Roman A. Blaheta

Robotic-assisted laparoscopic radical cystectomy and intra-abdominal formation of an orthotopic ileal neobladder.

PURPOSE To describe our technique of robotic-assisted laparoscopic radical cystectomy and intra-abdominal formation of an orthotopic neobladder (Hautmann) for treatment of transitional cell carcinoma of the bladder. METHODS We describe our surgical technique in the worldwide first attempt to perform a robotic-assisted laparoscopic radical cystectomy and completely intra-abdominal formation of an orthotopic neobladder. The DaVinci System (Intuitive Surgical, Mountain View, CA, USA) was utilized to perform the procedure. RESULTS Utilizing the DaVinci System the operation could be performed without any complications. Operating time was 8.5 hours, blood loss was 200 ml. The oncologic as well as the functional result of the reservoir were excellent. DISCUSSION We here demonstrated that sophisticated laparoscopic procedures like the intra-abdominal formation of an orthotopic neobladder are accomplishable with robotic assistance.

Valproate and valproate-analogues: potent tools to fight against cancer.

The branched-chain fatty acid valproic acid (VPA) is the most commonly used antiepileptic drug for treating generalized epilepsy. Although originally considered to be of low toxicity, VPA has proved to possess considerable teratogenic potential when applied to the pregnant epileptic women. During the last few years, it has become evident that some of the mechanisms which account for the malformations produced by VPA are related to distinct anti-tumor properties of this compound. This intriguing discovery opens novel aspects for the treatment of tumor patients. In the present review, the biological, biochemical and pharmacological properties of VPA are discussed. Analyses of structure-activity relationships can provide the necessary insight into the molecular structures responsible for the anti-tumor effects.

Mycophenolate mofetil modulates adhesion receptors of the beta1 integrin family on tumor cells: impact on tumor recurrence and malignancy

BackgroundTumor development remains one of the major obstacles following organ transplantation. Immunosuppressive drugs such as cyclosporine and tacrolimus directly contribute to enhanced malignancy, whereas the influence of the novel compound mycophenolate mofetil (MMF) on tumor cell dissemination has not been explored. We therefore investigated the adhesion capacity of colon, pancreas, prostate and kidney carcinoma cell lines to endothelium, as well as their beta1 integrin expression profile before and after MMF treatment.MethodsTumor cell adhesion to endothelial cell monolayers was evaluated in the presence of 0.1 and 1 μM MMF and compared to unstimulated controls. beta1 integrin analysis included alpha1beta1 (CD49a), alpha2beta1 (CD49b), alpha3beta1 (CD49c), alpha4beta1 (CD49d), alpha5beta1 (CD49e), and alpha6beta1 (CD49f) receptors, and was carried out by reverse transcriptase-polymerase chain reaction, confocal microscopy and flow cytometry.ResultsAdhesion of the colon carcinoma cell line HT-29 was strongly reduced in the presence of 0.1 μM MMF. This effect was accompanied by down-regulation of alpha3beta1 and alpha6beta1 surface expression and of alpha3beta1 and alpha6beta1 coding mRNA. Adhesion of the prostate tumor cell line DU-145 was blocked dose-dependently by MMF. In contrast to MMFs effects on HT-29 cells, MMF dose-dependently up-regulated alpha1beta1, alpha2beta1, alpha3beta1, and alpha5beta1 on DU-145 tumor cell membranes.ConclusionWe conclude that MMF possesses distinct anti-tumoral properties, particularly in colon and prostate carcinoma cells. Adhesion blockage of HT-29 cells was due to the loss of alpha3beta1 and alpha6beta1 surface expression, which might contribute to a reduced invasive behaviour of this tumor entity. The enhancement of integrin beta1 subtypes observed in DU-145 cells possibly causes re-differentiation towards a low-invasive phenotype.

Decreased neutrophil adhesion to human cytomegalovirus-infected retinal pigment epithelial cells is mediated by virus-induced up-regulation of Fas ligand independent of neutrophil apoptosis.

Human CMV (HCMV) retinitis frequently leads to blindness in iatrogenically immunosuppressed patients and in the end stage of AIDS. Despite the general proinflammatory potential of HCMV, virus infection is associated with a rather mild cellular inflammatory response in the retina. To investigate this phenomenon, the influence of HCMV (strains AD169 or Hi91) infection on C-X-C chemokine secretion, ICAM-1 expression, and neutrophil recruitment in cultured human retinal pigment epithelial (RPE) cells was studied. Supernatants from infected cultures contained enhanced levels of IL-8 and melanoma growth-stimulating activity/Gro α and induced neutrophil chemotaxis compared with supernatants from uninfected RPE cells. Despite HCMV-induced ICAM-1 expression on RPE cells, binding of activated neutrophils to HCMV-infected RPE cells and subsequent transepithelial penetration were significantly reduced. Reduced neutrophil adhesion to infected RPE cells correlated with HCMV-induced up-regulation of constitutive Fas ligand (FasL) expression. Functional blocking of FasL on RPE cells with the neutralizing mAbs NOK-1 and NOK-2 or of the Fas receptor on neutrophils with mAbB-D29 prevented the HCMV-induced impairment of neutrophil/RPE interactions. Fas-FasL-dependent impairment of neutrophil binding had occurred by 10 min after neutrophil/RPE coculture without apoptotic signs. Neutrophil apoptosis was first detected after 4 h. Treatment of neutrophils with a specific inhibitor of caspase-8 suppressed apoptosis, whereas it did not prevent impaired neutrophil binding to infected RPE. The current results suggest a novel role for FasL in the RPE regulation of neutrophil binding. This may be an important feature of virus escape mechanisms and for sustaining the immune-privileged character of the retina during HCMV ocular infection.

Cytomegalovirus- and interferon-related effects on human endothelial cells : cytomegalovirus infection reduces upregulation of HLA class II antigen expression after treatment with interferon-γ

Cultured human umbilical vein endothelial cells (HUVEs) were infected with human cytomegalovirus (HCMV) strain AD169. Up to 50% HUVEs proved to be positive for HCMV early nuclear antigens 24 hours after inoculation with virus. Following infection kinetics of surface expression of HLA class I and II, intercellular adhesion molecule (ICAM-1) and endothelial lymphocyte adhesion molecule (ELAM-1) on HUVEs were investigated by means of flow cytometry. A slight increase in HLA class I expression was observed, whereas expression of HLA class II (DR, DP, DQ) antigens was not induced by infection with HCMV. Furthermore, when compared with uninfected cells treated with interferon-gamma (IFN-gamma), reduced enhancement of HLA-DR expression was conspicuous in HCMV-infected cells treated with IFN-gamma. There is evidence that only a portion of HUVE is affected in its ability to upregulate HLA class II antigens. While expression of ICAM-1 was found to be enhanced between 8 and 20 hours after infection with a maximum at 12 hours after infection, no modulation of ELAM-1 was seen.

A rapid non-radioactive fluorescence assay for the measurement of both cell number and proliferation

Measuring the incorporation of radioactive thymidine into the cell nucleus gives important information as to cell activation and proliferation. In this study the DNA-intercalating fluorochromes, Hoechst 33342 and Hoechst 33258, were tested as an alternative to the classical [3H]thymidine assay. Mitogen and alloantigen stimulated lymphocytes as well as FK 506 and CsA inhibited lymphocytes were treated with the two dyes, and the cell number and proliferation rates by means of measured fluorescence values. Of these tested fluorochromes H33342 appears to be an appropriate alternative to the [3H]thymidine assay. It mirrors the cell number in a fast and convenient manner without any pretreatment of the cell suspension which can remain in the culture plates. The complete assay procedure including data analysis can be performed rapidly and the standard deviations are small. This dye may also prove to be of value in other assay procedures, e.g., adhesion experiments.

Development of resistance to vincristine and doxorubicin in neuroblastoma alters malignant properties and induces additional karyotype changes: a preclinical model.

Cytotoxic drug treatment of neuroblastoma often leads to the development of drug resistance and may be associated with increased malignancy. To study the effects of long‐term cytotoxic treatment on malignant properties of tumor cells, we established 2 neuroblastoma cell sublines resistant to vincristine (VCR) and doxorubicin (DOX). Both established cell lines (UKF‐NB‐2rVCR20 and UKF‐NB‐2rDOX100) were highly resistant to VCR, DOX and vice‐versa but retained their sensitivity to cisplatin. UKF‐NB‐2rVCR20 and UKF‐NB‐2rDOX100 expressed significant amounts of P‐glycoprotein, while parental cells were P‐glycoprotein negative. GD2 expression was upregulated, whereas NCAM expression was decreased in both resistant cells. Spectral karyotype (SKY) analysis revealed complex aberrant karyotypes in all cell lines and additional acquired karyotype changes in both resistant cells. All cell lines harbored high levels of N‐myc amplification. Compared to parental cells, UKF‐NB‐2rVCR20 and UKF‐NB‐2rDOX100 exhibited more than 2‐fold increase in clonal growth in vitro, accelerated adhesion and transendothelial penetration and higher tumorigenicity in vivo. We conclude that development of drug resistance and acquisition of certain karyotypic alterations is associated with an increase of additional malignant properties that may contribute to the poor prognosis in advanced forms of NB. The 2 novel neuroblastoma cell sublines also provide useful models for the study of drug resistance in aggressive forms of neuroblastoma.

Interleukin-4, interleukin-10, and interleukin-1-receptor antagonist but not transforming growth factor-β induce ramification and reduce adhesion molecule expression of rat microglial cells

The activity of microglial cells is strictly controlled in order to maintain central nervous system (CNS) immune privilege. We hypothesized that several immunomodulatory factors present in the CNS parenchyma, i.e., the Th2‐derived cytokines interleukin (IL)‐4 and IL‐10, interleukin‐1‐receptor‐antagonist (IL‐1‐ra), or transforming growth factor (TGF)‐β can modulate microglial morphology and functions. Microglial cells were incubated with IL‐4, IL‐10, IL‐1‐ra, TGF‐β, or with astrocyte conditioned media (ACM) and were analyzed for morphological changes, expression of intercellular adhesion molecule (ICAM)‐1, and secretion of IL‐1β or tumor necrosis factor (TNF)‐α. Whereas untreated controls showed an amoeboid morphology both Th2‐derived cytokines, IL‐1‐ra, and ACM induced a morphological transformation to the ramified phenotype. In contrast, TGF‐β‐treated microglial cells showed an amoeboid morphology. Even combined with the neutralizing antibodies against IL‐4, IL‐10, or TGF‐β ACM induced microglial ramification. Furthermore, ACM did not contain relevant amounts of IL‐4 and IL‐10, as measured by enzyme‐linked immunosorbent assay (ELISA). Flow cytometry showed that lipopolysaccharide (LPS)‐induced ICAM‐1‐expression on microglial cells was strongly suppressed by ACM, significantly modulated by IL‐4, IL‐10, or IL‐1‐ra, but not influenced by TGF‐β. The LPS‐induced secretion of IL‐1β and TNF‐α was only reduced after application of ACM, whereas IL‐4 or IL‐10 did not inhibit IL‐1β‐ or TNF‐α secretion. TGF‐β enhanced IL‐1β‐ but not TNF‐α secretion. In summary, we demonstrate that IL‐4, IL‐10, and IL‐1‐ra induce microglial ramification and reduce ICAM‐1‐expression, whereas the secretion of proinflammatory cytokines is not prevented. TGF‐β has no modulating effects. Importantly, unidentified astrocytic factors that are not identical with IL‐4, IL‐10, or TGF‐β possess strong immunomodulatory properties.

Inhibition of endothelial receptor expression and of T-cell ligand activity by mycophenolate mofetil

The novel immunosuppressive drug mycophenolate mofetil (CellCept, MMF) blocks DNA-synthesis by the inhibition of the enzyme inosine monophosphate dehydrogenase (IMDH). IMDH is also involved in the synthesis of adhesion receptors which are known to play an important role in the regulation of cell-cell contacts. Therefore, application of MMF might lead to a reduction of cellular infiltrates in the course of transplant rejection. To evaluate the therapeutic value of MMF, we investigated to what extent MMF blocks T-lymphocyte infiltration in vitro with regard to (a) adhesion to endothelial cells, (b) horizontal migration along these cells and (c) penetration through the endothelial cells. The results demonstrated a strong inhibition of both CD4+ and CD8+ T-cell adhesion and penetration by MMF. The ID50 value for CD4+ T-cell adhesion was calculated to be 0.03 microM and the ID50 value for CD4+ T-cell penetration 1.21 microM. MMF did not significantly influence the horizontal migration of T-lymphocytes along the human vascular endothelial cell (HUVEC) borders. FACS-analysis revealed a diminished E-selectin and P-selectin expression on endothelial cell membranes in the presence of MMF. Although MMF did not interfere with the synthesis of T-cell adhesion ligands, the binding activity of lymphocytic leucocyte function associated antigen 1 (LFA-1), very late antigen 4 (VLA-4) and PSGL-1 (P-selectin glycoprotein ligand 1) to immobilized intercellular adhesion molecule 1 (ICAM-1), vascular cell adhesion molecule 1 (VCAM-1) and P-selectin was impaired. Moreover, MMF prevented VLA-4 and PSGL-1 receptor accumulation on the membranes of T-cell pseudopodia. It can be concluded that MMF possesses potent infiltration blocking properties. MMF evoked down-regulation of specific endothelial membrane molecules and the loss of protein localization in the lymphocyte protrusions might be predominantly responsible for the observed blockade of cell adhesion and penetration.

Expression of Foxp3 in Colorectal Cancer but Not in Treg Cells Correlates with Disease Progression in Patients with Colorectal Cancer

Background Regulatory T cells (Treg) expressing the transcription factor forkhead-box protein P3 (Foxp3) have been identified to counteract anti-tumor immune responses during tumor progression. Besides, Foxp3 presentation by cancer cells itself may also allow them to evade from effector T-cell responses, resulting in a survival benefit of the tumor. For colorectal cancer (CRC) the clinical relevance of Foxp3 has not been evaluated in detail. Therefore the aim of this study was to study its impact in colorectal cancer (CRC). Methods and Findings Gene and protein analysis of tumor tissues from patients with CRC was performed to quantify the expression of Foxp3 in tumor infiltrating Treg and colon cancer cells. The results were correlated with clinicopathological parameters and patients overall survival. Serial morphological analysis demonstrated Foxp3 to be expressed in cancer cells. High Foxp3 expression of the cancer cells was associated with poor prognosis compared to patients with low Foxp3 expression. In contrast, low and high Foxp3 level in tumor infiltrating Treg cells demonstrated no significant differences in overall patient survival. Conclusions Our findings strongly suggest that Foxp3 expression mediated by cancer cells rather than by Treg cells contribute to disease progression.