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Biomedical subjects

A M Gressner

Publications and source records attributed to A M Gressner.

At least 19 recordsLinked to original sources
PubMed2006

[Elevated concentrations of fecal calprotectin in patients with liver cirrhosis].

UNLABELLED: BACKGROUND AND OBJECTION: Bacterial translocation from the gut lumen is considered to play an important role in the development of infectious complications in patients with liver cirrhosis. This translocation might be increased by inflammation of the gut mucosa. Calprotectin is a cytoplasmatic protein of neutrophilic granulocytes and is an established marker for the assessment of localized intestinal inflammation. It was the aim of the current study to systematically evaluate a localized intestinal inflammation in patients with liver cirrhosis by means of fecal calprotectin concentrations. PATIENTS AND METHODS: Fecal calprotectin concentrations were determined in 53 consecutive patients with liver cirrhosis and in 18 subjects without intestinal or liver diseases, who were comparable with respect to age and gender. Patients with diarrhoea, inflammatory bowel disease and a positive stool test for occult blood were excluded from the study. Fecal calprotectin concentrations were measured by a sandwich ELISA. The systemic inflammatory reaction of the patients was assessed by C-reactive protein, white blood cells counts and the serum concentrations of the cytokines IL-6, IL-8 and IL-10. RESULTS: Fecal calprotectin concentrations were significantly increased in patients with liver cirrhosis (median 37.0 mg/kg) compared to controls patients (median 2.2, P < 0.0001). There were no significant correlations of calprotectin concentrations with systemic inflammatory parameters, like CRP, white blood cell count or serum cytokines. However, fecal calprotectin concentrations were significantly associated with the stage of liver cirrhosis as expressed by the Child-Pugh score ( P < 0.001). A trend towards higher concentrations of calprotectin was found in patients with alcoholic liver cirrhosis ( P = 0.1). CONCLUSIONS: Patients with liver cirrhosis display elevated fecal calprotectin concentrations as a potential sign of intestinal inflammation. Further studies are warranted to establish a role of calprotectin for the risk assessment of infectious complications secondary to bacterial translocation in patients with liver cirrhosis.

Adult↗
PubMed2006

Fibroblast growth factor 16 and 18 are expressed in human cardiovascular tissues and induce on endothelial cells migration but not proliferation.

Endothelial cells line the blood vessel and precursor endothelial cells appear to have a pivotal effect on the organ formation of the heart, the embryonic development of the kidney, and the liver. Several growth factors including the fibroblast growth factors (FGF) seem to be involved in these processes. Ligands such as basic FGF produced and secreted by endothelial cells may also coordinate cellular migration, differentiation, and proliferation under pathological conditions including wound healing, tumorgenesis, and fibrogenesis in the adult. Recently we demonstrated the expression of two secreted FGFs, FGF16, and FGF18, in HUVEC and in rat aortic tissue. In the present report, we confirmed by RT-PCR analysis that FGF18 is wildly expressed in the cardiovascular tissue, while FGF16 showed a more restricted expression pattern. HUVEC clearly demonstrated chemotaxis towards FGF16 and FGF18. Both FGFs also enhanced cell migration in response to mechanical damage. However, recombinant FGF16 and FGF18 failed to induce endothelial cell proliferation or sprouting in a three-dimensional in vitro angiogenesis assay. Fgf18 expression was earlier reported in the liver, and we detected FGF18 expression in liver vascular and liver sinusoidal endothelial cells (LSECs), but not in hepatic parenchymal cells. Recombinant FGF18 stimulated DNA synthesis in primary hepatocytes, suggesting, that endothelial FGF18 might have a paracrine function in promoting growth of the parenchymal tissue. Interestingly, FGF2, which is mitogenic on endothelial cells and hepatocytes stimulates a sustained MAPK activation in both cell types, while FGF18 causes a short transient activation of the MAPK pathway in endothelial cells but a sustained activation in hepatocytes. Therefore, the difference in the time course of MAPK activation by the different FGFs appears to be the cause for the different cellular responses.

Animals↗
PubMed2006

Id1 is a critical mediator in TGF-beta-induced transdifferentiation of rat hepatic stellate cells.

Transforming growth factor (TGF)-beta is critically involved in the activation of hepatic stellate cells (HSCs) that occurs during the process of liver damage, for example, by alcohol, hepatotoxic viruses, or aflatoxins. Overexpression of the TGF-beta antagonist Smad7 inhibits transdifferentiation and arrests HSCs in a quiescent stage. Additionally, bile duct ligation (BDL)-induced fibrosis is ameliorated by introducing adenoviruses expressing Smad7 with down-regulated collagen and alpha-smooth muscle actin (alpha-SMA) expression. The aim of this study was to further characterize the molecular details of TGF-beta pathways that control the transdifferentiation process. In an attempt to elucidate TGF-beta target genes responsible for fibrogenesis, an analysis of Smad7-dependent mRNA expression profiles in HSCs was performed, resulting in the identification of the inhibitor of differentiation 1 (Id1) gene. Ectopic Smad7 expression in HSCs strongly reduced Id1 mRNA and protein expression. Conversely, Id1 overexpression in HSCs enhanced cell activation and circumvented Smad7-dependent inhibition of transdifferentiation. Moreover, knock-down of Id1 in HSCs interfered with alpha-SMA fiber formation, indicating a pivotal role of Id1 for fibrogenesis. Treatment of HSCs with TGF-beta1 led to increased Id1 protein expression, which was not directly mediated by the ALK5/Smad2/3, but the ALK1/Smad1 pathway. In vivo, Id1 expression and Smad1 phosphorylation were co-induced during fibrogenesis. In conclusion, Id1 is identified as TGF-beta/ALK1/Smad1 target gene in HSCs and represents a critical mediator of transdifferentiation that might be involved in hepatic fibrogenesis. Supplementary material for this article can be found on the HEPATOLOGY website (http://interscience.wiley.com/jpages/0270-9139/suppmat/index.html).

Animals↗
PubMed2005

Upregulation of pleiotrophin expression in rat hepatic stellate cells by PDGF and hypoxia: implications for its role in experimental biliary liver fibrogenesis.

Pleiotrophin (PTN) is a secretory heparin binding protein with various biological activities including mitogenesis, angiogenesis, and tissue repair after injury. Recent studies have shown that PTN is a strong mitogen of hepatocytes and involved in liver regeneration. In adult liver cells Ptn gene is mainly expressed by quiescent hepatic stellate cells (HSCs). Although we have been able to demonstrate mRNA and protein expression of the anaplastic lymphoma kinase-the receptor tyrosine kinase for PTN-on HSCs, PTN did not act as a mitogen of HSCs in contrast to hepatocytes. PTN immunoreactivity was markedly increased in experimental fibrogenesis by common bile duct ligation and observed in sinusoidal HSCs. In primary HSC cultures, Ptn transcription was significantly increased by PDGF-BB, and under hypoxic atmosphere. Mechanistically, hypoxia and PDGF mediated induction of PTN expression in sinusoidal HSCs may provide a strong mitogenic signal for hepatocytes to limit the damage to the parenchymal cells in biliary-type liver fibrogenesis.

Animals↗
PubMed2005

Identification of an unconventional nuclear localization signal in human ribosomal protein S2.

Ribosomal proteins must be imported into the nucleus after being synthesized in the cytoplasm. Since the rpS2 amino acid sequence does not contain a typical nuclear localization signal, we used deletion mutant analysis and rpS2-beta-galactosidase chimeric proteins to identify the nuclear targeting domains in rpS2. Nuclear rpS2 is strictly localized in the nucleoplasm and is not targeted to the nucleoli. Subcellular localization analysis of deletion mutants of rpS2-beta-galactosidase chimeras identified a central domain comprising 72 amino acids which is necessary and sufficient to target the chimeric beta-galactosidase to the nucleus. The nuclear targeting domain shares no significant similarity to already characterized nuclear localization signals in ribosomal proteins or other nuclear proteins. Although a Nup153 fragment containing the importinbeta binding site fused to VP22 blocks nuclear import of rpS2-beta-galactosidase fusion proteins, nuclear uptake of rpS2 could be mediated by several import receptors since it binds to importinalpha/beta and transportin.

Active Transport, Cell Nucleus↗
PubMed2005

Expression pattern of fibroblast growth factors (FGFs), their receptors and antagonists in primary endothelial cells and vascular smooth muscle cells.

Fibroblast growth factors (FGFs) are important angiogenic growth factors. While basic FGF (FGF2) is well established as a potent inducer of angiogenesis much less is known about other FGFs possibly expressed by EC. We investigated the expression of all known FGFs, their main tyrosine kinase receptors and antagonists by RT-PCR analysis in human umbilical vascular endothelial cells (HUVECs) to obtain a complete expression profile of this important growth factor system in model endothelial cells (EC). In addition to FGFR1IIIc, which is considered as the major FGF receptor in EC, HUVECs express similar levels of FGFR3IIIc, detectable amounts of FGFR2IIIc and a new FGF receptor without an intracellular kinase domain (FGFR5). HUVECs express several secreted FGFs, including FGF5, 7, 8, 16 and 18 and two members of the fibroblast growth factor homologous factors (FHFs), not yet reported to be expressed in EC. The expression panel was compared with that obtained from human vascular smooth muscle cells (VSMCs) and human aortic tissue. Human umbilical artery smooth muscle cells (HUASMCs) and HUVECs express the identical FGF receptor and ligand panel implicating that both cell types act, according the FGF signals more as an entity than as individual cell types. Expression of Fgf1, 2, 7, 16 and 18 and the antagonists Sprouty 2,3 and 4 was demonstrated for all analysed cDNAs. The IIIc isoforms of FGFR1 and 2 and the novel FGFR5 were expressed in the aorta, but expression of the FGF receptor 3 was not detected in cDNAs derived from aortic tissue. In the VSMC of rat aortic tissue and in HUASM cultured cells we could demonstrate FGF18 immunoreactivity in the nucleus of the cells. The expression of several secreted FGFs by EC may focus the view more on their paracrine effects on neighbouring cells during tissue regeneration or tumor formation.

Amino Acid Sequence↗
PubMed2005

Thrombospondin 1 acts as a strong promoter of transforming growth factor beta effects via two distinct mechanisms in hepatic stellate cells.

BACKGROUND AND AIMS: Thrombospondin 1 (TSP-1) is an important activator of latent transforming growth factor beta (TGF-beta) but little is known of the expression patterns and functions of TSP-1 in liver cells. We therefore analysed if and how TSP-1 acts on TGF-beta during fibrogenesis. METHODS AND RESULTS: Using reverse transcription-polymerase chain reaction, we demonstrated that hepatocytes from normal liver expressed no TSP-1 mRNA whereas Kupffer cells and sinusoidal endothelial cells did. TSP-1 mRNA and protein were detected in quiescent and activated cultured hepatic stellate cells (HSC) and TSP-1 expression was highly inducible by platelet derived growth factor BB (PDGF-BB) and, to a lesser extent, by tumour necrosis factor alpha in activated HSC. Furthermore, addition of PDGF-BB directly led to enhanced TGF-beta mRNA expression and a TSP-1 dependent increase in TGF-beta/Smad signalling. Using either a peptide specifically blocking the interaction of TSP-1 with latent TGF-beta or antibodies against TSP-1 not only abrogated activation of latent TGF-beta but also reduced the effects of the active dimer itself. CONCLUSIONS: Our data suggest that TSP-1 expression is important for TGF-beta effects and that it is regulated by the profibrogenic mediator PDGF-BB in HSC. Furthermore, the presence of TSP-1 seems to be a prerequisite for effective signal transduction by active TGF-beta not only in rat HSC but also in other cell types such as human dermal fibroblasts.

Animals↗
PubMed2004

Comparative analysis of adenoviral transgene delivery via tail or portal vein into rat liver.

The potential indications for gene therapy are expanding continuously. Currently, hepatotropic adenoviruses are useful vector systems for targeting liver in experimental animal models. Although this gene delivery technique is widely distributed, there is no common sense about how these viruses should be applied. In general, the local delivery into portal vein and the systemic application via tail vein induces above all substantial transgene expression. We here comparatively analysed both methods and found that the systemic administration of an adeno-virus expressing the green fluorescent protein resulted in a stronger infiltration, a more homogenous distribution, and a higher inter-individual reproducibility of reporter gene expression in rat liver than organ-specific administration via the portal vein.

Adenoviridae↗
PubMed2002

Quantitative monitoring of the mRNA expression pattern of the TGF-beta-isoforms (beta 1, beta 2, beta 3) during transdifferentiation of hepatic stellate cells using a newly developed real-time SYBR Green PCR.

Current methods to determine the mRNA of the TGF-beta-isoforms, beta 1, beta 2, and beta 3, are not sensitive enough to detect small alterations in the expression levels. Therefore, we established a SYBR Green I-based real-time quantitative PCR procedure with fragment-specific standards. The advantage of gene-specific quantification is the possibility to be abstain from the need to compare results with a house-keeping gene having a different sequence and PCR efficiency. Reproducibility of the results and analytical variances of the real-time PCR assays were tested. In transdifferentiating rat hepatic stellate cells (HSC) the TGF-beta 1-mRNA was found to be the predominant isoform expressed followed by TGF-beta 3 and low amounts of TGF-beta 2-mRNA. An alteration of the TGF-beta 1,-beta 2, and -beta 3 ratio during HSC transdifferentiation could not be detected. Furthermore, the GAPDH mRNA expression varied during HSC activation, and thus is not recommended as a standard in real-time PCR quantifications.

Animals↗
PubMed2002

The influence of HES on the filtration properties of capillary membrane plasmaseparation.

Plasmaseparation is a treatment under discussion for critically ill patients, especially in sepsis and multiorgan failure. These patients receive a variety of different fluid substitutes, including hydroxyethylstarch (HES). HES is known to influence rheological properties, but nothing is known about the possible interactions between HES and the plamaseparation procedure. We used an in vitro plasmaseparation circuit with heparinized porcine blood. Before priming the system, 2 liters of blood were supplemented by adding 100 ml of either NaCl 0.9% or HES (n=6 in each group). We monitored the transmembrane (TMP) and the filtration pressure (PF) and measured free plasma hemoglobin (free Hb) and platelet counts before and after the two hours plasmaseparation procedure. The final transmembrane pressure was significantly higher with HES substitution. In the HES group we found negative filtration pressures from the very beginning with a significant further decrease toward the end of the experiments. A significant increase in free Hb and decrease in platelet counts were noted only in the HES group. Volume substitution with HES leads to impaired filtration properties and deteriorated hemocompatibility in in vitro plasmaseparation. Further studies have yet to evaluate whether or not the effects described also occur under clinical conditions.

Animals↗
PubMed2001

LIM-domain protein cysteine- and glycine-rich protein 2 (CRP2) is a novel marker of hepatic stellate cells and binding partner of the protein inhibitor of activated STAT1.

Activation of hepatic stellate cells is considered to be the main step in the development of liver fibrosis, which is characterized by the transition of quiescent vitamin-A-rich cells to proliferative, fibrogenic and contractile myofibroblasts. The identification of regulatory genes during early cell activation and transdifferentiation is essential to extend our knowledge of hepatic fibrogenesis. In liver, the gene CSRP2 is exclusively expressed by stellate cells, whereas no transcripts are detectable in hepatocytes, sinusoidal endothelial cells or Kupffer cells. The early activation of stellate cells induced by platelet-derived growth factor is accompanied by an enhanced expression of CSRP2. During later stages of transdifferentiation, the expression of CSRP2 in these cells is suppressed in vitro and in vivo. The CSRP2-encoded cysteine- and glycine-rich double-LIM-domain protein (CRP)2 is proposed to function as a molecular adapter, arranging two or more as yet unidentified protein constituents into a macromolecular complex. To identify these proteins and assign a cellular function to CRP2, a human cDNA library was screened with full-length CRP2 as bait in a yeast two-hybrid screen. The protein inhibitor of activated STAT1 ('PIAS1') was shown to associate selectively with the C-terminal LIM domain of CRP2. Physical interaction of both proteins in the cellular environment was confirmed by co-localization experiments with confocal laser scanning microscopy and co-immunoprecipitation analysis. These results establish CRP2 as a potential new factor in the JAK/STAT-signalling pathway and suggest that the suppression of CSRP2 might be a prerequisite for the myofibroblastic transition of hepatic stellate cells.

Adaptor Proteins, Signal Transducing↗
PubMed2001

[Heterophile antibodies, lack of communication and the diagnostic dilemma].

BACKGROUND: Heterophilic antibodies represent a great danger to clinical care by producing false-positive values for certain markers. Too large confidence in specificity of laboratory markers together with lack of communication between clinicians and clinical chemists may lead to unnecessary interventional diagnostic and therapeutic procedures. The prevalence of heterophilic antibodies is probably much higher than assumed up till now and several markers can be affected. AIM: In this review for clinicians, we explain formation of heterophilic antibodies, mechanisms of interference and present clinical data about affected markers and "side effects" from the literature. Furthermore we discuss possible alternatives and measures against this phenomenon. We consider broad awareness of this problem among clinicians the most important action to avoid further harm to patients.

Antibodies, Heterophile↗
PubMed2001

Transforming growth factor beta signal transduction in hepatic stellate cells via Smad2/3 phosphorylation, a pathway that is abrogated during in vitro progression to myofibroblasts. TGFbeta signal transduction during transdifferentiation of hepatic stellate cells.

To current knowledge, transforming growth factor beta (TGFbeta) signaling is mandatory to establish liver fibrosis and various molecular interventions designed to affect the TGFbeta system were successfully used to inhibit fibrogenesis. Activated hepatic stellate cells (HSC), which are one important source of TGFbeta, are the major producers of extracellular matrix proteins in liver injury. We have previously shown that the TGFbeta response of this cell type is modulated during the transdifferentiation process. This work delineates the activation of TGFbeta downstream mediators, the Smads, in quiescent HSC and transdifferentiated myofibroblasts (MFB). The expression level of all Smads remained largely unchanged during this process. The response of HSC to TGFbeta, leading to, e.g., induction of alpha2 (I) collagen expression, is mediated by phosphorylation of Smad2 and Smad3 and subsequent nuclear translocation of a Smad containing complex. Neither TGFbeta-dependent nor endogenously phosphorylated Smad2/3 was detectable in comparable amounts in transdifferentiated MFB, indicating loss of TGFbeta sensitivity. Ectopic expression of Smad7 in HSC led to inhibition of Smad2 phosphorylation and abrogated TGFbeta response. In transdifferentiated MFB, expression of a constitutively active TGFbeta receptor I, but not treatment with TGFbeta1, resulted in transcriptional activation of a TGFbeta responsive promoter, thereby demonstrating completely restored TGFbeta signal transduction. Our data indicate that in contrast to a postulated mechanism of enduring autocrine TGFbeta signal transduction, early and late stages of HSC activation have to be distinguished, which is of importance for antifibrotic therapies.

3T3 Cells↗
PubMed2001

Expression of Smads during in vitro transdifferentiation of hepatic stellate cells to myofibroblasts.

TGFbeta is of crucial importance during transdifferentiation of resting retinoid-storing hepatic stellate cells (HSC) to extracellular matrix producing myofibroblasts (MFB) and consequently, inhibition of TGFbeta signal transduction is an effective means for preventing experimental fibrosis. We have shown that isolated HSC lose TGFbeta-dependent growth control during in vitro activation and that alpha2 (I) collagen production in transdifferentiated MFB is TGFbeta-independent. Furthermore, Smad complexes with SBE binding activity were only detected in early cultures of HSC, although TGFbeta receptor types I and II were significantly expressed in HSC and MFB. In the present report, we compared the expression pattern of TGFbeta downstream mediators, i.e., the Smads, in TGFbeta responsive HSC versus nonresponding MFB. The transdifferentiation process was monitored by morphology and increasing expression of TGFbeta and alpha-smooth muscle actin, and TGFbeta signaling was investigated by (CAGA)(9)-MLP-Luc. The expression level of all Smads remained essentially unchanged both during the activation process and after TGFbeta-treatment. Smad7 was transiently upregulated upon TGFbeta stimulation in quiescent HSC, indicating a negative feed back loop in responsive cells. In contrast, MFB neither displayed TGFbeta-inducible nor constitutively upregulated Smad7 expression. Instead, Smad3 mRNA was increased in MFB. Our data indicate that abrogation of the TGFbeta response in MFB versus HSC is not based on different regulation of Smad expression.

Alternative Splicing↗
PubMed2001

An unusual melting curve profile in LightCycler multiplex genotyping of the hemochromatosis H63D/C282Y gene mutations.

OBJECTIVES: Real time polymerase chain reaction followed by melting curve analysis using hybridization probes has become an important tool in routine diagnosis of the HFE mutations, which are associated with hereditary hemochromatosis. DESIGN AND METHODS: We used the LightCycler technology for simultaneous detection of the H63D and C282Y mutations of the HFE gene in patients with a higher prevalence for hemochromatosis. RESULTS: In our cohort we identified two siblings with a variant pattern of the HFE-LightCycler melting profiles preventing allelic discrimination. CONCLUSIONS: As a consequence, in these patients DNA sequencing or RFLP analysis is necessary to unequivocally assign the correct HFE genotype.

Amino Acid Substitution↗
PubMed2001

Expression of isoforms and splice variants of the latent transforming growth factor beta binding protein (LTBP) in cultured human liver myofibroblasts.

BACKGROUND/AIMS: The activation of hepatic stellate cells (HSC) to extracellular matrix (ECM) producing myofibroblasts (MFB) is the key pathogenetic event in human liver fibrogenesis. Latent transforming growth factor beta binding protein (LTBP), a component of the profibrogenic large latent transforming growth factor (TGF)-beta complex, is suggested to be important for secretion, latency, storage and activation of TGF-beta in the ECM. This study was performed to identify the expression profile of all hitherto known LTBP isoforms and LTBP splice variants in conjunction with that of TGF-beta isoforms in cultured human liver MFB. METHODS: Cultured human MFB were analyzed for TGF-beta and LTBP using reverse-transcription polymerase chain reaction (RT-PCR), sequence analysis, immunofluorescence staining, metabolic labeling, immunoprecipitation, and enzyme-linked immunosorbent assay (ELISA). RESULTS: Transcripts of all three TGF-beta isoforms, of all four LTBP isoforms and of nearly all splice variants of LTBP-1 and LTBP-4 so far known were detected. Metabolic labeling followed by immunoprecipitation with anti-LTBP-1 antibody revealed the synthesis of LTBP proteins. Secretion of free LTBP and LTBP integrated into the large latent TGF-beta complex was demonstrated by size-exclusion chromatography. Co-localization of LTBP-1 and -2 with fibronectin and collagen type I was observed by double immunofluorescence staining. CONCLUSION: The expression of a complete profile of hitherto known LTBP proteins by cultured human MFB suggests a role in modulating the bioactivity of TGF-beta in the diseased liver.

Alternative Splicing↗
PubMed2001

The expression and antigenicity identification of recombinant rat TGF-beta1 in bacteria.

In order to study structure-function details of TGF-beta1, the recombinant mature form of rat TGF-beta1 was expressed in bacteria. Synthesis of the 112 amino-acid carboxyl-terminal part of TGF-beta1 (amino acid 279-390) was controlled by an inducible gene expression system based on bacteriophage T7 RNA polymerase. This system allowed an active and selective synthesis of recombinant TGF-beta1. The molecular weight of expressed TGF-alpha1 monomer determined on SDS-polyacrylamide gel under reducing conditions was about 13 kD. Serial detergent washes combined with a single gel-filtration purification step were sufficient to purify the expression product to homogeneity. Amino-terminal sequencing revealed that the N-terminal of the recombinant protein was identical to the published data. In Western blot analysis the recombinant polypeptide showed excellent antigenicity against polyclonal TGF-beta1 antibody. The mature recombinant rat TGF-beta1 expressed in this study provides a useful tool for future detailed structural and functional studies.

Amino Acid Sequence↗