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K Lehnert

Publications and source records attributed to K Lehnert.

17 recordsLinked to original sources
PubMed2003

Magmatic and amagmatic seafloor generation at the ultraslow-spreading Gakkel ridge, Arctic Ocean.

A high-resolution mapping and sampling study of the Gakkel ridge was accomplished during an international ice-breaker expedition to the high Arctic and North Pole in summer 2001. For this slowest-spreading endmember of the global mid-ocean-ridge system, predictions were that magmatism should progressively diminish as the spreading rate decreases along the ridge, and that hydrothermal activity should be rare. Instead, it was found that magmatic variations are irregular, and that hydrothermal activity is abundant. A 300-kilometre-long central amagmatic zone, where mantle peridotites are emplaced directly in the ridge axis, lies between abundant, continuous volcanism in the west, and large, widely spaced volcanic centres in the east. These observations demonstrate that the extent of mantle melting is not a simple function of spreading rate: mantle temperatures at depth or mantle chemistry (or both) must vary significantly along-axis. Highly punctuated volcanism in the absence of ridge offsets suggests that first-order ridge segmentation is controlled by mantle processes of melting and melt segregation. The strong focusing of magmatic activity coupled with faulting may account for the unexpectedly high levels of hydrothermal activity observed.

Journal Article↗
PubMed2001

Angiostatin enhances B7.1-mediated cancer immunotherapy independently of effects on vascular endothelial growth factor expression.

Tumors must develop an adequate vascular network to meet their increasing demands for nutrition and oxygen. Angiostatin, a multiple kringle (1-4)-containing fragment of plasminogen, is an effective natural inhibitor of tumor angiogenesis. Here we show that gene transfer of angiostatin into small (0.1 cm in diameter) solid EL-4 lymphomas established in syngeneic C57BL/6 mice led to reduced tumor angiogenesis and weak inhibition of tumor growth. In contrast, when angiostatin gene therapy was preceded by in situ gene transfer of the T-cell costimulator B7.1, large (0.4 cm in diameter) tumors were rapidly and completely eradicated, whereas B7.1 and angiostatin monotherapies were ineffective. Combined gene transfer of B7.1 and angiostatin generated potent systemic antitumor immunity that was effective in eradicating a systemic challenge of 10(7) EL-4 cells. Gene transfer of angiostatin expression plasmids led to overexpression of angiostatin in tumors, increased apoptosis of tumor cells, and decreased density of tumor blood vessels, which may allow the immune system to overcome tumor immune resistance. The latter effects were not the result of a decrease in vascular endothelial growth factor expression, as tumoral vascular endothelial growth factor expression increased slightly after angiostatin gene transfer, presumably in response to increasing hypoxia. These results suggest that combining immunogene therapy with a vascular attack by angiostatin is a particularly effective approach for eliciting antitumor immunity.

Angiostatins↗
PubMed2001

Induction of systemic antitumor immunity by gene transfer of mammalian heat shock protein 70.1 into tumors in situ.

Heat shock proteins (hsps) chaperone cytosolic peptides, forming complexes that stimulate antitumor immunity. Hsps facilitate signal 1 in the two-signal model of T-cell costimulation, whereas cell adhesion molecules such as B7.1 provide secondary (signal 2) costimulatory signals. B7.1 gene transfer into tumors in situ has been shown to eradicate small (<0.3 cm in diameter) tumors in mice, and induce systemic antitumor immunity, but is ineffective against larger tumors. We examine whether mammalian hsps, as facilitators of T-cell costimulation, also exhibit this ability, and whether simultaneously stimulating both signal 1 (hsp-facilitated antigen presentation) and signal 2 (B7.1-mediated costimulation) enhances antitumor immunity compared to that achieved with either monotherapy. Prophylactic vaccination of mice with an hsp preparation from an EL-4 lymphoma weakly retarded tumor growth, to the same extent as that achieved with a single EL-4-derived peptide (AQHPNAELL), previously shown to induce antitumor immunity establishing that a preparation of EL-4 hsp-peptide complexes has antitumor activity. Here we show that injection of rat hsp70.1 into mouse tumors in situ causes the complete eradication of tumors, and generates potent systemic antitumor immunity mediated by CD4+ and CD8+ T cells. Unexpectedly, simultaneous gene transfer of hsp70.1 and B7.1 compromised the efficacy of hsp-mediated tumor rejection--a problem which could be partially overcome by the timed delivery of hsp70.1 and B7.1. Thus, gene transfer of hsp70 into tumors can be employed to generate potent systemic antitumor immunity, but further consideration is required if this approach is to be successfully combined with immunotherapies employing other T-cell costimulators.

Animals↗
PubMed2001

Gene transfer of antisense hypoxia inducible factor-1 alpha enhances the therapeutic efficacy of cancer immunotherapy.

Solid tumors meet their demands for nascent blood vessels and increased glycolysis, to combat hypoxia, by activating multiple genes involved in angiogenesis and glucose metabolism. Hypoxia inducible factor-1 (HIF-1) is a constitutively expressed basic helix-loop-helix transcription factor, formed by the assembly of HIF-1alpha and HIF-1beta (Arnt), that is stablized in response to hypoxia, and rapidly degraded under normoxic conditions. It activates the transcription of genes important for maintaining oxygen homeostasis. Here, we demonstrate that engineered down-regulation of HIF-1alpha by intratumoral gene transfer of an antisense HIF-1alpha plasmid leads to the down-regulation of VEGF, and decreased tumor microvessel density. Antisense HIF-1alpha monotherapy resulted in the complete and permanent rejection of small (0.1 cm in diameter) EL-4 tumors, which is unusual for an anti-angiogenic agent where transient suppression of tumor growth is the norm. It induced NK cell-dependent rejection of tumors, but failed to stimulate systemic T cell-mediated anti-tumor immunity, and synergized with B7-1-mediated immunotherapy to cause the NK cell and CD8 T cell-dependent rejection of larger EL-4 tumors (0.4 cm in diameter) that were refractory to monotherapies. Mice cured of their tumors by combination therapy resisted a rechallenge with parental tumor cells, indicating systemic antitumor immunity had been achieved. In summary, whilst intensive investigations are in progress to target the many HIF-1 effectors, the results herein indicate that blocking hypoxia-inducible pathways and enhancing NK-mediated antitumor immunity by targeting HIF-1 itself may be advantageous, especially when combined with cancer immunotherapy.

Animals↗
PubMed1999

Cloning, sequence analysis, and chromosomal localization of the novel human integrin alpha11 subunit (ITGA11).

The integrins are a large family of cell adhesion molecules consisting of noncovalently associated alphabeta heterodimers. We have cloned and sequenced the cDNA of a novel human integrin alpha-subunit, designated alpha11. The alpha11 cDNA encodes a mature protein with a large 1120-residue extracellular domain that contains an I-domain of 207 residues and is linked by a transmembrane domain to a short cytoplasmic domain of 24 amino acids. The deduced alpha11 protein shows the typical structural features of integrin alpha-subunits and is similar to a distinct group of alpha-subunits from collagen-binding integrins. However, it differs from most integrin alpha-chains by an incompletely preserved cytoplasmic GFFKR motif. The human ITGA11 gene was localized to bands q22.3-q23 on chromosome 15, and its transcripts were found in a variety of tissues, but predominantly in bone, cartilage, cardiac muscle, and skeletal muscle. Expression of a 5.5-kb alpha11 mRNA was detectable in small intestine.

Amino Acid Sequence↗
PubMed1999

Taking lessons from dendritic cells: multiple xenogeneic ligands for leukocyte integrins have the potential to stimulate anti-tumor immunity.

Expression of large numbers of different costimulatory integrin ligands (CILs) attributes dendritic cells with an ability to induce primary anti-tumor immune responses. Here, we show that optimized gene transfer of the xenogeneic (human) CILs VCAM-1, MAdCAM-1 and ICAM-1 causes rapid and complete rejection of established mouse EL-4 tumors, and generates prolonged systemic anti-tumor immunity; whereas human E-cadherin weakly slows tumor growth. In each case the immune response was mediated by CD8+ T cells and NK cells, accompanied by augmented tumor-specific cytolytic T cell (CTL) activity involving both the perforin and Fas-ligand pathways. Adoptive transfer of splenocytes from cured mice rapidly cleared established tumors in recipients. The mechanism for CIL-mediated immunity is unknown, but may involve CTL-facilitated tumor lysis, since CTLs were generally twice as efficient at killing CIL-transfected tumor cells than parental tumor cells. Optimized CIL-based gene therapy may provide an approach to complement or replace conventional DC adoptive cell therapy for suppressing tumor growth.

Animals↗
PubMed1999

Mouse M290 is the functional homologue of the human mucosal lymphocyte integrin HML-1: antagonism between the integrin ligands E-cadherin and RGD tripeptide.

Human mucosal lymphocyte antigen-1 (HML-1, alphaEbeta7) and E-cadherin, two members of unrelated cell adhesion superfamilies, have evolved to play cooperative roles in gut mucosal immunity. Human E-cadherin is self-ligand mediating intercellular adhesion of epithelial cells, as well as adhesion of intra-epithelial lymphocytes to intestinal enterocytes via an interaction with HML-1. Herein we report that both dimeric and monomeric forms of recombinant mouse E-cadherin-human immunoglobulin Fc chimera self-associate and support attachment of E-cadherin+ mouse colon epithelial cells. Both forms also support the adhesion of mouse MTC-1 T cells via M290, thereby establishing M290 as the functional mouse homologue of HML-1 and revealing that E-cadherin homophilic and heterophilic binding sites are distinct. Adhesion of MTC-1 cells to E-cadherin-Fc was inhibited by arginine-glycine-aspartate (RGD) peptides and vice versa cells bound to immobilized RGD polymer in an M290-dependent fashion, where adhesion was inhibitable with soluble E-cadherin-Fc. Hence, E-cadherin and RGD integrin ligands antagonize cell binding by one another, either by inducing integrin cross-talk or by binding to shared or overlapping sites within M290. Binding of E-cadherin-Fc by HML-1 costimulated the CD3-induced proliferation of purified CD4+ T cells, suggesting that E-cadherin expressed on dendritic cells may play a T cell costimulatory role in addition to facilitating dendritic cell-keratinocyte adhesion.

Amino Acid Sequence↗
PubMed1999

The integrin alpha10 subunit: expression pattern, partial gene structure, and chromosomal localization.

Herein we report the cloning of cDNAs and incompletely processed hnRNAs from endothelia and heart that encode the alpha10 subunit forming part of the novel collagen type II-binding integrin alpha10beta1 of chondrocytes. Analysis of hnRNA clones and reported expressed sequence tags revealed the positions of 17 putative intron-exon splice junctions shared with those of the p150,95 (ITGAX) gene. Human alpha10 transcripts of 5.4 and 1.8 kb were not restricted to chondrocytes but, instead, were widely expressed in a panel of 24 tissue types, where the highest expression was found in muscle and heart. The human alpha10 subunit gene (ITGA10) was localized to band q21 of chromosome 1.

Amino Acid Sequence↗
PubMed1998

LPAM-1 (integrin alpha 4 beta 7)-ligand binding: overlapping binding sites recognizing VCAM-1, MAdCAM-1 and CS-1 are blocked by fibrinogen, a fibronectin-like polymer and RGD-like cyclic peptides.

The alpha 4 integrin LPAM-1 (alpha 4 beta 7) mediates lymphocyte attachment within the extracellular matrix (ECM) by adhering to the connecting segment (CS)-1 site of fibronectin (FN). Here we reveal that very late antigen (VLA)-4 LPAM-1+ T cell lymphoma TK-1 cells bind via LPAM-1 to multiple copies of the RGD sequence engineered within an FN-like polymer. Further, the small conformationally restrained RGD-like cyclic peptides 1-adamantaneacetyl-Cys-Gly-Arg-Gly-Asp-Ser-Pro-Cys and Arg-Cys-Asp-thioproline-Cys inhibit the adhesion of TK-1 cells to immobilized CS-1 peptide, and to endothelial counterreceptors for LPAM-1, namely mucosal addressin cell adhesion molecule (MAdCAM)-1 and vascular cell adhesion molecule (VCAM)-1. Spontaneous adhesion of the VLA-4- LPAM-1+ B lymphoma cell line RPMI 8866 to CS-1 was likewise inhibited, confirming a previously undocumented ability of LPAM-1 to recognize the RGD tripeptide. The RGD-binding site in LPAM-1 either overlaps or is identical to sites required for interaction with MAdCAM-1, VCAM-1, and the CS-1. The binding of LPAM-1 and VLA-4 to RGD-containing ligands may have relevance in vivo given that fibrinogen at physiological concentrations is able to partially block the binding of TK-1 cells to MAdCAM-1. Hence fibrinogen and other vascular RGD-containing proteins may have mild anti-inflammatory activity required for maintaining effective homeostasis, analogous to the anti-thrombogenic activity of the vascular endothelium.

Amino Acid Sequence↗
PubMed1998

MAdCAM-1 costimulates T cell proliferation exclusively through integrin alpha4beta7, whereas VCAM-1 and CS-1 peptide use alpha4beta1: evidence for "remote" costimulation and induction of hyperresponsiveness to B7 molecules.

We have analyzed the effects of the alpha4 integrin ligands mucosal addressin cell adhesion molecule-1 (MAdCAM-1), vascular cell adhesion molecule-1 (VCAM-1), and the fibronectin CS-1 splice variant on T cell activation. Immobilized MAdCAM-1 and VCAM-1 IgG-Fc chimeras and a fibronectin CS-1 peptide efficiently costimulate T cell proliferation when antigen presentation is mimicked by anti-CD3 antibody. VCAM-1-Fc and fibronectin CS-1, which are adhesive ligands for both the alpha4beta1 and alpha4beta7 integrins, medicate T cell costimulation exclusively through integrin alpha4beta1, but not through alpha4beta7. The inability of VCAM-1-Fc to costimulate via alpha4beta7 suggests that cell adhesion per se is insufficient, and that exquisite recognition and activation events must be triggered. MAdCAM-1-Fc mediates costimulation exclusively via alpha4beta7, and can both synergize with and induce hyperresponsiveness to the classical costimulator B7-2. MAdCAM-1-Fc and VCAM-1-Fc, but not B7-2, effectively costimulate when immobilized on sites spatially distant from the anti-CD3 antibody ("remote" costimulation). In vitro, the relative potencies of the CAM were VCAM-1-Fc> ICAM-1-Fc> MAdCAM-1-Fc > B7-Fc, except at high concentrations where ICAM-1 was the most potent. Features of costimulatory CAM revealed by this study have important implications for the design of immunotherapeutic vaccine strategies to combat cancer and infection.

Abatacept↗
PubMed1996

Interaction of monocytoid cells with the mucosal addressin MAdCAM-1 via the integrins VLA-4 and LPAM-1.

The differentiation of myeloid cells into macrophages and granulocytes is accompanied by marked changes in adhesive phenotype. Here we seek to understand the regulation of expression and functionality of the VLA-4 (alpha 4 beta 1), LPAM-1 (alpha 4 beta 7) and HML-1 (alpha E beta 7) integrins on monocytes/macrophages and granulocytes, given that these integrins including LFA-1 (alpha L beta 2) mediate the entry, retention and signalling events of pathogenic leucocytes within chronically inflamed tissues. Phorbol ester-induced monocytic differentiation of the promyelocyte cell line HL60 led to increases in the steady-state levels of beta 2 and beta 7 mRNA transcripts, requiring a period of 10 and 24 h, respectively, of de novo protein synthesis. There was a parallel de novo expression of LPAM-1 on the cell surface, despite the fact that alpha 4 mRNA transcripts were rapidly down-regulated. At 72 h, HML-1 was not coexpressed with LPAM-1 on HL60 cells, although it was weakly expressed on peripheral blood monocytes/macrophages after a prolonged period of in vitro culture. Retinoic acid-induced granulocytic differentiation of HL60 cells led to the appearance of low levels of LPAM-1 at the cell surface. LPAM-1 was not found expressed on peripheral blood neutrophils, raising the possibility that it is transiently expressed during granulocyte differentiation. In accord with the above findings, differentiated monocytes and HL60 cells bound to recombinant MAdCAM-1 in an alpha 4- and beta 7-integrin-dependent fashion, whereas a population of undifferentiated HL60 cells and Mn(+2)-activated monocytes bound in an alpha 4-integrin-dependent beta 7-integrin-independent manner via VLA-4 expressed abundantly at all stages of differentiation. Four h after attachment, some of these VLA-4+ LPAM-1- HL60 cells could be seen to start spreading. These finding suggest that MAdCAM-1 can bind to VLA-4 when LPAM-1 is absent, and thus has the potential to recruit both VLA-4-bearing monocytes and VLA-4+ LPAM-1+ macrophages into chronically inflamed tissues.

Antigens, CD↗
PubMed1996

Cloning of the mucosal addressin MAdCAM-1 from human brain: identification of novel alternatively spliced transcripts.

The mucosal addressin cell adhesion molecule-1 (MAdCAM-1), expressed selectively on high endothelial venules (HEV) and lamina propria venules, directs lymphocyte traffic by binding the lymphocyte Peyer's patch adhesion molecule-1 (LPAM-1, alpha 4 beta 7). Full-length DNA encoding human MAdCAM-1 was obtained by combining sequences from an expressed sequence tag (EST) identified in an early stage human brain cDNA library, a polymerase chain reaction-derived clone, and a MAdCAM-1 genomic clone. The deduced amino acid sequence revealed an 18 amino acid signal peptide, two N-terminal immunoglobulin (Ig)-like domains conserved (59-65%) in sequence with those of the mouse homologue, an 86 amino acid mucin-like region rich in serine-threonine residues, a 20 amino acid transmembrane domain and a 43 amino acid charged cytoplasmic domain. No counterpart to the third IgA-like domain of mouse MAdCAM-1 was present; however, the serine-threonine-rich mucin domain was extended as two distinguishable major and minor mucin regions unrelated to the mouse domain. The major domain is formed from six tandem repeats of an eight amino acid sequence having the MUC-2-related consensus DTTSPEP/SP. Human MAdCAM-1 mRNA transcripts were restricted to small intestine, colon, spleen, pancreas and brain. Alternatively spliced MAdCAM-1 variants were identified that lack parts of the second Ig domain and all or part of the major mucin domain, indicating that the function of this vascular addressin is regulated by extensive modifications to its multi-domain structure.

Alternative Splicing↗
PubMed1995

Glycoprotein biosynthesis in Saccharomyces cerevisiae: ngd29, an N-glycosylation mutant allelic to och1 having a defect in the initiation of outer chain formation.

Outer chain glycosylation in Saccharomyces cerevisiae leads to heterogeneous and immunogenic asparagine-linked saccharide chains containing more than 50 mannose residues on secreted glycoproteins. Using a [3H]mannose suicide selection procedure a collection of N-glycosylation defective mutants (designated ngd) was isolated. One mutant, ngd29, was found to have a defect in the initiation of the outer chain and displayed a temperature growth sensitivity at 37 degrees C allowing the isolation of the corresponding gene by complementation. Cloning, sequencing and disruption of NGD29 showed that it is a non lethal gene and identical to OCH1. It complemented both the glycosylation and growth defect. Membranes isolated from an ngd29 disruptant or an ngd29mnn1 double mutant were no longer able, in contrast to membranes from wild type cells, to transfer mannose from GDPmannose to Man8GlcNAc2, the in vivo acceptor for building up the outer chain. Heterologous expression of glucose oxidase from Aspergillus niger in an ngd29mnn1 double mutant produced a secreted uniform glycoprotein with exclusively Man8GlcNAc2 structure that in wild type yeast is heavily hyperglycosylated. The data indicate that this mutant strain is a suitable host for the expression of recombinant glycoproteins from different origin in S. cerevisiae to obtain mammalian oligomannosidic type N-linked carbohydrate chains.

Alleles↗
PubMed1995

Construction and adhesive properties of a soluble MadCAM-1-Fc chimera expressed in a baculovirus system: phylogenetic conservation of receptor-ligand interaction.

MAdCAM-1 is a high endothelial venule adhesion molecule composed of immunoglobulin and mucin-like domains which binds the leucocyte integrin LPAM-1 (alpha 4 beta 7), and is largely responsible for the selective homing of lymphocytes to mucosal tissues. A novel soluble form of mouse MAdCAM-1 which is normally membrane bound has been produced by joining the extracellular region of the receptor to the Fc domain of human IgG1. The MAdCAM-1-Fc cDNA was inserted into the genome of Autographa californica nuclear polyhedrosis virus (AcNPV). Spodoptera frugiperda insect cells infected with the recombinant virus produced MAdCAM-1-Fc as a disulfide-linked homodimer of 82 kDa polypeptides, which was secreted into the culture medium at > 1 microgram/ml. The product purified by Protein G-Sepharose was identified as authentic MAdCAM-1-Fc by the anti-MAdCAM-1 monoclonal antibody (MoAb) MECA-367 using Western blot and ELISA analysis. When immobilized on glass it was fully functional in supporting the binding of mouse alpha 4 beta 1+ alpha 4 beta 7+ mesenteric lymph node lymphocytes, and the alpha 4 beta 1- alpha 4 beta 7+ TK1 T cell lymphoma. Binding was enhanced by Mn(++)-induced integrin activation, and specifically blocked by anti-integrin alpha 4 subunit and anti-MAdCAM-1 MoAbs. Binding was blocked by pretreatment of cells with sodium azide, and EDTA, indicating that binding is an energy-dependent process which requires divalent cations. Thus the mouse MAdCAM-1-Fc chimera produced in insect cells retains certain functional properties that typify the native receptor, and should be valuable in analysing the role of MAdCAM-1 in lymphocyte recirculation and emigration. However it was not sialylated despite being post-translational modified with N- and O-linked carbohydrate moieties, suggesting that the ability of MAdCAM-1 to support cell adhesion under static conditions is sialylation-independent. A rabbit polyclonal antibody raised against the entire cytoplasmic domain of the human integrin beta 7 subunit recognized LPAM-1-like molecules in human, rat, and mouse cells, suggesting a high degree of conservation of the MAdCAM-1 receptor across species. In agreement with this notion MAdCAM-1-Fc immobilized on glass was fully functional in supporting the cation-dependent binding of peripheral blood or spleen cells from a range of other species including human, rat, and guinea pig; and for human myeloid HL60 cells, binding was mediated by alpha 4 integrins.

Amino Acid Sequence↗
PubMed1994

Enzymes in diagnostics: achievements and possibilities of recombinant DNA technology.

We discuss, from an industrial point of view, the scope and possibilities of recombinant DNA technology for "diagnostic enzyme" production and application. We describe the construction of enzyme-overproducing strains and show how to simplify downstream processing, increase product quality and process profitability, improve diagnostic enzyme properties, and adjust enzymes to harsh assay conditions. We also consider some safety and environmental aspects of enzyme production. Other aspects of diagnostic enzymes that we cover are the facilitation of enzyme purification by attachment of short amino acid tails, the introduction of tails or tags for site-specific conjugation or oriented immobilization, the construction of bi- or multifunctional enzymes, and the production of enzyme-based diagnostic tests as demonstrated by the homogeneous immunoassay system of CEDIA tests. We use as examples of diagnostic enzymes glucose-6-phosphate dehydrogenase (EC 1.1.1.49), glucose oxidase (EC 1.1.3.4), alkaline phosphatase (EC 3.1.3.1), alpha-glucosidase (EC 3.2.1.20), pyruvate oxidase (EC 1.2.3.3), creatinase (EC 3.5.3.3), and beta-galactosidase (EC 3.2.1.23).

Amino Acid Sequence↗