The Androgen Hormone-Induced Increase in Androgen Receptor Protein Expression Is Caused by the Autoinduction of the Androgen Receptor Translational Activity.

The androgen receptor (AR) plays a central role in prostate, muscle, bone and adipose tissue. Moreover, dysregulated AR activity is a driving force in prostate cancer (PCa) initiation and progression. Consequently, antagonizing AR signalling cascades via antiandrogenic therapy is a crucial treatment option in PCa management. Besides, very high androgen levels also inhibit PCa cells' growth, so this effect could also be applied in PCa therapy. However, on the molecular and cellular level, these mechanisms have hardly been investigated so far. Therefore, the present study describes the effects of varying androgen concentrations on the viability of PCa cells as well as localization, transactivation, and protein stability of the AR. For this purpose, cell viability was determined via WST1 assay. Alterations in AR transactivity were detected by qPCR analysis of AR target genes. A fluorescent AR fusion protein was used to analyse AR localization microscopically. Changes in AR protein expression were detected by Western blot. Our results showed that high androgen concentrations reduce the cell viability in LNCaP and C4-2 cell lines. In addition, androgens have been reported to increase AR transactivity, AR localization, and AR protein expression levels. However, high androgen levels did not reduce these parameters. Furthermore, this study revealed an androgen-induced increase in AR protein synthesis. In conclusion, inhibitory effects on cell viability by high androgen levels are due to AR downstream signalling or non-genomic AR activity. Moreover, hormonal activation of the AR leads to a self-induced stabilization of the receptor, resulting in increased AR activity. Therefore, in clinical use, a therapeutic reduction in androgen levels represents a clinical target and would lead to a decrease in AR activity and, thus, AR-driven PCa progression.

Impact of Androgen Receptor Activity on Prostate-Specific Membrane Antigen Expression in Prostate Cancer Cells.

Prostate-specific membrane antigen (PSMA) is an essential molecular regulator of prostate cancer (PCa) progression coded by the FOLH1 gene. The PSMA protein has become an important factor in metastatic PCa diagnosis and radioligand therapy. However, low PSMA expression is suggested to be a resistance mechanism to PSMA-based imaging and therapy. Clinical studies revealed that androgen receptor (AR) inhibition increases PSMA expression. The mechanism has not yet been elucidated. Therefore, this study investigated the effect of activation and inhibition of androgen signaling on PSMA expression levels in vitro and compared these findings with PSMA levels in PCa patients receiving systemic therapy. To this end, LAPC4, LNCaP, and C4-2 PCa cells were treated with various concentrations of the synthetic androgen R1881 and antiandrogens. Changes in FOLH1 mRNA were determined using qPCR. Open access databases were used for ChIP-Seq and tissue expression analysis. Changes in PSMA protein were determined using western blot. For PSMA staining in patients' specimens, immunohistochemistry (IHC) was performed. Results revealed that treatment with the synthetic androgen R1881 led to decreased FOLH1 mRNA and PSMA protein. This effect was partially reversed by antiandrogen treatment. However, AR ChIP-Seq analysis revealed no canonical AR binding sites in the regulatory elements of the FOLH1 gene. IHC analysis indicated that androgen deprivation only resulted in increased PSMA expression in patients with low PSMA levels. The data demonstrate that AR activation and inhibition affects PSMA protein levels via a possible non-canonical mechanism. Moreover, analysis of PCa tissue reveals that low PSMA expression rates may be mandatory to increase PSMA by androgen deprivation.

The heat shock protein 70 inhibitor VER155008 suppresses the expression of HSP27, HOP and HSP90ß and the androgen receptor, induces apoptosis, and attenuates prostate cancer cell growth.

Heat shock proteins (HSPs) are molecular chaperones that play a pivotal role in correct folding, stabilization and intracellular transport of many client proteins including those involved in oncogenesis. HSP70, which is frequently overexpressed in prostate cancer (PCa), has been shown to critically contribute to tumor cell survival, and might therefore represent a potential therapeutic target. We treated both the androgen receptor (AR)-positive LNCaP and the AR-negative PC-3 cell lines with the pharmacologic HSP70 inhibitor VER155008. Although we observed antiproliferative effects and induction of apoptosis upon HSP70 inhibition, the apoptotic effect was more pronounced in AR-positive LNCaP cells. In addition, VER155008 treatment induced G1 cell cycle arrest in LNCaP cells and decreased AR expression. Further analysis of the HSP system by Western blot analysis revealed that expression of HSP27, HOP and HSP90ß was significantly inhibited by VER155008 treatment, whereas the HSP40, HSP60, and HSP90α expression remained unchanged. Taken together, VER155008 might serve as a novel therapeutic option in PCa patients independent of the AR expression status.

The formyl peptide receptor agonist Ac2-26 alleviates neuroinflammation in a mouse model of pneumococcal meningitis.

BACKGROUND: Bacterial meningitis is still a cause of severe neurological disability. The brain is protected from penetrating pathogens by the blood-brain barrier and the innate immune system. The invading pathogens are recognized by pattern recognition receptors including the G-protein-coupled formyl peptide receptors (FPRs), which are expressed by immune cells of the central nervous system. FPRs show a broad spectrum of ligands, including pro- and anti-inflammatory ones. Here, we investigated the effects of the annexin A1 mimetic peptide Ac2-26 in a mouse model of pneumococcal meningitis. METHODS: Wildtype (WT) and Fpr1- and Fpr2-deficient mice were intrathecally infected with Streptococcus pneumoniae D39 (type 2). Subsequently, the different mice groups were treated by intraperitoneal injections of Ac2-26 (1 mg/kg body weight) 2, 8, and 24 h post-infection. The extent of inflammation was analyzed in various brain regions by means of immunohistochemistry and real-time reverse transcription polymerase chain reaction (RT-PCR) 30 h post-infection. RESULTS: Ac2-26-treated WT mice showed less severe neutrophil infiltration, paralleled by a reduced induction of pro-inflammatory glial cell responses in the hippocampal formation and cortex. While meningitis was ameliorated in Ac2-26-treated Fpr1-deficient mice, this protective effect was not observed in Fpr2-deficient mice. Irrespective of Ac2-26 treatment, inflammation was more severe in Fpr2-deficient compared to Fpr1-deficient mice. CONCLUSIONS: In summary, this study demonstrates anti-inflammatory properties of Ac2-26 in a model of bacterial meningitis, which are mediated via FPR2, but not FPR1. Ac2-26 and other FPR2 modulators might be promising targets for the development of novel therapies for Streptococcus pneumoniae-induced meningitis.

Dirty deeds done dirt cheap: sensitization of prostate cancer cells to abiraterone treatment using hydroxylated polychlorinated biphenyls.

Effective targeting of androgen biosynthesis by the 17α-hydroxylase/17,20-lyase inhibitor abiraterone prolongs survival in a variety of prostate cancer patients. However, resistance to abiraterone treatment occurs frequently and the development of new drugs supporting or complementing abiraterone therapy is urgently needed. We recently reported antiproliferative and proapoptotic effects of hydroxylated polychlorinated biphenyls (PCBs) on various blood cell lines in vitro. Here we report the biological evaluation of the PCB28 derived OH-metabolites 3-OHCB28 or 3'-OHCB28 in prostate cancer cells. Depending on concentration, both metabolites inhibit the growth of PC3 cells, a cell line representing later stages of advanced prostate cancer. In addition 3'-OHCB28 reduced the necessary concentration of abiraterone required for the inhibition of PC3 cells by a factor of 4. Western blot analysis of cytoprotective heatshock proteins (HSP) implicated a significant reduction of HSP27 expression by 3'-OHCB28 in PC3 cells. Given the known HSP27 suppressive role of abiraterone, our results therefore suggest, that that the pharmacological interaction between abiraterone and 3'-OHCB28 in PC3 cells could be produced by the combined effect of both substances on the expression of HSPs, especially the expression of HSP27. Including the known dose response linkages and pharmacokinetic characteristics of the OH-metabolites described here, we conclude, that the use of hydroxylated PCBs can be supportive for the anti-proliferative treatment of prostate cancer and merits further investigation.

Cytokine adsorption as a promising option for septic shock and multiple organ failure due to Candida infection and decompensated type 1 diabetes mellitus.

Type 1 diabetes mellitus (T1DM) represents one of the most common chronic diseases in childhood. It is associated with high morbidity and mortality rates due to metabolic dysregulation, immunosuppressive effects, and a predisposition to fungal infections. Candidiasis is a severe infection and its prevalence has increased throughout the last decades. We report the case of a 19-year-old female patient admitted to our intensive care unit with T1DM and Candida infection associated with severe metabolic acidosis. In the absence of response to high dose catecholamine cardiovascular therapy and the presence of severe metabolic acidosis, a CytoSorb cartridge was implemented into the extracorporeal dialysis circuit resulting in a stabilization of hemodynamics accompanied by a tremendous decrease in vasopressor requirements, control of the hyperinflammatory response, as well as a resolution of metabolic acidosis and regeneration of renal function. Treatment with CytoSorb was safe and feasible without technical problems. Notably, this is the first case description reporting on the effects of CytoSorb in a patient with Candida infection as part of T1DM.

Cold Atmospheric Plasma Treatment of Chondrosarcoma Cells Affects Proliferation and Cell Membrane Permeability.

Chondrosarcoma is the second most common malign bone tumor in adults. Surgical resection of the tumor is recommended because of its resistance to clinical treatment such as chemotherapy and radiation therapy. Thus, the prognosis for patients mainly depends on sufficient surgical resection. Due to this, research on alternative therapies is needed. Cold atmospheric plasma (CAP) is an ionized gas that contains various reactive species. Previous studies have shown an anti-oncogenic potential of CAP on different cancer cell types. The current study examined the effects of treatment with CAP on two chondrosarcoma cell lines (CAL-78, SW1353). Through proliferation assay, the cell growth after CAP-treatment was determined. A strong antiproliferative effect for both cell lines was detected. By fluorescein diacetate (FDA) assay and ATP release assay, alterations in the cell membrane and associated translocation of low molecular weight particles through the cytoplasmic membrane were observed. In supernatant, the non-membrane-permeable FDA and endogenously synthesized ATP detected suggest an increased membrane permeability after CAP treatment. Similar results were shown by the dextran-uptake assay. Furthermore, fluorescence microscopic G-/F-actin assay was performed. G- and F-actin were selectively dyed, and the ratio was measured. The presented results indicate CAP-induced changes in cell membrane function and possible alterations in actin-cytoskeleton, which may contribute to the antiproliferative effects of CAP.

Inhibition of Angiogenesis by Treatment with Cold Atmospheric Plasma as a Promising Therapeutic Approach in Oncology.

BACKGROUND: Cold atmospheric plasma (CAP) is increasingly used in the field of oncology. Many of the mechanisms of action of CAP, such as inhibiting proliferation, DNA breakage, or the destruction of cell membrane integrity, have been investigated in many different types of tumors. In this regard, data are available from both in vivo and in vitro studies. Not only the direct treatment of a tumor but also the influence on its blood supply play a decisive role in the success of the therapy and the patient's further prognosis. Whether the CAP influences this process is unknown, and the first indications in this regard are addressed in this study. METHODS: Two different devices, kINPen and MiniJet, were used as CAP sources. Human endothelial cell line HDMEC were treated directly and indirectly with CAP, and growth kinetics were performed. To indicate apoptotic processes, caspase-3/7 assay and TUNEL assay were used. The influence of CAP on cellular metabolism was examined using the MTT and glucose assay. After CAP exposure, tube formation assay was performed to examine the capillary tube formation abilities of HDMEC and their migration was messured in separate assays. To investigate in a possible mutagenic effect of CAP treatment, a hypoxanthine-guanine-phosphoribosyl-transferase assay with non malignant cell (CCL-93) line was performed. RESULTS: The direct CAP treatment of the HDMEC showed a robust growth-inhibiting effect, but the indirect one did not. The MMT assay showed an apparent reduction in cell metabolism in the first 24 h after CAP treatment, which appeared to normalize 48 h and 72 h after CAP application. These results were also confirmed by the glucose assay. The caspase 3/7 assay and TUNEL assay showed a significant increase in apoptotic processes in the HDMEC after CAP treatment. These results were independent of the CAP device. Both the migration and tube formation of HDMEC were significant inhibited after CAP-treatment. No malignant effects could be demonstrated by the CAP treatment on a non-malignant cell line.

An Innovative Therapeutic Option for the Treatment of Skeletal Sarcomas: Elimination of Osteo- and Ewing's Sarcoma Cells Using Physical Gas Plasma.

Osteosarcoma and Ewing's sarcoma are the most common malignant bone tumors. Conventional therapies such as polychemotherapy, local surgery, and radiotherapy improve the clinical outcome for patients. However, they are accompanied by acute and chronic side effects that affect the quality of life of patients, motivating novel research lines on therapeutic options for the treatment of sarcomas. Previous experimental work with physical plasma operated at body temperature (cold atmospheric plasma, CAP) demonstrated anti-oncogenic effects on different cancer cell types. This study investigated the anti-cancer effect of CAP on two bone sarcoma entities, osteosarcoma and Ewing's sarcoma, which were represented by four cell lines (U2-OS, MNNG/HOS, A673, and RD-ES). A time-dependent anti-proliferative effect of CAP on all cell lines was observed. CAP-induced alterations in cell membrane functionality were detected by performing a fluorescein diacetate (FDA) release assay and an ATP release assay. Additionally, modifications of the cell membrane and modifications in the actin cytoskeleton composition were examined using fluorescence microscopy monitoring dextran-uptake assay and G-/F-actin distribution. Furthermore, the CAP-induced induction of apoptosis was determined by TUNEL and active caspases assays. The observations suggest that a single CAP treatment of bone sarcoma cells may have significant anti-oncogenic effects and thus may be a promising extension to existing applications.

The androgen receptor antagonist enzalutamide induces apoptosis, dysregulates the heat shock protein system, and diminishes the androgen receptor and estrogen receptor ß1 expression in prostate cancer cells.

Enzalutamide's accepted mode of action is by targeting the androgen receptor's (AR) activity. In clinical practice, enzalutamide demonstrates a good benefit-risk profile for the treatment of advanced prostate cancer (PC), even after poor response to standard antihormonal treatment. However, since both, well-established antiandrogens and enzalutamide, target AR functionality, we hypothesized that additional unknown mechanisms might be responsible for enzalutamide's superior anticancer activity. In the current study, PC cells were incubated with enzalutamide and enzalutamide-dependent modulation of apoptotic mechanisms were assessed via Western blot analysis, TDT-mediated dUTP-biotin nick end-labeling assay, and nuclear morphology assay. Alterations of heat shock protein (HSP), AR, and estrogen receptor (ER) expression were examined by Western blot analysis. Enzalutamide attenuated the proliferation of PC cells in a time- and dose-dependent manner. In the presence of enzalutamide, apoptosis occurred which was shown by increased BAX expression, decreased Bcl-2 expression, nuclear pyknosis, and genomic DNA fragmentation. Moreover, enzalutamide inhibited the expression of HSPs primarily involved in steroid receptor stabilization and suppressed AR and ERß1 expression. This study demonstrates for the first time that enzalutamide treatment of PC cells triggers varying molecular mechanisms resulting in antiproliferative effects of the drug. In addition to the well-characterized antagonistic inhibition of AR functionality, we have shown that enzalutamide also affects the intracellular synthesis of steroid receptor-associated HSPs, thereby diminishing the expression of AR and ERß1 proteins and inducing apoptotic pathways. According to an indirect attenuation of HSP-associated factors such as steroid receptors, endometrial carcinoma, uterine leiomyosarcoma, and mamma carcinoma cells also demonstrated inhibited cell growth in the presence of enzalutamide. Our data, therefore, suggest that enzalutamide's high efficacy is at least partially independent of AR and p53 protein expression, which are frequently lost in advanced PC.

Cabazitaxel inhibits prostate cancer cell growth by inhibition of androgen receptor and heat shock protein expression.

PURPOSE: Cabazitaxel, a semi-synthetic taxane of the third generation, inhibits prostate cancer (PC) cell growth by affecting the microtubule architecture. Since cabazitaxel has also been demonstrated to inhibit androgen receptor (AR) functionality, AR and AR-associated heat shock protein (HSP) expressions in the presence of cabazitaxel were characterized. METHODS: AR and HSP expressions were assessed via Western blotting utilizing a PC-cell-line in vitro system incubated with cabazitaxel. RESULTS: Incubation experiments with 0.3 nM cabazitaxel exhibited significantly reduced levels of AR and the AR-associated factors HSP90α, HSP40, and HSP70/HSP90 organising protein. Furthermore, expression of the anti-apoptotic factor HSP60 was suppressed. In contrast to other anticancer compounds, cabazitaxel did not alter the cytoprotective chemoresistance factor HSP27. CONCLUSIONS: Despite the deregulation of microtubule organisation, cabazitaxel has been shown to suppress the expression of HSP. Very notably, and may be as a result of down-regulated HSP, cabazitaxel additionally inhibits the expression of the AR in AR-positive PC cells. Thus, cabazitaxel bears an additional anti-proliferative activity which is at least in part specific for PC cells.

Lack of chemokine (C-C motif) ligand 3 leads to decreased survival and reduced immune response after bacterial meningitis.

Pneumococcal meningitis, caused by Streptococcus pneumoniae, is the most common type of bacterial meningitis. The clinical management of this disease has been challenged by the emergence of multidrug-resistant Streptococcus pneumoniae, requiring the urgent development of new therapeutic alternatives. Over the course of bacterial meningitis, pathogen invasion is accompanied by a massive recruitment of peripheral immune cells, especially neutrophil granulocytes, which are recruited under the coordination of several cytokines and chemokines. Here, we used chemokine (C-C motif) ligand 3 (Ccl3)-deficient mice to investigate the functional role of CCL3 in a mouse model of pneumococcal meningitis. Following intrathecal infection with Streptococcus pneumoniae Ccl3-deficient mice presented a significantly shorter survival and higher bacterial load than wildtype mice, paralleled by an ameliorated infiltration of neutrophil granulocytes into the CNS. Blood sample analysis revealed that infected Ccl3-deficient mice showed a significant decrease in erythrocytes, hemoglobin and hematocrit as well as in the number of banded neutrophils. Moreover, infected Ccl3-deficient mice showed an altered cytokine expression profile. Glial cell activation remained unchanged in both genotypes. In summary, this study demonstrates that CCL3 is beneficial in Streptococcus pneumoniae-induced meningitis. Pharmacological modulation of the CCL3 pathways might, therefore, represent a future therapeutic option to manage Streptococcus pneumoniae meningitis.

Expression, Intracellular Localization, and Prognostic Value of Plasminogen Activator Inhibitor 1 and PAI-1 RNA-Binding Protein 1 in Primary and Recurrent Ovarian Cancer: A Study of the Tumor Bank Ovarian Cancer Network.

BACKGROUND: The plasminogen activator system plays a key role in ovarian cancer (OC) tumor progression. The plasminogen activator inhibitor type 1 (PAI-1) and the recently identified PAI-1 RNA binding protein 1 (PAI-RBP1) are primary regulators of plasminogen activation and thus are putative biomarkers for OC progression. METHODS: One hundred fifty six OC patients were analyzed to identify the presence of PAI-1 and PAI-RBP1 and subsequently correlated to clinicopathological parameters. Primary cells obtained from OC patient samples were applied in fluorescence microscopy analysis for examination of PAI-1 and PAI-RBP1 distribution. RESULTS: PAI-1 and PAI-RBP1 have been found to be predictive markers for OC patients' outcome. PAI-1 levels significantly correlated with volume of ascites, FIGO staging, and lymph node status. PAI-RBP1 expression significantly correlated with age at first diagnosis, histological tumor type, presence of distant metastasis (pM), and recurrence. PAI-1 showed a trend toward association and PAI-RBP1 was significantly associated with progression-free survival. Notably, PAI-1 protein in recurrent OC tissues was exclusively localized in the nucleus. CONCLUSION: This study has shown that a combination of PAI-1 and PAI-RBP1 may represent novel prognostic factor for OC. Prospective trials are needed.

Physical plasma: a new treatment option in gynecological oncology.

Non-thermal application of physical plasma is rapidly gaining importance for the future therapy and prevention of chronic inflammatory diseases and tumors. Here, we outline the importance of this innovative and less invasive therapy option, particulary for the treatment and prevention of gynecological cancers.

Clinical Outcome of Neoadjuvant Radiochemotherapy in Locally Advanced Cervical Cancer: Results of an Open Prospective, Multicenter Phase 2 Study of the North-Eastern German Society of Gynecological Oncology.

OBJECTIVE: The aim of this study was to determine the response rate, toxicity, operability, and surgical complication rate of neoadjuvant concomitant radiochemotherapy (cRCH) (ifosfamide + carboplatin) followed by radical hysterectomy plus external-beam radiotherapy with curative intention in locally advanced primary inoperable stages IIB and IIIB squamous cell cervical cancer. METHODS: Patients with cervical cancer from 8 departments were enrolled. Patients received 3 cycles of ifosfamide 1.2 mg/m (+mesna 20%) plus carboplatin (area under the curve = 4), every 21 days, and concomitant external-beam radiotherapy (50.4 Gy [1.8 Gy/d]). Operability and remission were evaluated by clinical gynecological examination in general anesthesia (magnetic resonance imaging was optional), 4 weeks after the third cycle of cRCH. In case of achieved operability, a radical hysterectomy with pelvic lymphadenectomy was performed within 6 weeks after cRCH. If surgery was not performed because of incomplete remission or patient preferences, vaginal brachytherapy (15 Gy [5 Gy/d]) was given additionally. RESULTS: Forty-four patients were enrolled. Distribution of FIGO (International Federation of Gynecology and Obstetrics) tumor stage was as follows: IIB (19 patients) and IIIB (25 patients). All patients completed cRCH. Grade 3/4 hematologic toxicities (% of all cycles) were moderate: leukopenia, 7.3; thrombocytopenia, 2.4; and anemia, 3.2. In 13.8%, treatment cycles were delayed because of hematologic toxicity. Blood transfusions were given in 17.7% and granulocyte colony-stimulating factor in 39.5%. Overall, grade 3/4 nonhematologic toxicities were seldom (6.5%). Clinical overall response rate was 95.2%. Operability was achieved in 85.7%. Surgery was performed in 83.3%. Pathological response rates were as follows: pathological complete remission, 33.3%; partial remission, 63.3%; stable disease, 3.3%. CONCLUSIONS: Our study demonstrates that cRCH is an effective and tolerable regimen in locally advanced cervical cancer treatment.

Telomeres and Telomerase in Hematopoietic Dysfunction: Prognostic Implications and Pharmacological Interventions.

Leukocyte telomere length (TL) has been suggested as a marker of biological age in healthy individuals, but can also reflect inherited and acquired hematopoietic dysfunctions or indicate an increased turnover of the hematopoietic stem and progenitor cell compartment. In addition, TL is able to predict the response rate of tyrosine kinase inhibitor therapy in chronic myeloid leukemia (CML), indicates clinical outcomes in chronic lymphocytic leukemia (CLL), and can be used as screening tool for genetic sequencing of selected genes in patients with inherited bone marrow failure syndromes (BMFS). In tumor cells and clonal hematopoietic disorders, telomeres are continuously stabilized by reactivation of telomerase, which can selectively be targeted by telomerase-specific therapy. The use of the telomerase inhibitor Imetelstat in patients with essential thrombocythmia or myelofibrosis as well as the use of dendritic cell-based telomerase vaccination in AML patients with complete remissions are promising examples for anti-telomerase targeted strategies in hematologic malignancies. In contrast, the elevation in telomerase levels through treatment with androgens has become an exciting clinical intervention for patients with BMFS. Here, we review recent developments, which highlight the impact of telomeres and telomerase targeted therapies in hematologic dysfunctions.

Cold Atmospheric Plasma in the Treatment of Osteosarcoma.

Human osteosarcoma (OS) is the most common primary malignant bone tumor occurring most commonly in adolescents and young adults. Major improvements in disease-free survival have been achieved by implementing a combination therapy consisting of radical surgical resection of the tumor and systemic multi-agent chemotherapy. However, long-term survival remains poor, so novel targeted therapies to improve outcomes for patients with osteosarcoma remains an area of active research. This includes immunotherapy, photodynamic therapy, or treatment with nanoparticles. Cold atmospheric plasma (CAP), a highly reactive (partially) ionized physical state, has been shown to inherit a significant anticancer capacity, leading to a new field in medicine called "plasma oncology." The current article summarizes the potential of CAP in the treatment of human OS and reviews the underlying molecular mode of action.

Exosomal particles secreted by prostate cancer cells are potent mRNA and protein vehicles for the interference of tumor and tumor environment.

BACKGROUND: Remodeling of the tumor environment and the modulation of tumor associated non-malignant cells are essential events in tumor progression. Exosomes are small membranous vesicles of 50-150 nm in diameter, which are secreted into the extracellular space and supposedly serve as vehicles for signal and effector molecules to modulate adjacent target cells. We characterized the mRNA and protein composition as well as cellular functions of prostate cancer cell-derived exosomes. METHODS: Exosomes were prepared from prostate cancer cell culture supernatant by ultracentrifugation and subsequently characterized by dynamic light scattering and electron microscopy. Exosomal mRNA and protein composition were analyzed by DNA microarrays and gel electrophoresis coupled with mass spectrometry. Physiological effects of exosomes were studied by means of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and lactate dehydrogenase release cell assays. Using a SILAC approach, putative uptake of exosomal human proteins in canine cells and canine de novo synthesis of proteins specified by exosome-transferred human mRNA was analyzed in MDCK cells via mass spectrometry. RESULTS: Preparations of exosomes revealed typical cup shaped particles of 150 nm in diameter. Analysis of mRNA and protein composition of exosomes exhibited a wide range of mRNA and protein species. Interestingly, the packaging of at least small proteins into exosomes was apparently unspecific, as shown with the example of two model proteins. In cell culture incubation experiments exosomal preparations of prostate cancer cells caused anti-proliferative effects. MS analysis revealed the uptake of exosomal human proteins into canine cells after 6 hr of incubation. CONCLUSIONS: The results reveal a distinct exosomal functionality in the modulation of the prostatic tumor adjacent environment. The multitude of translocated factors implies the induction of numerous effects in tumor-associated target cells, including impact on cellular growth.

Lack of Proinflammatory Cytokine Interleukin-6 or Tumor Necrosis Factor Receptor-1 Results in a Failure of the Innate Immune Response after Bacterial Meningitis.

The most frequent pathogen that causes bacterial meningitis is the Gram-positive bacterium Streptococcus pneumoniae. By entering the brain, host cells will be activated and proinflammatory cytokines like interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) are released. The goal of the current study was to examine the interaction between IL-6 and TNFR1 as receptor for TNF-α and the innate immune response in vivo in a model of Streptococcus pneumoniae-induced meningitis. For the experiments IL-6(-/-), TNFR1(-/-), and TNFR1-IL-6(-/-) KO mice were used. Our results revealed higher mortality rates and bacterial burden after infection in TNFR1(-/-), IL-6(-/-), and TNFR1-IL-6(-/-) mice and a decreased immune response including lower neutrophil infiltration in the meninges of TNFR1(-/-) and TNFR1-IL-6(-/-) mice in contrast to IL-6(-/-) and wild type mice. Furthermore, the increased mortality of TNFR1(-/-) and TNFR1-IL-6(-/-) mice correlated with decreased glial cell activation compared to IL-6(-/-) or wild type mice after pneumococcal meningitis. Altogether, the results show the importance of TNFR1 and IL-6 in the regulation of the innate immune response. The lack of TNFR1 and IL-6 results in higher mortality by weakened immune defence, whereas the lack of TNFR1 results in more severe impairment of the innate immune response than the lack of IL-6 alone.