Ehrlich II –2nd World Conference on Magic Bullets

Can Erythropoietin be used to prevent brain injury in African Children with Cerebral Malaria? IDRO R^1,2, CASALS-PASCUAL C^3, DAY R^4, NEWTON C^2,5 ^1Makerere University, Kampala, Uganda; ^2Kenya Medical Research Council, Kilifi, Kenya;^3Medical Research Council Labs, Fajara, Gambia; ^4Oxford University, Oxford, UK; ^3UCL Institute of Child Health, London, UK. Background: Cerebral malaria is associated with high mortality and long-term neuro-cognitive deficits. Erythropoietin has shown neuro-protective properties in different neurological disorders. We hypothesized that the outcome of cerebral malaria is modified by the responses of this cytokine to hypoxia and that high plasma and cerebrospinal (CSF) levels would prevent neurological damage. AIM. Determine the relationship between plasma and CSF erythropoietin and the outcome of cerebral malaria in African children. Methods: We measured erythropoietin in paired samples of plasma and CSF of 124 children with cerebral malaria. Patients were categorized into 3: 76 without deficits, 32 with deficits and 16 who died. The median (IQR) plasma erythropoietin were compared. Logistic regression models were used to identify risk and protective factors associated with sequelae. Results: The median (IQR) plasma concentrations of erythropoietin were 123(29-1,726)U/L, 184(23-694)U/L and 278(96-1,852)U/L in children who died, survived with and without sequelae respectively. Conditional logistic regression analysis matching the 32 patients with sequelae to 64 patients without sequelae stratified for hemoglobin level estimated that plasma erythropoietin >200 U/L was associated with greater than 80% reduction in the risk of sequelae (adjusted OR 0.18, 95%CI 0.05-0.93). Both the level of erythropoietin and the protective effect of erythropoietin were greater in younger children. Conclusions: 1) High levels of erythropoietin were associated with reduced risk of neurological deficits. 2) The age-dependent erythropoietin response to anemia and the age-dependent protective effect may influence the clinical epidemiology of cerebral malaria. 3) These data support further study of erythropoietin as an adjuvant therapy in cerebral malaria.	Factors Affecting Toxicity and Efficacy of Injectable Nanomedicines: Significance on Particle Size IGARASHI E^1 ^1Nanosion Co., Ltd., Kashiwa, Japan. Background: Nanomedicine is the application of nanotechnology to medicine. Recently, the National Nanotechnology Initiative referred to nanotechnology as 'the understanding and control of matter at dimensions of roughly 1 to 100 nanometers, where unique phenomena enable novel applications'. The usefulness of cancer chemotherapy is restricted by the dose-limiting toxicity of cytotoxic anticancer drugs and the occurrence of side effects. Toxicity arises from the lack of efficient selectivity of traditional anticancer drugs for malignant cells. That is, there is no decrease in the distribution of drug in normal tissue (target organ) and concomitant increase in distribution in the tumor tissue with the use of chemotherapeutic drugs. The purpose of this article is to review the passive targeting properties of several injectable water-soluble polymeric nanomedicines that are between 1 and 100 nm in size, to illustrate the impact of polymeric nanomedicines on toxicology. I consider several biodegradable polymeric nanomedicines that are between 1 and 100 nm in size, and discuss the impact of this technology on efficacy, pharmacokinetics, toxicity and targeting. In addition to above previous discussion (Igarashi, 2008, published on Toxicol. Appl. Pharmacol., 229:121-134), I discuss the lower size limit around 5 nm rather than 1 nm for the passage of intact nanomedicines. Discussion: The degree of toxicity of polymeric nanomedicines is strongly influenced by the biological conditions of the local environment, which influence the rate of degradation or release of polymeric nanomedicines. The dissemination of polymeric nanomedicines in vivo depends on the capillary network, which can provide differential access to normal and tumor cells. The accumulation of nanomedicines in the microlymphatics depends upon retention time in the blood and extracellular compartments, as well as the type of capillary endothelium surrounding specific tissues. The toxicity or efficacy of intact nanomedicines is also dependent upon tissue type, i.e., non-endocrine or endocrine tissue, spleen, or lymphatics, as well as tumor type (Igarashi, 2008). Electron microsocopic and recent immunobiological studies of endothelial fenestral diaphragm showed that the lower size limit affecting the distribution of nanomedicines in normal tissue may depend on the size/shape of communicating channel spanned by diaphragm.
Suppression of Membrane Microvesiculation – a Possible Anticoagulant and Anti-Tumor Progression Effect of Heparin IGLIC A^1, SUSTAR V^2, JANSA V^2, SUSANJ P^2, KRALJ-IGLIC V^2 ^1Laboratory of Physics, Faculty of Electrical Engineering and ^2 Laboratory of Clinical Biophysics, Faculty of Medicine, University of Ljubljana, Ljubljana, Slovenia Background: Heparin was found a successful drug not only in preventing thromboembolic events but also in slowing down the development of some types of cancer. As microvesiculation of membranes is enhanced both in hypercoagulabile states as well as in cancer, a hypothesis of anticoagulant and anti-tumor-progression effects of heparin is put forward: heparin mediates an attractive interaction between membranes and suppresses microvesiculation by adhesion of buds to the mother membrane before they become free vesicles. Fig.1. Suppression of phospholipid membrane vesiculation by the mediating effect of the solution. Bar = 10m. Methods: Giant phospholipid vesicles were created by electroformation and observed under the phase contrast microscope. Blood plasma and heparin were added to the suspension of vesicles. A consequent adhesion between vesicles was assessed by measuring the average contact angle between the adhered vesicles. Results: Addition of 10 l of therapeutic concentration of Fraxiparine, GlaxoSmithKline, UK, mixed and preincubated with plasma, into 55 l of vesicle suspension caused stronger mediating effect than addition of plasma alone. The average contact angle 30 minutes after the addition of the sample was 98 degrees, while it was 86 degrees due to plasma alone, the difference being statistically significant (p<0.001). The same concentration of heparin without plasma did not cause adhesion of vesicles. Conclusion: Heparin in therapeutic concentrations enhances the ability of plasma to cause adhesion between membranes and may thereby suppress microvesiculation. This represents a possible biophysical anticoagulant mechanism of heparin.	The impact of non-fucosylated therapeutic antibodies in humans in vivo. SHIGERU IIDA, MITSUO SATOH Antibody Research Laboratories, Kyowa Hakko Kogyo Co., Ltd., Tokyo, Japan Background: Antibody-dependent cellular cytotoxicity (ADCC) has recently attracted attention as an important mechanism of the clinically effective therapeutic antibody. However, the ADCC of currently licensed fucosylated therapeutic IgG1s has been found to be strongly inhibited by plasma IgG through competition for binding of the therapeutics to FcγRIIIa on NK cells, which causes such a high dose requirement in antibody therapies. Here, we investigated whether or not ADCC of non-fucosylated therapeutic IgG1 is also influenced by plasma IgG. Methods: Ex vivo ADCC upon CD20-positive human B cells was induced by incubation of human whole blood with non-fucosylated and/or fucosylated anti-CD20 IgG1s rituximab, and quantified by measuring the remaining CD19-positive human B cells using flow cytometry. Results: Non-fucosylated anti-CD20 showed markedly higher ex vivo B-cell depletion activity than its fucosylated forms in the presence of plasma IgG. The efficacy of fucosylated anti-CD20 was greatly diminished in plasma, resulting in the need for a high concentration (over 1 g/mL) to achieve saturated efficacy. In contrast, non-fucosylated anti-CD20 reached saturated ADCC at lower concentrations (0.01 to 0.1 g/mL) with much higher efficacy than fucosylated counterparts through improved FcRIIIa binding. Importantly, the high ADCC efficacy of non-fucosylated therapeutic antibodies has been shown to be inhibited by the fucosylated forms through the competition for binding to the antigen on target cells. Conclusions: Our data showed that non-fucosylated IgG1, not including fucosylated forms, can evade the inhibitory effect of plasma IgG on ADCC through its high FcγRIIIa binding, and elicit saturable ADCC. Hence, the application of non-fucosylated antibodies is expected to be a promising approach as next-generation therapeutic antibodies with improved efficacy, even when administrated at low doses in humans in vivo. Clinical trials using non-fucosylated antibody therapeutics are currently underway.
Expression of Estrogen Receptors during Postnatal Development of the Brain IKEDA Y^1, IKEDA M-A^2 ^1Yokohama City University School of Medicine, Yokohama, Japan; ^2Tokyo Medical and Dental University Graduate School, Tokyo, Japan. Background: Estrogen receptors (ERs) mediate effects of estrogens, which are important for regulating neuroendocrine, physiological, and reproductive functions in matured and developing mammalian brains. The two ER subtypes, ER and ERß, are expressed in many brain regions of rodents. However, the expression pattern is different between ER and ERß, and the expression of each receptor can change during development. The purpose of this study is to compare the expression level and cellular distribution between the two ERs during postnatal development of the rat cerebellum. Methods: Expression levels of ER and ERß mRNAs were examined using quantitative real-time RT-PCR (qRT-PCR), at postnatal days 7 (P7), P14, P21, and in adulthood, in the rat cerebellum. Furthermore, distributions of mRNA and protein for both ERs in the developing cerebellum were analyzed using in situ hybridization and immunohistochemistry. Double-label immunofluorescence for ER and ERß was utilized to determine whether the two receptors were expressed within the same cell. Results: QRT-PCR demonstrated that levels of cerebellar ER mRNA in neonatal pups were significantly higher than those in adults. In contrast, expression levels of cerebellar ERß mRNA remained significantly unchanged during postnatal development. In situ hybridization and immunohistochemistry demonstrated that ER mRNA and protein were predominantly expressed by Purkinje cells. ER-immunoreactive Purkinje cells were distributed in most lobes at P14 and P21, and some of them were co-localized with ERß. However, only a few ER-immunoreactive cells were observed in the adult cerebellum. ERß expression occurred in Golgi type neurons in the granular layer at P7, Purkinje cells at P14, and basket cells in the molecular layer at P21, and was detected in all the cell types in the adult cerebellum. Conclusions: 1) ER expression was transiently increased during the time when Purkinje cell dendritic growth and synapse formation proceed, suggesting that a role for ER in Purkinje cell differentiation. 2) The expression profile of ERß suggests a role for ERß associated with neuronal differentiation and maintenance. 3) The discrete expression profiles for ER and ERß in the developing cerebellum suggest the two ERs play distinct roles in cerebellar development.	Improvement of PGH-Synthase and Papain Stability in Pectin Contained Systems Used for Healing of Skin Wounds ILYINA A.^1, SEGURA C. E. P.^1, GREGORIO J.K.M.^1, MARTÍNEZ H.J.L.^1, VARGAS D.C.I.^2 ^1Universidad Autónoma de Coahuila, Fax: [phone], E-mail: [e-mail]. ^2Clinica Magisterio Secc. 38, Saltillo, Coah. Mexico. Background: Prostaglandin H synthase (PGHS, EC 1.14.99.1) mediates a variety of physiological processes. Papain (EC 3.4.22.2) is protease that is used as debridement agent. The goal of the present study was to obtain materials applicable for treatment of skin wounds by means of these enzymes immobilization in biopolymer pectin. Methods: PGHS was isolated from bovine vesicular glands. Both enzymes were immobilized on pectin films and tested for storage stability. Papain was also prepared in spray with 6% pectin gel to form a thick film on a skin. The papain stability in aerosol spray was defined at 65°C under pressure of air and N2 (up to 90 psi) as well as during its storage. The effectiveness of immobilized PGHS and papain on tissue repair was tested in mice and rabbits, respectively, by measuring linear size of wound. Prostaglandin E2 (PGE2) was measured by HPLC. Moreover, applications of pectin/papain films on wounds of voluntary patients were performed. Results: Immobilized PGHS retained 41 and 12% after 60 days of storage at -15º and 4ºC, respectively, while in solution it was inactivated to 14% after 24 h. Papain retained 98% of activity on pectin film and only 8% in spray after 6 months of its storage at 4ºC, while in solution it was inactivated after 1 week. The papain activity and stability were decreased under high propellant pressure in solution and were increased in pectin gel. Application of immobilized enzymes accelerated animal wound healing. The PGE2 level and specific rate of healing progression, calculated as a tangent of lines obtained in semi-logarithmic coordinate, were increased under PGHS treatment. A kinetic scheme was proposed to describe the effect of PGHS. Wound healing of treated patients was accelerated without any negative secondary effects. Conclusions: 1. Immobilization on pectin contained systems allows for enzymes stabilization and simplifies their application on skin, it may be considered for other drugs. 2. Prostaglandins level regulation, for example by PGHS addition, is a mechanism to influence skin wound repair. 3. Kinetics of the healing process of a linear surgical wound may be described by the logistic curve that led to kinetic model describing the effect of PGHS on wound repair process.
Molecular investigation of drug resistant Neisseria gonorrhoeae clinical isolates ILINA EN¹, BOROVSKAYA AD¹, SIDORENKO SV^2, KUBANOVA AA^2, MAIER T^3, KOSTRZEWA M^3, GOVORUN VM¹. ¹Res Inst Physical-Chemical Medicine, Moscow, Russia ^2Centr Res Inst of Dermatology and Venereology, Moscow, Russia ^3Bruker Daltonik GmbH, Leipzig, Germany Background: The introduction of new adequate methods for correct species identification and susceptibility testing of N. gonorrhoeae (NG) remains an actual problem of sexual transmitted diseases monitoring. Aims: 1) to show the suitability of the MALDI Biotyper, a system based on mass spectrometry (MS) profiling, for NG species identification, 2) to determine known genetic markers of penicillin (PEN), tetracycline (TET) and fluoroquinolones (FQ) resistance in gonococci, 3) to study the relation between genetic markers of drug resistance and the susceptibility profile of clinical NG strains. Methods: This study included NG clinical isolates collected from different regions of Russia. Susceptibility testing to PEN, TET and FQ was performed by the agar dilution method according to CLSI. Single colonies of fresh bacterial cultures were tested by the MALDI Biotyper system (www.bruker.com) using a Microflex LT MALDI-TOF mass spectrometer. Total genomic DNA from NG strains was isolated using the “DNA express” kit (Lytech Ltd, Russia). The presence of bla and tet(M) genes was analyzed by PCR. Mutations in NG genes (rpsJ, por1, penA, ponA, gyrA, parC, mtrR, norM) associated with drug resistance were detected by primer extension reaction followed by MALDI-TOF MS analysis or by sequencing. Results: Totally, 293 bacterial isolates previously identified as NG were investigated. Susceptibility levels to PEN, TET and FQ were found to be 26 %, 36 %, and 54 %, respectively. For 280 samples the MS profiles were matched to strain NG ATCC 49226. The other MS profiles (n=13) were similar to each other but rather different from NG as well as from N. meningitidis, non-pathogenic Neisseria and further 1671 different microorganisms stored in the MALDI Biotyper library. Further 16S RNA sequencing referred them as unknown species from the genus Ralstonia. The distribution of genetic drug resistance markers was studied for certain susceptibility groups of NG strains. Their positive predictive value was found to be different for FQ (90 %), PEN (91 %) and TET (82 %). Conclusions: 1) The MALDI Biotyper system is a highly suitable tool for the correct NG species identification, 2) the surveillance of genetic markers may be useful for NG monitoring.	Organ protective effects of tunicamycin as an endoplasmic reticulum stress inducer INAGI R, NANGAKU M Div Nephrol & Endocrinal, Univ Tokyo Shc Med, Tokyo, Japan. Background: Various disturbances cause the endoplasmic reticulum (ER) dysfunction and thereby induce the accumulation of unfolded proteins in the ER, in turn leading to “ER stress”. ER stress induces unfolded protein response (UPR), which is aimed at ameliorating the cell stress buttriggers apoptosis when ER stress is excessive and/or prolonged. Tunicamycin (TUN) is an ER stress chemical inducer and has renal toxicity by causing ER stress-induced apoptosis. Here we hypothesized that a low dose of TUN, which induces an adaptive UPR rather than proapoptotic UPR, is protective against ER stress-related kidney diseases. Thus, we investigated if therapeutic approach targeting ER stress is effective in a model of glomerulonephritis (GN). Methods & Results: ER stress status was assessed in GN rats by detection of UPR: 1) expression of ER stress-inducible chaperones, 2) intracellular signaling for shut down of translation. By immunohistochemistry and Western blot analysis, UPR was significantly increased in damaged glomeruli of GN rats and it was associated with the disease manifestations. We then assessed if preconditioning of ER stress prevents the disease progression. TUN at non-nephritogenic dose (0.3mg/kg) was injected into the rats 4 days before GN induction for ER stress preconditioning. TUN pretreatment barely increased UPR in glomeruli of normal rats without affecting glomerular morphology and renal function. Interestingly, our histological analyses showed that disease progression of GN was dramatically improved by TUN (p<0.05). These histological improvements were associated with amelioration of proteinuria (39.4+10.5 v.s. 126.1+18.1 mg/day; P<0.01). Of note, the protective effect of TUN on the glomerular damage was associated with the modulation of UPR: excessive increase in ER stress-inducible chaperon expression and the signaling for translation shut down observed in glomeruli of GN was reduced by TUN, demonstrating that amelioration of excessive ER stress by preconditioning may contribute to improvement of glomerular injury. Conclusions: Glomerulonephropathy is associated with excessive ER stress. ER stress preconditioning with non-nephritogenic dose of TUN ameliorates manifestations of glomerulonephropathy, suggesting the possibility of therapeutic approach targeting ER stress in the kidney. (Grant, JSPS-19590939 to RI and -19390228 to MN)
Pathophysiological Approach against Resistant Bacteria-causing Infectious Diseases; - from Autopsy Findings to Clinical Applications – INAI K^1, Noriki S^2, IWASAKI H^3, YAMASHITA M^4, UEDA T^3, NAIKI H^1 ^1Div Mol Pathol, ^2Div Tumor Pathol, ^3Div Hematol & Cardiol, ^4Div Clin Inspect, Univ. FUKUI, Fukui, Japan. Background: Autopsy is a powerful tool for analyzing the cause of a patient’s death, progression of the disease, and the therapeutic response. However, it is difficult to identify the bacterial characteristics using morphological analysis only, especially in cases of resistant-bacterial strains. Methods: To elucidate the pathogenesis of resistant bacteria-causative infectious diseases, intrapulmonary sputum was harvested by directly inserting a swab into a resected lung at autopsy, and the bacterial composition was analyzed using both pathological and microbiological techniques from 15 patients with hematological malignancies, and the results were compared with those from 25 patients with other medical and surgical diseases. Then, we established an in vivo infection model to investigate kinetics of pathogens or effects of antimicrobials on slide sections by using autoradiography methods. Results: Among the 54 bacteria strains isolated from the 40 patients, multi-drug resistant strains were significantly more prevalent in hematological group than in other diseases (16/21 vs. 11/33, p=0.002). E. faecium was preferentially isolated from the hematological patients, whereas the methicillin-resistant S.aureus was predominantly found in the non-hematological group. Even coagulase-negative S. epidermidis strains in hematological diseases may be diagnosed as causative bacteria of pneumonia by both bacterial and pathological techniques. In mice or rats infected bacteria or fungus, the administration of ^3H-labeled cell wall substrates brought about accumulation of the compounds in the microorganisms with very low backgrounds on the pathological sections (Rt-figure represents that the deposition of RI compounds can visualize Aspergillus on slide sections using both pathological and autoradiography techniques). Conclusions: Traditional pathological approach such as autopsy would extend a further analytical tool of resistant-bacteria causing infectious diseases in combination with microbiological or radiological techniques. Our new animal model could also contribute to develop new antimicrobials as well as clinical applications.	»Carbon bullet«: Fullerenol C60(OH)24 as organo-protector against doxorubicin-induced toxicity INJAC R^1, ŠTRUKELJ B^1, ĐORĐEVIĆ A^2 ^1Univ. Ljubljana, Ljubljana, Slovenia; ^2Univ. Novi Sad, Novi Sad, Serbia Background: Fullerenes are a type of carbon molecule first discovered in the mid-1980s. The most well-known of these is the buckyball, which is a closed spherical molecule, shaped like a soccer ball. Many aggressive studies are presently in progress to search for pharmaceutical products that can capitalize on the excellent bioactivity of fullerenes and their derivatives. Thus far, different fullerene properties have been put to use, such as its ability to eliminate radicals as well as its particular molecular structure. Studies have found it to be efficient in the suppression of metastases, the treatment of cerebral conditions such as Alzheimer's and Parkinson's diseases, type-C hepatitis therapy, and HIV treatment. A polyhydroxilated derivative of fullerene, named fullerenols C60(OH)n is being extensively studied due to its great potential as an antioxidant. It is proposed that fullerenols may act as a free radical scavenger in biological systems, in xenobiotics, as well as radioactive irradiation-induced oxidative stress. It has demonstrated protective effects against cytotoxicity of doxorubicin (Dox) in animal models, especially by fullerenol C60(OH)24. Methods: Sprague Dawley outbred rats with chemically induced mammary carcinomas were used to investigate a potential protective role of fullerenol against cardio-, hepato-, nephro- and pulmo- toxicity induced by Dox. According to preliminary studies on healthy adult Wistar rats our research group confirmed that 100 mg/kg (i.p.) of fullerenol administered 30 min before Dox has a protective influence on heart and liver tissue. Therefore that dose was chosen as effective against acute Dox-induced toxicity in further examinations. Results: In our recently published paper an in vivo – in vitro study was examined to confirm the potential protective role of fullerenol C60(OH)24 on Dox-induced liver toxicity.The in vivo results (Sprague-Dawley rats) showed that treatment with Dox alone caused significant changes in the serum levels of ALT, AST, LDH and α-HBDH, as well as in the levels of MDA, GSH, GSH-Px, TAS, GR, CAT, and SOD in the liver tissue. These effects were drastically reduced for all investigated parameters by pre-treatment with fullerenol, although not for the MDA and GSH level. On the other hand, the human hepatocellular carcinoma (HepG2) cell line was continuously treated with fullerenol for 12, 24, 48 and 96 h, at concentrations of 10 and 44 mg/mL. With the aim of evaluating the modulating activity of fullerenol on Dox-induced hepatotoxicity, the cell line was concurrently treated with Dox (1 μM; 5 μM) and fullerenol (10 μg/mL; 44μg/mL) in different combinations. When the cells are treated with 5 μM Dox along with the fullerenol, a significant development of cell capability during the entire timeline can be seen. It was concluded that fullerenol has cytotoxic effects on HepG2 by itself, but when the oxidative stress is too high, the cytotoxic effects of fullerenol are overcome by its protective role as a strong antioxidant compound. Damage to the heart muscle after Dox administration was also confirmed by changes in the ultra-structural pathology results and SOD, MDA, CAT, GSSG, GR, and TAS levels, as was a potential cardioprotective influence of fullerenol as a pretreatment agent for Dox therapy in the acute phase. Fullerenol itself, in a dose of 100 mg/kg, did not affect heart injury in rats with breast cancer. The presented results suggested that fullerenol might be a potential cardioprotector in Dox-treated individuals. Conclusions: The key benefit of fullerenol, in contrast to other known antioxidants, is its dual function as radio-protector and organo-protector during the anticancer therapy (radio- and chemo-). However, there is a need to carry out further studies, including a chronic investigation in animals (this study was done and will be also presented) and human trials.