Vitellogenin synthesis in primary cultures of fish liver cells as endpoint for in vitro screening of the (anti)estrogenic activity of chemical substances

Concern over possible adverse effects of endocrine-disrupting compounds on fish has caused the development of appropriate testing methods. In vitro screening assays may provide initial information on endocrine activities of a test compound and thereby may direct and optimize subsequent testing. Induction of vitellogenin (VTG) is used as a biomarker of exposure of fish to estrogen-active substances. Since VTG induction can be measured not only in vivo but also in fish hepatocytes in vitro, the use of VTG induction response in isolated fish liver cells has been suggested as in vitro screen for identifying estrogenic-active substances. The main advantages of the hepatocyte VTG assay are considered its ability to detect effects of estrogenic metabolites, since hepatocytes in vitro remain metabolically competent, and its ability to detect both estrogenic and anti-estrogenic effects. In this article, we critically review the current knowledge on the VTG response of cultured fish hepatocytes to (anti)estrogenic substances. In particular, we discuss the sensitivity, specificity, and variability of the VTG hepatocyte assay. In addition, we review the available data on culture factors influencing basal and induced VTG production, the response to natural and synthetic estrogens as well as to xenoestrogens, the detection of indirect estrogens, and the sources of assay variability. The VTG induction in cultured fish hepatocytes is clearly influenced by culture conditions (medium composition, temperature, etc.) and culture system (hepatocyte monolayers, aggregates, liver slices, etc.). The currently available database on estrogen-mediated VTG induction in cultured teleost hepatocytes is too small to support conclusive statements on whether there exist systematic differences of the VTG response between in vitro culture systems, VTG analytical methods or fish species. The VTG hepatocyte assay detects sensitively natural and synthetic estrogens, whereas the response to xenoestrogens appears to be more variable. The detection of weak estrogens can be critical due to the overshadow with cytotoxic concentrations. Moreover, the VTG hepatocyte assay is able to detect antiestrogens as well as indirect estrogens, i.e substances which require metabolic activation to induce an estrogenic response. Nevertheless, more chemicals need to be analysed to corroborate this statement. It will be necessary to establish standardized protocols to minimize assay variability, and to develop a set of pass-fail criteria as well as cut-offs for designating positive and negative responses.

Comparison of drug retention rates and causes of drug discontinuation between anti-tumor necrosis factor agents in rheumatoid arthritis

OBJECTIVE: Tumor necrosis factor (TNF) inhibitors have revolutionized the treatment of severe rheumatoid arthritis (RA), yet drug discontinuation is common. The aim of this study was to compare treatment retention rates and specific causes of anti-TNF discontinuation in a population-based RA cohort. METHODS: All patients treated with etanercept, infliximab, or adalimumab within the Swiss Clinical Quality Management RA cohort between 1997 and 2006 were included in the study. Causes of treatment discontinuation were broadly categorized as adverse events (AEs) or nontoxic causes, and further subdivided into specific categories. Specific causes of treatment interruption were analyzed using a Cox proportional hazards model and adjusted for potential confounders. RESULTS: A total of 2,364 anti-TNF treatment courses met the inclusion criteria. Treatment discontinuation was reported 803 times: 309 with etanercept, 249 with infliximab, and 245 with adalimumab. Drug inefficacy represented the largest single cause of treatment discontinuation (55.8% of cases). The median time of receiving anti-TNF therapy was 37 months, but discontinuation rates differed between the 3 anti-TNF agents (P < 0.001), with shorter retention rates for infliximab (hazard ratio [HR] 1.24, 99% confidence interval [99% CI] 1.01-1.51). The specific causes of treatment discontinuation revealed an increased risk of AEs with infliximab (HR 1.4, 99% CI 1.003-1.96), mostly due to an increased risk of infusion or allergic reactions (HR 2.11, 99% CI 1.23-3.62). Other discontinuation causes were equally distributed between the anti-TNF agents. CONCLUSION: In this population, infliximab was associated with higher overall discontinuation rates compared with etanercept and adalimumab, which is mainly due to an increased risk of infusion or allergic reactions.

Anti-eosinophil activity and clinical efficacy of the CRTH2 antagonist OC000459 in eosinophilic esophagitis

BACKGROUND: Eosinophilic esophagitis (EoE) is a chronic, Th2-type inflammatory disease. Chemoattractant receptor-homologous molecule on Th2 cells (CRTH2) is a prostaglandin D(2) (PGD(2)) receptor, expressed by Th2 cells and other inflammatory cells, including eosinophils and basophils, that mediates chemotaxis and activation. OC000459 is a selective CRTH2 antagonist and would be expected to suppress eosinophilic tissue inflammation. The purpose of this study was to evaluate the efficacy and safety of an OC000459 monotherapy in adult patients with active, corticosteroid-dependent or corticosteroid-refractory EoE. METHODS: In this randomized, double-blind, placebo-controlled trial, 26 adult patients (m/f = 22/4; mean age 41 years, range 22-69 years) with active EoE, dependent or resistant to corticosteroids, were treated either with 100 mg OC000459 (n = 14) or placebo (n = 12) twice daily. Pre- and post-treatment disease activity was assessed clinically, endoscopically, histologically, and via biomarkers. The primary end point was the reduction in esophageal eosinophil infiltration. RESULTS: After an 8-week OC000459 treatment, the esophageal eosinophil load decreased significantly, from 114.83 to 73.26 eosinophils per high-power field [(eos/hpf), P = 0.0256], whereas no reduction was observed with placebo (102.80-99.47 eos/hpf, P = 0.870). With OC000459, the physician's global assessment of disease activity improved from 7.13 to 5.18 (P = 0.035). OC000459 likewise reduced extracellular deposits of eosinophil peroxidase and tenascin C, the effects not seen with placebo. No serious adverse events were observed. CONCLUSIONS: An 8-week treatment with the CRTH2-antagonist, OC000459, exerts modest, but significant, anti-eosinophil and beneficial clinical effects in adult patients with active, corticosteroid-dependent or corticosteroid-refractory EoE and is well tolerated.

Impact of MET targeting on tumor-associated angiogenesis and growth of MET mutations-driven models of liver cancer

Deregulated expression of the MET receptor tyrosine kinase has been reported in up to 50% of patients with hepatocellular carcinoma, the most abundant form of liver cancers, and is associated with decreased survival. Consequently, MET is considered as a molecular target in this malignancy, whose progression is highly dependent on extensive angiogenesis. Here we studied the impact of MET small molecule inhibitors on angiogenesis-associated parameters and growth of xenograft liver models consisting of cells expressing MET-mutated variants M1268T and Y1248H, which exhibit constitutive kinase activity. We demonstrate that MET mutations expression is associated with significantly increased production of vascular endothelial growth factor, which is blocked by MET targeting only in cells expressing the M1268T inhibitor-sensitive but not in the Y1248H inhibitor-resistant variant. Decrease in vascular endothelial growth factor production is also associated with reduction of tyrosine phopshorylation of the vascular endothelial growth factor receptor 2 expressed on primary liver sinusoidal endothelial cells and with inhibition of vessel formation. Furthermore, MET inhibition demonstrated an efficient anti-tumor activity and considerable reduction in microvessel density only against the M1268T-derived intrahepatic tumors. Collectively, our data support the role of targeting MET-associated angiogenesis as a major biological determinant for liver tumor growth control.

A comprehensive understanding of thioTEPA metabolism in the mouse using UPLC-ESI-QTOFMS-based metabolomics

ThioTEPA, an alkylating agent with anti-tumor activity, has been used as an effective anticancer drug since the 1950s. However, a complete understanding of how its alkylating activity relates to clinical efficacy has not been achieved, the total urinary excretion of thioTEPA and its metabolites is not resolved, and the mechanism of formation of the potentially toxic metabolites S-carboxymethylcysteine (SCMC) and thiodiglycolic acid (TDGA) remains unclear. In this study, the metabolism of thioTEPA in a mouse model was comprehensively investigated using ultra-performance liquid chromatography coupled with electrospray ionization quadrupole time-of-flight mass spectrometry (UPLC-ESI-QTOFMS) based-metabolomics. The nine metabolites identified in mouse urine suggest that thioTEPA underwent ring-opening, N-dechloroethylation, and conjugation reactions in vivo. SCMC and TDGA, two downstream thioTEPA metabolites, were produced from thioTEPA from two novel metabolites 1,2,3-trichloroTEPA (VII) and dechloroethyltrichloroTEPA (VIII). SCMC and TDGA excretion were increased about 4-fold and 2-fold, respectively, in urine following the thioTEPA treatment. The main mouse metabolites of thioTEPA in vivo were TEPA (II), monochloroTEPA (III) and thioTEPA-mercapturate (IV). In addition, five thioTEPA metabolites were detected in serum and all shared similar disposition. Although thioTEPA has a unique chemical structure which is not maintained in the majority of its metabolites, metabolomic analysis of its biotransformation greatly contributed to the investigation of thioTEPA metabolism in vivo, and provides useful information to understand comprehensively the pharmacological activity and potential toxicity of thioTEPA in the clinic.

Targeting the insulin-like growth factor-1 receptor in human cancer

The insulin-like growth factor (IGF) signaling system plays a crucial role in human cancer and the IGF-1 receptor (IGF-1R) is an attractive drug target against which a variety of novel anti-tumor agents are being developed. Deregulation of the IGF signaling pathway frequently occurs in human cancer and involves the establishment of autocrine loops comprising IGF-1 or IGF-2 and/or IGF-1R over-expression. Epidemiologic studies have documented a link between elevated IGF levels and the development of solid tumors, such as breast, colon, and prostate cancer. Anti-cancer strategies targeting the IGF signaling system involve two main approaches, namely neutralizing antibodies and small molecule inhibitors of the IGF-1R kinase activity. There are numerous reports describing anti-tumor activity of these agents in pre-clinical models of major human cancers. In addition, multiple clinical trials have started to evaluate the safety and efficacy of selected IGF-1R inhibitors, in combination with standard chemotherapeutic regimens or other targeted agents in cancer patients. In this mini review, I will discuss the role of the IGF signaling system in human cancer and the main strategies which have been so far evaluated to target the IGF-1R.

Prolactin upregulates sphingosine kinase-1 expression and activity in the human breast cancer cell line MCF7 and triggers enhanced proliferation and migration

Sphingosine kinases (SK) catalyze the formation of sphingosine-1-phosphate (S1P) which plays a crucial role in cell growth and survival. Here, we show that prolactin (PRL) biphasically activates the SK-1, but not the SK-2 subtype, in the breast adenocarcinoma cell-line MCF7. A first peak occurs after minutes of stimulation and is followed by a second delayed activation after hours of stimulation. A similar biphasic effect on SK-1 activity is seen for 17beta-estradiol (E(2)). The delayed activation of SK-1 derives from an upregulated mRNA and protein expression and is due to increased SK-1 promoter activity and mechanistically involves STAT5 activation as well as protein kinase C and the classical mitogen-activated protein kinases. Furthermore, glucocorticoids also block both hormone-induced SK-1 expression and activity. Functionally, long-term stimulation of MCF7 cells with PRL or E(2) is well known to trigger increased cell proliferation and migration. Both hormone-induced cell responses critically involve SK-1 activation since the depletion of SK-1, but not SK-2, by siRNA transfection abolishes the hormone-induced cell proliferation and migration. In summary, our data show that PRL and E(2) cause a pronounced delayed SK-1 activation which is due to increased gene transcription, and critically determines the capability of cells to grow and move. Thus, the SK-1 may represent a novel attractive target for anti-tumor therapy.

Profound activity of the anti-cancer drug bortezomib against Echinococcus multilocularis metacestodes identifies the proteasome as a novel drug target for cestodes.

A library of 426 FDA-approved drugs was screened for in vitro activity against E. multilocularis metacestodes employing the phosphoglucose isomerase (PGI) assay. Initial screening at 20 µM revealed that 7 drugs induced considerable metacestode damage, and further dose-response studies revealed that bortezomib (BTZ), a proteasome inhibitor developed for the chemotherapy of myeloma, displayed high anti-metacestodal activity with an EC50 of 0.6 µM. BTZ treatment of E. multilocularis metacestodes led to an accumulation of ubiquinated proteins and unequivocally parasite death. In-gel zymography assays using E. multilocularis extracts demonstrated BTZ-mediated inhibition of protease activity in a band of approximately 23 kDa, the same size at which the proteasome subunit beta 5 of E. multilocularis could be detected by Western blot. Balb/c mice experimentally infected with E. multilocularis metacestodes were used to assess BTZ treatment, starting at 6 weeks post-infection by intraperitoneal injection of BTZ. This treatment led to reduced parasite weight, but to a degree that was not statistically significant, and it induced adverse effects such as diarrhea and neurological symptoms. In conclusion, the proteasome was identified as a drug target in E. multilocularis metacestodes that can be efficiently inhibited by BTZ in vitro. However, translation of these findings into in vivo efficacy requires further adjustments of treatment regimens using BTZ, or possibly other proteasome inhibitors.

Amicrobial pustulosis-like rash in a patient with Crohn's disease under anti-TNF-alpha blocker

Amicrobial pustulosis of the folds (APF) is a recently described entity characterized by relapsing pustular lesions predominantly involving the cutaneous flexures and scalp. This disease typically occurs in association with systemic lupus erythematosus and a variety of other autoimmune diseases. We here describe an APF-like pustular eruption predominantly affecting the scalp, face and trunk, occurring during long-term infliximab treatment for Crohn's disease. Immunohistochemical staining of skin biopsy specimens for myxovirus resistance protein A, a marker for type 1 interferon-inducible proteins, showed increased staining in the epidermis and dermal mononuclear inflammatory infiltrate. Our observation further extends the spectrum of cutaneous adverse reactions potentially related to anti-tumor necrosis factor-α, the clinical context in which APF can occur as well as its clinical presentations.

Quassinoids: From traditional drugs to new cancer therapeutics

Quassinoids are a group of compounds extracted from plants of the Simaroubaceae family, which have been used for many years in folk medicine. These molecules gained notoriety after the initial discovery of the anti-leukemic activity of one member, bruceantin, in 1975. Currently over 150 quassinoids have been isolated and classified based on their chemical structures and biological properties investigated in vitro and in vivo. Many molecules display a wide range of inhibitory effects, including anti-inflammatory, anti-viral, anti-malarial and anti-proliferative effects on various tumor cell types. Although often the exact mechanism of action of the single agents remains unclear, some agents have been shown to affect protein synthesis in general, or specifically HIF-1α and MYC, membrane polarization and the apoptotic machinery. Considering that future research into chemical modifications is likely to generate more active and less toxic derivatives of natural quassinoids, this family represents a powerful source of promising small molecules targeting key prosurvival signaling pathways relevant for diverse pathologies. Here, we review available knowledge of functionality and possible applications of quassinoids and quassinoid derivatives, spanning traditional use to the potential impact on modern medicine as cancer therapeutics.

Induction of tachyzoite egress from cells infected with the protozoan Neospora caninum by nitro- and bromo-thiazolides, a class of broad-spectrum anti-parasitic drugs

Neospora caninum represents an important pathogen causing stillbirth and abortion in cattle and neuromuscular disease in dogs. Nitazoxanide (NTZ) and its deacetylated metabolite tizoxanide (TIZ) are nitro-thiazolyl-salicylamide drugs with a broad-spectrum anti-parasitic activity in vitro and in vivo. In order to generate compounds potentially applicable in food and breeding animals, the nitro group was removed, and the thiazole-moiety was modified by other functional groups. We had shown earlier that replacement of the nitro-group by a bromo-moiety did not notably affect in vitro efficacy of the drugs against N. caninum. In this study we report on the characterization of two bromo-derivatives, namely Rm4822 and its de-acetylated putative metabolite Rm4847 in relation to the nitro-compounds NTZ and TIZ. IC(50) values for proliferation inhibition were 4.23 and 4.14 microM for NTZ and TIZ, and 14.75 and 13.68 microM for Rm4822 and Rm4847, respectively. Complete inhibition (IC(99)) was achieved at 19.52 and 22.38 microM for NTZ and TIZ, and 18.21 and 17.66 microM for Rm4822 and Rm4847, respectively. However, in order to exert a true parasiticidal effect in vitro, continuous culture of infected fibroblasts in the presence of the bromo-thiazolide Rm4847 was required for a period of 3 days, while the nitro-compound TIZ required 5 days continuous drug exposure. Both thiazolides induced rapid egress of N. caninum tachyzoites from their host cells, and egress was inhibited by the cell membrane permeable Ca(2+)-chelator BAPTA-AM. Host cell entry by N. caninum tachyzoites was inhibited by Rm4847 but not by TIZ. Upon release from their host cells, TIZ-treated parasites remained associated with the fibroblast monolayer, re-invaded neighboring host cells and resumed proliferation in the absence of the drug. In contrast, Rm4847 inhibited host cell invasion and respective treated tachyzoites did not proliferate further. This demonstrated that bromo- and nitro-thiazolides exhibit differential effects against the intracellular protozoan N. caninum and bromo-thiazolides could represent a valuable alternative to the nitro-thiazolyl-salicylamide drugs.

Anti-inflammatory properties of alpha- and gamma-tocopherol

Natural vitamin E consists of four different tocopherol and four different tocotrienol homologues (alpha,beta, gamma, delta) that all have antioxidant activity. However, recent data indicate that the different vitamin E homologues also have biological activity unrelated to their antioxidant activity. In this review, we discuss the anti-inflammatory properties of the two major forms of vitamin E, alpha-tocopherol (alphaT) and gamma-tocopherol (gammaT), and discuss the potential molecular mechanisms involved in these effects. While both tocopherols exhibit anti-inflammatory activity in vitro and in vivo, supplementation with mixed (gammaT-enriched) tocopherols seems to be more potent than supplementation with alphaT alone. This may explain the mostly negative outcomes of the recent large-scale interventional chronic disease prevention trials with alphaT only and thus warrants further investigation.

Antimicrobial activity of gentamicin in experimental enterococcal endocarditis

The in vitro activity of gentamicin was compared with its therapeutic efficacy in rabbits with Streptococcus faecalis endocarditis. The test strain was resistant to gentamicin as measured by MICs and MBCs determined in Mueller-Hinton broth alone or in broth supplemented with 50% rabbit serum. Gentamicin also failed to manifest anti-enterococcal activity when evaluated by time-kill studies in broth. However, the addition of serum to the medium did enhance the activity of gentamicin. In the therapy of experimental endocarditis, gentamicin used alone demonstrated anti-enterococcal activity equivalent to that of ampicillin used alone. Vegetation titers in animals treated with gentamicin alone were lower than those of untreated controls (P less than 0.01) and comparable to those in animals treated with ampicillin alone. Thus, gentamicin demonstrated anti-enterococcal activity in vivo despite the resistance observed in vitro, as measured by conventional assays to determine MICs and MBCs.

Immunoglobulin M-enriched intravenous immunoglobulin inhibits classical pathway complement activation, but not bactericidal activity of human serum

Acute or even hyperacute humoral graft rejection, mediated by classical pathway complement activation, occurs in allo- and xenotransplantation due to preformed anti-graft antibodies. Intravenous immunoglobulin (IVIg) preparations can prevent complement-mediated tissue injury and delay hyperacute xenograft rejection. It is known that IgM-enriched IVIg (IVIgM) has a higher capacity to block complement than IVIgG. Different IVIgs were therefore tested for specificity of complement inhibition and effect on anti-bacterial activity of human serum. IVIgM-I (Pentaglobin), 12% IgM), IVIgM-II (IgM-fraction of IVIgM-I, 60% IgM), and three different IVIgG (all >95% IgG) were used. The known complement inhibitor dextran sulfate was used as control. Hemolytic assays were performed to analyze pathway-specificity of complement inhibition. Effects of IVIg on complement deposition on pig cells and Escherichia coli were assessed by flow cytometry and cytotoxicity as well as bactericidal assays. Complement inhibition by IVIgM was specific for the classical pathway, with IC50 values of 0.8 mg/ml for IVIgM-II and 1.7 mg/ml for IVIgM-I in the CH50 assay. Only minimal inhibition of the lectin pathway was seen with IVIgM-II (IC50 15.5 mg/ml); no alternative pathway inhibition was observed. IVIgG did not inhibit complement in any hemolytic assay. Classical pathway complement inhibition by IVIgM was confirmed in an in vitro xenotransplantation model with PK15 cells. In contrast, IVIgM did not inhibit (mainly alternative pathway mediated) killing of E. coli by human serum. In conclusion, IgM-enriched IVIg is a specific inhibitor of the classical complement pathway, leaving the alternative pathway intact, which is an important natural anti-bacterial defense, especially for immunosuppressed patients.