibit neointimal proliferation but have no apparent effect on the uninjured artery. Thus, the up-regulation of fibroblast growth factor receptors in vascular injury suggests new therapeutic possibilities for such refractory conditions as restenosis following balloon angioplasty.

Amino Acid Sequence↗