MIF-CD74 signaling impedes microglial M1 polarization and facilitates brain tumorigenesis.

Microglial cells in the brain tumor microenvironment are associated with enhanced glioma malignancy. They persist in an immunosuppressive M2 state at the peritumoral site and promote the growth of gliomas. Here, we investigated the underlying factors contributing to the abolished immune surveillance. We show that brain tumors escape pro-inflammatory M1 conversion of microglia via CD74 activation through the secretion of the cytokine macrophage migration inhibitory factor (MIF), which results in a M2 shift of microglial cells. Interruption of this glioma-microglial interaction through an antibody-neutralizing approach or small interfering RNA (siRNA)-mediated inhibition prolongs survival time in glioma-implanted mice by reinstating the microglial pro-inflammatory M1 function. We show that MIF-CD74 signaling inhibits interferon (IFN)-γ secretion in microglia through phosphorylation of microglial ERK1/2 (extracellular signal-regulated protein kinases 1 and 2). The inhibition of MIF signaling or its receptor CD74 promotes IFN-γ release and amplifies tumor death either through pharmacological inhibition or through siRNA-mediated knockdown. The reinstated IFN-γ secretion leads both to direct inhibition of glioma growth as well as inducing a M2 to M1 shift in glioma-associated microglia. Our data reveal that interference with the MIF signaling pathway represents a viable therapeutic option for the restoration of IFN-γ-driven immune surveillance.

Definition of K(trans) and FA thresholds for better assessment of experimental glioma using high-field MRI: a feasibility study.

PURPOSE: To define K(trans) and fractional anisotropy (FA) thresholds in correlation to histology for improved magnetic resonance imaging (MRI) tumor assessment in an animal model of brain glioma. METHODS: Twelve rats underwent 4.7 T MRI at day 10 after tumor implantation. Anatomical scans (T2, T1 at 8 min after double dose contrast application) as well as dynamic contrast-enhanced (DCE) imaging with calculation of K(trans) and diffusion tensor imaging (DTI) with calculation of FA were performed. T2- and T1-derived tumor volumes were calculated and thresholds for K(trans) and FA were defined for best MRI tumor assessment correlated to histology. RESULTS: Tumor volumes were 159 ± 14 mm(3) (histology), 126 ± 26 mm(3) (T1 with contrast, r=0.76), and 153 ± 12 mm(3) (T2, r=0.84), respectively. K(trans)- and FA-derived tumor volumes were 160 ± 16 mm(3) (for K(trans ≥ 0.04 min(-1), r=0.94), and 159 ± 14 mm(3) (for FA £0.14, r=0.96), respectively. CONCLUSIONS: DCE-MRI and DTI with calculation of K(trans) and FA maps allow very precise brain glioma assessment comparable to histology if established thresholds for the given tumor model are used.

Towards evidence-based use of serum tumour marker requests: an audit of use in a tertiary hospital.

BACKGROUND: Serum tumour markers (TM) are often measured in hospital patients. The reasons for their use and their benefits with regards to earlier cancer diagnosis and patient management are not known. AIMS: To identify the patterns of TM use in a tertiary hospital and to determine the usefulness and appropriateness of requests in this setting. METHODS: A cross-sectional, retrospective study of TM ordered over a 3-month period was conducted. Data were obtained from patient records. CA-125, CA 15-3, CA 19-9, carcinoembryonic antigen (CEA), and alpha-fetoprotein (AFP) were studied. Prostate specific antigen was not separately investigated. The reasons for ordering, usefulness and appropriateness of use were defined prior to analysis. RESULTS: A total of 476 TM was ordered in 373 patients. One hundred and six (22%) of all results were abnormal by laboratory criteria. AFP was the most popular test ordered. Forty-seven per cent of patients had no cancer diagnosis. Oncological units (ONC) ordered 27% of tests. The most popular reasons for TM ordering were for screening (36%) followed by diagnostic aid (19%). ONC units ordered TM mainly for monitoring disease status, as opposed to non-ONC units who ordered TM usually for diagnostic aid. TM were deemed appropriately ordered in 69% of cases. Twenty-nine per cent of TM were helpful in patient management. Only four results (<1%) aided in diagnosis. CONCLUSIONS: The reasons and appropriateness of TM use varied depending on the specialization of the requesting clinician. The current serum TM are most useful as aids in cancer patients, rather than for diagnosis (P <0.0001). Apart from AFP, these TM seem to have limited use in the general medical, non-oncological patients. Guidelines for their use in this setting are needed.

Pro-EMAP II is not primarily cleaved by caspase-3 and -7.

Endothelial monocyte-activating polypeptide (EMAP) II is a unique cytokine, also known as p43, the active mature form of which exhibits antiangiogenic properties in vivo and in vitro. The proteolytic enzymes associated with the cleavage and release of the active mature form, however, remain unclear. Here we show that, in contrast to prior observations, purified pro-EMAP II is not cleaved by either caspase-3 or -7 in vivo or in vitro. Thus other proteolytic processes, which allow it to induce apoptosis via caspase-3 activation in migrating and dividing endothelium, may be involved in the release of the active mature EMAP II.

Rapid chiral on-chip separation with simplified amperometric detection.

The enantiomers of adrenaline, noradrenaline, ephedrine and pseudoephedrine were separated by capillary electrophoresis on a micromachined device. Detection was carried out with a new two-electrode amperometric detector, eliminating the need for individual counter and reference electrodes. Separation of the isomers was achieved by employing carboxymethyl-beta-cyclodextrin as chiral selector in the buffer, partly with the additional inclusion of the crown ether 18-crown-6. Plate numbers of up to 20,000, chiral resolutions of 2.5 and detection limits of the order of 10(-7) M were achieved. All separations were completed in less then 3 min.

Control of stromal keratitis by inhibition of neovascularization.

Stromal keratitis resulting from ocular infection with herpes simplex virus is a common cause of blindness. This report investigates the role of neovascularization in the pathogenesis of stromal keratitis by measuring the outcome of treatment with the potent anti-angiogenesis cytokine endothelial monocyte-activating polypeptide II (EMAP II). We show that systemic and topical administration of EMAP II from the outset of infection resulted in markedly diminished levels of herpes simplex virus-induced angiogenesis and significantly reduced the severity of stromal keratitis lesions. EMAP II treatment had no demonstrable pro-inflammatory or toxic effects and failed to express antiviral activity. The mechanism of action of EMAP II was shown to proceed by causing apoptosis in vascular endothelial cells. Our data document for the first time the essential role of angiogenesis in the pathogenesis of stromal keratitis and also indicate that the therapy of herpetic stromal keratitis could benefit by procedures that diminish angiogenesis.

The management of testicular cancer in Victoria, 1988-1993. Urology Study Committee of the Victorian Co-operative Oncology Group.

OBJECTIVES: To evaluate the patterns of care and management of testicular cancer in Victoria. DESIGN AND SETTING: Retrospective analysis of all cases of testicular cancer in Victoria from 1988 to 1993 identified through the Victorian Cancer Registry. MAIN OUTCOME MEASURES: Description of patient characteristics, staging investigations, initial management, and outcome. RESULTS: 667 eligible cases of testicular cancer were identified and questionnaires were returned for 633 of these patients (94.9% response rate). There were 357 (56.4%) patients with pure seminoma; 271 (42.8%) with non-seminomatous germ cell tumours, 3 (0.5%) with stromal tumours, and 2 (0.3%) with other tumours. The median age was 32 years (range, 0-80 years). Preoperative marker levels were not available for 8% of patients, and initial staging was considered inadequate in 6%. Surveillance programs used for patients with Stage I disease were considered inadequate in most. Relative survival at five years was 99% for patients with seminoma and 91% for non-seminoma. CONCLUSIONS: There was considerable variation in the investigation, treatment, and follow-up of these patients, which is likely to have resulted in unnecessary morbidity. Clinical practice guidelines should be developed and implemented to promote optimal management.

A two-electrode configuration for simplified amperometric detection in a microfabricated electrophoretic separation device.

The simplified amperometric detection scheme demonstrated is based on the amperometric working and electrophoretic ground electrodes only. The latter serves as counter and pseudo-reference as well. It is shown via the successful determination of neurotransmitters, ascorbic acid and phenols on gold or platinum working electrodes that this approach is feasible for detection on a channel based electrophoretic separation device. Also presented is the detection of carbohydrates and amino acids with copper electrodes. The results were found to be similar to those obtained with conventional capillary systems with amperometric detection, albeit at much reduced analysis times.

Acute lung injury: cellular mechanisms and derangements.

The clinical course of acute lung injury (ALI) is a complex and variable process accompanied by severe lung dysfunction, which persists for a long period of time with variable recovery of pulmonary function. The extent and severity of the lung disease associated with ALI varies with those patients having the most severe manifestations of lung disease being grouped as acute respiratory distress syndrome (ARDS). The pathological injury associated with this disease process, termed diffuse alveolar damage (DAD), has three overlapping phases (exudative, proliferative and fibrotic) which are the consequences of severe injury to the alveolar-capillary unit. There is no uniformity to the progression and length of each stage. This review explores those cellular mechanisms and derangements involved in the progression of ARDS. Those areas that demonstrate the major advances within the field are highlighted because of the diverse and vast nature of the cellular components involved in the process of ALI. We are beginning to identify those processes that contribute to the cellular derangements which are the hallmark of ALI. By expanding our understanding of those factors, we should in the future be able to construct therapeutic interventions that address the aetiology of ALI.

Recent developments in detection methods for microfabricated analytical devices.

Sensitive detection in microfluidic analytical devices is a challenge because of the extremely small detection volumes available. Considerable efforts have been made lately to further address this aspect and to investigate techniques other than fluorescence. Among the newly introduced techniques are the optical methods of chemiluminescence, refraction and thermooptics, as well as the electrochemical methods of amperometry, conductimetry and potentiometry. Developments are also in progress to create miniaturized plasma-emission spectrometers and sensitive detectors for gas-chromatographic separations.

Antiangiogenic treatment enhances photodynamic therapy responsiveness in a mouse mammary carcinoma.

Photodynamic therapy (PDT) is a promising cancer treatment that induces localized tumor destruction via the photochemical generation of cytotoxic singlet oxygen. PDT-mediated oxidative stress elicits direct tumor cell damage as well as microvascular injury within exposed tumors. Reduction in vascular perfusion associated with PDT-mediated microvascular injury produces tumor tissue hypoxia. Using a transplantable BA mouse mammary carcinoma, we show that Photofrin-mediated PDT induced expression of the hypoxia-inducible factor-1alpha (HIF-1alpha) subunit of the heterodimeric HIF-1 transcription factor and also increased protein levels of the HIF-1 target gene, vascular endothelial growth factor (VEGF), within treated tumors. HIF-1alpha and VEGF expression were also observed following tumor clamping, which was used as a positive control for inducing tissue hypoxia. PDT treatment of BA tumor cells grown in culture resulted in a small increase in VEGF expression above basal levels, indicating that PDT-mediated hypoxia and oxidative stress could both be involved in the overexpression of VEGF. Tumor-bearing mice treated with combined antiangiogenic therapy (IM862 or EMAP-II) and PDT had improved tumoricidal responses compared with individual treatments. We also demonstrated that PDT-induced VEGF expression in tumors decreased when either IM862 or EMAP-II was included in the PDT treatment protocol. Our results indicate that combination procedures using antiangiogenic treatments can improve the therapeutic effectiveness of PDT.

Endothelial monocyte activating polypeptide II inhibits lung neovascularization and airway epithelial morphogenesis.

Neovascularization is crucial to lung development and is mediated through a variety of angiogenic and anti-angiogenic factors. Herein, we show that excess Endothelial Monocyte Activating Polypeptide (EMAP) II, an anti-angiogenic protein, not only inhibits fetal lung neovascularization, but also significantly alters airway epithelial morphogenesis. In a murine allograft model of lung neovascularization and morphogenesis, embryonic lungs transplanted under the skin of immunocompromised mice receiving intraperitoneal EMAP II, had a 56% reduction in vessel density (P<0.0001) compared to control. EMAP II treated lung transplants also exhibited a marked alteration in lung morphogenesis, including lack of type II alveolar cell formation, determined by markedly decreased expression of surfactant protein C, and increased apoptosis. In contrast, lung implants in animals receiving an EMAP II blocking antibody had an increase in vessel density of 50% (P<0.0001) and increased expression of surfactant protein C mRNA in distal epithelium. These studies demonstrate that EMAP II negatively modulates lung neovascularization as well as leading to the arrest of lung airway epithelial morphogenesis and apoptosis.

Temporo-spatial distribution of endothelial-monocyte activating polypeptide II, an anti-angiogenic protein, in the mouse embryo.

We describe the temporo-spatial distribution of Endothelial-Monocyte Activating Polypeptide (EMAP) II in order to better understand what role this anti-angiogenic proteins may play in fetal development. In situ hybridization, immunohistochemistry, and Western analysis were performed on fetal, neonatal, and adult tissue. EMAP II was first detected only within the central nervous system on 9 days postcoitum (dpc). Subsequently, at 11 through 18 dpc, EMAP II expression was detected in the respiratory, central nervous, cardiovascular, urogenital systems, sense organs, and digestive tract. EMAP II mRNA and protein was localized to the epithelium, with its highest expression in neurons, blood vessels, and at sites of epithelial-mesenchymal interaction. The temporo-spatial distribution of EMAP II suggests that it could play an important role in morphogenesis of the vertebrate embryo.

Angiogenesis and morphogenesis of murine fetal distal lung in an allograft model.

Neovascularization is crucial to lung morphogenesis; however, factors determining vessel growth and formation are poorly understood. The goal of our study was to develop an allograft model that would include maturation of the distal lung, thereby ultimately allowing us to study alveolar development, including microvascular formation. We transplanted 14-day gestational age embryonic mouse lung primordia subcutaneously into the back of nude mice for 3.5-14 days. Lung morphogenesis and neovascularization were evaluated by light microscopy, in situ hybridization, and immunohistochemical techniques. Embryonic 14-day gestational age control lungs had immature structural features consistent with pseudoglandular stage of lung development. In contrast, 14 days after subcutaneous transplantation of a 14-day gestational age lung, the allograft underwent significant structural morphogenesis and neovascularization. This was demonstrated by continued neovascularization and cellular differentiation, resulting in mature alveoli similar to those noted in the 2-day postnatal neonatal lung. Confirmation of maturation of the allograft was provided by progressive type II epithelial cell differentiation as evidenced by enhanced local expression of mRNA for surfactant protein C and a threefold (P < 0.008) increase in vessel formation as determined by immunocytochemical detection of platelet endothelial cell adhesion molecule-1 expression. Using the tyrosine kinase Flk-1 receptor (flk-1) LacZ transgene embryos, we determined that the neovascularization within the allograft was from the committed embryonic lung endothelium. Therefore, we have developed a defined murine allograft model that can be used to study distal lung development, including neovascularization. The model may be useful when used in conjunction with an altered genetic background (knockout or knock in) of the allograft and has the further decided advantage of bypassing placental barriers for introduction of pharmacological agents or DNA directly into the lung itself.

Affinity capillary electrophoresis in pharmaceutics.

The technique of Affinity capillary electrophoresis (ACE) is a useful tool to characterize complexation and partition equilibria. A wide range of applications in pharmaceutics has been presented: the determination of pKA values, of association constants between drugs and cyclodextrins or amphiphilic compounds as well as the characterization of drug affinities to several vehicle systems, such as micelles, microemulsions and liposomes. Due to the widespread use of capillary electrophoresis in drug analysis, the technical equipment is quite common. ACE is advantageous as a rapid and simple screening method which provides quantitative results about various interactions with a minimum of substance consumption and time spent.

Wheatgerm agglutinin-mediated toxicity in pancreatic cancer cells.

Lectin binding specificities for carbohydrate allow phenotypic and functional characterization of membrane-associated glycoproteins expressed on cancer cells. This analysis examined wheatgerm agglutinin binding to pancreatic cancer cells in vitro and the resulting toxicity. Membrane preparations of nine human pancreatic carcinoma cell lines were studied for lectin binding using wheatgerm agglutinin (WGA), concanavalin A (ConA) and phytohaemagglutinin-L (PHA-L) in a lectin blot analysis. Cell proliferation in vitro was measured by thymidine incorporation in the absence or presence of lectins at various concentrations. Sialic acid binding lectins or succinyl-WGA (succWGA) served as controls. WGA toxicity was tested after swainsonine or neuraminidase pretreatment. Binding and uptake of fluorescein-labelled lectins was studied under fluorescence microscopy. All pancreatic cell lines displayed high WGA membrane binding, primarily to sialic acid residues. Other lectins were bound with weak to moderate intensity only. Lectin toxicity corresponded to membrane binding intensity, and was profound in case of WGA (ID50 at 2.5-5 microg ml(-1)). WGA exposure induced chromatin condensation, nuclear fragmentation and DNA release consistent with apoptosis. Important steps for WGA toxicity included binding to sialic acid on swainsonine-sensitive carbohydrate and lectin internalization. There was rapid cellular uptake and subsequent nuclear relocalization of WGA. In contradistinction to the other lectins studied, WGA proved highly toxic to human pancreatic carcinoma cells in vitro. WGA binding to sialic acid residues of N-linked carbohydrate, cellular uptake and subsequent affinity to N-acetyl glucosamine appear to be necessary steps. Further analysis of this mechanism of profound toxicity may provide insight relevant to the treatment of pancreatic cancer.

Endothelial-monocyte activating polypeptide II, a novel antitumor cytokine that suppresses primary and metastatic tumor growth and induces apoptosis in growing endothelial cells.

Neovascularization is essential for growth and spread of primary and metastatic tumors. We have identified a novel cytokine, endothelial-monocyte activating polypeptide (EMAP) II, that potently inhibits tumor growth, and appears to have antiangiogenic activity. Mice implanted with Matrigel showed an intense local angiogenic response, which EMAP II blocked by 76% (P < 0.001). Neovascularization of the mouse cornea was similarly prevented by EMAP II (P < 0.003). Intraperitoneally administered EMAP II suppressed the growth of primary Lewis lung carcinomas, with a reduction in tumor volume of 65% versus controls (P < 0.003). Tumors from human breast carcinoma-derived MDA-MB 468 cells were suppressed by >80% in EMAP II-treated animals (P < 0.005). In a lung metastasis model, EMAP II blocked outgrowth of Lewis lung carcinoma macrometastases; total surface metastases were diminished by 65%, and of the 35% metastases present, approximately 80% were inhibited with maximum diameter <2 mm (P < 0.002 vs. controls). In growing capillary endothelial cultures, EMAP II induced apoptosis in a time- and dose-dependent manner, whereas other cell types were unaffected. These data suggest that EMAP II is a tumor-suppressive mediator with antiangiogenic properties allowing it to target growing endothelium and limit establishment of neovasculature.

Metastasis correlates with production of vascular endothelial growth factor in a murine model of human Wilms' tumor.

BACKGROUND/PURPOSE: The growth and spread of solid tumors are critically dependent on the induction of angiogenesis. We hypothesized that vascular endothelial growth factor (VEGF) would be detected in Wilms' tumors, and that both growth and metastasis would parallel VEGF levels in a murine model. METHODS: Primary tumors were established in the right kidneys of nude mice (n = 21). Mice were killed at 3, 4.5, or 6 weeks. Tumor-bearing and control kidneys were subjected to enzyme-linked immunosorbent assay (ELISA) for VEGF. Representative sections were assessed by histology and immunohistochemistry. Lungs were examined for metastases. Clinical specimens of Wilms' tumor (n = 12) also were assayed for VEGF. RESULTS: The authors detected VEGF by ELISA with increasing frequency, and in increasing quantity, as experimental Wilms' tumors were grown over time. Immunohistochemistry demonstrated accumulation of VEGF in areas of viable tumor. Lung metastases occurred in 8 of 10 animals with VEGF-positive tumors, but in only 3 of 11 animals with VEGF-negative tumors, an association that was statistically significant. VEGF was found in 10 of 12 clinical Wilms' tumor specimens tested. CONCLUSIONS: VEGF is present in both clinical and experimental Wilms' tumors. In a murine model, absolute VEGF levels increase as primary tumors grow, and VEGF production is significantly associated with tumor metastasis.

Characterization of micelles by capillary electrophoresis.

A practical approach for the characterization of pure micelles, and binary or ternary mixed micelles by capillary electrophoretic methods is presented. Interest is focused on the determination of mobility and composition of the micelles. The investigations are performed with bile salts, phosphatidylcholines and fatty acids. Pure bile salt micelles are characterized with the help of marking and displacement methods. Binary bile salt/phospholipid and ternary bile salt/phospholipid/fatty acid micelles are analyzed using capillary electrophoresis (CE) techniques with standard and improved UV detection, laser-induced fluorescence and electrospray ionization-mass spectrometry (ESI-MS). For both the binary and the ternary systems, only one stable mixed micellar phase is found with a high negative mobility.

Interaction between food components and drugs. Part 6: Influence of starch degradation products on propranolol transport.

Interactions between different starch degradation products and propranolol (P) were studies using permeation experiments and affinity capillary electrophoresis (ACE). In the presence of maltooligosaccharides (MO), the transport of P across artificial lipid membranes was retarded. The most intensive interaction with the drug was found in the presence of the not-debranched MO from maize starch. The electrophoretic mobility mu of the drug was decreased in dependence of the MO concentration. However calculation of equilibrium binding constants from the ACE experiments was not possible because of the relatively weak influence of starch degradation products on the electrophoretic behaviour of the drug. With both methods the same tendency in strength of interactions between the different MO and P was found. Molecular parameters of the MO and their viscosity play a role in the interactions and in the decreased permeation of the drug.