Wnt/ß-Catenin Signalling and Its Cofactor BCL9L Have an Oncogenic Effect in Bladder Cancer Cells.

Bladder cancer (BC) is characterised by a high recurrence and progression rate. However, the molecular mechanisms of BC progression remain poorly understood. BCL9L, a coactivator of ß-catenin was mutated in the 5' and 3' untranslated regions (UTRs). We assessed the influence of UTRs mutations on BCL9L, and the role of BCL9L and Wnt/ß-catenin signalling in BC cells. UTR mutations were analysed by a luciferase reporter. BCL9L protein was assessed by immunohistochemistry in BC tissues. Cell proliferation was examined by crystal violet staining and by the spheroid model. Moreover, migration and invasion were analysed in real-time using the xCelligence RTCA system. The A > T mutation at 3' UTR of BCL9L reduces the luciferase reporter mRNA expression and activity. BCL9L is predominantly increased in dysplastic urothelial cells and muscle-invasive BC. Knockdown of BCL9L and inhibition of Wnt/ß-catenin signalling significantly repress the proliferation, migration and invasion of Cal29 and T24. In addition, BCL9L knockdown reduces mRNA level of Wnt/ß-catenin target genes in Cal29 but not in T24 cells. BCL9L and Wnt/ß-catenin signalling play an oncogenic role in bladder cancer cells and seems to be associated with BC progression. Nevertheless, the involvement of BCL9L in Wnt/ß-catenin signalling is cell-line specific.

Reduced IQGAP2 Promotes Bladder Cancer through Regulation of MAPK/ERK Pathway and Cytokines.

The progression of non-muscle-invasive bladder cancer (NMIBC) to muscle-invasive bladder cancer (MIBC) is a major challenge in urologic oncology. However, understanding of the molecular processes remains limited. The dysregulation of IQGAP2 is becoming increasingly evident in most tumor entities, and it plays a role in multiple oncogenic pathways, so we evaluated the role of IQGAP2 in bladder cancer. IQGAP2 was downregulated in tumors compared with normal urothelium tissues and cells. IQGAP2 effectively attenuated bladder cancer cell growth independently from apoptosis. Reduced IQGAP2 promoted EMT in bladder cancer cells via activation of the MAPK/ERK pathway. In addition, IQGAP2 might influence key cellular processes, such as proliferation and metastasis, through the regulation of cytokines. In conclusion, we suggest that IQGAP2 plays a tumor-suppressing role in bladder cancer, possibly via inhibiting the MAPK/ERK pathway and reducing cytokines.

Combined assessment of 3q26 amplification and promoter methylation in patients with high grade cervical lesions show age specific differences.

A considerable proportion of high grade cervical intraepithelial lesions (CIN2/3) are known to resolve on their own especially among young women. However, since reliable prognostic markers are still lacking, the diagnosis "CIN3" is still an indication for surgery which may result in overtreatment. It is conceivable that a combination of different, ideally independent molecular markers may provide more reliable results. In the present cross-sectional study two established triage markers, 3q26 amplification and a methylation signature, were evaluated in an age-dependent manner. The patient cohort comprised 60 patients with histologically confirmed CIN2/3 in two equally sized age groups (<30 years, ≥30 years). Cervical scrapes were analyzed by interphase fluorescence in situ hybridization for 3q26 amplification and methylation specific PCR (GynTect®) for six different genome regions. Both assays showed a significantly different pattern of test outcome independent of age (P = .001). Moreover, the combination of both assays differed significantly for double positive and double negative cases when comparing the two age groups: In patients <30 years there were clearly less cases with positive methylation signature and amplification of 3q26 as in women ≥30 years (23% vs 63%, Bonferroni adjusted P = .016). Of particular interest is the finding that double negative results were exclusive for the young age group (0% vs 27%, Bonferroni adjusted P = .020). Since regression of CIN2/3 characteristically occurs among young women it is tempting to speculate that a double negative test result could be prognostic for regression of CIN2/3. This will have to be investigated further in a prospective longitudinal intervention study.

4-miRNA Score Predicts the Individual Metastatic Risk of Renal Cell Carcinoma Patients.

BACKGROUND: In order to improve individual prognostication as well as stratification for adjuvant therapy in patients with clinically localized clear cell renal cell carcinoma (ccRCC), reliable prognostic biomarkers are urgently needed. In this study, microRNAs (miRNAs) have emerged as promising candidates. We investigated whether a combination of differently expressed miRNAs in primary tumors can predict the individual metastatic risk. METHODS: Using two prospectively collected biobanks of academic centers, 108 ccRCCs were selected, including 57 from patients with metastatic disease at diagnosis or during follow-up and 51 without evidence of metastases. Fourteen previously identified candidate miRNAs were tested in 20 representative formalin-fixed and paraffin embedded samples in order to select the best discriminators between metastatic and nonmetastatic ccRCC. These miRNAs were approved in 108 tumor samples. We evaluated the association of altered miRNA expression with the metastatic potential of tumors using quantitative polymerase chain reaction. A prognostic 4-miRNA model has been established using a random forest classifier. Cox regression analyses were performed for correlation of the miRNA model and clinicopathological parameters to metastasis-free and overall survival. RESULTS: Nine miRNAs indicated significant expression alterations in the small cohort. These miRNAs were validated in the whole cohort. The established 4-miRNA score (miR-30a-3p/-30c-5p/-139-5p/-144-5p) has been identified as a superior predictor for metastasis-free survival (hazard ratio 12.402; p = 7.0E-05) and overall survival (p = 1.1E-04) compared with clinicopathological parameters, and likewise in the Leibovich score subgroups. CONCLUSIONS: We identified a 4-miRNA model that was found to be superior to clinicopathological parameters in accurately predicting individual metastatic risk and can support patient selection for risk-stratified follow-up and adjuvant therapy studies.

Laparoscopic radical hysterectomy with transvaginal closure of vaginal cuff - a multicenter analysis.

OBJECTIVE: Laparoscopic/robotic radical hysterectomy has been historically considered oncologically equivalent to open radical hysterectomy for patients with early cervical cancer. However, a recent prospective randomized trial (Laparoscopic Approach to Cervical Cancer, LACC) has demonstrated significant inferiority of the minimally invasive approach. The aim of this study is to evaluate the oncologic outcomes of combined laparoscopic-vaginal radical hysterectomy. METHODS: Between August 1994 and December 2018, patients with invasive cervical cancer were treated using minimally-invasive surgery at the Universities of Jena, Charité Berlin (Campus CCM and CBF) and Cologne and Asklepios Clinic Hamburg. 389 patients with inclusion criteria identical to the LACC trial were identified. In contrast to the laparoscopic/robotic technique used in the LACC trial, all patients in our cohort underwent a combined transvaginal-laparoscopic approach without the use of any uterine manipulator. RESULTS: A total of 1952 consecutive patients with cervical cancer were included in the analysis. Initial International Federation of Gynecology and Obstetrics (FIGO) stage was IA1 lymphovascular space invasion (LVSI+), IA2 and IB1/IIA1 in 32 (8%), 43 (11%), and 314 (81%) patients, respectively, and histology was squamous cell in 263 (68%), adenocarcinoma in 117 (30%), and adenosquamous in 9 (2%) patients. Lymphovascular invasion was confirmed in 106 (27%) patients. The median number of lymph nodes was 24 (range 2-86). Lymph nodes were tumor-free in 379 (97%) patients. Following radical hysterectomy, 71 (18%) patients underwent adjuvant chemoradiation or radiation. After a median follow-up of 99 (range 1-288) months, the 3-, 4.5-, and 10-year disease-free survival rates were 96.8%, 95.8%, and 93.1 %, and the 3-, 4.5-, and 10-year overall survival rates were 98.5%, 97.8%, and 95.8%, respectively. Recurrence location was loco-regional in 50% of cases with recurrence (n=10). Interestingly, 9/20 recurrences occurred more than 39 months after surgery. CONCLUSION: The combined laparoscopic-vaginal technique for radical hysterectomy with avoidance of spillage and manipulation of tumor cells provides excellent oncologic outcome for patients with early cervical cancer. Our retrospective data suggest that laparoscopic-vaginal surgery may be oncologically safe and should be validated in further randomized trials.

A Promising New Approach for the Treatment of Inflammatory Pain: Transfer of Stem Cell-Derived Tyrosine Hydroxylase-Positive Cells.

OBJECTIVES: The appearance of endogenous tyrosine hydroxylase-positive cells (TH+ cells) in collagen-induced arthritis was associated with an anti-inflammatory effect. Here we investigated putative anti-inflammatory and antinociceptive effects of the transfer of induced, bone marrow stem cell-derived TH+ cells (iTH+ cells) on murine antigen-induced arthritis (AIA). METHODS: Bone marrow-derived stem cells were differentiated into iTH+ cells. These cells were transferred to mice immunized against methylated bovine serum albumin (mBSA) 2 days before AIA was induced by injection of mBSA into one knee joint. In AIA control mice and iTH+-treated mice the severity of AIA, pain-related behavior, humoral and cellular responses, and the invasion of macrophages into the dorsal root ganglia were assessed. RESULTS: The intravenous transfer of iTH+ cells before AIA induction did not cause a sustained suppression of AIA severity but significantly reduced inflammation-evoked pain-related behavior. The iTH+ cells used for transfer exhibited enormous production of interleukin-4. A major difference between AIA control mice and iTH+-treated AIA mice was a massive invasion of the dorsal root ganglia by iNOS-negative, arginine 1-positive macrophages corresponding to an M2 phenotype. The differences in other cellular and humoral immune parameters such as release of cytokines from stimulated lymphocytes between AIA control mice and iTH+-treated mice were small. CONCLUSIONS: The transfer of iTH+ cells may cause a long-lasting reduction of arthritis-induced pain even if it does not ameliorate inflammation. The invasion of M2 macrophages into the dorsal root ganglia is likely to be an important mechanism of antinociception.

Tribbles 2 mediates cisplatin sensitivity and DNA damage response in epithelial ovarian cancer.

Aim was to identify methylated genes with functional involvement in cisplatin-resistance development of epithelial ovarian cancer (EOC). Genome-wide analyses of hypermethylated CpG-islands in resistant cell lines in combination with qRT-PCR analyses were used to identify epigenetically silenced genes. EOC-Type-II tumors were analyzed for gene methylation and expression and TCGA data were interrogated in-silico. Experiments revealed 37 commonly hypermethylated genes in resistant cells of which Tribbles 2 (TRIB2) showed the most pronounced downregulation on mRNA level and was characterized further. TRIB2 showed a reactivation after 5'-Aza-Cytidine treatment in resistant cells but a cisplatin-dependent, prominent upregulation on mRNA level in sensitive cells, only. Re-expression in resistant A2780 cells increased the sensitivity to cisplatin and other DNA-damaging agents, but not taxanes. Contrary, knockdown of TRIB2 increased resistance to cisplatin in sensitive cells. TRIB2 was involved in the induction of a cisplatin-dependent cell cycle arrest and apoptosis by influencing p21 and survivin expression. An increased Pt-DNA-adduct formation in TRIB2 re-expressing cells did not translate in higher levels of dsDNA damage (yH2AX-foci). Thus, TRIB2 is potentially involved in the signal transduction from nucleotide excision repair of intrastrand cross links. Importantly, patient stratification of two homogenous cohorts of EOC-Type-II patients from Jena (n = 38) and the TCGA (n = 149) by TRIB2 mRNA expression consistently revealed a significantly decreased PFS for patients with low TRIB2 levels (log-rank p < 0.05). Tumors from resistant patients expressed the lowest levels of TRIB2. Downregulation of TRIB2 contributes to platin-resistance and TRIB2 expression should be validated as prognostic and predictive marker for EOC.

Gene expression analysis combined with functional genomics approach identifies ITIH5 as tumor suppressor gene in cervical carcinogenesis.

Progression from human papillomavirus-induced premalignant cervical intraepithelial neoplasia (CIN) to cervical cancer (CC) is driven by genetic and epigenetic events. Our microarray-based expression study has previously shown that inter-α-trypsin-inhibitor heavy chain 5 (ITIH5) mRNA levels in CCs were significantly lower than in high-grade precursor lesions (CIN3s). Therefore, we aimed to analyze in depth ITIH5 expression during cervical carcinogenesis in biopsy material and cell culture. Moreover, functional analyses were performed by ectopic expression of ITIH5 in different cell lines. We were able to confirm the validity of our microarray differential expression data by qPCR, demonstrating a clear ITIH5 downregulation in CC as compared with CIN2/3 or normal cervix. ITIH5 protein loss, evaluated by immunohistochemistry, was evident in 81% of CCs, whereas ITIH5 showed weak to moderate cytoplasmic staining in 91% of CIN2/3 cases. In addition, ITIH5 was strongly reduced or absent in seven CC cell lines and in three immortalized keratinocyte cell lines. Moreover, ITIH5 mRNA loss was associated with ITIH5 promoter methylation. ITIH5 expression could be restored in CC cell lines by pharmacological induction of DNA demethylation and histone acetylation. Functionally, ITIH5 overexpression significantly suppressed proliferation of SW756 cells and further resulted in a significant reduction of colony formation and cell migration in both CaSki and SW756 tumor models, but had no effect on invasion. Remarkably, ITIH5 overexpression did not influence the phenotype of HeLa cells. Taken together, ITIH5 gene silencing is a frequent event during disease progression, thereby providing evidence for a tumor suppressive role in cervical carcinogenesis.

Stress-resistant Translation of Cathepsin L mRNA in Breast Cancer Progression.

The cysteine protease cathepsin L (CTSL) is often thought to act as a tumor promoter by enhancing tumor progression and metastasis. This goes along with increased CTSL activity in various tumor entities; however, the mechanisms leading to high CTSL levels are incompletely understood. With the help of the polyoma middle T oncogene driven breast cancer mouse model expressing a human CTSL genomic transgene, we show that CTSL indeed promotes breast cancer metastasis to the lung. During tumor formation and progression high expression levels of CTSL are maintained by enduring translation of CTSL mRNA. Interestingly, human breast cancer specimens expressed the same pattern of 5' untranslated region (UTR) splice variants as the transgenic mice and the human cancer cell line MDA-MB 321. By polyribosome profiling of tumor tissues and human breast cancer cells, we observe an intrinsic resistance of CTSL to stress-induced shutdown of translation. This ability can be attributed to all 5' UTR variants of CTSL and is not dependent on a previously described internal ribosomal entry site motif. In conclusion, we provide in vivo functional evidence for overexpressed CTSL as a promoter of lung metastasis, whereas high CTSL levels are maintained during tumor progression due to stress-resistant mRNA translation.

Amelioration of experimental arthritis by stroke-induced immunosuppression is independent of Treg cell function.

OBJECTIVES: Clinical evidence suggests that neurological lesions can protect from arthritis. Acute cerebral ischaemia induces severe immunosuppression, resulting in enhanced susceptibility to infections. We aimed to determine if stroke-induced immunosuppression can ameliorate arthritis and to delineate the immunological mechanisms involved. METHODS: Unilateral cerebral ischaemia was induced in mice by occlusion of one middle cerebral artery (MCAO) at different time points after induction of G6PI-induced arthritis in mice. Clinical and histological signs of arthritis were assessed. Regulatory T cells were specifically depleted by injection of diphtheria toxin into transgenic DEREG mice. Immunological correlates of MCAO were determined by flow cytometry and serological methods. RESULTS: MCAO reduced the clinical and histological signs of arthritis significantly. To be effective, stroke had to be induced during the induction phase or the early clinical stage of arthritis. MCAO induced a global loss of leucocytes. Despite the reduced absolute number of lymphocytes, the functional differentiation of T helper cells into Th1/17 cells and the production of autoantibodies were unimpaired. Depletion experiments showed that regulatory T cells were dispensable for the protective effect of MCAO. CONCLUSIONS: MCAO ameliorates arthritis. The correlate of protection from arthritis is not the reduction of a particular pathogenic leucocyte subset or the preferential expansion or emergence of a protective cell population but the global reduction of leucocytes during arthritis.

MicroRNAs with prognostic potential for metastasis in clear cell renal cell carcinoma: a comparison of primary tumors and distant metastases.

BACKGROUND: MicroRNAs (miRNAs) are regulators of gene expression in tumor development and progression. However, their influence on metastasis of clear cell renal cell carcinoma (ccRCC) is less understood. To determine the role of miRNAs in metastatic progression, miRNA expression in primary ccRCC was compared to distant metastases. METHODS: Total RNA of 53 primary ccRCCs, 35 distant metastases from lung, bone, brain, and abdomen, as well as 17 normal kidney tissues was isolated from fresh frozen tissue and formalin-fixed paraffin-embedded (FFPE) samples. The miRNA microarrays were performed based on fresh frozen tissue. Results were validated by quantitative reverse transcription-polymerase chain reaction (qRT-PCR) on fresh frozen tissue and FFPE samples. Real-time cell analyses and transwell invasion assays were carried out after transient transfection of microRNA-30c (miR-30c) in cell line 786-O. RESULTS: There were 14 miRNAs differently expressed in metastatic primary ccRCC and distant metastases compared to non-metastatic primary tumors. A strong correlation of miRNAs to progression-free- and cancer-specific 5-year-survival was determined. Specific miRNAs were differently expressed in distant metastases compared to primary ccRCC. A miRNA signature distinguished lung metastases from other metastatic sites. Overexpression of miR-30c increased adherence and decreased migration and invasion in the ccRCC cell line. CONCLUSIONS: MiRNAs are deregulated in metastatic primary ccRCC and could be promising prognostic markers for an early prediction of metastasis. Alterations in miRNA expression characterize distant metastases of different metastatic sites. Furthermore, our study suggests a functional role of miR-30c in metastasis. The miRNAs could be a helpful tool for individual follow-up prediction and personalized therapy selection.

Histamine 4 receptor plays an important role in auto-antibody-induced arthritis.

Rheumatoid arthritis is a widespread autoimmune disease. In the murine K/B×N arthritis model, anti-GPI (anti-glucose 6-phosphate isomerase) antibodies lead to the formation of immune complexes. In the course of pathogenesis, these complexes activate the immune system and induce degranulation of mast cells, which are essential in this model of rheumatoid arthritis. A major mediator in mast cell granules is histamine, which is proven to be indispensable for joint inflammation in K/B×N mice. Histamine is known to bind to four different receptors (HR1-4), which have different expression profiles and exert a variety of different functions, including activation of the immune system. To analyze the contribution of the different histamine receptors, we employed histamine receptor antagonists (cetirizine, ranitidine, thioperamide and clozapine) blocking the receptors in C57BL/6 mice. Arthritis was induced via K/B×N serum injection. The results demonstrated that mice treated with all four histamine receptor antagonists simultaneously showed no arthritic symptoms, while positive control mice injected with K/B×N serum and vehicle suffered from severe symptoms. When antagonists specific for HR1-4 were applied individually, only the HR4 antagonist clozapine could protect mice from arthritis, reflecting its expression and functionality in the immune system.

Breast pathology after cryotherapy. Histological regression of breast cancer after cryotherapy.

A breast saving treatment is contemporary the preferred method of treatment with comparable results in comparing with mastectomy. In this study were evaluated the effects of cryotherapy by histological verification of changes in post treatment resection specimens. Fifty-three patients in age of 38-81 year with histologically confirmed breast cancer in needle biopsies were managed by cryotherapy between 1999 and 2007. The patients were operated between day 1 and 35 after cryotherapy. The histologic examination of operation materials showed in all cases at least partial tumor destruction. In general in 54.7% of all handled cases (29 patient) there was no residual tumor. In 6 cases (22.2%) from group 1 and in 23 cases (88.5%) of group 2 no tumor rest was found. Cryotherapy can lead to complete destruction of tumoral tissue. In our study all 29 (54.7%) of tumor-free cases after cryotherapy were those with cT1 stage. The experience of operator and the correct selection of appropriate patients (primarily taking the tumor size into account) play the most important role for achieving the best results.

Association between survival in patients with primary invasive breast cancer and computer aided MRI.

PURPOSE: To identify the potential of semi-quantitative enhancement-analysis in breast MRI to predict disease-related death in primary breast cancer patients. MATERIALS AND METHODS: The present study was planned and conducted according to international recommendations. All patients referred for pretherapeutic staging of primary breast cancer during 24 consecutive months were included into the study collective. They were followed-up by our multidisciplinary breast center. For semi-quantitative MRI-analysis dedicated CAD-software (computer assisted diagnosis) was used. Association between enhancement parameters and disease-related survival was investigated using Cox proportional-hazards -regression (CR). RESULTS: A total of 115 patients were eligible for CR analysis. Median follow-up time was 52 months. In 15 patients, disease-related death occurred. CR analysis identified four enhancement parameters as independent and significant (P < 0.001) predictors of the endpoint. Coefficients were "Initial enhancement" (B = 0.0166), "Time to peak-enhancement" (B = 1.0573), "Tumor volume" (B = 0.0175), and proportion of "tumor volume" showing "slow initial enhancement" followed by a "persistent" curve-type (B = -0.0586). CONCLUSION: This study demonstrates the significant relationship between semi-quantitative enhancement analysis in breast MRI and disease-related death of breast cancer patients. As results were extracted from a routine staging examination, MRI noninvasively provides not only diagnostic information but also outcome data at one step. Future studies should address the impact of these findings on patient management and therapeutic approach.

Mechanisms and functional consequences of liver failure substantially differ between endotoxaemia and faecal peritonitis in rats.

BACKGROUND: Many of the concepts describing molecular mechanisms of sepsis-induced liver failure are derived from endotoxin models. However, the biological significance of such models is questionable as the complexity of clinical sepsis and associated organ failure is only partially replicated. AIMS: Comparison of cytokine response, leucocyte recruitment, oxidative stress and markers of hepatic organ dysfunction in rat models of endotoxaemia or peritoneal contamination and infection (PCI). METHODS: Endotoxemia and polymicrobial sepsis were induced in rats by intraperitoneal injection of lipopolysaccharide (LPS) or stool suspension, respectively. RESULTS: Both insults produced clinical and laboratory signs of multiple organ dysfunction, including hepatic excretory dysfunction. However, TNF alpha, oxidative stress responses and the degree of cell death were significantly higher in endotoxaemia compared to PCI (e.g. serum TNF levels (pg/ml) at 1.5 h post-insult: sham 5 ± 1.4, LPS 1 mg/kg bw 2176.92 ± 373.78, sepsis below detection limit; P P < 0.05). Cholestasis was significantly more pronounced in polymicrobial sepsis whereas serum bilirubin in endotoxaemic animals did not differ from sham-operated controls (plasma levels of bilirubin (µmol/L) at 15 h after the insult: sham 7.1 ± 0.6, LPS 30 mg/kg 9.1 ± 0.6, sepsis 15.2 ± 1.3). CONCLUSIONS: Polymicrobial sepsis produces profound hepatocellular dysfunction in the absence of traditional cytokine-mediated mechanisms of cellular injury. This questions the central role of cytokines and the ensuing oxidative stress as key molecular events in mediating liver dysfunction.

The role of interleukin-1ß in arthritic pain: main involvement in thermal, but not mechanical, hyperalgesia in rat antigen-induced arthritis.

OBJECTIVE: Interleukin-1ß (IL-1ß) is considered a pronociceptive cytokine, but its role in the generation of arthritic pain is unknown. The aim of this study was to investigate the role of IL-1ß in arthritic pain and to explore the antinociceptive potential of the IL-1 receptor type I (IL-1RI) antagonist anakinra. METHODS: Antigen-induced arthritis (AIA) was induced in rats. Expression of IL-1RI in the dorsal root ganglia (DRGs) was determined, and the effects of anakinra on inflammation, pain-related behavior, and receptor expression were assessed. In cultured DRG neurons, the effect of IL-1ß on the expression of the transient receptor potential vanilloid 1 (TRPV-1) ion channel was examined. Recordings of action potentials from joint nociceptors were made after intraarticular injection of IL-1ß into the rat knee joints. RESULTS: AIA generated pronounced and persistent mechanical and thermal hyperalgesia, and IL-1RI expression in the lumbar DRGs was significantly up-regulated. Treatment with anakinra did not significantly reduce the severity of arthritis or mechanical hyperalgesia, but did result in a pronounced reduction in thermal hyperalgesia. In cultured DRG neurons, IL-1ß up-regulated the expression of TRPV-1, a major transduction molecule involved in thermal hyperalgesia. During AIA, anakinra treatment down-regulated the expression of TRPV-1, consistent with the pronounced reduction in thermal hyperalgesia. IL-1ß increased the mechanosensitivity of C-fibers of the joint, but reduced the mechanosensitivity of Aδ-fibers, thus having opposite effects on these mechanonociceptive nerve fibers. CONCLUSION: In the context of arthritic knee pain, IL-1ß and IL-1 receptors appear to be involved in thermal, rather than mechanical, hyperalgesia. Therefore, neutralization of IL-1ß may be mainly antinociceptive in disease states characterized by thermal hyperalgesia, but not in disease states mainly characterized by mechanical hyperalgesia.

Macrophage-derived, macrophage migration inhibitory factor (MIF) is necessary to induce disease in the K/BxN serum-induced model of arthritis.

Rheumatoid arthritis (RA) is characterized by the interaction of multiple mediators, among the most important of which are cytokines. In recent years, extensive studies demonstrate a pivotal role for one cytokine, macrophage migration inhibitory factor (MIF), in fundamental events in innate and adaptive immunity. MIF has now been demonstrated to be involved in the pathogenesis of many diseases, but in the case of RA the evidence for a role of MIF is very strong. MIF is abundantly expressed in the sera of RA patients and in RA synovial tissue correlating with disease activity. MIF-deficient mice were used to induce arthritis by serum transfer from K/BxN mice. K/BxN serum transfer arthritis was markedly attenuated in MIF(-) mice, with reduction in clinical index and histological severity as well as decrease in synovial cytokines. Macrophage transfers were done to investigate the specific role of macrophage-derived MIF. We show that adoptive transfer of wild-type macrophages into MIF(-) mice restores the sensitivity of MIF(-) mice to arthritis development, and this affect was associated with a restoration in serum IL-1ß and IL-6 production. These results indicate that MIF plays a critical role in inflammation and joint destruction in K/BxN serum-induced arthritis and that the systemic expression of MIF by a subpopulation of macrophages is necessary and sufficient for the full development of arthritis.

Synergistic antitumor effects of combined cathepsin B and cathepsin Z deficiencies on breast cancer progression and metastasis in mice.

The lysosomal cysteine proteases cathepsin B (Ctsb) and cathepsin Z (Ctsz, also called cathepsin X/P) have been implicated in cancer pathogenesis. Compensation of Ctsb by Ctsz in Ctsb (-/-) mice has been suggested. To further define the functional interplay of these proteases in the context of cancer, we generated Ctsz null mice, crossed them with Ctsb-deficient mice harboring a transgene for the mammary duct-specific expression of polyoma middle T oncogene (PymT), and analyzed the effects of single and combined Ctsb and Ctsz deficiencies on breast cancer progression. Single Ctsb deficiency resulted in delayed detection of first tumors and reduced tumor burden, whereas Ctsz-deficient mice had only a prolonged tumor-free period. However, only a trend toward reduced metastatic burden without statistical significance was detected in both single mutants. Strikingly, combined loss of Ctsb and Ctsz led to additive effects, resulting in significant and prominent delay of early and advanced tumor development, improved histopathologic tumor grading, as well as a 70% reduction in the number of lung metastases and an 80% reduction in the size of these metastases. We conclude that the double deficiency of Ctsb and Ctsz exerts significant synergistic anticancer effects, whereas the single deficiencies demonstrate at least partial reciprocal compensation.

Expression of the E-cadherin repressors Snail, Slug and Zeb1 in urothelial carcinoma of the urinary bladder: relation to stromal fibroblast activation and invasive behaviour of carcinoma cells.

Epithelial-mesenchymal transition (EMT) is regulated by interaction of carcinoma and stromal cells and crucial for progression of urinary bladder carcinoma (UBC). Therefore, the influence of activated fibroblasts on the expression of E-cadherin repressors as well as EMT and invasion in UBC was investigated. A correlative analysis of the immunohistochemical expression of fibroblast (ASMA, S100A4, FAP, SDF1, PDGFRß) and EMT (Snail, Slug, Zeb1, E-cadherin) markers was performed on 49 UBC cases of different stages. The impact of distinguishable growth factor stimulated fibroblasts on invasion, EMT, and E-cadherin repressor expression was investigated in an invasion model. In situ, invasiveness was significantly correlated to the loss of membranous E-cadherin (E-cad_m) and increased Snail, Slug, Zeb1 in tumour cells, as well as to increased ASMA, S100A4, and PDGFRß in stromal cells. A significant correlation to nodal metastasis could be evidenced for the loss of E-Cad_m, and for an increase in S100A4 and PDGFRß. Comparison of stromal and EMT markers revealed significant correlations of ASMA to Snail and Slug; of S100A4 to the loss of E-cad_m and Zeb1; and of PDGFRß to the loss of E-Cad_m, Slug and Zeb1. In vitro, TGFß1 induced myofibroblasts were the strongest attractants, while aFGF or TGFß1/aFGF stimulated fibroblasts were the most potent EMT inductors. As shown here for the first time, distinct sub-populations of fibroblasts are to various extents associated with EMT and tumour progression in UBC. These relevant findings might be the basis for the identification of new diagnostic markers and therapeutic targets selectively affecting tumour supporting CAF effects.

Differential vascular expression and regulation of oncofetal tenascin-C and fibronectin variants in renal cell carcinoma (RCC): implications for an individualized angiogenesis-related targeted drug delivery.

The study was aimed at determining the vascular expression of oncofetal fibronectin (oncfFn) and tenascin-C (oncfTn-C) isoforms in renal cell carcinoma (RCC) and its metastases which are well-known targets for antibody-based pharmacodelivery. Furthermore, the influence of tumour cells on endothelial mRNA expression of these molecules was investigated. Evaluation of vascular ED-A(+) and ED-B(+) Fn as well as A1(+) and C(+) Tn-C was performed after immunofluorescence double and triple staining using human recombinant antibodies on clear cell, papillary and chromophobe primary RCC and metastases. The influence of hypoxic RCC-conditioned medium on oncfFn and oncfTn-C mRNA expression was examined in human umbilical vein endothelial cells (HUVEC) by real time RT-PCR. There are RCC subtype specific expression profiles of vascular oncfFn and oncfTn-C and corresponding patterns when comparing primary tumours and metastases. Within one tumour, there are different vessel populations with regard to the incorporation of oncfTn-C and oncfFn into the vessel wall. In vitro tumour-derived soluble mediators induce an up regulation of oncfTn-C and oncfFn mRNA in HUVEC which can be blocked by Avastin(®). Vascular expression of oncFn and oncTn-C variants depends on RCC subtype and may reflect an individual tumour stroma interaction or different stages of vessel development. Therefore, oncFn or oncTn-C variants can be suggested as molecular targets for individualized antibody based therapy strategies in RCC. Tumour-derived VEGF could be shown to regulate target expression.