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Multiple representatives of eulipotyphlan mammals such as shrews have oral venom systems. Venom facilitates shrews to hunt and/or hoard preys. However, little is known about their venom composition, and especially the mechanism to hoard prey in comatose states for meeting their extremely high metabolic rates. A toxin (BQTX) was identified from venomous submaxillary glands of the shrew Blarinella quadraticauda. BQTX is specifically distributed and highly concentrated (~ 1% total protein) in the organs. BQTX shares structural and functional similarities to toxins from snakes, wasps and snails, suggesting an evolutional relevancy of venoms from mammalians and non-mammalians. By potentiating thrombin and factor-XIIa and inhibiting plasmin, BQTX induces acute hypertension, blood coagulation and hypokinesia. It also shows strong analgesic function by inhibiting elastase. Notably, the toxin keeps high plasma stability with a 16-h half-life in-vivo, which likely extends intoxication to paralyze or immobilize prey hoarded fresh for later consumption and maximize foraging profit., (© 2022. The Author(s), under exclusive licence to Springer Nature Switzerland AG.)

Epidemiologic studies have shown that sleep disorders are associated with the development of hypertension. The present study investigated dynamic changes in sleep patterns during the development of hypertension across the lifespan in spontaneously hypertensive rats (SHRs) and the neural mechanism that underlies these comorbidities, with a focus on the orexinergic system. Blood pressure in rats was measured using a noninvasive blood pressure tail cuff. Sleep was monitored by electroencephalographic and electromyographic recordings. Immunohistochemistry was used to detect the density and activity of orexinergic neurons in the perifornical nucleus. Hcrt2-SAP (400 or 800 ng) was microinjected in the lateral hypothalamus to lesion orexinergic neurons. Compared with Wistar-Kyoto rats, SHRs exhibited various patterns of sleep disturbances. In SHRs, dynamic changes in hypersomnia in the rats' active phase was not synchronized with the development of hypertension, but hyperarousal in the inactive phase and difficulties in falling asleep were observed concurrently with the development of hypertension. Furthermore, the density and activity of orexinergic neurons in the perifornical nucleus were significantly higher in SHRs than in age-matched Wistar-Kyoto rats. The reduction of orexinergic neurons in the lateral hypothalamus partially ameliorated the development of hypertension and prevented difficulties in falling asleep in SHRs. These results indicate that although the correlation between sleep disturbances and hypertension is very complex, common mechanisms may underlie these comorbidities in SHRs. Overactivity of the orexin system may be one such common mechanism., Competing Interests: Declaration of Competing Interest The authors declare no conflicts of interest., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Lysolipid-containing thermosensitive liposomes (LTSL) are clinically-relevant drug nanocarriers which have been used to deliver small molecule cytostatics to tumors in combination with local hyperthermia (42 °C) to trigger local drug release. The objective of this study was to investigate the feasibility of LTSL for encapsulation and triggered release of macromolecular drugs such as plant-derived cytotoxins. As therapeutic protein we used Mistletoe lectin-1 (ML1) - a ribosome-inactivating protein with potent cytotoxic activity in tumor cells. Model macromolecules (dextrans, albumin) and ML1 were encapsulated in small unilamellar LTSL with varying lipid compositions by the thin film hydration method and extrusion. LTSLs showed molecular weight dependent heat-triggered release of the loaded cargo. The most promising composition, ML1 formulated in LTSL composed of 86:10:4 %mol DPPC:MSPC:DSPE-PEG 2000 , was further studied for bioactivity against murine CT26 colon carcinoma cells. Confocal live-cell imaging showed uptake of released ML1 after mild hyperthermia at 42 °C, subsequently leading to potent cytotoxicity by LTSL-ML1. Our study shows that LTSL in combination with localized hyperthermia hold promise as local tumor delivery strategy for macromolecular cytotoxins., (Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.)

Due to the limitations and safety issues of the two currently approved live attenuated rotavirus (RV) vaccines "RotaTeq and Rotarix," studies on nonreplicating sources of RV vaccines and search for proper RV antigens are actively carried out. The adjuvant activity of NSP4 and highly immunogenic properties of RV VP6 protein prompted us to consider the construction of a NSP4 112-175 -VP6 fusion protein and to assess the anti-VP6 IgG, IgA, and IgG subclass responses induced by Escherichia coli-derived NSP4-VP6 fusion protein compared to that of VP6 protein with/without formulation in Montanide ISA 50V2 (M50) in BALB/c mice. Results indicated to the proper expression of the fused NSP4-VP6 and VP6 proteins in E. coli. Intraperitoneal immunization by M50 formulated NSP4-VP6 fusion protein (M5+NSP4-VP6) induced the highest titration of VP6-specific IgG and IgA responses compared to the other groups. Indeed, the presence of NSP4 resulted to the induction of stronger humoral immune responses against the fused protein compared to that elicited by administration of VP6 protein alone (with/without M50 formulation), implying the adjuvant properties of NSP4 for the fused protein. Moreover, the "M50+NSP4-VP6" formulation induced higher serum IgG2a titers than IgG1 and increased Interferon-γ levels, despite unchanged interleukin-4 amounts compared to other groups, indicating Th1-oriented responses with a possible role of NSP4. In conclusion, this study further highlights the potentiality of NSP4-VP6 fusion protein as an efficient and cost-effective immunogen in the field of RV vaccine development.

Animals using toxic peptides and proteins for predation or defense typically depend on specialized morphological structures, like fangs, spines, or a stinger, for effective intoxication. Here we show that amphibian poisons instead incorporate their own molecular system for toxin delivery to attacking predators. Skin-secreted peptides, generally considered part of the amphibian immune system, permeabilize oral epithelial tissue and enable fast access of cosecreted toxins to the predator's bloodstream and organs. This absorption-enhancing system exists in at least three distantly related frog lineages and is likely to be a widespread adaptation, determining the outcome of predator-prey encounters in hundreds of species.

Viscumin (mistletoe lectin I, MLI) in concentrations of 10 -11 -10 -7 M causes endoplasmic reticulum stress and triggers unfolded protein response, a modulator of antitumor immunity, in target cells.

Some patients use complementary medicine. We present a patient with Hodgkin's lymphoma, scanned with 18 F-FDG PET/CT for evaluation of response after chemotherapy, who was self-administering mistletoe as a homeopathic medicine product. The careful review of the images of the entire scan and patient collaboration in anamnesis were crucial to avoid a false positive result. A review of the published scientific data on the effects of mistletoe is also presented., (Copyright © 2016 Elsevier España, S.L.U. y SEMNIM. All rights reserved.)

The Annonaceous acetogenin annonacin is an environmental neurotoxin identified in the pulp of several fruits of the Annonaceae family, whose consumption was linked to the occurrence of sporadic atypical Parkinsonism with dementia. A method for its quantification in Rat brain homogenates by UPLC-MS/MS in selected reaction monitoring (SRM) mode was developed and validated. This method was applied to the quantitation of annonacin in Rat brain after intravenous (0.5 mg/kg) and oral (10 mg/kg, 100 mg/kg) administration., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Indoxyl sulfate and p-cresol sulfate have been suggested to induce kidney tissue remodeling. This study aimed to clarify the molecular mechanisms underlying this tissue remodeling using cultured human proximal renal tubular cells and half-nephrectomized mice treated with indoxyl sulfate or p-cresol sulfate as study models. Molecular docking results suggested that indoxyl sulfate and p-cresol sulfate dock on a putative interdomain pocket of the extracellular EGF receptor. In vitro spectrophotometric analysis revealed that the presence of a synthetic EGF receptor peptide significantly decreased the spectrophotometric absorption of indoxyl sulfate and p-cresol sulfate. In cultured cells, indoxyl sulfate and p-cresol sulfate activated the EGF receptor and downstream signaling by enhancing receptor dimerization, and increased expression of matrix metalloproteinases 2 and 9 in an EGF receptor-dependent manner. Treatment of mice with indoxyl sulfate or p-cresol sulfate significantly activated the renal EGF receptor and increased the tubulointerstitial expression of matrix metalloproteinases 2 and 9. In conclusion, indoxyl sulfate and p-cresol sulfate may induce kidney tissue remodeling through direct binding and activation of the renal EGF receptor., (Copyright © 2015 by the American Society of Nephrology.)

Interferon (IFN)-γ-driven and CD8+ T cell-dependent inflammatory injury to extrahepatic biliary epithelium (EHBE) is likely to be involved in the development of biliary atresia (BA). We previously showed that viral protein NSP4 is the pathogenic immunogen that causes biliary injury in BA. In this study, NSP4 or four synthetic NSP4 (NSP4(157-170), NSP4(144-152), NSP4(93-110), NSP4(24-32)) identified by computer analysis as candidate CD8+ T cell epitopes were injected into neonatal mice. The pathogenic NSP4 epitopes were confirmed by studying extrahepatic bile duct injury, IFN-γ release and CD8+ T cell response against EHBE. The results revealed, at 7 days postinjection, inoculation of glutathione S-transferase (GST)-NSP4 caused EHBE injury and BA in neonatal mice. At 7 or 14 days postinject, inoculation of GST-NSP4, NSP4(144-152), or NSP4(157-170) increased IFN-γ release by CD8+ T cells, elevated the population of hepatic memory CD8+ T cells, and augmented cytotoxicity of CD8+ T cells to rhesus rotavirus (RRV)-infected or naive EHBE cells. Furthermore, depletion of CD8+ T cells in mice abrogated the elevation of GST-NSP4-induced serum IFN-γ. Lastly, parenteral immunization of mouse dams with GST-NSP4, NSP4(144-152), or NSP4(157-170) decreased the incidence of RRV-induced BA in their offspring. Overall, this study reports the CD8+ T cell response against EHBE is activated by epitopes within rotavirus NSP4 in experimental BA. Neonatal passive immunization by maternal vaccination against NSP4(144-152) or NSP4(157-170) is effective in protecting neonates from developing RRV-related BA., (Copyright © 2014 the American Physiological Society.)

Purpose: Organic anion-transporting polypeptide (OATP) 1B1 and OATP1B3 contribute to hepatic uptake of numerous drugs. Thus, reduced OATP1B1 and OATP1B3 activity in chronic kidney disease (CKD) may have a major impact on the hepatic clearance of drugs. The effect of drug-uremic toxin interactions on OATP1B1 and OATP1B3 has not been well studied. In the present study, we examine the inhibitory effects of uremic toxins on OATP1B1 and OATP1B3 transport activity to evaluate the interactions between drugs and uremic toxins in patients with chronic kidney disease. METHODS. [3H]Estron-3-sulfate, [3H]taurocholate uptake and [3H]methotrexate by OATP1B1 and OATP1B3 expressing HEK293 cells were performed to evaluate the inhibitory effect of uremic toxins. To clarify whether the uremic toxins that interact with OATP1B1 and/or OATP1B3 were substrates for these transporters, we performed uptake studies. RESULTS. Four uremic toxins, kynurenic acid, indole-3-acetic acid, indoxyl sulfate, and p-cresol, inhibited OATP1B1- and OATP1B3-mediated transport in a concentration-dependent manner, with IC50 values of 180, 770, 2700, and 4600 µM, respectively, for OATP1B1 and 180, 1100, 1300, and 1700 µM, respectively, for OATP1B3. [3H]Methotrexate uptake by OATPs was also inhibited by the four uremic toxins in a dose-dependent manner. Uptake studies revealed that kynurenic acid is a substrate for both the OATP1B1 and OATP1B3. Moreover, OATP1B3 was involved in the transport of indoxyl sulfate. Indole-3-acetic acid and p-cresol were not significantly transported by OATP1B1 and OATP1B3. CONCLUSIONS. We showed that some uremic toxins inhibit OATP-mediated uptake in a concentration-dependent manner, and clarified OATPs contribution to uremic toxin handling in the liver. Thus, we provided basic information to estimate the inhibitory effects of uremic toxins on OATPs in CKD patients. These data suggest that the dose of drugs excreted via renal and non-renal pathways should be carefully adjusted in CKD patients.

Phase I oncology clinical trials are designed to identify the optimal dose that will be recommended for phase II trials. This dose is typically defined as the dose associated with a certain probability of severe toxicity during the first cycle of treatment, although toxicity is repeatedly measured over cycles on an ordinal scale. We propose a new adaptive dose-finding design using longitudinal measurements of ordinal toxic adverse events, with proportional odds mixed-effect models. Likelihood-based inference is implemented. The optimal dose is then the dose producing a target rate of severe toxicity per cycle. This model can also be used to identify cumulative or late toxicities. The performances of this approach were compared with those of the continual reassessment method in a simulation study. Operating characteristics were evaluated in terms of correct identification of the target dose, distribution of the doses allocated and power to detect trends in the risk of toxicities over time. This approach was also used to reanalyse data from a phase I oncology trial. Use of a proportional odds mixed-effect model appears to be feasible in phase I dose-finding trials, increases the ability of selecting the correct dose and provides a tool to detect cumulative effects., (Copyright © 2013 John Wiley & Sons, Ltd.)

Drug-induced liver injury (DILI) is a significant consideration for drug development. Current preclinical DILI assessment relying on histopathology and clinical chemistry has limitations in sensitivity and discordance with human. To gain insights on DILI pathogenesis and identify potential biomarkers for improved DILI detection, we performed untargeted metabolomic analyses on rats treated with thirteen known hepatotoxins causing various types of DILI: necrosis (acetaminophen, bendazac, cyclosporine A, carbon tetrachloride, ethionine), cholestasis (methapyrilene and naphthylisothiocyanate), steatosis (tetracycline and ticlopidine), and idiosyncratic (carbamazepine, chlorzoxasone, flutamide, and nimesulide) at two doses and two time points. Statistical analysis and pathway mapping of the nearly 1900 metabolites profiled in the plasma, urine, and liver revealed diverse time and dose dependent metabolic cascades leading to DILI by the hepatotoxins. The most consistent change induced by the hepatotoxins, detectable even at the early time point/low dose, was the significant elevations of a panel of bile acids in the plasma and urine, suggesting that DILI impaired hepatic bile acid uptake from the circulation. Furthermore, bile acid amidation in the hepatocytes was altered depending on the severity of the hepatotoxin-induced oxidative stress. The alteration of the bile acids was most evident by the necrosis and cholestasis hepatotoxins, with more subtle effects by the steatosis and idiosyncratic hepatotoxins. Taking together, our data suggest that the perturbation of bile acid homeostasis is an early event of DILI. Upon further validation, selected bile acids in the circulation could be potentially used as sensitive and early DILI preclinical biomarkers., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Acute lymphoblastic leukemia (ALL) is the most common malignancy affecting children and a major cause of mortality from hematopoietic malignancies in adults. A substantial number of patients become drug resistant during chemotherapy, necessitating the development of alternative modes of treatment. rGel (recombinant Gelonin)/BlyS (B-lymphocyte stimulator) is a toxin-cytokine fusion protein used for selective killing of malignant B-cells expressing receptors for B-cell-activating factor (BAFF/BLyS) by receptor-targeted delivery of the toxin, Gelonin. Here, we demonstrate that rGel/BLyS binds to ALL cells expressing BAFF receptor (BAFF-R) and upon internalization, it induces apoptosis of these cells and causes downregulation of survival genes even in the presence of stromal protection. Using an immunodeficient transplant model for human ALL, we show that rGel/BLyS prolongs survival of both Philadelphia chromosome-positive and negative ALL-bearing mice. Furthermore, we used AMD3100, a CXCR4 antagonist, to mobilize the leukemic cells protected in the bone marrow (BM) microenvironment and the combination with rGel/BLyS resulted in a significant reduction of the tumor load in the BM and complete eradication of ALL cells from the circulation. Thus, a combination treatment with the B-cell-specific fusion toxin rGel/BLyS and the mobilizing agent AMD3100 could be an effective alternative approach to chemotherapy for the treatment of primary and relapsed ALL.

This work describes the application of an impedance-based measurement for the real time evaluation of targeted tumor therapies in cell culture (HeLa cells). We used a treatment procedure that is well established in cells and mice. Therein, tumor cells are treated with a combination of an epidermal growth factor-based targeted toxin named SE and particular plant glycosides called saponins. In the present study HeLa cells were seeded in different numbers onto interdigitated electrode structures integrated into the bottom of a 96 well plate. The cells were treated with SE in the presence and absence of the saponin SpnS-1 (isolated from Saponaria officinalis roots). The impedance was directly correlated with the viability of the cells. As expected from known end point measurements, a concentration dependent enhancement of toxicity was observed; however, with the impedance measurement we were for the first time able to trace the temporal changes of cell death during the combination treatment. This substantially added to the understanding of initial cellular mechanisms in the augmentation of the toxicity of targeted toxins by saponins and indicated the superiority of real time monitoring over end point assays. The method is less labor intensive and label-free with ease of monitoring the effects at each time point., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Biological drugs are among the most exciting drugs of the future, offering better treatment options for patients than ever before but they need an appropriate delivery vehicle. Carrier erythrocytes are one of the most promising drug-delivery systems. Application of erythrocytes as containers for various drugs minimizes toxicity, decreasing the risk of side effects and pathologic immune reactions against encapsulated agents as well as improving their efficacy, leading to better patient compliance. This review discusses the rationale for the use of erythrocytes as a vehicle for biopharmaceuticals and summarizes the categories of these new encapsulable compounds that are currently under investigation. The authors' intent is to describe the development of this delivery system to give the reader an overview of the remarkable potential of erythrocytes as naturally designed carriers and their versatility in the field of biologics for the treatment of various pathological conditions.

Ricin, isolated from the castor bean plant Ricinus communis, is included on the Centers for Disease Control and Prevention Category B list of bioterrorism agents, indicating that the toxin is moderately easy to disseminate and could result in moderate morbidity rates. This study evaluated two promising recombinant ricin subunit vaccines, one made using an Escherichia coli codon-optimized gene and the other using a yeast codon-optimized gene in E. coli-based fermentations. Rabbits were vaccinated four times over a period of 6 months and challenged with ∼10 to 30 times the median lethal dose of aerosolized ricin. All unvaccinated control rabbits were either found dead or humanely euthanized within 30 h postchallenge, while the rabbits vaccinated with either vaccine survived the exposure without adverse clinical signs. When the protective antibody responses were analyzed, no significant difference was seen between the two vaccines. However, there was a significant difference in the immune response over time for both vaccines tested. Although clinical pathology was unremarkable, significant histological lesions in the control animals included fibrinonecrotic pneumonia, acute necrotizing lesions in the upper respiratory tract, and necrotizing lymphadenitis in the lymph nodes draining the upper and lower respiratory tract. Vaccine-treated rabbits exhibited resolving lesions associated with ricin exposure, namely chronic inflammation in the upper respiratory tract and lungs, fibrosis, type II pneumocyte hyperplasia, and bronchiolitis obliterans. This study confirmed the safety and efficacy of two recombinant ricin subunit vaccines in rabbits, offering potential protection to warfighters and select populations.

Background: Two hormetic modifications of a monotonically decreasing log-logistic dose-response function are most often used to model stimulatory effects of low dosages of a toxicant in plant biology. As just one of these empirical models is yet properly parameterized to allow inference about quantities of interest, this study contributes the parameterized functions for the second hormetic model and compares the estimates of effective dosages between both models based on 23 hormetic data sets. Based on this, the impact on effective dosage estimations was evaluated, especially in case of a substantially inferior fit by one of the two models., Methodology/principal Findings: The data sets evaluated described the hormetic responses of four different test plant species exposed to 15 different chemical stressors in two different experimental dose-response test designs. Out of the 23 data sets, one could not be described by any of the two models, 14 could be better described by one of the two models, and eight could be equally described by both models. In cases of misspecification by any of the two models, the differences between effective dosages estimates (0-1768%) greatly exceeded the differences observed when both models provided a satisfactory fit (0-26%). This suggests that the conclusions drawn depending on the model used may diverge considerably when using an improper hormetic model especially regarding effective dosages quantifying hormesis., Conclusions/significance: The study showed that hormetic dose responses can take on many shapes and that this diversity can not be captured by a single model without risking considerable misinterpretation. However, the two empirical models considered in this paper together provide a powerful means to model, prove, and now also to quantify a wide range of hormetic responses by reparameterization. Despite this, they should not be applied uncritically, but after statistical and graphical assessment of their adequacy.

Targeted toxins, also known as immunotoxins or cytotoxins, are recombinant molecules that specifically bind to cell surface receptors that are overexpressed in cancer and the toxin component kills the cell. These recombinant proteins consist of a specific antibody or ligand coupled to a protein toxin. The targeted toxins bind to a surface antigen or receptor overexpressed in tumors, such as the epidermal growth factor receptor or interleukin-13 receptor. The toxin part of the molecule in all clinically used toxins is modified from bacterial or plant toxins, fused to an antibody or carrier ligand. Targeted toxins are very effective against cancer cells resistant to radiation and chemotherapy. They are far more potent than any known chemotherapy drug. Targeted toxins have shown an acceptable profile of toxicity and safety in early clinical studies and have demonstrated evidence of a tumor response. Currently, clinical trials with some targeted toxins are complete and the final results are pending. This review summarizes the characteristics of targeted toxins and the key findings of the important clinical studies with targeted toxins in malignant brain tumor patients. Obstacles to successful treatment of malignant brain tumors include poor penetration into tumor masses, the immune response to the toxin component and cancer heterogeneity. Strategies to overcome these limitations are being pursued in the current generation of targeted toxins.

Aims: Indoxyl sulfate (IS) is a uraemic toxin found at high concentration in patients with chronic kidney disease (CKD) co-morbid with chronic heart failure (CHF). The aim of this study was to determine direct effects of IS on cardiac cells as well as the pro-inflammatory effect of IS., Methods and Results: Indoxyl sulfate significantly increased neonatal rat cardiac fibroblast collagen synthesis (by 145.7% vs. control, P < 0.05) and myocyte hypertrophy (by 134.5% vs. control, P < 0.001) as determined by (3)H-proline or (3)H-leucine incorporation, respectively. Indoxyl sulfate stimulated tumour necrosis factor-alpha, interleukin-6 (IL-6), and IL-1beta mRNA expression in THP-1 cells as quantified by RT-PCR. Both p38 (RWJ-67657) and MEK1/2 (U0126) inhibitors suppressed all these effects by IS. Furthermore, western blot analysis showed that IS activated mitogen-activated protein kinase (MAPK) (p38, p42/44) and nuclear factor-kappa B (NFkappaB) pathways. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay showed that IS exerted its effects without affecting cell viability., Conclusion: This study has, for the first time, demonstrated that IS has pro-fibrotic, pro-hypertrophic, and pro-inflammatory effects, indicating that IS might play an important role in adverse cardiac remodelling mediated via activation of the p38 MAPK, p42/44 MAPK, and NFkappaB pathways. Targeting reduction of IS and/or the pathways it activates may represent a novel therapeutic approach to the management of CHF with concomitant CKD.

Diffuse large B-cell lymphoma (DLBCL) is an aggressive subtype of B-cell non-Hodgkin lymphoma (NHL) and accounts for 30%to 40%of NHL. Molecules targeting nuclear factor-kappaB (NF-kappaB) are expected to be of therapeutic value in those tumors where NF-kappaB seems to play a unique survival role such as activated B-cell (ABC)-subtype DLBCL. We previously generated a rGel/BLyS fusion toxin for receptor-mediated delivery of the rGel toxin specifically to malignant B cells. In this study, we examined this fusion toxin for its ability to suppress DLBCL growth in vitro and in vivo. rGel/BLyS was specifically cytotoxic to DLBCL lines expressing all three BLyS receptors and constitutively active NF-kappaB. Treatment with rGel/BLyS induced down-regulation of the phosphorylation of inhibitory subunit of NF-kappaB (IkappaB-alpha), inhibition of NF-kappaB DNA-binding activity, and accumulation of IkappaB-alpha. In agreement with these results, we additionally found that rGel/BLyS downregulated levels of several NF-kappaB targets including Bcl-xL, Mcl-1, survivin, and x-chromosome linked inhibitor-of-apoptosis. Treatment also induced up-regulation of Bax and apoptosis through caspase-3 activation and poly ADP-ribose polymerase cleavage. Importantly, rGel/BLyS significantly inhibited tumor growth (P < .05) in a DLBCL xenograft model. Thus, our results indicate that rGel/BLyS is an excellent candidate for the treatment of aggressive NHLs that are both dependent on NF-kappaB and are resistant to conventional chemotherapeutic regimens.

Rotavirus nonstructural protein 4 (NSP4) is a protein with pleiotropic properties. It functions in rotavirus morphogenesis, pathogenesis, and is the first described viral enterotoxin. Since many bacterial toxins function as potent mucosal adjuvants, we evaluated whether baculovirus-expressed recombinant simian rotavirus SA11 NSP4 possesses adjuvant activity by co-administering NSP4 with keyhole limpet hemocyanin (KLH), tetanus toxoid (TT) or ovalbumin (OVA) as model antigens in mice. Following intranasal immunization, NSP4 significantly enhanced both systemic and mucosal immune responses to model immunogens, as compared to the control group, in an antigen-specific manner. Both full-length and a cleavage product of SA11 NSP4 had adjuvant activity, localizing this activity to the C-terminus of the protein. NSP4 forms from virulent and avirulent porcine rotavirus OSU strain, and SA11 NSP4 localized within a 2/6-virus-like particle (VLP) also exhibited adjuvant effects. These studies suggest that the rotavirus enterotoxin NSP4 can function as an adjuvant to enhance immune responses for a co-administered antigen., (Copyright 2010 Elsevier Ltd. All rights reserved.)

Measurements of ion channels are important for scientific, sensing and pharmaceutical applications. Reconstitution of ion channels into lipid vesicles and planar lipid bilayers for measurement at the single molecule level is a laborious and slow process incompatible with the high throughput methods and equipment used for sensing and drug discovery. A recently published method of lipid bilayer formation mechanically combines lipid monolayers self-assembled at the interfaces of aqueous and apolar phases. We have expanded on this method by vertically orienting these phases and using gravity as the driving force to combine the monolayers. As this method only requires fluid dispensation, it is trivially integrated with high throughput automated liquid-handling robotics. In a proof-of-concept demonstration, we created over 2200 lipid bilayers in 3h. We show single molecule measurements of technologically and physiologically relevant ion channels incorporated into lipid bilayers formed with this method.

Safety of aviscumine by subcutaneous route was assessed in patients with advanced cancer refractory to chemotherapy. Patients with progressive disease received escalating doses twice weekly. Treatment of the accrued 26 patients (10 colorectal cancer (CRC), 6 soft tissue sarcoma (STS), 5 melanoma (MM), 5 others) was well tolerated without substance-related grade 3 or 4 toxicities. Grade 1/2 toxicities were predominantly injection site reactions. Aviscumine lacked dose-limiting toxicity (DLT) up to a maximal dose of 10 ng/kg. An increase of interleukin-1 beta and interferon-gamma from baseline was seen in the patient's plasma between the 1st and 11th injection. Highest release of both cytokines was in the dose range of 4-5.9 ng/kg. Interferon-gamma was not detected after doses higher than 6 ng/kg. Eight patients (5 CRC, 1 MM, 1 STS, 1 RCC) had disease stabilisation for 79-250 days (median122 days) associated with an increase of interleukin (IL)-1 beta and interferon (IFN)-gamma. Aviscumine was well tolerated and appeared to possess clinical activity at a biologically active dose between 4 and 6 ng/kg.

This study investigates the effects of mistletoe lectin-I (ML-I) on melanoma growth and spread in vivo. The human melanoma cell line MV3 was xenografted into severe combined immunodeficient mice and vehicle solution or purified ML-I was administered at 30, 150 and 500 ng per kg body weight (20 mice per group) daily. After 19 days, mice were killed, primary tumours (PTs) and lungs were dissected out, and tumour weights, number of lung metastases (LMs), number of tumour-infiltrating dendritic cells (DCs), and apoptosis rates in the melanoma cells and in the DCs were assessed. A 35% reduction of PT weight (P=0.03) and a 55% decrease in number of LMs (P=0.016) were evident for low-dose ML-I (30 ng kg(-1)) treatment but not for higher doses. Mistletoe lectin-I increased apoptosis rates in the melanoma cells of PTs at all doses, while no induction of apoptosis was noted in the LMs. Low-dose ML-I significantly increased the number of DCs infiltrating the PTs (P<0.0001) and protected DCs against apoptosis, while higher doses induced apoptosis in the DCs (P<0.01). Our results demonstrate that low-dose ML-I reduced melanoma growth and number of metastases in vivo, primarily due to immunomodulatory effects.

Currently used targeted toxins are recombinant molecules specifically binding to surface receptors overexpressed on tumor cells. These recombinant proteins consist of a tumor-selective ligand coupled to a truncated peptide toxin. Ligands may bind to tumor-associated molecules with receptor signaling properties, such as epidermal growth factor receptor, transferrin receptor, and interleukin-13 or interleukin-4 receptors. The toxin part of the molecule in all clinically used toxins is a modified bacterial polypeptide fused to one of the above ligands. Targeted toxins are very effective against tumor cells resistant to radiation and chemotherapy. They are far more potent than any known chemotherapy drug. Targeted toxins have shown an acceptable profile of toxicity and safety in early clinical studies and have demonstrated some evidence for tumor response. Currently, phase 3 trials with some targeted toxins are underway and final results are still pending. This review summarizes the study protocols and key findings of the most important clinical studies with targeted toxins in malignant glioma patients. It offers in addition an outlook into future areas of development of targeted toxins, such as improved delivery modes and non-invasive imaging of toxin distribution.

The central anti-nociception of BmK IT2, a sodium channel modulator from scorpion Buthus martensi Karsh (BmK) was investigated in this study. It was found that the formalin-induced rat spontaneous flinches and spinal c-Fos expression could be significantly suppressed by intrathecal BmK IT2 pre- or post-formalin injection in a dose-dependent manner. The time course of inhibitory effect exerted by intrathecal BmK IT2 on spontaneous flinches was longer in the pre-treatment group than in post-treatment group. This was consistent with the stronger suppression on spinal c-Fos expression exerted by intrathecal BmK IT2 pre-treatment. In addition, the suppression by intrathecal BmK IT2 on formalin-induced c-Fos expression in superficial laminae was more significant than that in deeper laminae. These results indicate that BmK IT2 can induce central anti-nociceptive response and might thus be a valuable molecular tool for the understanding of pain mechanisms.

Urinary metabolites of tobacco smoke toxins are often used as biomarkers for the evaluation of active and passive exposure to cigarette smoke toxins. In a study of healthy smokers, we investigated concentrations of urinary biomarkers in relation to concentrations of selected toxins in mainstream cigarette smoke as determined by machine smoking of cigarettes in a manner that mimics an individual's smoking behavior (topography). Concentrations of nicotine, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone, and benzo(a)pyrene, in mainstream smoke determined under human smoking conditions, and their urinary metabolites cotinine, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol, and 1-hydroxypyrene were established for 257 individuals who smoked low-yield (0.1-0.8 mg Federal Trade Commission nicotine/cigarette; mean, 0.66; n = 87), medium-yield (0.9-1.2 mg nicotine/cigarette; mean, 1.1; n = 109), and high-yield cigarettes (nicotine, >1.3 mg nicotine/cigarette; mean, 1.41; n = 61). Levels of urinary metabolites expressed per unit of delivered parent compounds decreased with increased smoke emissions. In smokers of low-, medium-, and high-yield cigarettes, the respective cotinine (ng/mg creatinine)-to-nicotine (mg/d) ratios were 89.4, 77.8, and 57.1 (low versus high; P = 0.06); the 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (pmol/mg creatinine)-to-4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (ng/d) ratios were 0.81, 0.55, and 0.57 (low versus high; P = 0.05); and the 1-hydroxypyrene (pg/mg creatinine)-to-benzo(a)pyrene (ng/d) ratios were 1.55, 1.13, and 0.97 (low versus high; P = 0.008). Similarly, means of cotinine per unit of delivered nicotine in smokers who consumed <20 cigarettes per day was 3.5-fold higher than in those who smoked >20 cigarettes per day. Likewise, a negative correlation was observed between cotinine-to-nicotine ratios and delivered doses of nicotine in subgroups of smokers who used the identical brand of cigarette, namely a filter tip-vented Marlboro (r = -0.59), which is a popular brand among Euro-Americans, and Newport (r = -0.37), a menthol-flavored cigarette without filter tip vents that is preferred by African-Americans. Thus, the intensity of the exposures significantly affects the levels of urinary biomarkers of exposure and should be taken into account in the evaluation of human exposure to cigarette smoke toxins.

Background: The NFkappaB signalling pathway plays an important role in chemoresistance and decreased apoptosis. One indirect way to inhibit the NFkappaB pathway is to slow down the proteasomal degradation of its inhibitor IkappaB, thus preventing NFkappaB from translocation into the nucleus. Hence, the effect of the proteasome inhibitor bortezomib (Velcade) on the cell proliferation of the MV3, FemX-1 and G361 human melanoma cell lines and its action in combination with the PPAR-gamma agonist rosiglitazone or the mistletoe lectin ML-I, both having anti-proliferative effects on melanoma cells in single agent use, was investigated., Materials and Methods: Proliferation of melanoma cells under the different treatment regimes over a broad concentration range (0.0001-100 microg/ml) was assessed by means of the XTT cell proliferation assay., Results: At a concentration of 0.1 microg/ml bortezomib significantly reduced the proliferation rate of all melanoma cells to 1-13% of the control, which was mediated through increased apoptosis and inhibition of NFkappaB expression. Furthermore, the combination of bortezomib and rosiglitazone was the most potent and increased the effectiveness against melanoma cell growth by 63-71% (compared to single use of rosiglitazone) and by 27-39% (compared to single use of bortezomib), respectively., Conclusion: This combination strategy might be a promising approach for future melanoma therapy.

Traditional anti-cancer treatments consist of chemotherapeutic drugs that effectively eliminate rapidly dividing tumor cells. However, in many cases chemotherapy fails to eliminate the tumor and even when chemotherapy is successful, its systemic cytotoxicity often results in detrimental side effects. To overcome these problems, many laboratories have focused on the design of novel therapies that exhibit tumor specific toxicity. The transferrin receptor (TfR), a cell membrane-associated glycoprotein involved in iron homeostasis and cell growth, has been explored as a target to deliver therapeutics into cancer cells due to its increased expression on malignant cells, accessibility on the cell surface, and constitutive endocytosis. The TfR can be targeted by direct interaction with conjugates of its ligand transferrin (Tf) or by monoclonal antibodies specific for the TfR. In this review we summarize the strategies of targeting the TfR in order to deliver therapeutic agents into tumor cells by receptor-mediated endocytosis.

Many clinicians often do not feel comfortable with the Continual Reassessment Method (CRM). This article reviews its implementation, showing the characteristics, advantages and limitations of this method in Phase I studies as an alternative to the classical 'Fibonacci' escalation schema. A two center, dose escalation phase I study of rViscumin was carried out. Thirty-seven patients were included at 14 different dose-levels (10 to 6400 ng/kg). The complete clinical results are presented elsewhere. A 2-step CRM design enables one to speed-up the study and most importantly to obtain an accurate estimate of the maximum tolerated dose (MTD). Different management issues related to a multicenter study are illustrated and we show how the method can go wrong when severe toxicity, or dose limiting toxicity (DLT), is not considered by the clinician as being sufficient to limit dose escalation (here a grade 3 asthenia related to the drug). This would have affected any dose finding methods. We believe that CRM is a good alternative to the standard method from both a statistical and a practical point of view but further methodological research is necessary to address the issues related to the composite nature of the endpoint.

In grazing systems, forage availability is a function of herbivore density, which can influence an animal's ability to be selective. In turn, the influence of food availability on selectivity has the potential to influence plant biodiversity. We hypothesized that the ability of herbivores to mix toxin-containing foods in their diets is a function of the availability of nontoxic foods and the nutritional characteristics of the toxin-containing foods. We evaluated this hypothesis in two trials simulating different diet qualities (high-quality foods in trial 1, low-quality foods in trial 2). Within each trial, the four treatment groups were offered with different amounts of nutritious, familiar foods-10, 30, 50, and 70% of ad libitum intake-but were offered with ad libitum access to toxin-containing foods. Each lamb was presented with five foods, including three toxin-containing unfamiliar foods (terpenes, tannins, and oxalates) and two nutritious familiar foods (alfalfa and barley). In trial 1, as the availability of nutritious familiar foods decreased, animals ate more of all three toxin-containing foods. As the availability of nutritious alternatives increased, the pattern of selection shifted from terpenes to tannins and oxalates. In trial 2, animals also ate more toxins as the availability of nutritious alternatives decreased, but their pattern of diet mixing changed. Low availability of nutritious alternatives resulted in the animals eating more oxalates. During preference tests when all five foods were offered ad libitum, animals fed with 10, 30, 50, and 70% of ad libitum intake from trial 1 ate 49, 47, 41, and 38% of the three toxin-containing foods, respectively. The lower diet quality in trial 2 affected intake of the toxin-containing foods such that animals fed with 10, 30, 50, and 70% of ad libitum intake ate 37, 36, 29, and 27%, respectively, of the three toxin-containing foods. Thus, the quality of toxin-containing foods and the availability of nutritious alternatives interacted to modify the pattern of diet mixing by lambs.

The objective of this randomised, multicentre, double-blind clinical trial was to investigate the impact of PS76A2, an aqueous mistletoe extract standardised to mistletoe lectins, on quality of life (QoL) in breast cancer patients. A total of 352 patients were randomly allocated to 2 groups receiving PS76A2 (15 ng mistletoe lectin/0.5 ml) or matching placebo twice weekly for 4 to 6 cycles of CMF (cyclophosphamide, methotrexate, fluorouracil) chemotherapy followed by 2 months follow-up. The primary efficacy end-point was the change from baseline of 3 FACT-G subscales (physical, emotional and functional well-being) during the fourth CMF cycle. Secondary measures included GLQ-8 (8 linear analogue self-assessment scales), Spitzer's uniscale and haematological variables. The main variables of safety analysis were adverse events, including injection site reactions and clinical laboratory tests. The results showed that physical, emotional and functional well-being improved upon PS76A2, but deteriorated following placebo. The treatment differences were statistically significant for the 3 subscales as well as for the summary score FACT-G, which was analysed as O'Brien's rank sum of its 3 subscales: The total score increased by 4.40 +/- 11.28, indicating a higher QoL after PS76A2, but decreased by 5.11 +/- 11.77 with placebo (p<0.0001). The GLQ-8 sum of 8 LASA scales was analysed as a summary score of GLQ-5 (sum of item nos. 1, 5, 6, 7, 8) and GLQ-3 (sum of item nos. 2, 3, 4). GLQ-5 characterises typical aspects of QoL, while GLQ-3 consists of 3 side-effects of CMF (feeling sick, numbness or pins and needles, loss of hair). GLQ-5 decreased by 42.9 +/- 125.0 upon PS76A2, indicating an improvement in QoL, but increased by 60.3 +/- 94.0 upon placebo (p<0.0001). GLQ-3 deteriorated in both groups (PS76A2: 13.9 +/- 52.4; placebo: 34.5 +/- 57.0), but the differences in favour of PS76A2 were, nevertheless, statistically significant (p=0.0007). The total score GLQ-8 improved by 28.9 +/- 154.6 after PS76A2 and deteriorated by 94.8 +/- 141.1 after placebo (p<0.0001). Spitzer's uniscale improved by 12.2 +/- 30.7 upon PS76A2 and deteriorated by 10.8 +/- 26.1 with placebo (p<0.0001). After follow-up without chemotherapy, a significant treatment difference in favour of PS76A2 was determined by means of FACT-G, GLQ-8 and Spitzer's uniscale. PS76A2 was well tolerated in this trial, with the exception of slight local reactions in 17.6% of the PS76A2 group. In conclusion, PS76A2 (15 ng mistletoe lectin/0.5 ml twice weekly) was shown to be safe and effective in improving QoL in breast cancer patients during chemotherapy and follow-up.

The castor-oil plant Ricinus communis A-B dimeric toxin B subunit (RTB) was genetically linked at its N-terminus with a 90 amino acid peptide from simian rotavirus SA-11 non-structural protein NSP4(90) and produced in Escherichia coli BL21 cells. Biologically active recombinant NSP4(90)-RTB fusion protein was shown to bind glycoprotein asialofetuin receptor molecules in an in vitro enzyme-linked immunosorbent assay (ELISA). Oral inoculation of the purified NSP4(90)-RTB ligand-antigen fusion protein delivered the chimeric protein to intestinal epidermal cells for mucosal immunization against rotavirus infection. Mice fed the NSP4(90)-RTB fusion protein generated higher humoral and intestinal antibody titers than mice inoculated with NSP4(90) alone. Titers of serum IgG2a antibodies were significantly higher than IgG1 titers suggesting a dominant Th1 lymphocyte immune response. ELISA measurement of cytokines secreted from splenocyte isolated from immunized mice confirmed NSP4(90)-RTB fusion protein stimulates a strong Th1 cell-mediated immune response. The experimental results demonstrate that the ricin toxin B subunit N-terminus can be used as a site for delivery of virus antigens to the gut associated lymphoid tissues for RTB-mediated immune stimulation of antiviral mucosal immune responses.

The neuropeptide hypocretin, also known as orexin, has been implicated in waking since its deletion leads to the sleep disorder narcolepsy. Hypocretin neurons project to major arousal areas, and in an effort to determine which region is responsible for the changes in sleep-wake architecture we have developed the neurotoxin hypocretin2-saporin, which lesions hypocretin receptor bearing neurons. Here, in rats, we investigate the effects of hypocretin2-saporin lesions of the substantia nigra and ventral tegmental area in the regulation of sleep and wakefulness. Bilateral injection of hypocretin2-sap into both the ventral tegmental area and substantia nigra (92 and 184 ng/microl, 0.25 microl in the ventral tegmental area and 0.5 microl in the substantia nigra) or into the substantia nigra alone (184 ng/microl, 0.5 microl) produced insomnia. The insomnia seemed to be associated with a large increase in locomotion on days 4 and 6 postinjection, as hyperactivity and stereotypic movements were consistently observed on the video recordings in all lesioned rats. In these rats, a nearly complete loss of both tyrosine hydroxylase and neuron-specific nuclear protein (neuronal nuclei) immunoreactive cells in the substantia nigra as well as diminution of tyrosine hydroxylase-immunoreactive fibers in the caudate putamen was found. Following bilateral injection of hypocretin2-sap at a lower concentration (46 ng/microl, 0.25 microl in the ventral tegmental area and 0.5 microl in the substantia nigra), very little reduction in the number of tyrosine hydroxylase- and neuronal nuclei-immunoreactive neurons and only a temporary increase in wakefulness (17.4% increase during light-off period on day 6 postinjection) were observed. Ventral tegmental area lesions (184 ng/mul of hypocretin2-sap, 0.25 microl, bilateral injections) did not produce significant changes in sleep, although most of the tyrosine hydroxylase- and neuronal nuclei-immunoreactive neurons in the ventral tegmental area were destroyed. Insomnia following hypocretin2-sap lesions of the substantia nigra could be secondary to increased motor activity resulting from reduction of tonic inhibitory control by the substantia nigra.

A fusion protein containing the shiga toxin-1 B subunit (STB) linked to a 90 amino acid peptide (aa residues 86--175) from simian rotavirus (SA--11) nonstructural protein NSP4 was synthesized in Escherichia coli. Mice orally inoculated with 60 microg of STB::NSP4(90) fusion protein per dose generated higher humoral and intestinal antibody titers than mice inoculated with 30 microg of NSP4 alone. Serum anti-NSP4 IgG2a isotype titers were substantially greater than IgG1 titers, suggesting a dominant Th1 immune response. ELISA measurement of cytokines secreted from splenocytes isolated from immunized mice confirmed the STB::NSP4(90) fusion protein stimulation of a strong Th1 cell mediated immune response. Diarrhea in SA-11 rotavirus challenged neonates suckling from STB::NSP4 immunized dams was significantly reduced in severity and duration in comparison with virus challenged neonates from unimmunized mice. Together, our experiments demonstrate for the first time that the shiga toxin B subunit provides ligand mediated delivery of virus antigens to the gut-associated lymphoid tissues for enhanced stimulation of humoral and cellular responses against rotavirus gastroenteritis.

Folate targeted drug delivery has emerged as an alternative therapy for the treatment and imaging of many cancers and inflammatory diseases. Due to its small molecular size and high binding affinity for cell surface folate receptors (FR), folate conjugates have the ability to deliver a variety of molecular complexes to pathologic cells without causing harm to normal tissues. Complexes that have been successfully delivered to FR expressing cells, to date, include protein toxins, immune stimulants, chemotherapeutic agents, liposomes, nanoparticles, and imaging agents. This review will summarize the applications of folic acid as a targeting ligand and highlight the various methods being developed for delivery of therapeutic and imaging agents to FR-expressing cells., (Copyright (c) 2005 Wiley-Liss, Inc. and the American Pharmacists Association)

Voltage gated potassium channels open and inactivate in response to changes of the voltage across the membrane. After removal of the fast N-type inactivation, voltage gated Shaker K-channels (Shaker-IR) are still able to inactivate through a poorly understood closure of the ion conduction pore. This, usually slower, inactivation shares with binding of pore occluding peptide toxin two important features: i), both are sensitive to the occupancy of the pore by permeant ions or tetraethylammonium, and ii), both are critically affected by point mutations in the external vestibule. Thus, mutual interference between these two processes is expected. To explore the extent of the conformational change involved in Shaker slow inactivation, we estimated the energetic impact of such interference. We used kappa-conotoxin-PVIIA (kappa-PVIIA) and charybdotoxin (CTX) peptides that occlude the pore of Shaker K-channels with a simple 1:1 stoichiometry and with kinetics 100-fold faster than that of slow inactivation. Because inactivation appears functionally different between outside-out patches and whole oocytes, we also compared the toxin effect on inactivation with these two techniques. Surprisingly, the rate of macroscopic inactivation and the rate of recovery, regardless of the technique used, were toxin insensitive. We also found that the fraction of inactivated channels at equilibrium remained unchanged at saturating kappa-PVIIA. This lack of interference with toxin suggests that during slow inactivation the toxin receptor site remains unaffected, placing a strong geometry-conservative constraint on the possible structural configurations of a slow inactivated K-channel. Such a constraint could be fulfilled by a concerted rotation of the external vestibule.

Aviscumine is a ribosome-inactivating protein with potent antitumour activity in vitro and in vivo and is an Escherichia coli-derived recombinant counterpart of natural mistletoe lectin-I. The current study was performed to determine the safety profile, dose-limiting toxicity (DLT) and maximum tolerated dose (MTD) of a prolonged infusion of aviscumine in cancer patients. Aviscumine was given once weekly as a 24 h central intravenous infusion in patients with advanced, refractory progressive solid malignant tumours. Fourteen fully eligible patients (11 male, 3 female) with a median age 58 yrs (range 41-77) were enrolled. They had histologically verified disease, were 18 yrs old, had an ECOG PS 2 and adequate bone marrow, liver and renal function. DLT was defined as any non-haematological grade 3-4 toxicity (Common Toxicity Criteria [CTC] version 2.0), neutrophil count <500/ microl for 7 days, febrile neutropenia or thrombocytopenia grade 4. The MTD was defined as the dose level below the dose at which 2 patients per dose level experienced a DLT during the first treatment cycle. Colorectal cancer, soft tissue sarcoma and pancreatic cancer were the most common tumour types. Dose levels of aviscumine ranged from 4 to 6 microg/kg. The median number of cycles was 2.8 (range, 2-8). Common side effects in cycle 1 were fatigue, fever, nocturia, urticaria, erythema and pruritus. DLTs occurred in 2/3 patients on the 6 microg/kg dose level and consisted of increases in ASAT grade 3, ALAT grade 3, gammaGT grade 3/4, hypokalemia grade 3 and fatigue grade 3. No DLTs were observed on dose levels 4 and 5 microg/kg. The best response (RECIST) was stable disease in 4 pts, lasting for 4-8 cycles. Pharmacokinetics indicated that potentially active plasma levels of the compound were maintained during the entire infusion. We conclude that the recommended dose for weekly 24 h infusions of Aviscumine should be 5 microg/kg.

Background: In single case observations, tumour remissions after intratumoral injections of mistletoe extracts have been described., Materials and Methods: We investigated the antitumour activity of intratumorally (i.t.)-injected lectin-rich mistletoe extract at different dosages and i.t.-injected mistletoe lectin I in comparison to intravenous (i.v.) Gemcitabine and i.t. treatment with placebo in a human pancreatic cancer xenograft., Results: In a preliminary dose-response experiment, the most marked tumour inhibition was induced when mistletoe extract was given at 8 mg/kg body weight (BW) and mistletoe lectin I at 5.3 microg/kg BW. In a second experiment, bi-weekly i.t. injections of mistletoe extract over 8 weeks resulted in a very high antitumour activity with an optimal T/C value (=median relative tumour volume of the test group vs. the control) of 0.4% combined with 3/8 partial and 3/8 complete remissions. Gemcitabine was less active with 2/8 partial and 1/8 complete remissions and an optimal TIC of 4.6%., Conclusion: I.t.-injected lectin-rich mistletoe extract should be further evaluated in patients with inoperable locally advanced pancreatic cancer.

The utilisation of nonviral gene delivery methods has been increasing steadily, however, a drawback has been the relative low efficiency of gene transfer with naked DNA compared with viral delivery methods. In vivo electroporation, which has previously been used clinically to deliver chemotherapeutic agents, also enhances the delivery of plasmid DNA and has been used to deliver plasmids to several tissue types, particularly muscle and tumour. Recently, a large number of preclinical studies for a variety of therapeutic modalities have demonstrated the potential of electrically mediated gene transfer. Although clinical trials using gene transfer with in vivo electroporation have not as yet been realised, the tremendous growth of this technology suggests that the first trials will soon be initiated.

Background: Aviscumine is an Escherichia coli-derived recombinant type II ribosome-inactivating protein with potent antitumor activity in vitro and in vivo. It is the recombinant counterpart of natural mistletoe lectin-I. The current study was performed to determine the safety profile, dose-limiting toxicity (DLT) and maximum tolerated dose (MTD) of the intravenous (i.v.) administration of aviscumine in cancer patients. Translational research included the evaluation of pharmacokinetics and monitoring of plasma cytokine and anti-aviscumine antibody induction after administration of the drug., Patients and Methods: Aviscumine was given twice weekly as a 1 h central i.v. infusion in patients with advanced, refractory progressive, solid malignant tumors who had not been previously exposed to natural mistletoe preparations. They had histologically or cytologically verified disease, were > or =18 years old, had an Eastern Cooperative Oncology Group performance status < or =2 and adequate bone marrow, liver and renal function. DLT was defined as any non-hematological grade 3-4 toxicity (National Cancer Institute Common Toxicity Criteria version 2.0), neutrophil count <500/microl for > or =7 days, febrile neutropenia or thrombocytopenia grade 4. The MTD was defined as the dose at which >20% of patients experienced DLT during the first treatment cycle. The Continual Reassessment Method was used to determine the number of patients required per dose level., Results: Forty-one fully eligible patients (19 male, 22 female) with a median age of 56 years (range 37-74) were enrolled. Colorectal, ovarian, renal cell and breast cancer were the most common tumor types. Dose levels of aviscumine ranged from 10 to 6400 ng/kg. The median number of cycles was two (range one to eight). Common clinical toxicities in cycle 1 were fatigue, fever, nausea, vomiting and allergic reactions. Fatigue grade 3 was dose limiting in one of six patients at 4000 ng/kg and reversible grade 3 liver toxicity (elevation in alkaline phosphatase, transaminases and/or gamma-glutamyltransferase) occurred in one of 10 patients at 4800 ng/kg and in two of five patients at 6400 ng/kg. The best response (RECIST criteria) was stable disease in 11 patients, lasting for two to eight cycles. The pharmacokinetic evaluation revealed a short alpha half-life of 13 min and linear kinetics on dose levels > or =1600 ng/kg. Aviscumine stimulated the immune system with a release of cytokines such as interleukin (IL)-1beta, IL-6 and interferon-gamma, and induced immunoglobulin (Ig) G- and/or IgM-anti-aviscumine antibodies of uncertain clinical relevance., Conclusions: The recommended dose for further clinical trials is 5600 ng/kg twice weekly. Based on the short half-life of the recombinant protein observed in this trial, the exploration of prolonged infusion schedules of aviscumine is warranted.

Patients with breast cancer receiving adjuvant chemotherapy frequently suffer from a restricted quality of life (QoL) due to the side-effects of chemotherapy and the consequences of coping with the diagnosis. Therefore, the objective of this clinical study was to investigate the impact of PS76A2, an aqueous mistletoe extract standardised to the galactoside-specific mistletoe lectin, on QoL by performing a placebo-controlled trial. Overall, 272 patients with breast cancer receiving adjuvant CMF chemotherapy (cyclophosphamide-methotrexate-fluorouracil) were enrolled and randomised to groups receiving placebo or PS76A2 at concentrations of 10, 30 or 70 ng mistletoe lectin (ML) per ml. The patients received 0.5 ml study medication twice weekly subcutaneously for 15 consecutive weeks (4 CMF cycles). Primary variables were the self-assessment QoL scores GLQ-8 (Global Life Quality) and Spitzer's uniscale. As a result, statistically significant effects on QoL were obtained with the medium dose (15 ng ML/0.5 ml). The treatment difference between the medium dose and placebo with regard to the GLQ-8 sum was 60.8 mm (95% confidence interval: 19.3 to 102.0 mm). The treatment effect for Spitzer's uniscale between the medium dose and placebo was 16.4 mm (95% confidence interval: 6.3 to 26.6 mm). The results on QoL were supported by an increase of T helper lymphocytes (CD4+) and the CD4+/CD8+ ratio (p<0.05). Overall, PS76A2 was well tolerated. Local reactions at the injection sites occurred dose-dependently, but were mild at the low and medium dose levels.

Hypercreatinuria is a well-known feature of liver and testicular toxicity and we have recently proposed that hepatotoxin-induced hypercreatinuria would arise as a consequence of increased cysteine synthesis associated with the provision of protective substances (glutathione and/or taurine). Here a direct relationship between hepatotoxin-induced hypercreatinaemia and hypercreatinuria is shown and the possible relationships of hepatotoxin-induced hypercreatinaemia and hypercreatinuria to hepatic damage and to weakened nutritional status are examined. Male Sprague-Dawley rats were dosed with a variety of model hepatotoxins at two dose levels per toxin. Blood plasma samples taken at 24 h post-dosing and urine samples collected from 24-31 h post-dosing were analysed by (1)H NMR spectroscopy. Both hypercreatinaemia and hypercreatinuria were found in rats dosed with allyl formate (75 mg/kg), chlorpromazine (30 and 60 mg/kg), alpha-naphthylisothiocyanate (ANIT, 100 mg/kg) and thioacetamide (200 mg/kg), whilst significant hypercreatinuria, but not hypercreatinaemia, was found after dosing with thioacetamide (50 mg/kg). Neither hypercreatinaemia nor hypercreatinuria were found after dosing with allyl formate (25 mg/kg), ethionine (300 and 1000 mg/kg) or ANIT (30 mg/kg). Reduced feeding is known to cause hypercreatinuria in rats and, of the four hepatotoxins that induced hypercreatinaemia and hypercreatinuria at the given time-points, two, chlorpromazine and ANIT, also affected nutritional status with ketosis being clearly identifiable from the plasma (1)H NMR spectra. Thus, the creatine changes induced by ANIT and chlorpromazine are potentially attributable, in whole or in part, to reduced feeding rather than to liver effects alone and, consequently, the results were examined with and without inclusion of the ANIT and chlorpromazine data. With all of the data included, there were eight out of ten points of correspondence between the incidence of hypercreatinaemia and/or hypercreatinuria and the incidence of increases in plasma alanine aminotransferase (ALT) activity. At the same time there were nine out of ten points of correspondence between the incidence of hypercreatinaemia and/or hypercreatinuria and the incidence of increases in plasma aspartate aminotransferase (AST) activity. However, with the ANIT and chlorpromazine data excluded there was complete (six out of six points) correspondence between the incidence of hypercreatinaemia and/or hypercreatinuria and the incidence of increases in plasma AST and ALT in the remaining data. Likewise, with all of the data included, there was some apparent correlation (correlation coefficient, r=0.80) between the group mean levels of plasma AST and plasma creatine when expressed relative to the mean values for controls sampled at the same time-point. However, with the ANIT and chlorpromazine data excluded, that correlation coefficient was increased to 0.95. The findings of these studies suggest that the ANIT- and chlorpromazine-induced creatine changes may have been caused by reduced feeding rather than by liver toxicity. The allyl formate and thioacetamide data indicate that hepatocellular necrosis is accompanied by increases in plasma and urinary creatine, and suggest the possibility of a quantitative relationship between the increases in plasma AST and the increases in plasma creatine that are associated with hepatocellular necrosis. The ethionine and ANIT data suggest that fatty liver (steatosis) and cholestatic damage may not be associated with hypercreatinaemia and hypercreatinuria.

Principal component analysis (PCA) has been applied to three nuclear magnetic resonance (NMR) spectral editing methods, namely, the Carr-Purcell-Meiboom-Gill spin-echo, diffusion editing, and skyline projection of a two-dimensional J-resolved spectrum, obtained from high-resolution magic-angle spinning NMR spectroscopy of liver tissues, to distinguish between control and hydrazine-treated rats. The effects of the toxin on rat liver biochemistry were directly observed and characterized by depleted levels of liver glycogen, choline, taurine, trimethylamine N-oxide, and glucose and by elevated levels of lipids and alanine. The highly unsaturated omega-3-type fatty acid was observed for the first time in hydrazine-treated rat liver. The contributions of the metabolites to the separation of control from dosed liver tissues varied depending on the type of spectral editing method used. We have shown that subtle changes in the metabolic profiles can be selectively amplified using a metabonomics approach based on the different NMR spectral editing techniques in conjunction with PCA.

Pulmonary delivery is an attractive alternative route to deliver protein drugs that are currently delivered by injection. Inhalation therapy via nebulizers is a well accepted way for pulmonary application of proteins considering the formulation difficulties of MDIs or DPIs. This research presents the effect of variable excipients on the stability and aerosol performance of freeze-dried aviscumine after reconstitution and nebulization. Aviscumine formulations containing 100 mmol/L Tris buffer, 0.1% (w/v) Polysorbate 80, 0.01% (w/v) Na(2)-EDTA and 8% (w/v) Hydroxyethyl starch have been lyophilized and reconstituted with a buffered isotonic solution pH 8. The aviscumine activity was determined by a binding assay directly after reconstitution and after nebulization with a PariBoy air-jet nebulizer, a Multisonic and a Systam ultrasonic nebulizer. The stabilization of aviscumine by the addition of variable buffer salts to the reconstitution medium, such as 50, 100, and 200 mmol/L Tris buffer, 20 and 100 mmol/L phosphate buffer, and 20 and 100 mmol/L Tricine buffer, was studied. About 50% of aviscumine activity was lost after 20 min nebulization time without any additives. Nevertheless, higher buffer concentrations confer greater stability. About 70% of the aviscumine activity could be retained by the addition 0.03% octanoyl-N-methylglucamide and 100 mmol/L Tricine to the reconstitution medium.

The pulmonary drug delivery of proteins present an alternative to parenteral and oral administration. Nebulization of aqueous protein solutions is an ideal method for pulmonary application of therapeutic proteins considering the difficulties of their formulation as MDIs or DPIs. This research presents the effect of variable excipients on the stability of freeze-dried aviscumine after reconstitution and nebulization. Formulations containing different lyoprotectants have been lyophilized and reconstituted with isotonic salt solution. The loss of aviscumine activity in the nebulizer reservoir and after nebulization with a PariBoy air-jet nebulizer, a Multisonic ultrasonic nebulizer and a Systam ultrasonic nebulizer was determined by a binding assay. The effect of variable lyoprotectants such as 8% (w/v) Dextran T1, HES130, HES450, HP-beta-CD and 6% (w/v) HES450 plus 2% (w/v) mannitol on the stability of aviscumine to air-jet and ultrasonic nebulization has been evaluated. Only 50% of aviscumine activity was retained after 20 min nebulization, where 8% (w/v) HES450 was shown to be the best stabilizer. Stabilization of aviscumine by the addition of variable surfactants as 0.01 and 0.1% (w/v) Poloxamer 188, 0.03 and 0.1% (w/v) PEG 8000, 0.03 and 0.1% (w/v) Solutol HS15 and 0.03 and 0.1% (w/v) octanoyl-N-methyl-glucamide to the reconstitution solution has also been studied. By the addition of 0.03% (w/v) octanoyl-N-methyl-glucamide, 70% of the activity was retained after 20 min nebulization.

Background: Many cancer patients use alternative therapies in addition to conventional treatment. In a survey among such patients, we assessed the prevalence of and the motivation for alternative therapy use in a rural area of Switzerland., Methods: From 1st February to 30th November 2001, we interviewed 108 patients treated in the oncology outpatient clinic of the Langenthal District General Hospital, Switzerland, using a structured questionnaire. 77% of the patients were female. 49% of the patients (i.e., 64% of the female patients) suffered from breast cancer., Results: 42 (39%) of all patients had used an alternative therapy in addition to conventional treatment at least once. Mistletoe preparations were by far the most popular with a prevalence of 74%. Homeopathy (24%) and cancer diets (12%) were used less often. 79% of the patients seeking help from alternative treatment informed their treating oncologist and/or medical practitioner accordingly. 57% of the doctors encouraged their patients to continue the alternative treatment, none discouraged the patient to do so. The main reasons for the use of alternative therapy were: the desire to feel more hopeful (83%); "to do as much as possible myself to cure the disease" (83%); and to harness mental energy (62%). Only 19% of the patients hoped to be cured of cancer by alternative therapy., Conclusions: The motivation to seek help from alternative treatment is not based on a distrust of conventional care. Maintaining hope and taking an active role in self-care are the main stimuli for using alternative medicine.

Constraints on rates of detoxification and elimination of plant toxins are thought to be responsible for limiting dietary specialization in mammalian herbivores. This hypothesis, known as the detoxification limitations hypothesis, suggests that most mammalian herbivores are generalists to avoid overdosing on toxins from a single plant species. The hypothesis also predicts that the few mammalian specialists that exist should have adaptations for rapid detoxification and elimination of plant secondary compounds. We took a pharmacological approach to test whether specialists eliminate toxins from the bloodstream faster than generalists. We compared elimination rate and total exposure of alpha-pinene in closely related dietary specialist and generalist woodrats, Neotoma stephensi and N. albigula, respectively. Animals were orally gavaged with alpha-pinene, a plant secondary compound present in the natural diets of both woodrat species. We collected venous blood at 3, 6, 10, 15, and 20 min post-ingestion of alpha-pinene. Blood was analyzed for alpha-pinene concentration using gas chromatography. We found that specialist and generalist woodrats did not differ in elimination rates of alpha-pinene. However, specialists had lower exposure levels of alpha-pinene than generalists due to lower initial delivery of alpha-pinene to the general circulation. The levels of alpha-pinene detected in the bloodstream of specialists were 4.7-5.3x lower over all time intervals than generalists. Thus, specialists encounter a functionally lower dose of toxin than generalists. We suggest that the lower exposure level of specialist woodrats may be due to mechanisms in the gut that decrease toxin absorption. Regardless of mechanism, lower exposure to plant toxins may allow specialists to forage on diets with high toxin concentrations thereby facilitating dietary specialization.

The centralisation and globalisation of the world food supply presents opportunities for massive epidemics of food borne illness. Food processing generally decreases exposure to naturally occurring toxicants. A significant number of deaths are caused in both developed and developing countries from the consumption of naturally occurring toxicants and this exceeds the numbers attributable to artificial food contaminants and pollutants. The risk posed by naturally occurring toxicants is greater in developing countries where food choice and facilities for food processing are limited. The main risk in developed countries is from food important from areas where food safety controls are inadequate. Toxins derived from marine algae present a specific threat to health especially as seafood is being traded from areas where poisoning by algal toxins is known to occur. A systematic approach to food safety using HACCP methodology and identity preservation of the food are crucial in the prevention of food related illness. There is also a need for international agreed standards for tolerable levels of naturally occurring toxicants in foodstuffs but these should derived not be set unreasonably low as this would threaten food security in developing countries. The occurrence of a number of fatalities in developing countries among people consuming herbal teas and dietary supplements underscores the need to assess the safety of these products before they are placed on the market.