Georg Steiner

Differentiation between cell death modes using measurements of different soluble forms of extracellular cytokeratin 18.

Cytokeratins are released from carcinoma cells by unclear mechanisms and are commonly used serum tumor markers (TPA, TPS, and CYFRA 21–1). We here report that soluble cytokeratin-18 (CK18) is released from human carcinoma cells during cell death. During necrosis, the cytosolic pool of soluble CK18 was released, whereas apoptosis was associated with significant release of caspase-cleaved CK18 fragments. These results suggested that assessments of different forms of CK18 in patient sera could be used to examine cell death modes. Therefore, CK18 was measured in local venous blood collected during operation of patients with endometrial tumors. In most patient sera, caspase-cleaved fragments constituted a minor fraction of total CK18, suggesting that tumor apoptosis is not the main mechanism for generation of circulating CK18. Monitoring of different CK18 forms in peripheral blood during chemotherapy of prostate cancer patients showed individual differences in the patterns of release. Importantly, several examples were observed where the increase of apoptosis-specific caspase-cleaved CK18 fragments constituted only a minor fraction of the total increase. These results suggest that cell death of epithelially derived tumors can be assessed in patient serum and suggest that tumor apoptosis may not necessarily be the dominating death mode in many tumors in vivo.

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-γ and IL-2, -4, -5, -6, -10, and -13, as well as TNF-α and -β by four-color flow cytometry. Lymphokine release was tested using Th1/Th2 cytokine bead arrays. The amount of IFN-γ and IL-2, -4, -13, and TGF-β 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-γ+/IL-4− Th1 cells, 10 ± 2% were IFN-γ−/IL-4+ Th2, and 12 ± 6% were IFN-γ+/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-γ, and TNF-α > 4 μg; of IL-4 > 2 μg; and of IL-10 > 1 μg/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-γ and the increase in TGF-β mRNA expression. Infiltrated BPH specimens with BPH histology expressed significantly more IFN-γ (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-γ 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.

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

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.

Coexpression of gonadotropic hormones and their corresponding FSH- and LH/CG-receptors in the human prostate.

Benign prostatic hyperplasia (BPH) affects the majority of elderly men, and prostate cancer is the most common male cancer. Prostatic growth and function are thought to be regulated by steroid hormones, primarily androgens and estrogens, but nonandrogenic hormones must also be considered. The increasing evidence of para/autocrine functions of the gonadotropic glycoprotein‐hormones (GPH), their allocation to the superfamily of cystine‐knot growth factors, and luteinizing hormone (LH)/chorionic gonadotropin (CG)‐receptor (R) gene expression in nongonadal tissues led us to investigate intraprostatic GPH and GPH‐R gene expression.

Phenotype and Function of Peripheral and Prostatic Lymphocytes in Patients with Benign Prostatic Hyperplasia

In a previous report, we demonstrated intense lymphocytic infiltration of all benign prostatic hypertrophy (BPH) tissues analyzed in conjunction with HLA-DR expression on normally MHC-class-II-negative prostate epithelial cells. The composition of these infiltrates (70 to 80% CD3+ T-cells, but no granulocytes) resembles the situation seen in immune responses against altered self or self rather than against foreign antigens (infection). In the present study, phenotypic and functional immunoassays were used in order to investigate whether T-cells in BPH are indeed activated, and whether this activation is systemic or restricted locally to the prostate. Analysis of T-cell activation marker expression and proliferation requirements provided substantial evidence that these infiltrating lymphocytes, in contrast to their peripheral counterparts, are chronically activated. Since local accumulation of activated lymphocytes can cause tissue destruction, high concentrations of cytokines, and consequently tissue rebuilding, this process might contribute to the pathogenesis of BPH.

Role of bacteria in the development of kidney stones.

Currently, only struvite stones are regarded as deriving from bacteria. Recent work has suggested that calcium-based stones might also have an infectious origin. Nanobacteria, small intracelluar bacteria found in human kidney stones, are capable of forming a calcium phosphate shell, and thus could serve as crystallization centres for renal calculi formation. Until now, however, all trials performed to confirm the presence of nanobacteria in human calculi, serum or urine have failed. In a hyperoxaluric rat model, tissue-residing macrophages were able to remove interstitial crystals and thus may not be primarily engaged in defending against micro-organisms, if present.

High Expression of a CD38-Like Molecule in Normal Prostatic Epithelium and its Differential Loss in Benign and Malignant Disease

PURPOSE We characterize an undescribed antigen on prostatic epithelial cells, which is recognized by anti-CD38. MATERIALS AND METHODS Normal (3 cases), benign hyperplastic (10) and carcinomatous (10) prostatic tissues were analyzed using immunohistochemistry, immuno-electron microscopy and the Western blot test. RESULTS Comparison of prostatic and lymphatic CD38 antigen revealed identical bands at 45 kD. Electron microscopy demonstrated anti-CD38 reactivity on the cytoplasmic membrane and within the secretory vacuoles. Double labeling with anti-cytokeratin types 5/15 and 8/18 confirmed that basal and secretory normal prostatic epithelial cells express CD38. By contrast, a complete loss was found in malignant (52.8%), tumor-surrounding nonmalignant (16.3%) and benign prostatic hyperplasia derived glands (3.7%). CONCLUSIONS CD38 is a novel prostatic antigen. Its role in intracellular calcium mobilization may contribute to smooth muscle cell contraction and/or sperm motility.

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

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.

Automated data acquisition by confocal laser scanning microscopy and image analysis of triple stained immunofluorescent leukocytes in tissue

The aim of the study was to develop a novel system permitting automated analysis of multicolor immunofluorescence-staining of cells in solid tissues which would be comparable to the analytical capacity of flow cytometry. In the user friendly automated data acquisition and image processing system which was established, the software includes a set of pre-defined processing steps for improved object identification and can be either interactive or fully automatic. As with multi color flow cytometry, stained cells can be analyzed in a fully automated manner regardless of tissue type. The software organizes computerized sample movement, autofocus, laser readjustment, data capture and storage as well as calculation. Data are presented as histograms indicating the staining intensity and frequency of each antigen, and in dotplots with each channel plotted against the other. The calculated statistics give information about how many of the cells are single-, double- or triple-reactive and how intensely they react with the respective antibodies. Comparison of data generated by this automated fluorescence confocal laser scanning microscopy (AF-CLSM) with flow cytometry using triple stained peripheral blood lymphocytes revealed a highly significant correlation between the methods (P<0.001). A correlation was also observed when sections triple stained for anti-CD45, -CD3 and -CD8 were analyzed by AF-CLSM and the data were compared to visual/manual cell counting (P<0.01). The AF-CLSM system permits for the first time, fast (online) and reproducible analysis of immunofluorescence staining of an unlimited number of cells in tissue sections. The software, including a manual, is available for a small fee to cover the costs of printing and postage.

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.