Biomedical subjects

Gerd Birkenmeier

Publications and source records attributed to Gerd Birkenmeier.

12 recordsLinked to original sources

PubMed2006

Expression of alpha2-macroglobulin, neutrophil elastase, and interleukin-1alpha differs in early-stage and late-stage atherosclerotic lesions in the arteries of the circle of Willis.

Different types of atherosclerotic (AS) lesions can be distinguished histologically and represent different stages of AS plaque development. Late-stage lesions more frequently develop complications such as plaque rupture and thrombosis with vessel occlusion than early AS lesions. To clarify whether protective, destructive, and inflammatory proteins are differentially expressed in early-stage and late-stage AS plaques we examined the proteinase inhibitor alpha(2)-macroglobulin (A2M), the neutrophil elastase (NE)-an enzyme degrading elastin and collagen fibers-and the proinflammatory protein interleukin-1alpha (IL-1alpha) in all types of AS plaques in the arteries of the circle of Willis from 78 human autopsy cases of both genders (61-91 years of age). Paraffin sections of AS plaques were immunostained with antibodies directed against A2M, NE and IL-1alpha. In initial AS lesions A2M was found, whereas NE and IL-1alpha were absent. NE and IL-1alpha became detectable as soon as a significant number of macrophages occurred within AS lesions. With increasing histopathological type of AS lesions, a marked increase of the area of the plaque exhibiting NE and IL-1alpha was observed. The area which exhibits A2M in AS plaques, on the other hand, did not vary significantly between the different stages. Thus, our results indicate a disproportionately high increase of the destructive enzyme NE and the proinflammatory protein IL-1alpha in relation to A2M with the progression of the grade of AS lesions pointing to the transgression of the protective capacity of A2M by NE and IL-1alpha in late-stage plaques. Therefore, our findings support the hypothesis that NE-induced tissue damage in late-stage AS plaques contributes to the development of plaque rupture and subsequent thrombosis.

Aged↗

PubMed2006

Polymyxin B-conjugated alpha 2-macroglobulin as an adjunctive therapy to sepsis: Modes of action and impact on lethality.

A drug targeting both the inflammatory initiators (lipopolysaccharide; LPS) and mediators [tumor necrosis factor-alpha (TNF-alpha)] should have advantage over a "single-factor targeting strategy" in sepsis prevention trials. We have prepared conjugates of polymyxin B (PMB) and the cytokine binding protein alpha2-macroglobulin (A2M). The conjugate binds TNF-alpha as well as LPS as studied by electrophoresis and phase partitioning. Compared with free PMB, the conjugate is nontoxic to cells and does not affect the viability of human monocytes. The A2M-PMB conjugate binds to the A2M receptor (CD91/low-density lipoprotein receptor-related protein 1) with affinity similar to that of the nonmodified protein. Fluorescein isothiocyanate-labeled LPS in the presence of A2M-PMB is rapidly transported into fibroblasts for degradation via receptor-mediated endocytosis. In vitro, A2M-PMB demonstrated inhibition of LPS-induced secretion of TNF-alpha from isolated monocytes as well as in the whole blood assay. The efficacy of the drug was tested in mice after induction of acute inflammation (LPS model) and after induction of a polymicrobial sepsis by cecal ligation and puncture (CLP) model. Treatment of mice with A2M-PMB up to 250 microg/g body weight was not toxic to the animal. When the drug was administered 30 min before or 30 min after the LPS challenge, a survival rate of 90 and 70%, respectively, was obtained compared with the placebo control group (5%). A2M-PMB also protected mice after induction of polymicrobial sepsis when administered 30 min before CLP. These results support our hypothesis that A2M-PMB acts as a polyvalent drug to target different host mediators as well as sepsis inducer at the same time.

Animals↗

PubMed2006

Method for preparing single-stranded DNA templates for Pyrosequencing using vector ligation and universal biotinylated primers.

In Pyrosequencing, the addition of nucleotides to a primer-template hybrid is monitored by enzymatic conversion of chemical energy into detectable light. The technique yields both qualitative and quantitative sequence information because the chemical energy is released by a stoichiometric split off of pyrophosphates from incorporated deoxynucleotide triphosphates and a defined nucleotide dispensation order is given. Because Pyrosequencing works best if single-stranded DNA templates are used, template generation usually requires PCR with a target-specific biotinylated primer and a subsequent purification involving interaction of the biotin label with immobilized streptavidin. To circumvent the need for numerous and expensive template-specific biotinylated primers, we developed a method that uses the ligation of amplified DNA fragments into a plasmid vector, thereby facilitating subsequent PCR using a universal vector-specific biotinylated primer. This approach allows easy and straightforward isolation of single-stranded templates of any PCR product. As a proof of principle, we used the method for genotyping two single-nucleotide polymorphisms in the human genes CARD15 and A2M and for characterization of four multisite variations in the human DEFB104 gene.

Biotinylation↗

PubMed2006

Binding properties of the cerebral alpha4beta2 nicotinic acetylcholine receptor ligand 2-[18F]fluoro-A-85380 to plasma proteins.

INTRODUCTION: To determine the availability of nicotinic acetylcholine receptors in different human brain regions using the positron emission tomography (PET) radioligand 2-[18F]fluoro-A-85380 (2-[18F]FA) and invasive approaches for quantification, it is important to correct the arterial input function as well for plasma protein binding (PPB) of the radioligand as for radiolabeled metabolites accumulating in blood. This study deals with some aspects of PPB of 2-[18F]FA. METHODS: Patients with different neurological disorders (n=72), such as Parkinson's disease, Alzheimer's disease and multiple sclerosis, and a group of healthy volunteers (n=15) subjected for PET imaging were analyzed for their PPB level of 2-[18F]FA using ultrafiltration. Protein gel electrophoresis of plasma samples was performed to identify the binding protein of 2-[18F]FA. The dependency of PPB on time and on free ligand concentration was analyzed to obtain the binding parameters Bmax and Kd. RESULTS: Albumin was identified to be the binding protein of 2-[18F]FA. PPB of 2-[18F]FA was low at 17+/-4% and did not show significant differences between the groups of patients. Corresponding to this, a narrow range of plasma albumin of 0.62+/-0.05 mM was observed. Bmax was determined as twice the albumin concentration, which indicates two binding sites for 2-[18F]FA on the protein. No time dependence of the PPB could be observed. By relating PPB to Bmax, an average Kd value of 6.0+/-1.5 mM was obtained. CONCLUSION: This study shows the dependency of PPB of 2-[18F]FA on human albumin plasma concentration. An equation utilizing Bmax and Kd to easily estimate PPB is presented.

Azetidines↗

PubMed2005

The human FGF2 level is influenced by genetic predisposition.

BACKGROUND: The fibroblast growth factor 2 (FGF2) is involved in various processes possibly leading to the development of complex diseases such as atherosclerosis. In recent studies, its cardioprotective properties, due to its ability to stimulate the proliferation of collateral vessels, could be shown. STUDY DESIGN: In this clinical study, the relation between clinical risk markers, a genomic variant of FGF2, namely the c.223C>T polymorphism, and the in vivo FGF2 expression was evaluated. Therefore, 198 clinically well-characterized probands, all of Caucasian origin, were included. The FGF2 mRNA level was determined in monocytes by competitive RT-PCR, whereas the plasma level of circulating FGF2 protein was analysed by ELISA. By considering the angiographically proven stenotic state of the patient, a significant increase in FGF2 mRNA, but not in protein level, could be shown for patients with significant stenosis. Apart from this, no influence on FGF2 expression was found in the case of all of the clinical and biochemical markers investigated. However, in the case of the c.223C>T polymorphism, a significant increase in the individual FGF2 mRNA and protein level in CC-carriers was shown. In multivariate analysis, this relation was independent of all other risk markers investigated. CONCLUSIONS: Our results suggest that an increase in FGF2 mRNA expression, related to coronary atherosclerosis, may be necessary for the maintenance of the individual FGF2 plasma level. Since the individual FGF2 mRNA and protein level are, to a large extent, triggered off by genetic background, the FGF2 expression cannot be referred to as an independent clinical marker for CAD.

Coronary Artery Disease↗

PubMed2005

Effect of alpha2-macroglobulin on retinal glial cell proliferation.

BACKGROUND: Activation of the receptor for alpha2-macroglobulin (alpha2 M), the low-density lipoprotein-related protein (LRP1; CD91), has been suggested to represent a possible strategy for the inhibition of uncontrolled retinal cell proliferation via stimulation of the clearance of alpha2 M-bound growth factors and proteinases from the extracellular space. In order to prove this assumption, we investigated the effect of alpha2 M on the proliferation of Müller glial cells in vitro. METHODS: Proliferation assays using bromodeoxyuridine were carried out on cultured Müller glial cells of the guinea pig in the absence and presence of alpha2 M. RESULTS: Activated alpha2 M evoked a slight increase of the cell proliferation at control conditions. Addition of alpha2 M to the culture medium inhibited the proliferation evoked by agonists of G-protein-coupled receptors [adenosine 5'-triphosphate (ATP), neuropeptide Y]. However, alpha2 M did not diminish the proliferation evoked by agonists of receptor tyrosine kinases (epidermal and platelet-derived growth factors) and by serum, respectively. Inhibition of LRP1 by a neutralizing antibody did not alter the ATP-evoked proliferation while it increased the proliferation in the presence of alpha2 M. CONCLUSION: It is concluded that alpha2 M inhibits the proliferation evoked by agonists of G-protein-coupled receptors, possibly via enhanced growth factor clearance by LRP.

Adenosine Triphosphate↗

PubMed2004

Characterization of novel monoclonal antibodies for prostate-specific antigen (PSA) with potency to recognize PSA bound to alpha 2-macroglobulin.

BACKGROUND: Different molecular forms of prostate-specific antigen (PSA) have been used to differentiate between benign prostatic hyperplasia and prostate cancer. Detecting PSA bound to endogenous inhibitors such as alpha(1)-antichymotrypsin (ACT) and alpha(2)-macroglobulin (alpha(2)M) is often difficult because of epitope masking or sensitivity problems. Here we report the characterization of four novel mouse monoclonal antibodies (mabs) obtained by immunization with PSA-alpha(2)M complexes. Their ability to detect free PSA and PSA-inhibitor complexes was shown, and their epitopes were analyzed by phage display technology. METHODS: The properties of the mabs were studied by competition and sandwich assays and by Western blotting. Epitope mapping was performed by screening of a phage display peptide library. RESULTS: All four mabs recognized free PSA, PSA-ACT, and PSA-alpha(2)M complexes, but to various degrees. With different combinations of mabs in competition experiments, antibodies were identified that enhance binding of other mabs to PSA, forming the molecular basis of a very sensitive assay for the detection of PSA and PSA-ACT complexes. Mabs with highest reactivity for PSA-alpha(2)M were selected to establish an immunoassay for that complex. Western blot analysis revealed that all mabs recognized conformational epitopes of PSA. These findings were supported by phage display results demonstrating mimotopes in the PSA molecule. CONCLUSION: The results presented here could aid in the further development of clinically relevant assays for PSA and PSA-alpha(2)M complexes.

Animals↗

PubMed2004

Genetic and functional characteristics of the human in vivo LRP1/A2MR receptor suggested as a risk marker for Alzheimer's disease and other complex (degenerative) diseases.

LDL receptor-related protein/alpha2-macroglobulin receptor (LRP1/A2MR) a multiligand receptor is considered as not only being a possible risk factor of neurodegenerative diseases like Alzheimer's disease but also as determining the progression of other complex diseases like atherosclerosis and cancer. Although a large number of in vitro studies have highlighted its functional importance, as yet not enough is known about the clinical importance of the genetic background of LRP1 in human diseases. The aim of this ex vivo/in vivo study of 448 subjects was to present data on genetic LRP1 variants of healthy European Caucasians from Central Germany. Genotype-dependent LRP1 expression was analyzed in a representative subgroup (gene expression: n = 127, protein expression: n = 44). These data were evaluated in comparison to other published clinical LRP1 studies. For 15 functionally interesting genetic variants the genotype and allele distributions of the German Caucasians were presented in relation to their in vivo LRP1 gene and protein expression. A direct influence of the LRP1 promoter polymorphism c.1-25C>G on the human in vivo LRP1 expression level was demonstrated. In an analysis of 48 further studies genomic and functional results were evaluated. The analysis especially on Alzheimers's disease partly highlighted contradictory results, but suggested that ethnic as well as genomic characteristics determine LRP1 expression and must be considered in clinical investigations on human LRP1.

Adult↗

PubMed2004

Expression of LRP1 in retinal pigment epithelial cells and its regulation by growth factors.

PURPOSE: The retinal pigment epithelial (RPE) cells are mitotically inactive under normal conditions, but play a pivotal role in the pathogenesis of proliferative vitreoretinopathy (PVR). Triggered by changes in the concentrations of growth factors, RPE cells reenter the cell cycle, proliferate, and migrate onto the retinal surface, into the subretinal space, and into the vitreous. The receptor for alpha(2)-macroglobulin (low-density lipoprotein receptor-related protein [LRP1], or CD91) is known to be involved in the processes of cell migration and invasion, as well as in the regulation of growth factor homeostasis. The purpose of this study was to investigate the expression of this receptor and its regulation, at the protein and mRNA levels, in human (h)RPE cells. METHODS: The cell surface expression of the receptor was studied by immunocytochemistry and flow cytometry. The endocytosis-related activity of LRP1 in hRPE cells was examined by assessing the uptake of FITC-labeled, methylamine (MA)-treated alpha(2)-M (alpha(2)-M-MA). LRP1 mRNA expression was analyzed by means of the RNase protection assay (RPA) after the hRPE cells were stimulated with the growth factors TGF-beta1, TGF-beta2, PDGF, VEGF (each 10 ng/mL), or bFGF (5 ng/mL). RESULTS: hRPE cells expressed LRP1 on their cell surface. The receptor mediated rapid binding and endocytosis of FITC-labeled alpha(2)-M-MA. The expression of LRP1 mRNA strongly increased on stimulation of the cells with TGF-beta1, TGF-beta2, or VEGF, whereas PDGF or bFGF elicited only minor effects. CONCLUSIONS: The expression of functionally active LRP1 in hRPE cells suggests that the receptor may be involved in cell migration and invasion, as reported for other LRP1-expressing cells. Thus, certain growth factors may control RPE cell migration and invasion in vivo through a regulation of LRP1 expression. As LRP1 mediates the clearance of alpha(2)-M, known to regulate the homeostasis of many cytokines and growth factors, this receptor may be a promising target for therapeutic intervention in PVR.

Aged↗

PubMed2003

Mast cell-derived exosomes induce phenotypic and functional maturation of dendritic cells and elicit specific immune responses in vivo.

Mast cells (MCs) are considered major players in IgE-mediated allergic responses, but have also recently been recognized as active participants in innate as well as specific immune responses. Recent work provided evidence that MCs are able to activate B and T lymphocytes through the release of vesicles called exosomes. Here we demonstrate that exosomes, which are located in the endocytic pathway, harbor exogenous Ags that associate with other molecules endowed with immunomodulatory functions, including 60- and 70-kDa heat shock proteins. Administration to naive mice of Ag-containing exosomes in the absence of conventional adjuvants elicits specific Ab responses across the MHC II haplotype barrier. We demonstrate that MC-exosomes induce immature dendritic cells (DCs) to up-regulate MHC class II, CD80, CD86, and CD40 molecules and to acquire potent Ag-presenting capacity to T cells. Uptake and processing of Ag-associated exosomes by endogenous DCs were also demonstrated. Finally, exosome-associated heat shock proteins are critical for the acquisition by DCs of the Ag-presenting function. This work demonstrates a heretofore unrecognized collaborative interaction between MCs and DCs leading to the elicitation of specific immune responses.

Animals↗

PubMed2003

Role of LDL receptor-related protein (LRP) in coronary atherosclerosis.

The development and progression of coronary atherosclerosis is influenced by a variety of genetic and environmental factors. Among the genetic factors, the cell surface receptor LRP/A2MR (LDL receptor-related protein/alpha2-macroglobulin receptor) was shown to be involved in a variety of biological processes leading to atherosclerotic plaque formation. That is why the individual expression of this receptor may, therefore, be considered as an evident predictor for coronary atherosclerosis. In this clinical ex vivo study the expression was measured by competitive RT-PCR and macroarray analysis in native monocytes. Both methods were first tested in an in vitro model using different human cells and cell lines (fibroblasts: chorion, skin; endothelial cells from umbilical cord vein; monocyte cell line: Mono-Mac-6): after stimulation with an LRP/A2MR ligand, leptin, the anticipated direct effect of this ligand, namely an increase in both receptor mRNA and protein expression, was confirmed. In disease-related ex vivo studies the mRNA and protein-expression of LRP/A2MR was investigated in 36 male patients suffering from myocardial infarction. In comparison to the control group (36 healthy male blood donors), a significant up-regulation of mRNA was detected in the myocardial infarction patient group (control: 122.3 ag/cell versus patients: 223 ag/cell; P<0.001). Investigating the LRP/A2MR protein expression a significant down-regulation of protein expression was determined in the patient group (control: 6 pg/cell versus patients: 1.6 pg/cell; P<0.001). The ratio of LRP/A2MR mRNA and protein expression is obviously an evident marker for coronary atherosclerosis, recommendable for the assessment of the individual coronary risk profile.

Blood Donors↗

PubMed2002

The LDL receptor-related protein (LRP1/A2MR) and coronary atherosclerosis--novel genomic variants and functional consequences.

The LDL receptor-related protein/alpha 2-macroglobulin receptor (LRP1/A2MR) is a multifunctional cell-surface glycoprotein that endocytoses several structurally and functionally distinct ligands. In clinical studies different genomic variants of the LRP1/A2MR and its role in the development of degenerative diseases like atherosclerosis or Alzheimer's disease were studied. We screened for novel genomic variants of LRP1/A2MR and investigated the importance of these variants in 214 coronary patients suffering from myocardial infarction as well as in 224 healthy controls. We detected a novel C>G polymorphism at position -25 in the functionally important promoter region of LRP1/A2MR. This polymorphism (c.1-25C>G) leads to the creation of a new GC-box, recognized by the constitutively expressed SP 1 transcription factor. Investigating the LRP1/A2MR gene expression with respect to this polymorphism, carriers of the mutant G-allele were found to have a higher mRNA expression level. A novel polymorphism in exon 22 (c.4012C>T), and two novel polymorphisms in intron 24 (IVS24+123C>A and IVS24+690G>A) associated with a previously described polymorphism in exon 61 (c.10249G>A), were related to the development of myocardial infarction. Two novel rare genetic variants of exon 88 (c.13933C>T) and intron 88 (IVS88+15G>A) were identified in four patients with severe coronary symptoms. However, the LRP1/A2MR gene expression was found to be independent of all identified novel genomic variants as well as other previously described changes (A217V, A775P, D2080N, D2632E, G4379S) except the promoter polymorphism.

Adult↗