Biomedical subjects

K Friedrich

Publications and source records attributed to K Friedrich.

At least 19 recordsLinked to original sources

PubMed1998

Activation of STAT6 is not dependent on phosphotyrosine-mediated docking to the interleukin-4 receptor and can be blocked by dominant-negative mutants of both receptor subunits.

Stimulation of susceptible cells by interleukin-4 leads to activation of signal transducer and activator of transcription (STAT6) through tyrosine phosphorylation and dimerisation, thus directing it to the cell nucleus and rendering it a sequence-specific transcription factor. We functionally reconstituted human interleukin-4 receptor complexes with intracellular truncations of either the alpha or gamma subunits and demonstrate the requirement for elements from both receptor chains for STAT6 activation induced by interleukin-4. By assaying the signalling properties of human interleukin-4-receptor alpha-chain-deletion constructs in both Ba/F3 cells and COS-7 cells, we show that all its cytoplasmic tyrosine residues can be removed without affecting the capability of the receptor complex to trigger STAT6 function with regard to tyrosine phosphorylation, DNA binding, and specific gene transcription. The activation of both STAT6 and janus kinase 1 (JAK1) by the interleukin-4 receptor was completely abolished by disruption of the membrane-proximal 'box1' motif in the interleukin-4 receptor alpha chain. Our results indicate a redundant role of the previously defined phosphotyrosine-containing STAT6 docking site and suggest a mechanism of immediate activation of STAT6 by receptor-associated janus kinase(s). In addition, we demonstrate that dominant negative versions of both interleukin-4 receptor subunits are able to block interleukin-4 induced signalling via STAT6.

Animals↗

PubMed1997

Receptor-associated constitutive protein tyrosine phosphatase activity controls the kinase function of JAK1.

Exposure of cells to protein tyrosine phosphatase (PTP) inhibitors causes an increase in the phosphotyrosine content of many cellular proteins. However, the level at which the primary signaling event is affected is still unclear. We show that Jaks are activated by tyrosine phosphorylation in cells that are briefly exposed to the PTP inhibitor pervanadate (PV), resulting in tyrosine phosphorylation and functional activation of Stat6 (in addition to other Stats). Mutant cell lines that lack Jak1 activity fail to support PV-mediated [or interleukin 4 (IL-4)-dependent] activation of Stat6 but can be rescued by complementation with functional Jak1. The docking sites for both Jak1 and Stat6 reside in the cytoplasmic domain of the IL-4 receptor alpha-chain (IL-4Ralpha). The glioblastoma-derived cell lines T98G, GRE, and M007, which do not express the IL-4Ralpha chain, fail to support Stat6 activation in response to either IL-4 or PV. Complementation of T98G cells with the IL-4Ralpha restores both PV-mediated and IL-4-dependent Stat6 activation. Murine L929 cells, which do not express the gamma common chain of the IL-4 receptor, support PV-mediated but not IL-4-dependent Stat6 activation. Thus, Stat6 activation by PV is an IL-4Ralpha-mediated, Jak1-dependent event that is independent of receptor dimerization. We propose that receptor-associated constitutive PTP activity functions to down-regulate persistent, receptor-linked kinase activity. Inhibition or deletion of PTP activity results in constitutive activation of cytokine signaling pathways.

Amino Acid Sequence↗

PubMed1997

X-SCID B cell responses to interleukin-4 and interleukin-13 are mediated by a receptor complex that includes the interleukin-4 receptor alpha chain (p140) but not the gamma c chain.

This study investigates the effect of interleukin (IL)-4 mutant proteins and a monoclonal antibody to the IL-4 receptor alpha chain on IL-4 and IL-13 response by B cells from X-linked severe combined immunodeficiency (X-SCID) patients in which the common gamma chain (gamma c chain) gene mutations have been fully characterized and no gamma c chain expression was detected. In this gamma c chain gene knockout model, it was confirmed that the gamma c chain is essential for B cell responses to IL-2 but not for IL-4 or IL-13. Dose-response curves for X-SCID and normal B cell responses to IL-4 were indistinguishable, showing that the loss of the gamma c chain did not diminish the sensitivity of B cells to IL-4. The mutant protein IL-4(Y124D) and an antibody to the IL-4R alpha chain both inhibited responses of X-SCID B cells to IL-4 and IL-13, showing that X-SCID B cell responses to these cytokines are mediated by a receptor complex that includes the IL-4R alpha chain but not the gamma c chain. Another mutant protein, IL-4(R88D), which has greatly reduced affinity for IL-4R alpha, was found to inhibit responses by normal B cells to IL-4 but not to IL-13. IL-4(R88D), did not, however, inhibit X-SCID B cell responses to IL-4. This result is consistent with IL-4(R88D) inhibition of responses mediated by receptor complexes that include the gamma c chain. We propose that X-SCID B cells responses to IL-4 are mediated by an IL-13 receptor complex comprised of the IL-4R alpha chain associated with the recently cloned IL-13R binding protein. This model has major implications for understanding normal B cell responses to IL-4.

Animals↗

PubMed1997

Electron beam dose calculations with the VMC algorithm and the verification data of the NCI working group.

The accuracy of the Monte Carlo algorithm for fast electron dose calculation, VMC, is demonstrated by comparing calculations with measurements performed by a working group of the National Cancer Institute (NCI) of the USA. For both energies investigated, 9 and 20 MeV, the measurements in water are taken to determine the energy spectra of the Varian Clinac 1800 accelerator. For the majority of the experiments a good agreement is found between measurements and VMC calculations. However, in some cases deviations have been observed, which could be explained by the incompletely known geometry on the one hand and by inconsistent data on the other hand. As a reference, dose distributions calculated by the MDAH pencil-beam algorithm are also presented. It is shown that, especially near low- or high-density inhomogeneities, large dose overestimations and underestimations are calculated by using a pencil-beam approach, whereas VMC is able to reproduce the correct doses for these cases also.

Algorithms↗

PubMed1997

Comparison of the transactivation domains of Stat5 and Stat6 in lymphoid cells and mammary epithelial cells.

Stat (signal transducers and activators of transcription) and Jak (Janus kinases) proteins are central components in the signal transduction events in hematopoietic and epithelial cells. They are rapidly activated by various cytokines, hormones, and growth factors. Upon ligand binding and cytokine receptor dimerization, Stat proteins are phosphorylated on tyrosine residues by Jak kinases. Activated Stat proteins form homo- or heterodimers, translocate to the nucleus, and induce transcription from responsive genes. Stat5 and Stat6 are transcription factors active in mammary epithelial cells and immune cells. Prolactin activates Stat5, and interleukin-4 (IL-4) activates Stat6. Both cytokines are able to stimulate cell proliferation, differentiation, and survival. We investigated the transactivation potential of Stat6 and found that it is not restricted to lymphocytes. IL-4-dependent activation of Stat6 was also observed in HC11 mammary epithelial cells. In these cells, Stat6 activation led to the induction of the beta-casein gene promoter. The induction of this promoter was confirmed in COS7 cells. The glucocorticoid receptor was able to further enhance IL-4-induced gene transcription through the action of Stat6. Deletion analysis of the carboxyl-terminal region of Stat6 and recombination of this region with a heterologous DNA binding domain allowed the delimitation and characterization of the transactivation domain of Stat6. The potencies of the transactivation domains of Stat5, Stat6, and viral protein VP16 were compared. Stat6 had a transactivation domain which was about 10-fold stronger than that of Stat5. In pre-B cells (Ba/F3), the transactivation domain of Stat6 was IL-4 regulated, independently from its DNA binding function.

Amino Acid Sequence↗

PubMed1997

Morphological heterogeneity of p53 positive and p53 negative nuclei in breast cancers stratified by clinicopathological variables.

The study was aimed to detect differences in nuclear morphology between nuclear populations as well as between tumours with different p53 expression in breast cancers with different clinicopathological features, which also reflect the stage of tumour progression. The p53 immunohistochemistry was performed on paraffin sections from 88 tumour samples. After the cells had been localised by means of an image cytometry workstation and their immunostaining had been categorised visually, the sections were destained and stained by the Feulgen protocol. The nuclei were relocated and measured cytometrically by the workstation. There were significant differences in the nuclear features between tumours as well as between nuclear populations with different p53 expression in the most subgroups. The variability of nuclear shape in tumour groups, classified by the tumour size or the lymph node status, increase with the p53 immunoreactive score, whereas in tumours grouped by the Bloom-Richardson grade features of the chromatin distribution were different between the p53 staining categories. The nuclear subpopulations showed differences in the amount and distribution of chromatin in most subgroups. The results demonstrate the relationship between the nuclear morphology and the p53 expression in different stages of breast cancers. The p53 status is an important factor of the biological behaviour but not the only one.

Breast Neoplasms↗

PubMed1997

Correlation between p53 status, DNA ploidy, proliferation rate and nuclear morphology in breast cancer. An image cytometric study.

The study was designed to detect differences in the nuclear morphology of tumours and tumour cell populations with different p53 expression in correlation with DNA ploidy and proliferation rate. The paraffin sections from routinely processed samples of 88 breast cancers were immunostained with the monoclonal p53-antibody DO-1. After localization and evaluation with a scoring system the sections were destained and stained by the Feulgen method. The nuclei were relocated automatically and measured by means of the image cytometry workstation. Significant differences between the tumours and tumour cell populations with different p53 expression were found in the euploid tumours as well as in the aneuploid tumours and in the breast cancers with a high proliferation rate. The breast cancers with a low immunoreactive score (IRS 1-4) differ from the negative cancers as well as from the cancers with a higher immunoreactive score (IRS 5-12). Evaluating the nuclear populations of the p53 positive cancers, there were differences in the features of the chromatin amount and distribution in the groups of the euploid breast cancers and in cancer with a high proliferation rate. In contrast, the nuclear populations of the aneuploid cancers did not show any differences in their nuclear morphology. The results showed the different impacts of the p53 expression, DNA ploidy and the proliferation rate on the nuclear morphology in breast cancer.

Adult↗

PubMed1997

Nuclear image analysis of p53-positive and -negative cells in breast carcinoma.

OBJECTIVE: To detect differences in nuclear morphology in breast carcinoma cells with different p53 expression. STUDY DESIGN: Immunohistochemical reactions were performed on paraffin sections from 88 breast cancers with the monoclonal antibody DO-1. By means of an image cytometry workstation, 500 nuclei were localized and categorized in an immunohistochemical staining scoring system in each case. After destaining and Feulgen staining, the nuclei were relocated and measured by means of the workstation. RESULTS: P53-positive nuclei showed greater irregularity of the chromatin pattern and stronger granularity of the chromatin. There were also differences in the chromatin texture and nuclear shape between tumors different in their p53 expression. The p53-negative nuclear population from the p53-positive tumors and the whole nuclear population of the p53-negative tumors were morphologically different, also. CONCLUSION: Nuclear image analysis of immunohistochemically characterized cells allows further insight into the relationship of structure and function in malignant tumor cells.

Adult↗

PubMed1996

Homodimerization of interleukin-4 receptor alpha chain can induce intracellular signaling.

The possible role of homodimerization events in intracellular signal transduction triggered by the bipartite human interleukin-4 receptor was addressed. We generated cell lines functionally expressing derivatives of the two receptor subunits alpha and gamma, which allow for a specific and background-free experimental induction of intracellular homo- and heterodimers. A heterodimer of alpha and gamma released an intracellular signal, whereas a gamma-gamma homodimer did not. Unexpectedly, we found the intracellular domain of interleukin-4 receptor alpha chain to evoke cell proliferation and activation of tyrosine kinase Jak1 as well as of transcription factor Stat6 upon homodimerization. Both recruitment of the common gamma chain and activation of kinase Jak3 were shown to be dispensible for these processes.

Animals↗

PubMed1996

A chemiluminescence-based method for the detection and quantification of antigen-antibody interactions on the surface of eukaryotic cells.

Enhanced chemiluminescence was applied to detect the binding of monoclonal antibodies to surface antigens on intact cells. The fast and simple assay is performed in the microtiter scale and thus allows for the simultaneous processing of a large number of samples with a sensitivity comparable to conventionally used techniques such as cytometry or Western blot analysis. In two model experiments, we demonstrate (a) the detection of a heterologously expressed cytokine receptor subunit on the surface of suspension cells and (b) the screening of hybridoma clones for the production of antibodies specifically recognizing surface antigens on a tumor cell line. Moreover, the assay is shown to be suitable for the determination of antibody affinities and of antibody binding sites per cell.

Animals↗

PubMed1996

[Liver transplantation--preparing patients, early postoperative care and occupational rehabilitation].

Authors summarize the results of liver transplantation first of all in the view of rehabilitation. Role of rehabilitation experts is discussed in the period before and after transplantation. The necessity of information for the patients is specially underlined already from the first raise of the possibility of liver transplantation. They express that after successful operation it is needed for the patients to return gradually to the used activities of everyday life. All the questions of immunosuppressive treatment, hygienic rules, physical activities, patient care at home and work place rehabilitation are discussed.

Adult↗

PubMed1996

Nuclear image analysis of immunohistochemically stained cells in breast carcinomas.

Hitherto, the relationship between malignancy-associated morphological features in single tumour cells and the expression of markers indicating functional properties of these cells remained widely unknown. This study was aimed at describing differences in the size, shape and chromatin structure between tumour cells with different marker expression for progesterone receptors (PgR) and p53. Two series of breast cancers, consisting of 50 PgR-positive, and 39 p53-negative and 49 p53-positive mammary carcinomas, were investigated. The immunohistochemical staining was performed on paraffin sections using 3-amino-9-ethylcarbazole as the chromogenic substrate. By means of a cytometry workstation equipped with a computer-controlled motorised scanning stage, about 500 positive and negative tumour cells in each case were localised in the microscope and categorised by a scoring system for their staining intensity. After destaining, the tissue sections were Feulgen-stained. Then, all the tumour cells were relocated automatically and analysed by high resolution image cytometry. Among the numerous size, shape, and texture features used in the system, several variables of the nuclear contour and chromatin structure were found to be significantly different between the positive and negative tumour cell populations. Nuclei without PgR had more malignancy-associated morphological features than PgR-positive cells. Whereas p53-negative nuclei had a higher degree of regularity, their positive counterparts exhibited higher DNA ploidy values.

Biomarkers, Tumor↗

PubMed1996

Biological activity of IL-4 and IL-13 on human endothelial cells: functional evidence that both cytokines act through the same receptor.

The cytokine IL-4 has unique effects on human endothelial cells. It specifically increases expression of vascular cell adhesion molecule (VCAM)-1 promoting adhesion of lymphocytes but not neutrophils, and causes profound effects on the morphology of endothelial monolayers characterized by formation of cell clusters and the appearance of holes in the cultured monolayer. In this study we show that the effects of IL-13 on human umbilical vein endothelial cells (HUVEC) are indistinguishable from those of IL-4. Both cytokines induce the same morphological changes in cultured HUVEC monolayers which are distinct from any other cytokine. In addition, IL-13 and IL-4 stimulate comparable levels of VCAM-1 expression with similar time kinetics, but at doses 10-fold less than those required for B cell activation and proliferation. Using a combination of mutant IL-4 antagonists and mAb to the IL-4R alpha chain (CD124), we show that expression of IL-4R alpha is essential for HUVEC responses to both IL-4 and IL-13, consistent with this receptor subunit being a component of the receptors for both cytokines. In contrast, the common gamma chain (gamma c), which is a component of the classical IL-4 receptor, was not detected on endothelial cells by flow cytometry or immunogold histochemistry. In addition, RT-PCR showed extremely low or absent gamma c mRNA, consistent with the absence of detectable surface protein. These results strongly suggest that the cytokines IL-4 and IL-13 are both important in modulating endothelial cell function, and may act through a single receptor complex on human endothelial cells that includes the IL-4R alpha chain but not the gamma c chain.

Antibodies, Monoclonal↗

PubMed1996

3D electron dose calculation using a Voxel based Monte Carlo algorithm (VMC).

A new model for calculating electron beam dose has been developed. The algorithm is based on a two- or three-dimensional geometry defined by computerized tomography (CT) images. The Monte Carlo technique was used to solve the electron transport equation. However, in contrast to conventional Monte Carlo models (EGS4) several approximations and simplifications in the description of elementary electron processes were introduced reducing in this manner the computational time by a factor of about 35 without significant loss in accuracy. The Monte Carlo computer program does not need any precalculated data. The random access memory required is about 16 Mbytes for a 128(2) X 50 matrix, depending on the resolution of the CT cube. The Voxel Monte Carlo model (VMC) was tested in comparison to calculations by EGS4 and the "Hogstrom algorithm" (MDAH) using several fictive phantoms. In all cases a good coincidence has been found between EGS4 and VMC, especially near tissue inhomogeneities, whereas the MDAH algorithm has produced dose underestimations of up to 40%.

Algorithms↗

PubMed1995

Different human interleukin-4 mutants preferentially activate human or murine common receptor gamma chain.

Interleukin-4 (IL-4) shows species-specific activity due to species-restricted interaction with the IL-4 receptor alpha (IL-4R alpha) chain. The second subunit of a functional IL-4 receptor, the common gamma chain (gamma c), is more promiscuous, since human IL-4 is able to activate IL-4 receptor complexes containing either human or murine common gamma receptor chain (gamma c). We have stably transfected factor-dependent mouse cells of myeloid and lymphoid origin with combinations of human IL-4R alpha and gamma c derivatives. In these cell lines, both human and murine gamma c receptors as well as IL-4R alpha chains from both species are simultaneously expressed. Both versions of gamma c readily form ternary complexes with either human IL-4 and human IL-4R alpha or murine IL-4 and murine IL-4R alpha. Due to distinct ligand-binding properties of human and murine gamma c, the two receptor complexes can be activated preferentially by different mutant variants of human IL-4. The contribution of murine common gamma chain to human IL-4-induced signal transduction is suppressed by an inhibitory antibody directed to the extracellular domain of the mouse gamma c. We present evidence that the two IL-4R complexes functionally interfere with each other and compete for response-limiting signalling components.

Animals↗

PubMed1995

Species-specific agonist/antagonist activities of human interleukin-4 variants suggest distinct ligand binding properties of human and murine common receptor gamma chain.

Interleukin-4 (IL-4) is a pleiotropic cytokine eliciting various responses in target cells upon binding to its receptor. Activation of the IL-4 receptor probably involves interaction of the ligand with both the IL-4 receptor alpha subunit (IL-4R alpha) and the common gamma chain (c gamma). Although human and murine IL-4 receptor alpha chains are specific for IL-4 from the same species, murine c gamma can form a signal-competent complex with human IL-4R alpha (hIL-4R alpha) and human IL-4 (hIL-4). We have generated a hIL-4 responsive murine myeloid cell line (FDC-4G) expressing a chimera comprising the extracellular domain of human IL-4R alpha and the intracellular domain of human granulocyte colony-stimulating factor receptor (hG-CSFR). This hybrid receptor was shown to form a complex with hIL-4 and the murine c gamma-chain. Biological activities of human IL-4 variants on murine FDC-4G cells and on the human erythroleukemic cell line TF-1 displayed a strikingly different pattern. Single amino acid replacements at two different positions in the C-terminal helix of hIL-4, the region of the previously defined "signaling site," lead to an inverse agonist/antagonist behavior of the resulting cytokines in the two cellular systems. From these findings we conclude that upon formation of the activated IL-4 receptor complex murine and human c gamma interact with hIL-4 in a geometrically different fashion.

Amino Acid Sequence↗

PubMed1995

Expression of p53 and bcl-2 in correlation to clinicopathological parameters, hormone receptor status and DNA ploidy in breast cancers.

The expression of p53 and bcl-2 was immunohistochemically investigated in 61 formalin-fixed, paraffin-embedded invasive breast carcinomas. The study was aimed to elucidate the relationship between both markers and the correlation of p53 and bcl-2, respectively, to clinicopathological variables, to hormone receptor status and to DNA-ploidy. Twenty tumors showed a positive reaction with the monoclonal antibody DO-1 against p53 protein. Its immunohistochemical demonstration was significantly correlated with a tumor size larger than 2 cm, a low estrogen receptor status and DNA-aneuploidy. Bcl-2 was demonstrated in 51 breast cancers. Bcl-2 was preferably seen in low grade and hormone receptor positive tumors. We found a negative correlation between the immunoreactive scores of p53 and bcl-2, but in 17 carcinomas a coexpression of both proteins was seen. Cases with this coexpression did not differ significantly from the other tumors in clinicopathological parameters. In eight of these cases more than 10% of the cells were found to be positive for both markers. In four cases we could show many cells to exhibit both markers as it was assessed by an immunofluorescence double labeling technique.

Breast Neoplasms↗

PubMed1995

A potent human interleukin-4 antagonist stimulates the proliferation of murine cells expressing the human interleukin-4 binding chain.

A single-amino-acid substitution mutant form of human interleukin-4 (hIL-4), Y124D.hIL-4, has been described previously as an antagonist of the effects of hIL-4 on various human cells. The murine T-cell leukemic cell line CT.h4S, which expresses the human IL-4 receptor, proliferates in response to both hIL-4 and murine IL-4. Although Y124D.hIL-4 antagonizes the proliferative effects of hIL-4 on human phytohaemagglutinin-stimulated peripheral blood mononuclear cells, Y124D.hIL-4 is a potent stimulator for CT.h4S cells. Molecular modelling studies were performed to investigate the stability of different conformations of residue 124 as well as the efficiency of different molecular mechanics force fields in homology modelling. We suggest that the aspartate substitution alters the C-terminal end of the D-helix in such way that the analogue still binds to the human IL-4 receptor alpha-chain and signals through the murine gamma c-chain. In contrast, the Y124D.hIL-4/IL-4 receptor complex cannot signal through the human gamma c-chain.

Amino Acid Sequence↗