Ehrlich II –2nd World Conference on Magic Bullets

Counteracting drug resistance by nutritional management and genetic improvement of disease resistance - lessons from in-silico studies for nematode infections of sheep
DOESCHL-WILSON AB¹, VAGENAS D¹, BISHOP SC², KYRIAZAKIS I^3
1Scottish Agric. College, Edinburgh, UK; ²Roslin Biocentre, Edinburgh, UK; ³Univ. of Thessaly, Karditsa, Greece
Background: Gastrointestinal parasitism (GIP) is a major challenge to animal production worldwide. Parasite resistance to drugs impedes the control of the disease and raises the need for alternative methods, such as host nutrition and breeding for resistance. Field study results on the efficacy of the two methods are however contradictory and give rise to much speculation. Aims: (1) to explore, in silico, the interactive effect of host genotype and nutrition on performance and GIP and relevant genetic parameters of sheep infected with nematode parasites. (2) to determine under what circumstances nutritional control and / or selective breeding become feasible alternatives to chemoprophylaxis.

Methods: A mechanistic model, describing the growth, nutrient utilization, development of immunity and levels of nematode infections in lambs challenged with GI nematodes was developed. Host genetic variation was assumed in the ability to grow and to resist GI parasites. Simulated breeds differing in growth and resistance genotypes, were infected daily with a trickle challenge of 3000 L3s of Teladorsagia circumcinta nematodes and given access to either good or poor quality grass.

Results: Mean values for production and resistance traits 50 days post infection are shown in the table. Means, heritabilities (fraction of variability due to genetic variation) and correlations for growth and resistance traits changed markedly over time, and were affected more by nutrition than by host genotype. Genetic parameter estimates from field studies were only reproduced when underlying growth and resistance mechanisms were genetically related.

Conclusions: Nutritional management and selective breeding are valuable alternatives to anthelmintics in the control of GIP, but their efficicacy depends on the interaction between host genotype and the nutritional environment.

Rituximab Maintenenance Therapy in CD20+ B-Cell Non-Hodgkin-Lymphoma – First results of a multicenter prospective randomised Phase II study WITZENS-HARIG M, HENSEL M, SCHMIER JW, NEBEN K, BENNER A, DREGER P, KUHN C, SCHMIDT-WOLF I, KRÄMER A, HO AD Clinical and pharmacokinetic data suggest that the effect of rituximab could be improved by prolonged exposure to the drug. To test for this hypothesis we performed a prospective randomized trial of rituximab maintenance therapy in patients with CD20+ B-cell Non-Hodgkins-Lymphoma. After completion of standard treatment patients were randomized to either observation or maintenance therapy with rituximab (375 mg/m^2) every 3 months for 2 years. Patients after first line therapy as well as relapse patients were included in the study. Patients with aggressive lymphoma were enrolled if they had achieved a complete response (CR) after initial treatment. Patients with aggressive lymphoma with residual tumor mass were examined with positrone emission tomography (PET) and qualified for randomization if PET showed no signs of tumor activity. Patients with indolent lymphoma qualified for the study if at least a partial response (PR) was achieved. After recruitment of 172 patients a planed interim analysis was performed. Complete data sets of 162 patients (pts) with CD20+ B-cell Non-Hodgkins-Lymphoma were evaluable for analysis. Histological subtypes included diffuse large cell lymphoma (69 pts), follicular lymphoma (41 pts), mantle cell lymphoma (18 pts), primary mediastinal lymphoma (15 pts), marginal zone lymphoma (9 pts), Burkitt’s lymphoma (3 pts), immunocytoma (2 pts), primary intestinal lymphoma (1 pt), hairy cell leukemia (1 pt), chronic lymphocytic leukemia (1 pt) and unclassified B-cell lymphoma (2 pts). The interim analysis showed that event free survival was significantly prolonged in the rituximab maintenance group compared to the observation group (p<0.05). However, no difference in overall survival between the two groups was observed so far. Two patients in the treatment group developed WHO grade III adverse events (1 leucopenia, 1 infection). Both pts recovered shortly after appropriate treatment. We conclude that rituximab maintenance therapy is feasable, safe and well tolerated in patients with CD20+ B-cell Non-Hodgkins-Lymphoma and may prolong event free survival in this patient population.
Reloading Ehrlich’s Magic Redox Bullets: Targeting the Redox Achilles Heel of Melanoma Using Phenothiazinium Dyes WONDRAK GT, TUCSON AZ University of Arizona, College of Pharmacy & Arizona Cancer Center, USA The use of redox dyes as vital stains and metabolic probes was pioneered by Paul Ehrlich, who associated their cytotoxicity with spontaneous autoxidation of the bio-reductively generated leuco-form. Altered redox signaling and regulation in cancer cells represent a chemical vulnerability that can be targeted by selective chemotherapeutic intervention. Here, we demonstrate that 3,7-diaminophenothiazinium-based redoxcyclers (PRC) including methylene blue and toluidine blue O induce selective cancer cell apoptosis by NAD(P)H:quinone oxidoreductase (NQO1)-dependent bioreductive generation of cellular oxidative stress. Using PRC lead compounds against human metastatic G361 melanoma cells, apoptosis occurred with phosphatidylserine-externalization, loss of mitochondrial transmembrane potential, cytochrome C release, caspase-3 activation, and massive ROS production. Consistent with reductive activation and subsequent redoxcycling as the mechanism of PRC cytotoxicity, co-incubation with catalase achieved cell protection, whereas reductive antioxidants enhanced PRC-cytotoxicity. In contrast, human A375 melanoma cells were resistant to PRC-induced apoptosis, and PRC-sensitive G361 cells were protected by preincubation with the NQO1-inhibitor dicoumarol. Indeed, NQO1 specific enzymatic activity was nine fold higher in G361 than in A375 cells. The critical role of NQO1 in PRC-bioactivation and cytotoxicity was confirmed, when NQO1-transfected breast cancer cells stably overexpressing active NQO1 displayed strongly enhanced PRC-sensitivity as compared to vector-control transfected cells with base line NQO1 activity. Based on pilot studies performed in mouse xenograft models and the known overexpression of NQO1 in various tumors including melanoma and lung cancer these findings suggest the feasibility of developing PRC lead compounds into tumor-selective bioreductive chemotherapeutics. Supported in part by grants from NIH (R01CA122484; ES06694) and ABRC (0721).	Applications of recombinant human epidermal growth factor in the treatment of hard-to-heal wounds WONG WKR Hong Kong University of Science and Technology, Department of Biochemistry, Clear Water Bay, Kowloon, Hong Kong Background: Human epidermal growth factor (hEGF) is a 53 amino acid polypeptide found in our duodenum and salivary glands. It is capable of stimulating cell proliferation and differentiation of various epidermal tissues and has been applied to promote the repair of duodenal ulcer, hepatic injury and eye damage. Our group at the Hong Kong University of Science and Technology has been interested in the production and applications of hEGF in cosmetic and skin care industries. In this presentation, the various skin care applications of hEGF will be discussed. Methods: Topical application of cream products supplemented with 0.02% (wt/wt) and 0.04% (wt/wt) of recombinant hEGF, which was produced by a proprietary Escherichia coli excretion system^1 (www.gene-vinate.com), onto skin wounds has been reported previously^2. Results: Our recent study^2 of using hEGF cream products to treat diabetic patients suffering from chronic ulcers revealed that 0.04% (wt/wt) hEGF caused more ulcers to heal over a 12-week period and it was 38% to 53% more effective than 0.02% (wt/wt) hEGF and the negative control, respectively, in healing diabetic foot ulcers. Further studies of the applications of hEGF in the treatment of other hard-to-heal wounds, including drug-induced Steven Johnson syndrome^3, scalded skin, surgical wounds, and psoriasis all resulted in an enhanced healing effect. The results support our view that topical application of hEGF, when administered at an effective dosage, may offer a simple and effective treatment to the management of various skin wounds. Conclusions: 1) Topical applicationof hEGF-containing cream to skin wounds helps enhance the healing effect and reduce the healing time. 2) Topical application of hEGF presents a simple and effective treatment to the management of a broad range of skin wounds. References: 1. Wong, W.K.R. et al. (2001) Biotechnology & Genetic Engineering Reviews, 18:51-71. 2. Tsang M.W. and Wong, W.K.R. et al. (2003) Diabetes Care, 26(6): 1856-61. 3. Tsang, M.W. and Wong, W.K.R. et al. (2004) Derm. Ontline J., 10 (1): Article 25.
Feasibility of Mapping Brain pH Using 31P MR Spectroscopy WU RH^1^,2, CHEN YW^1, LIU WW^1, WANG H^3, QIU QC^1, TERBRUGGE K^2, MIKULIS DJ^2 ^1Shantou University Medical College, Shantou, China; ^2University of Toronto, Toronto, Canada; ^3Southeast University, Nanjing, China Background: Magnetic resonance (MR) spectroscopy is a valuable method for the noninvasive investigation of metabolic processes. Although brain adenosinetriphosphate (ATP) studies can be found in multi-voxel ^31P MR spectroscopy, previous studies of intracellularbrain “potential of hydrogen” pH was conducted in single-voxel ^31P MR spectroscopy. Aims: To explore the feasibility of mapping brain ATP and brain pH by using multivoxel ^31P MR spectroscopy. Methods: Phantom studies were carried out by using a GE 3T scanner firstly. Many available sequences were tested using phantom and the two dimensional (2D) Point RESolved Spectroscopy Chemical Shift Imaging (PRESSCSI) sequence was selected because of better signal to noise ratio. Time of repetition (TR) was 1000 msec and time of echo (TE) 144 msec with 128 scan averages. The acquisition matrix was 16 x 16 phase encodings over a 24-cm field of view (FOV). Slice thickness was 10 mm. Then a healthy volunteer from MR research team was studied. Data were processed offline using the Spectroscopic Analysis of General Electric / Interface Definition Language (SAGE/IDL) software. Baseline and phase corrections were performed. Multivoxel spectra and brain ATP map were analyzed. Brain pH values were calculated from the difference in chemical shifts between inorganic phosphate (Pi) and phosphocreatine (PCr) resonances. Color scaling map was generated using MatLab software. Results: Multivoxel ^31P spectra were obtained for phantom and the healthy volunteer. PCr map was obtained in phantom. At this moment, peaks of PCr were not homogeneous in phantom studies. There was noise for multivoxel ^31P spectra in volunteer study. Phosphomonoester (PME) peak, Pi peak, phosphodiester (PDE) peak, PCr peak, γATP peak, αATP peak, and βATP peak can be identified. Preliminary brain ATP map and brain pH map were generated in the volunteer. Conclusions: It is feasible to map brain ATP and brain pH using multivoxel ^31P MR spectroscopy. However, endeavors should be made to improve quality of multivoxel ^31P MR spectroscopy.	The G-rich promoter and G-rich coding sequence of basic fibroblast growth factor are the targets of thalidomide in glioma MEI SC^1, WU RT^1,2 ^1Institute of Biopharmaceutical Science and ^2Research Center for Drug Discovery, School of Pharmaceutical Science, National Yang-Ming University, Shih-Pai, Taipei, Taiwan, Republic of China Background: Despite the very high risk of teratogenicity, thalidomide is emerging as a treatment for cancer and inflammatory diseases. Thalidomide is considered to be an effective drug for treating refractory multiple myeloma due to its antiangiogenic and immunomodulatory activities. In addition to myelomas, thalidomide has been widely tested on various types of tumors, such as renal cell carcinoma, prostate cancer, glioma, and Kaposi’s sarcoma. Clinical efficacy in some inflammatory conditions, including graft-versus-host disease after allogeneic bone marrow transplantation and renal transplantation, further supports the immunomodulatory properties of thalidomide. Although current data provide much promise for the use of thalidomide in the treatment of these diseases, its mechanism of action is still incompletely understood. Earlier clinical studies have found that patients responding to this drug often had high plasma levels of basic fibroblast growth factor (bFGF). This cytokine is a proangiogenic factor overexpressed in many tumors and is also a regulator of limb development; hence, it might be a target of thalidomide. bFGF belongs to the FGF gene family and is a potent autocrine and paracrine mitogen that is ubiquitously expressed. Secretion of bFGF is independent of the traditional ER-Golgi pathways. Besides the secreted form, which is translated using the first AUG codon, there exist four nuclear target forms of bFGF. These four forms are translated differently from upstream in-frame CUG codons through an internal ribosome entry site (IRES)-dependent mechanism. Due to the different intracellular distribution and the NH2-terminal extension of high molecular weight (HMW) bFGFs, the functions of these HMW bFGFs compared with the low molecular weight (LMW) bFGFs are believed to be different. bFGF is overexpressed in various types of tumors, such as glioma and renal cancer, and increased expression of bFGF has been found to be correlated with disease progression. The expression of bFGF transcripts is under the control of a G-rich promoter. In addition to transcriptional regulation by the G-rich promoter, the NH2-terminal–extended bFGF coding sequence is also G-rich, which may function to regulate translation of different isoforms. This RNA region may also serve as a target for some DNA-binding drugs and consequently modulate expression of the isoforms. Drucker et al. reported that thalidomide at relative high concentrations (>25 μg/mL) could downregulate transcription for genes with GC-rich promoters.Additionally, Stephens et al. proposed that thalidomide may inhibit insulin-like growth factor–induced and bFGF-induced limb genesis because both genes were under the control of GC-rich promoters. The transcriptional and the translational regulation of bFGF are under the control of G-rich–containing sequences, which can interact with thalidomide. Considering that bFGF is a ubiquitous growth factor involved in many biological activities, we hypothesized that the G-rich sequences of bFGF are the major targets of thalidomide. The present study was to determinate the possible molecular mechanism of thalidomide as well as to highlight the feasibility of using low-dose thalidomide to treat tumors as glioma in a clinical setting. Methods: To examine the antitumor effect of thalidomide, we used U-87 MG cells, which are a high-grade human glioblastoma cell line expressing high basal levels of bFGF Real-time reverse transcription-PCR analysis was used to assess the mRNA levels of bFGF in U-87 MG cells with and without thalidomide treatment. Because the 5’-end of the bFGF transcript is GC-rich, we postulated that translation of bFGFmay also be affected by thalidomide. Western blot analysis was therefore done to analyze the levels of the various bFGF isoforms. Because bFGF has been shown to promote cell transformation, a soft agar colony formation assay and hanging drop technique were used to assess the effects of thalidomide on anchorage-independent and three-dimensional growth abilities of U-87 MG cells, respectively. we next examined whether the tumorigenicity of these cells was reduced by down-regulation of bFGF expression. Three different bFGF shRNAs (#1, #2, and #3) or control shRNA was introduced into U-87 MG cells by lentivirus infection to generate three bFGF knockdown clones. To evaluate the effect of thalidomide on transcription driven by the bFGF promoter, a pbFGF-EGFP plasmid, containing a portion of the bFGF promoter to drive the expression of EGFP, was stably transfected in U-87 MG cells to give U-87-bFGF-EGFP cells. Cells were treated with thalidomide (0.1–10 μg/mL) for different time intervals, and then transcript levels and relative fluorescence indices were measured to evaluate the effect of thalidomide on the expression of EGFP. Because downregulation of the HMW bFGFs by thalidomide was more significant than LMW bFGF, we asked whether IRESdependent translation of bFGF, which was reported to regulate the expression of different isoforms, was also affected by this drug. HMW and LMW bFGF IRES fragments were inserted into bicistronic vectors to generate pHMW-IRES and pLMW-IRES plasmids. Both plasmids were then stably transfected into U-87 MG cells to give U-87-HMW-IRES cells and U-87-LMW-IRES cells. After treating with liposome encapsulated thalidomide for 12 h, IRES activity was estimated by calculating the ratio between firefly luciferase activity and Renilla luciferase activity. The former represented the translational efficiency of the upstream cistron, and the latter represented that of the individual bFGF IRES. To examine whether thalidomide interacted preferentially with the G-rich coding sequence of bFGF, we measured the UV-VIS absorbance of thalidomide after incubation with a G-rich (nucleotides 363–428) or a non–G-rich (nucleotides 673–768) DNA fragment derived from bFGF. We hypothesized that the absorbance of thalidomide may be diminished more dramatically by the secondary structure of a DNA fragment that was bound more tightly by this drug. Results: Using U-87 MG cell lines, we found that thalidomide, especially when encapsulated in a liposome, down-regulated the transcription and translation of the FGF-2 gene by interacting with G-rich regions present in the promoter and the internal ribosome entry site of its transcript at concentrations much lower than therapeutic serum concentrations. Thalidomide treatment also dramatically suppressed the anchorage-independent growth of U-87 MG and other glioma cells by over a thousand fold without affecting its anchorage-dependent growth, which may be accomplished by knocking down endogenous bFGF expression in these cells. Accordingly, the addition of recombinant bFGF partially restored the anchorage independent growth of these cells. In addition, the absorbance at 230 nm was quenched to a greater extent on thalidomide treatment when it was incubated with a G-rich DNA fragment, suggesting that this drug may bind preferentially with nucleic acids that have a high content of guanosine.Our data suggest that by targeting the G-rich regions of bFGF, thalidomide (at 0.1 μg/mL) can reduce cellular bFGF levels and affect tumor anchorage-independent growth, the hallmark of tumorigenicity.Our results are promising for future clinical investigations using low doses of thalidomide. Authors’ disclosure statement (not counting towards the character count): Low-dose thalidomide : novel mechanism & new insight in cancer therapy Although the mechanism of action responsible for the effects of thalidomide remains unclear, this drug is currently under investigation for the treatment of several disease types, ranging from inflammatory conditions to cancer. Using liposome encapsulation for preventing the rapid hydrolysis of thalidomide, Mei and Wu found that low concentration of thalidomide as 0.1μg/ml target the G rich region of bFGF in glioma thereby diminishing cellular bFGF levels and suppressing anchorage- independent tumor growth. The data may have important implications for understanding the mechanistic rationale for low-dose thalidomide use in the clinical realm of cancer therapy.
Two vaccines too far: the poliovaccine fiasco of 1935 WYATT HV University of Leeds, Leeds, U.K Background: Animal models are useful – but may be misleading. The Kolmer and Park-Brodie vaccines were based on inadequate basic knowledge. It was assumed that both vaccines contained live poliovirus. A more subtle explanation does not exclude this possibility, although the virus was probably non-infectious by the route used and did not cause the few paralyses and deaths. As well as virus, the vaccines contained monkey spinal cord tissue which causes inflammatory reaction when injected into humans. This inflammation would have mimicked provocation. Other polioviruses (of low virulence) were circulating and the coincident inflammation would have increased their apparent virulence in a few vaccinees and caused the few cases of polio. There had been good evidence for the theory of genetic susceptibility to polio, but the vaccine fiasco seemed to suggest that a safe vaccine was not possible. The theory was forgotten. Instead it was thought that while small doses of virus gave immunity, large doses resulted in polio and that paralytic cases excreted large quantities of virus (resulting in family cases) – neither of these theories could be tested. Conclusions: However plausible, theories unsupported by evidence can be wrong. Later research has shown that there are two groups that are genetically susceptible. Ehrlich’s pioneering work of monitoring vaccine use was forgotten until the 1950’s.