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L Witte

Publications and source records attributed to L Witte.

At least 19 recordsLinked to original sources
PubMed2005

Cell-based selection of internalizing fully human antagonistic antibodies directed against FLT3 for suppression of leukemia cell growth.

FMS-like tyrosine kinase 3 (FLT3) receptor is highly expressed in an array of hematological malignancies including approximately 90% of acute myelogenous leukemia (AML). Ligand stimulation of the receptor promotes the survival and proliferation of leukemia cells. Strategies targeting FLT3 using monoclonal antibodies may therefore constitute an effective therapeutic approach for these leukemia. Towards this, we selected a naïve antibody phage display library on both recombinant FLT3 receptor protein and FLT3-expressing leukemia cells using a tailored selection scheme that was designed to isolate antagonistic phage antibodies that not only interfere with receptor/ligand binding but also trigger receptor internalization upon cell surface binding. Phage antibodies were screened first for their ability to bind to cell surface receptor and induce receptor internalization, followed by their activity in blocking ligand-receptor interaction and neutralizing ligand-stimulated receptor activation and cell proliferation. We identified three fully human antibodies, EB10, A2IN, and D4-3, which bound specifically to both soluble and cell surface-expressed FLT3. All three antibodies were shown to be internalized upon binding to cell surface-expressed receptor in a time-dependent fashion. EB10 and D4-3 blocked ligand binding to the receptor with IC(50)s of 14 and 7 nM, respectively. Further, EB10 and D4-3 inhibited FLT3 ligand-induced receptor phosphorylation and cell proliferation in EOL-1 leukemia cells. Taken together, these results suggest that both EB10 and D4-3 may represent excellent therapeutic candidates for the treatment of FLT3-expressing human leukemia, both as unmodified antibodies and as conjugates of cytotoxic agents.

Antibodies, Monoclonal↗
PubMed2003

Biochemical processing of plant acquired pyrrolizidine alkaloids by the neotropical leaf-beetle Platyphora boucardi.

Leaf beetles of the genus Platyphora, feeding on plant species containing pyrrolizidine alkaloids of the lycopsamine type, not only sequester these alkaloids and concentrate them in their exocrine defensive secretions, but also specifically process the plant acquired alkaloids. Using P. boucardi as subject, three mechanisms were studied: (i). utilization of host plant alkaloids that are not sequestered per se; (ii). elucidation of the mechanism of the already documented C-7 epimerization of heliotridine O(9)-monoesters; (iii). the specificity of insect catalyzed necine base esterification. P. boucardi does not sequester the triester parsonsine, the principal alkaloid of its host plant Prestonia portobellensis (Apocynaceae). Beetles fed with a purified mixture of nor-derivatives of parsonsine, obtained from Parsonsia laevigata, did not sequester the triesters but transformed them by partial degradation into monoesters that are accumulated in the defensive secretions. The mechanism of the previously described transformation of rinderine into intermedine by C-7 epimerization was elucidated by feeding C-7 deuterated heliotrine (3'-methylrinderine). The transformation of heliotrine into epiheliotrine (3'-methylintermedine) catalyzed by P. boucardi is accompanied by complete loss of deuterium, indicating the same mechanism of an oxidation-reduction process via a ketone intermediate as recently demonstrated in a pyrrolizidine alkaloid sequestering lepidopteran. P. boucardi is able to form ester alkaloids from five different necine bases fed as radioactively labeled substrates. However, besides C-7 epimerization the beetles are not able to convert simple necine bases into retronecine. The functional importance of the various alkaloid transformations is discussed in comparison to striking parallels of analogous reactions known from pyrrolizidine alkaloid sequestering Lepidoptera.

Animals↗
PubMed2003

Inhibition of human leukemia in an animal model with human antibodies directed against vascular endothelial growth factor receptor 2. Correlation between antibody affinity and biological activity.

Vascular endothelial growth factor (VEGF) and its receptors (VEGFR) have been implicated in promoting solid tumor growth and metastasis via stimulating tumor-associated angiogenesis. We recently showed that certain 'liquid' tumors such as leukemia not only produce VEGF, but also express functional VEGFR, resulting in an autocrine loop for tumor growth and propagation. A chimeric anti-VEGFR2 (or kinase insert domain-containing receptor, KDR) antibody, IMC-1C11, was shown to be able to inhibit VEGF-induced proliferation of human leukemia cells in vitro, and to prolong survival of nonobese diabetic-severe combined immune deficient (NOD-SCID) mice inoculated with human leukemia cells. Here we produced two fully human anti-KDR antibodies (IgG1), IMC-2C6 and IMC-1121, from Fab fragments originally isolated from a large antibody phage display library. These antibodies bind specifically to KDR with high affinities: 50 and 200 pM for IMC-1121 and IMC-2C6, respectively, as compared to 270 pM for IMC-1C11. Like IMC-1C11, both human antibodies block VEGF/KDR interaction with an IC(50) of approximately 1 nM, but IMC-1121 is a more potent inhibitor to VEGF-stimulated proliferation of human endothelial cells. These anti-KDR antibodies strongly inhibited VEGF-induced migration of human leukemia cells in vitro, and when administered in vivo, significantly prolonged survival of NOD-SCID mice inoculated with human leukemia cells. It is noteworthy that the mice treated with antibody of the highest affinity, IMC-1121, survived the longest period of time, followed by mice treated with IMC-2C6 and IMC-1C11. Taken together, our data suggest that anti-KDR antibodies may have broad applications in the treatment of both solid tumors and leukemia. It further underscores the efforts to identify antibodies of high affinity for enhanced antiangiogenic and antitumor activities.

Animals↗
PubMed2002

Pyrrolizidine alkaloids in Pulmonaria obscura.

Leaves, roots and rhizomes, and inflorescenses of Pulmonaria obscura from two localities and of two different growth forms within one site were analyzed by GC-MS for their content in pyrrolizidine alkaloids (PAs). In roots and rhizomes PAs of the lycopsamine type typical for the Boraginaceae could be detected, e. g. intermedine, lycopsamine, and their O(7)-derivatives. The total PA concentrations in roots and rhizomes lay between 0.026 and 0.158 mg/g dry weight. In leaves and inflorescenses, on the other hand, only trace amounts of PAs (below 0.4 ng/mg dry weight) could be detected. No significant differences in total concentration of PAs could be found between the two sites.

Plant Extracts↗
PubMed2001

Complete inhibition of vascular endothelial growth factor (VEGF) activities with a bifunctional diabody directed against both VEGF kinase receptors, fms-like tyrosine kinase receptor and kinase insert domain-containing receptor.

Vascular endothelial growth factor (VEGF) binds to and mediates its activity mainly through two tyrosine kinase receptors, VEGF receptor 1 [or fms-like tyrosine kinase receptor (Flt-1)] and VEGF receptor 2 [or kinase insert domain-containing receptor (KDR)]. Numerous studies have shown that overexpression of VEGF and its receptor plays an important role in tumor-associated angiogenesis and hence in both tumor growth and metastasis. We demonstrated previously that antagonistic antibodies to KDR specifically inhibited VEGF-stimulated receptor activation, cell migration, and endothelial cell mitogenesis. Here we constructed a recombinant bifunctional diabody that is capable of blocking both Flt-1 and KDR from binding to their ligands, including VEGF and placenta growth factor (PlGF). The diabody was expressed in Escherichia coli and purified by single-step affinity chromatography. The diabody retained the capacity to bind both KDR and Flt-1 and effectively blocked interaction between KDR and VEGF, Flt-1 and VEGF, and Flt-1 and PlGF. Furthermore, the diabody is a stronger inhibitor than its parent antibodies to VEGF-stimulated mitogenesis of human endothelial cells, as well as both VEGF- and PlGF-induced migration of human leukemia cells. Taken together, our results suggest that dual receptor blockade with the bifunctional diabody may prove to be a more efficient approach in inhibiting VEGF-stimulated angiogenesis.

3T3 Cells↗
PubMed2001

Inhibition of both paracrine and autocrine VEGF/ VEGFR-2 signaling pathways is essential to induce long-term remission of xenotransplanted human leukemias.

Antiangiogenic agents block the effects of tumor-derived angiogenic factors (paracrine factors), such as vascular endothelial growth factor (VEGF), on endothelial cells (EC), inhibiting the growth of solid tumors. However, whether inhibition of angiogenesis also may play a role in liquid tumors is not well established. We recently have shown that certain leukemias not only produce VEGF but also selectively express functional VEGF receptors (VEGFRs), such as VEGFR-2 (Flk-1, KDR) and VEGFR1 (Flt1), resulting in the generation of an autocrine loop. Here, we examined the relative contribution of paracrine (EC-dependent) and autocrine (EC-independent) VEGF/VEGFR signaling pathways, by using a human leukemia model, where autocrine and paracrine VEGF/VEGFR loops could be selectively inhibited by neutralizing mAbs specific for murine EC (paracrine pathway) or human tumor (autocrine) VEGFRs. Blocking either the paracrine or the autocrine VEGF/VEGFR-2 pathway delayed leukemic growth and engraftment in vivo, but failed to cure inoculated mice. Long-term remission with no evidence of disease was achieved only if mice were treated with mAbs against both murine and human VEGFR-2, whereas mAbs against human or murine VEGFR-1 had no effect on mice survival. Therefore, effective antiangiogenic therapies to treat VEGF-producing, VEGFR-expressing leukemias may require blocking both paracrine and autocrine VEGF/VEGFR-2 angiogenic loops to achieve remission and long-term cure.

Animals↗
PubMed2001

Cloning, genetic characterization, and chromosomal mapping of the mouse VDUP1 gene.

VDUP1 encodes a vitamin D3-inducible gene product that has been shown to be down-regulated in chemically-induced mammary tumors in rats. It has recently been reported to negatively regulate thioredoxin expression and function. We have cloned the mouse VDUP1 gene and characterized its genomic locus. The VDUP1 coding region spans eight exons within a total length of 2.3 kb located on mouse chromosome 3. Consensus sites for polyadenylation were identified 1.3 kb downstream of the gene, defining a long 3' untranslated region. The minimal functional VDUP1 promoter contains TATA and CCAAT boxes and transcription is initiated from two major start sites downstream. A direct repeat element located proximal to the TATA with homology to the USF binding site was identified as a potential regulator of VDUP1gene expression. Expression analysis determined that VDUP1 mRNA was markedly induced in myeloma cells in high density cell culture, but not in sub-confluent cells arrested by serum deprivation. All samples of a panel of mouse immortalized or transformed cell lines were shown to express abundant levels of VDUP1 mRNA.

Animals↗
PubMed2001

Vascular endothelial growth factor and angiopoietin-1 stimulate postnatal hematopoiesis by recruitment of vasculogenic and hematopoietic stem cells.

Tyrosine kinase receptors for angiogenic factors vascular endothelial growth factor (VEGF) and angiopoietin-1 (Ang-1) are expressed not only by endothelial cells but also by subsets of hematopoietic stem cells (HSCs). To further define their role in the regulation of postnatal hematopoiesis and vasculogenesis, VEGF and Ang-1 plasma levels were elevated by injecting recombinant protein or adenoviral vectors expressing soluble VEGF(165), matrix-bound VEGF(189), or Ang-1 into mice. VEGF(165), but not VEGF(189), induced a rapid mobilization of HSCs and VEGF receptor (VEGFR)2(+) circulating endothelial precursor cells (CEPs). In contrast, Ang-1 induced delayed mobilization of CEPs and HSCs. Combined sustained elevation of Ang-1 and VEGF(165) was associated with an induction of hematopoiesis and increased marrow cellularity followed by proliferation of capillaries and expansion of sinusoidal space. Concomitant to this vascular remodeling, there was a transient depletion of hematopoietic activity in the marrow, which was compensated by an increase in mobilization and recruitment of HSCs and CEPs to the spleen resulting in splenomegaly. Neutralizing monoclonal antibody to VEGFR2 completely inhibited VEGF(165), but not Ang-1-induced mobilization and splenomegaly. These data suggest that temporal and regional activation of VEGF/VEGFR2 and Ang-1/Tie-2 signaling pathways are critical for mobilization and recruitment of HSCs and CEPs and may play a role in the physiology of postnatal angiogenesis and hematopoiesis.

Adenoviridae↗
PubMed2001

Monoclonal antibody strategies to block angiogenesis.

Antibodies represent a unique class of therapeutics because of their high specificity towards a defined target antigen. Recent clinical success with antibody-based cancer therapeutics has led to an increase in the clinical development of these agents. Antibody therapeutics offer a promising approach for inhibiting new blood vessel formation (angiogenesis), which is associated with a variety of diseases, including cancer. In this review we will focus on angiogenesis-related mechanisms targeted by antibody-based therapeutics, with an emphasis on those studies where pre-clinical in vivo data is available.

Journal Article↗
PubMed2001

Vascular trauma induces rapid but transient mobilization of VEGFR2(+)AC133(+) endothelial precursor cells.

Bone marrow (BM)-derived circulating endothelial precursor cells (CEPs) are thought to play a role in postnatal angiogenesis. Emerging evidence suggests that angiogenic stress of vascular trauma may induce mobilization of CEPs to the peripheral circulation. In this regard, we studied the kinetics of CEP mobilization in two groups of patients who experienced acute vascular insult secondary to burns or coronary artery bypass grafting (CABG). In both burn and CABG patients, there was a consistent, rapid increase in the number of CEPs, determined by their surface expression pattern of vascular endothelial growth factor receptor 2 (VEGFR2), vascular endothelial cadherin (VE-cadherin), and AC133. Within the first 6 to 12 hours after injury, the percentage of CEPs in the peripheral blood of burn or CABG patients increased almost 50-fold, returning to basal levels within 48 to 72 hours. Mobilized cells also formed late-outgrowth endothelial colonies (CFU-ECs) in culture, indicating that a small, but significant, number of circulating endothelial cells were BM-derived CEPs. In parallel to the mobilization of CEPs, there was also a rapid elevation of VEGF plasma levels. Maximum VEGF levels were detected within 6 to 12 hours of vascular trauma and decreased to baseline levels after 48 to 72 hours. Acute elevation of VEGF in the mice plasma resulted in a similar kinetics of mobilization of VEGFR2(+) cells. On the basis of these results, we propose that vascular trauma may induce release of chemokines, such as VEGF, that promotes rapid mobilization of CEPs to the peripheral circulation. Strategies to improve the mobilization and incorporation of CEPs may contribute to the acceleration of vascularization of the injured vascular tissue.

AC133 Antigen↗
PubMed2001

Non-toxic pyrrolizidine alkaloids from Eupatorium semialatum.

The leaves of Eupatorium semialatum were investigated for the occurrence of pyrrolizidine alkaloids. Although this type of alkaloids generally occurs in the Eupatorieae, only unusual non-toxic pyrrolizidines of the tussilagin type were identified. All compounds are methyl esters of the corresponding beta-amino acids.

Journal Article↗
PubMed2001

Sequestration, metabolism and partial synthesis of tertiary pyrrolizidine alkaloids by the neotropical leaf-beetle Platyphora boucardi.

Platyphora boucardi leaf-beetles sequester tertiary pyrrolizidine alkaloids of the lycopsamine type acquired from their host-plant Prestonia portobellensis (Apocynaceae) and synthesize their own alkaloids from exogenous retronecine and aliphatic 2-hydroxy acids. Tracer studies with [14C]rinderine and its N-oxide revealed that P. boucardi sequesters both alkaloidal forms with the same efficiency, but accumulates exclusively tertiary alkaloids. There is no substantial alkaloid accumulation in the body outside the defensive glands. Feeding studies with [2H][14C]rinderine confirmed that P. boucardi specifically epimerizes rinderine to its stereoisomers intermedine and lycopsamine. Feeding studies with [2H][14C]retronecine proved the ability of P. boucardi to synthesize O7- and O9-(2-hydroxyisovaleryl)-retronecine and O7-lactyl-O9-(2-hydroxyisovaleryl)-retronecine. Both, alkaloids of the lycopsamine type and self-synthesized retronecine esters accumulate in the defensive secretions at concentrations up to 38 mM and 33 mM, respectively. The different biochemical strategies to maintain pro-toxic pyrrolizidine alkaloids and to prevent self-poisoning, developed by specialized insects, are compared. There are two major findings: (1) the chemical defense mediated by plant acquired pyrrolizidine alkaloids in the taxonomically related palaearctic Oreina and neotropical Platyphora leaf beetles have been evolved independently, since the biochemical mechanisms of storing and maintaining the alkaloids is completely different in the two genera; (2) unexpected parallels exist between taxonomically unrelated Coleoptera and Lepidoptera in their ability to synthesize the same retronecine esters and to catalyze the same site-specific epimerizations of the lycopsamine stereoisomers.

Animals↗
PubMed2001

New insecticidal rocaglamide derivatives and related compounds from Aglaia oligophylla.

Organic-soluble extracts of the twigs of Aglaia oligophylla collected in Vietnam yielded four insecticidal cyclopentatetrahydrobenzofurans of the rocaglamide type including one new natural product (compound 4). Moreover, two cyclopentatetrahydrobenzopyran derivatives, belonging to the aglain and aglaforbesin types, respectively, were also isolated. The aglaforbesin derivative 6 proved likewise to be a new natural product. All isolated rocaglamide, aglain, and aglaforbesin derivatives have a characteristic methylenedioxy substituent linked to C-6 and C-7 or to C-7 and C-8, respectively. Structure elucidation of the new natural products and the determination of the absolute configuration of compound 1 by calculation of its CD spectrum with molecular dynamics simulation are described. All isolated rocaglamide derivatives exhibited strong insecticidal activity toward neonate larvae of the polyphageous pest insect Spodoptera littoralis when incorporated into an artificial diet, with LC(50) values varying between 2.15 and 6.52 ppm.

Animals↗
PubMed2001

Merrekentrones A-D, ipomeamarone-like furanosesquiterpenes from Merremia kentrocaulos.

Four new furanosesquiterpenes, merrekentrones A (1), B (2), C (3), and D (4), were isolated from the roots and rootstocks of Merremia kentrocaulos. Their structures were elucidated on the basis of spectroscopic data interpretation. In contrast to ipomeamarone (5), the well-known phytoalexin of Ipomoea batatas, 1-4 seem to be normal plant constituents. Merrekentrone A (1) was also detected in the roots of M. guerrichii and M. aurea.

Chromatography, High Pressure Liquid↗
PubMed2001

A simple method to extract essential oils from tissue samples by using microwave radiation.

A microwave protocol to extract lipophilic substances from tissue was modified to extract essential oils (EOs) from plant tissue and insect feculae. The material, in a solvent transparent to microwave radiation, is exposed for a short time to steam in a microwave oven. EO extracts are analyzed directly by GC or GC-MS when plant material is fresh and terpenes contained in glandular structures on leaf surfaces are readily released into the solvent. For dried material or insect feculae, mechanical means are utilized first to break up tissue; however, the complete procedure is carried out inside the same vial to reduce losses. Statistical analysis shows that the reproducibility of the modified method is high. Several samples can be run within an hour with this method.

Animals↗
PubMed2001

Involvement of VEGFR-2 (kdr/flk-1) but not VEGFR-1 (flt-1) in VEGF-A and VEGF-C-induced tube formation by human microvascular endothelial cells in fibrin matrices in vitro.

Different forms of vascular endothelial growth factor (VEGF) and their cellular receptors (VEGFR) are associated with angiogenesis, as demonstrated by the lethality of VEGF-A, VEGFR-1 or VEGFR-2 knockout mice. Here we have used an in vitro angiogenesis model, consisting of human microvascular endothelial cells (hMVEC) cultured on three-dimensional (3D) fibrin matrices to investigate the roles of VEGFR-1 and VEGFR-2 in the process of VEGF-A and VEGF-C-induced tube formation. Soluble VEGFR-1 completely inhibited the tube formation induced by the combination of VEGF-A and TNF alpha (VEGF-A/TNF alpha). This inhibition was not observed when tube formation was induced by VEGF-C/TNF alpha or bFGF/TNF alpha. Blocking monoclonal antibodies specific for VEGFR-2, but not antibodies specifically blocking VEGFR-1, were able to inhibit the VEGF-A/TNF alpha-induced as well as the VEGF-C/TNF alpha-induced tube formation in vitro. P1GF-2, which interacts only with VEGFR-1, neither induced tube formation in combination with TNF alpha, nor inhibited or stimulated by itself the VEGF-A/TNF alpha-induced tube formation in vitro. These data indicate that VEGF-A or VEGF-C activation of the VEGFR-2, and not of VEGFR-1, is involved in the formation of capillary-like tubular structures of hMVEC in 3D fibrin matrices used as a model of repair-associated or pathological angiogenesis in vitro.

Animals↗
PubMed2001

Impaired recruitment of bone-marrow-derived endothelial and hematopoietic precursor cells blocks tumor angiogenesis and growth.

The role of bone marrow (BM)-derived precursor cells in tumor angiogenesis is not known. We demonstrate here that tumor angiogenesis is associated with recruitment of hematopoietic and circulating endothelial precursor cells (CEPs). We used the angiogenic defective, tumor resistant Id-mutant mice to show that transplantation of wild-type BM or vascular endothelial growth factor (VEGF)-mobilized stem cells restore tumor angiogenesis and growth. We detected donor-derived CEPs throughout the neovessels of tumors and Matrigel-plugs in an Id1+/-Id3-/- host, which were associated with VEGF-receptor-1-positive (VEGFR1+) myeloid cells. The angiogenic defect in Id-mutant mice was due to impaired VEGF-driven mobilization of VEGFR2+ CEPs and impaired proliferation and incorporation of VEGFR1+ cells. Although targeting of either VEGFR1 or VEGFR2 alone partially blocks the growth of tumors, inhibition of both VEGFR1 and VEGFR2 was necessary to completely ablate tumor growth. These data demonstrate that recruitment of VEGF-responsive BM-derived precursors is necessary and sufficient for tumor angiogenesis and suggest new clinical strategies to block tumor growth.

Animals↗