Biomedical subjects

Georg E Steiner

Publications and source records attributed to Georg E Steiner.

11 recordsLinked to original sources

PubMed2006

An improved method for discrimination of cell populations in tissue sections using microscopy-based multicolor tissue cytometry.

BACKGROUND: In tissue context, researchers and pathologists lack a generally applicable standard for quantitative determination of cytological parameters. Increasing knowledge of disease-specific markers calls for an appropriate in situ tissue cytometry. METHODS: Microscopy-based multicolor tissue cytometry (MMTC) permits multicolor analysis of single cells within tissue context. RESULTS: Tissue specimens stained for CD45/CD3/CD4/CD8 were analyzed. Specificity as well as reproducibility of MMTC is demonstrated and a novel MMTC-based function to improve visual discrimination of subpopulations is introduced. CONCLUSIONS: Our data demonstrate that MMTC constitutes an important step toward automated and quantitative fluorometry of solid tissues and cell monolayers.

Algorithms↗

PubMed2006

3D parallel coordinate systems--a new data visualization method in the context of microscopy-based multicolor tissue cytometry.

BACKGROUND: Presentation of multiple interactions is of vital importance in the new field of cytomics. Quantitative analysis of multi- and polychromatic stained cells in tissue will serve as a basis for medical diagnosis and prediction of disease in forthcoming years. A major problem associated with huge interdependent data sets is visualization. Therefore, alternative and easy-to-handle strategies for data visualization as well as data meta-evaluation (population analysis, cross-correlation, co-expression analysis) were developed. METHODS: To facilitate human comprehension of complex data, 3D parallel coordinate systems have been developed and used in automated microscopy-based multicolor tissue cytometry (MMTC). Frozen sections of human skin were stained using the combination anti-CD45-PE, anti-CD14-APC, and SytoxGreen as well as the appropriate single and double negative controls. Stained sections were analyzed using automated confocal laser microscopy and semiquantitative MMTC-analysis with TissueQuest 2.0. The 3D parallel coordinate plots are generated from semiquantitative immunofluorescent data of single cells. The 2D and 3D parallel coordinate plots were produced by further processing using the Matlab environment (Mathworks, USA). RESULTS: Current techniques in data visualization primarily utilize scattergrams, where two parameters are plotted against each other on linear or logarithmic scales. However, data evaluation on cartesian x/y-scattergrams is, in general, only of limited value in multiparameter analysis. Dot plots suffer from serious problems, and in particular, do not meet the requirements of polychromatic high-context tissue cytometry of millions of cells. The 3D parallel coordinate plot replaces the vast amount of scattergrams that are usually needed for the cross-correlation analysis. As a result, the scientist is able to perform the data meta-evaluation by using one single plot. On the basis of 2D parallel coordinate systems, a density isosurface is created for representing the event population in an intuitive way. CONCLUSIONS: The proposed method opens new possibilities to represent and explore multidimensional data in the perspective of cytomics and other life sciences, e.g., DNA chip array technology. Current protocols in immunofluorescence permit simultaneous staining of up to 17 markers. Showing the cross-correlation between these markers requires 136 scattergrams, which is a prohibitively high number. The improved data visualization method allows the observation of such complex patterns in only one 3D plot and could take advantage of the latest developments in 3D imaging.

Humans↗

PubMed2004

Response to sublethal heat treatment of prostatic tumor cells and of prostatic tumor infiltrating T-cells.

BACKGROUND: To investigate the possibilities offered by high intensity focused ultrasound (HIFU) in the field of tumor vaccination, we analyzed how prostatic cancer (CaP) cells react towards heat treatment and whether increased access to CaP cells by the immune system would be the result. METHODS: Heat/stress response of CaP cells in situ and of CaP cell lines was analyzed by immunohistochemistry, Western blotting, and Atlas array. A heat-induced change in immune recognition was analyzed functionally using human T-helper (Th)1 and Th2-cytokine release with tumor infiltrating T-lymphocytes (TIL) as responder and autologous CaP cells either heated or untreated as stimulator cells. RESULTS: Transcription of 68 out of 500 genes was upregulated by sublethal heat in LNCaP and PC3 cells. Significantly upregulated stress protein (SP) expression (HSP-72, -73, GRP-75, -78) was seen at the border zone of HIFU treatment. Remarkably, even untreated benign prostatic hyperplasia (BPH) specimens revealed relative overexpression of heat shock protein (HSP)-72, -73 and glucose regulated protein (GRP)-75, -78. Heated CaP cells increased Th1-cytokine (IL-2, IFN-gamma, TNF-alpha) release but decreased Th2-cytokine (IL-4, -5, -10) release of TIL. CONCLUSIONS: HIFU treatment may alter the presentation of prostate tissue and tumor antigens and this presentation is most likely stimulatory. HSP-72/73 overexpression in untreated BPH may suggest a mechanism by which BPH can incite inflammation.

Antigens, Neoplasm↗

PubMed2004

Microscopy-based multicolor tissue cytometry at the single-cell level.

Cytomics is a novel perspective from which to look at life. As with genomics and proteomics before, this discipline requires novel and innovative techniques and technologies to focus on its substrate of research--the cytome. With cytomics being the discipline that analyzes cellular systems and their interdependencies, advanced microscopy represents a key technology in cytomics research. Yet, conventional microscopy-based investigations, i.e., "look and conclude" analyses, do not meet the major cytomics criteria of 1) relating multiple parameters to each other, 2) within large populations of cells, 3) on a single-cell basis, and 4) in a quantitative and observer-independent manner. However, emerging improvements in the fields of fluorophore technology, sensitive fluorescence detection devices, and sophisticated image analysis procedures, are important and necessary steps into the cytomics era. Tissue represents an important class of cytomes, hence tissue cytometry--on the single cell level--can be expected to become an important cytomics technology. In this report, the techniques and technologies of microscopy-based multicolor tissue cytometry (MMTC) are outlined and applications are discussed, including the phenotypic characterization of tissue infiltrating leukocytes, in situ quantification of proliferation markers and tumor suppressors, and in situ quantification of apoptosis.

Apoptosis↗

PubMed2004

Application of spectral imaging microscopy in cytomics and fluorescence resonance energy transfer (FRET) analysis.

BACKGROUND: Specific signal detection has been a fundamental issue in fluorescence microscopy. In the context of tissue samples, this problem has been even more pronounced, with respect to spectral overlap and autofluorescence. METHODS: Recent improvements in confocal laser scanning microscopy combine sophisticated hardware to obtain fluorescence emission spectra on a single-pixel basis and a mathematical procedure called "linear unmixing" of fluorescence signals. By improving both the specificity of fluorescence acquisition and the number of simultaneously detectable fluorochromes, this technique of spectral imaging (SI) allows complex interrelations in cells and tissues to be addressed. RESULTS: In a comparative approach, SI microscopy on a quantitative basis was compared to conventional bandpass (BP) filter detection, demonstrating substantial superiority of SI with respect to detection accuracy and dye combination. An eight-color immunofluorescence protocol for tissue sections was successfully established. Moreover, advanced use of SI in fluorescence resonance energy transfer (FRET) applications using enhanced green fluorescence protein (EGFP) and enhanced yellow fluorescence protein (EYFP) in a confocal set up could be demonstrated. CONCLUSIONS: This novel technology will help to perform complex multiparameter investigations at the cellular level by increasing the detection specificity and permitting simultaneous use of more fluorochromes than with classical techniques based on emission filters. Moreover, SI significantly extends the possibilities for specialized microscopy applications, such as the visualization of macromolecular interactions or conformational changes, by detecting FRET.

Flow Cytometry↗

PubMed2003

Expression and function of pro-inflammatory interleukin IL-17 and IL-17 receptor in normal, benign hyperplastic, and malignant prostate.

INTRODUCTION AND OBJECTIVES: To investigate factors involved in inflammation of the prostate besides IL-15, we screened prostatic cells and tissues for IL-17 and IL-17 receptor expression. METHODS: Normal prostate (n = 1), BPH (n = 19), and carcinoma (CaP, n = 12) specimens were screened for IL-17, IL-17 receptor, CD45, IL-6, and IL-8 mRNA expression. The carcinoma cell lines DU145, PC3, LNCaP, and BPH-epithelial (EC), stromal cell (SC) preparations, and BPH-T-cell lines were analyzed for IL-17 production by RT-PCR and ELISA. The effect of IL-17 on IL-6, IL-8, TGF-beta1, and fibroblast growth factor (FGF-2) mRNA expression and/or release of SC was analyzed using real-time PCR and/or ELISA. Immunohistochemistry was used to localize both IL-17 and IL-17 receptor. RESULTS: In the normal prostate, IL-17 expression was very weak and restricted to lymphocytes. In 79% of BPH and 58% of CaP specimens, IL-17 mRNA and protein expression was increased. IL-17 mRNA expression could be shown for activated BPH-T-cells and to some extend for BPH-EC. Expression of IL-17 receptor was ubiquitous. Release of IL-17 was shown only for activated BPH-T-cells. IL-17 stimulated expression of IL-6 (13-fold) and IL-8 (26-fold) by prostatic BPH-SC. In situ, however, the amount of IL-17mRNA in BPH-tissue did not correlate with the amount of IL-6 and IL-8 mRNA. In CaP tissue, significant correlation was found only between the amount of IL-6 and IL-8 mRNA. CONCLUSIONS: Activated BPH-T-cells abundantly express IL-17. The increase of IL-17 in BPH-tissues goes hand in hand with elevated levels of IL-15, a pro-inflammatory cytokine with T-cell growth factor properties. A clinical relevance of increased IL-17 expression under pathological conditions is suggested by the demonstration of significant upregulation of IL-6 and IL-8 production of prostatic SC by IL-17.

Adult↗

PubMed2003

Vascular endothelial growth factor C and vascular endothelial growth factor receptor 3 expression in squamous cell carcinomas of the head and neck.

BACKGROUND AND METHODS: VEGF proteins and their receptors are involved in tumor vessel neoformation. The third VEGF receptor, VEGFR3 (flt-4) is important during both blood vessel development and lymphatic vessel formation. Because HNSCC preferentially metastasizes to regional lymph nodes, we investigated the expression of VEGFR3 and its ligand VEGF-C in head and neck squamous cell carcinomas by semiquantitative RT-PCR (4 HNSCC cells lines and 6 HNSCC specimens) and by immunohistochemistry (18 HNSCC specimens). VEGFR3 protein expression was confirmed by Western blotting in four HNSCC cell lines and six HNSCC specimens. RESULTS: Semiquantitative mRNA analysis showed VEGF-C mRNA expression in three (SCC9, SCC25, LFFR) of four HNSCC cell lines and all six HNSCC specimens. VEGFR3 mRNA was found in two HNSCC cell lines (JPPA and SCC25) and only weakly detected in the other two HNSCC cell lines (SCC9 and LFFR). High amounts of VEGFR3 mRNA were shown in all six patients' tumor specimens. VEGFR3 Western blot analysis yielded a distinct band at the predicted size of 210 kD in JPPA and SCC9 and hardly detectable bands in SCC25 and LFFR cell lines. All six HNSCC specimens displayed strong VEGFR3 protein bands. Immunohistochemistry in 18 HNSCC specimens assigned strong to mediate VEGF-C IR and minor VEGFR3 IR to tumor cells and strong VEGF-C and VEGFR3 IR to tumor surrounding vessels. In addition, intense VEGF-C immunostaining was observed on perivascular and mononuclear cells in the tumor surrounding stroma. Subtyping of VEGFR3+ microvascular tumor vessels revealed partially double immunolabeling with CD34 and flk-1, indicating a common origin of blood and lymphatic vessels. The expression of VEGF-C on tumor cells could be correlated with recurrences, and larger primary tumors had more VEGF-C-positive vessels. CONCLUSIONS: The broad expression of VEGF C and VEGFR3 in HNSCC suggests involvement in tumor lymph angiogenesis and vascular angiogenesis, promoting tumor growth and propagation of cancer cells. This implies that inhibitors of lymph angiogenesis could become effective therapeutic options similar to classical angiogenesis inhibitors.

Adult↗

PubMed2003

Cytokine expression pattern in benign prostatic hyperplasia infiltrating T cells and impact of lymphocytic infiltration on cytokine mRNA profile in prostatic tissue.

The aim of the study is to characterize the type of immune response in benign prostatic hyperplasia (BPH) tissue. BPH tissue-derived T cells (n = 10) were isolated, activated (PMA + ionomycin), and analyzed for intracellular reactivity with anti-IFN-gamma and IL-2, -4, -5, -6, -10, and -13, as well as TNF-alpha and -beta by four-color flow cytometry. Lymphokine release was tested using Th1/Th2 cytokine bead arrays. The amount of IFN-gamma and IL-2, -4, -13, and TGF-beta mRNA expressed in normal prostate (n = 5) was compared with that in BPH tissue separated into segments with normal histology (n = 5), BPH histology with (n = 10) and without (n = 10) lymphocytic infiltration, and BPH nodules (n = 10). Expression of lymphokine receptors was analyzed by immunohistology, flow cytometry, and RT-PCR. We found that 28 +/- 18% of BPH T helper cells were IFN-gamma(+)/IL-4(-) Th1 cells, 10 +/- 2% were IFN-gamma(-)/IL-4(+) Th2, and 12 +/- 6% were IFN-gamma(+)/IL-4(+) Th0 cells. In relation, cytotoxic and double-negative BPH T lymphocytes showed a slight decrease in Th1 and Th0 in favor of Th2. In double-positive BPH T lymphocytes, the trend toward Th2 (35 +/- 15%) was significant (Th1: 12 +/- 7%; Th0: 5 +/- 4%). Lymphokine release upon stimulation was found in the case of IL-2, IL-5, IFN-gamma, and TNF-alpha > 4 microg; of IL-4 > 2 microg; and of IL-10 > 1 microg/ml. Expression of lymphokine mRNA in tissue was increased (2- to 10-fold) in infiltrated BPH specimens with and without BPH histology. The infiltrated BPH specimens with normal histology differed from those with BPH histology, most evident by the significant decrease in IFN-gamma and the increase in TGF-beta mRNA expression. Infiltrated BPH specimens with BPH histology expressed significantly more IFN-gamma (5-fold), IL-2 (10-fold), and IL-13 (2.8-fold) when compared with noninfiltrated BPH specimens. BPH nodules, however, showed the highest level of expression of IL-4 and IL-13, with only intermediate levels of IFN-gamma and very low levels of IL-2 mRNA. Immune response in histologically less transformed BPH specimens is primarily of type 1, whereas in chronically infiltrated nodular BPH and especially within BPH nodules, it is predominantly of type 0 or type 2.

Adult↗

PubMed2002

The arylhydrocarbon receptor (AhR), but not the AhR-nuclear translocator (ARNT), is increased in hearts of patients with cardiomyopathy.

The objective of this study was to investigate the expression of the arylhydrocarbon receptor (AhR) and its partner AhR-nuclear translocator (ARNT) in left ventricle specimens from explanted hearts from patients with cardiomyopathy (CMP). Explanted hearts from 16 patients with ischemic (n=9, age 63+/-12 years) and dilative (n=7, age 54+/-12 years) CMP, undergoing heart transplantation were examined. Healthy donor hearts from five accident victims served as controls. As these donors were of younger age (32+/-11 years), additionally, donor hearts from three older accident victims (age 48+/-15 years) without clinical symptoms but with signs of ventricular hyperthrophy (n=1) or atherosclerotic lesions (n=2) were included ("pathological controls"). Expression of AhR and ARNT was analyzed using semi-quantitative immunohistochemistry, and in selected samples, Western blot- and reverse-transcription polymerase chain reaction analysis were performed to confirm AhR and ARNT expression. Immunohistological analysis revealed weak to intermediate staining of anti-AhR in control, but weak to intense staining in CMP- and "pathologic control" specimens, indicating significantly increased AhR levels in the diseased heart. Moreover, in CMP specimens, the percentage of AhR-positive cells was strongly increased. Higher anti-AhR staining was also seen in two atherosclerotic "pathologic control" specimens. In all groups, the intensity of anti-ARNT staining was more pronounced than AhR staining, but significant differences or any age-related alterations were not observed. In conclusion, the increased cellular content of AhR in left ventricular specimens from CMP patients suggests a role for AhR in heart disease.

Adult↗

PubMed2002

Increased expression of lymphocyte-derived cytokines in benign hyperplastic prostate tissue, identification of the producing cell types, and effect of differentially expressed cytokines on stromal cell proliferation.

BACKGROUND: Benign prostatic hyperplasia (BPH) frequently exhibit infiltration of CD4 (+)/CD45RO (+) memory-T-lymphocytes. Expression and impact of lymphocyte-derived growth factors on prostatic stromal cell (PSC) growth were investigated. METHODS; Lymphokine synthesis in normal prostate tissues (n = 3), BPH-tissues (n = 13), BPH-derived T-cells (n = 6), BPH-derived epithelial cells (BPH-EC) (n = 5), normal prostate-derived (n = 3) and BPH-derived stromal cell lines (BPH-SC) (n = 6), and prostate cancer (CaP) lines (n = 3) was analyzed by RT-PCR and Southern-blotting. The effect of interleukin (IL)-2, -4, -7, and interferon-gamma (IFN-gamma) on normal and BPH-SC growth was investigated by (3)H-thymidine incorporation assays. RESULTS: All BPH-tissues and, to a lesser degree, normal prostates, expressed significant amounts of IFN-gamma mRNA. However, only BPH-tissues contained IL-2 and IL-4 mRNA (ratio: 10:13). BPH-T-cell lines were heterogeneous in composition and expressed significant amounts of IFN-gamma, IL-2, and IL-4 mRNA. Low level expression of these lymphokines was also observed in BPH-EC, CaP lines, and PSC lines. IL-2, -7 and IFN-gamma stimulated the proliferation of BPH-PSC lines but not that of normal PSC, while IL-4 inhibited BPH-PSC growth. CONCLUSIONS: Chronic inflammation may induce an increased growth pattern of fibromuscular tissue in BPH similar to that of wound healing.

Adolescent↗

PubMed2002

The picture of the prostatic lymphokine network is becoming increasingly complex.

The immunology of the prostate has recently developed into a new field of research in urology. Although we do not yet understand why the leukocyte population increases, we know that most resected prostate tissue shows signs of an inflammatory reaction. Different types of inflammation exist, and must be distinguished carefully according to distribution and location of leukocytes and histology of the surrounding tissue. This article reviews recent findings and discusses the complex mechanisms involved in the prostatic inflammatory response. The roles of estrogen, interleukin (IL)-6, IL-8, IL-15, and IL-17 are examined.

Journal Article↗