SALEH F Faculty of Medicine, Kuwait University, Kuwait
Kuwait ranks sixth in the world in terms of oil reserve and ranks third in terms of oil exporter worldwide. The population of Kuwait reached only one million in 2008. Accordingly, Kuwait is a very wealthy country, and the average income of the Kuwaitis is one of the highest in the world.
Despite the above wealth, the incidence of cancer in the country increased by 300% in the past 10 years. Surgery, and chemo and radiation therapy are still the traditional methods used to treat the disease in Kuwait. Immunotherapy is still lacking in this country. Accordingly, and in an attempt to introduce such therapy, I have been working for the past four years on establishing the immune profiles of the neoplasm excised from the patients, as well as the immune profiles of the patients_ peripheral blood. Some interesting findings were seen during my work, and they sound appealing to start a nucleus of cancer immunotherapy in Kuwait.
Based on my findings, I have contacted the senior Kuwaiti officials enquiring about possible funding of the above nucleus. The feedback was extremely positive, and I was officially promised to be given the funding needed and more.
Therefore, I would like to use this coming prestigious EHRLICH II, 2nd World Conference on Magic Bullets as a venue to invite pharmaceutical and non-pharmaceutical international institutions to participate in establishing cancer immunotherapy in Kuwait. I believe this will add to the international efforts which have been aiming at making the dream of Paul Ehrlich come true.
Glomerular nephrotoxicity of aminoglycosides SALGADO CM Unidad de Investigacion, Hospital Universitario de Salamanca, and
Unidad de Fisiopatologia Renal y Cardiovascular, Departamento de Fisiologia y Farmacologia, Universidad de Salamanca
The body defends itself against potentially harmful compounds like drugs, toxic compounds, and their metabolites by elimination, in which the kidney plays an important role. Nephrotoxicity is a major side effect in clinical practice, frequently leading to acute renal failure. Many physiological mechanisms have been implicated in drug-induced renal injury. Aminoglycoside antibiotics are the most commonly used antibiotics worldwide in the treatment of Gram-negative bacterial infections. However, aminoglycosides induce nephrotoxicity in 10-20% of therapeutic courses
Gentamicin-induced nephrotoxicity is characterized by slow rises in serum creatinine, tubular necrosis (due to their partial reabsorption by proximal tubular cells) and marked decreases in glomerular filtration rate and in the ultrafiltration coefficient (Kf). Kf regulation depends on the activity of intraglomerular mesangial cells. Tubular nephrotoxicity has been intensively reviewed previously, but glomerular toxicity has received less attention; thus, this presentation describes the glomerular nephrotoxic mechanisms of action of the aminoglycoside antibiotic gentamicin, with the aim to provide an actualized and mechanistic vision of pathways involved in glomerular toxic effects of aminoglycosides.
Vitamin utilisation pathways as antimalarial drug targets SALIBA KJ The Australian National University, Canberra, ACT, 0200, Australia
Background: Growth of the intraerythrocytic stage of the human malaria parasite, Plasmodium falciparum, is dependent on a constant supply of nutrients. The pathways by which the parasite utilises these nutrients are under investigation in our laboratory as viable antimalarial drug targets. The parasite has an absolute requirement for an extracellular supply of pantothenate (vitamin B5), the precursor of the important enzyme cofactor, coenzyme A (CoA). We have previously shown that a pantothenate analogue, pantothenol, kills malaria parasites both in vitro and in vivo, but its exact mechanism of action is still unclear. Recently, it has been shown that the parasite also requires an extracellular supply of thiamine (vitamin B1), but whether an extracellular supply of other vitamins is also required remains unclear. We have therefore set out to: (i) investigate the mechanism of action of pantothenol, (ii) establish whether an extracellular supply of riboflavin (vitamin B2), is required by the parasite and (iii) establish whether a natural riboflavin analogue, roseoflavin, inhibits the growth of malaria parasites in vitro and in vivo.
Methods: Parasite metabolism of [14C]pantothenol was monitored by HPLC. In vitro parasite growth was assessed by the [3H]hypoxanthine incorporation assay. In vivo antimalarial activity was monitored using the 4-day suppression test in mice infected with P. vinckei.
Results: Pantothenol was metabolised by the parasite into phosphopantothenol and at least two additional metabolites. Parasite proliferation was significantly impaired when cultured in erythrocytes depleted of riboflavin. Roseoflavin inhibited parasite growth in vitro in the sub-M range at physiological [riboflavin]. Increasing the extracellular [riboflavin] antagonised the antiplasmodial activity of roseoflavin, consistent with roseoflavin acting by inhibiting riboflavin utilisation by the parasite. Administration of roseoflavin to mice infected with P. vinckei reduced the parasitemia and prolonged the survival of the mice.
Conclusions: (i) Pantothenol acts as a substrate (rather than an inhibitor) of CoA biosynthesis enzymes, possibly producing non-functional metabolites. (ii) An extracellular supply of riboflavin is essential for the prolonged survival of the intraerythrocytic stage of P. falciparum. (iii) Roseoflavin inhibits the growth of malaria parasites both in vitro and in vivo.
Glucose Transporter Type 2 - Does It Pave the Way to Sporadic Alzheimer's Disease? SALKOVIC-PETRISIC M1, GRÜNBLATT E2, OSMANOVIC J1, HOYER S3, RIEDERER P2 1 Dept. Pharmacol., Sch. Med., Univ. Zagreb, Croatia; 2Dept. Clin. Neurochem., Univ. Dept. Psychiat. Psychother., Univ. Würzburg, Würzburg, Germany; 3Dept. Pathol., Univ. Heidelberg, Heidelberg, Germany
Background: Sporadic type of Alzheimer’s disease (sAD) is associated with brain insulin receptor (IR) signalling abnormalities. The sequence of these events in relation to the beta amyloid (Aβ) pathology can be traced only in sAD animal model, rats treated intracerebroventricularly with streptozotocin (STZ-icv). STZ enters the cell through glucose transporter type 2 (GLUT2) and selectively damages insulin producing/secreting cells and IR, as well as GLUT2. The time course of brain insulin system dysfunction following damage induced by icv application of GLUT2- and IR-toxic drug was investigated in this sAD model.
Methods: Male Wistar rats (3-4 month old, 6-8 per group) were treated bilaterally icv with STZ (1-3 mg/kg) and followed after 1, 3 and 6 months for the memory (Morris Water Maze Swimming Test), hippocampal neurochemistry (RT-PCR for insulin-1 /Ins-1/, IR and insulin degrading enzyme /IDE/, immunoblotting for IR, protein kinase B /Akt/PKB/, glycogen synthase kinase 3 /GSK-3/, IDE, tau protein, and Elisa assay for tyrosine kinase /TK/), and histology (immunohistochemistry and Congo red staining for Aβ). Data were analysed by Kruskal Wallis median and Mann Whitney U test.
Results: One month following the STZ-icv treatment IR mRNA and protein were decreased (p<0.05). Three months following the STZ-icv treatment Ins-1 and IR mRNA were decreased (p<0.05) and IR-TK activity increased (p<0.05). This was followed by alterations (p<0.05) of downstream IR signaling elements, decreased expression of Akt/PKB and p-GSK-3/GSK-3 ratio, increased expression of hyperphosphorylated tau protein, and decreased expression of IDE mRNA and protein. Aβ-like congophilic capillary aggregates (cerebral amyloid angiopathy) and Aβ1-42 intraneuronal aggregates were found after 3 months. Decreased expression of Ins-1, IR and IDE mRNA, IR and IDE protein were found 6 months after STZicv treatment when Aβ1-42 primitive plaques were found in hippocampal/cortical regions. Cognitive deficits were found at each time point.
Conclusions: STZ-icv induced damage of brain GLUT2 and IR leads to insulin resistant brain state eventually triggering Aβ pathology in animal sAD model.
Supported by DAAD and MZOS.
Nicotinic acetylcholine receptor containing an alpha6 subunit: target for a magic bullet? SALMINEN O1), GRADY SR 2), MARKS MJ 2) , COLLINS AC 2) 1) University of Helsinki, Faculty of Pharmacy, Finland
2) University of Colorado, Institute for Behavioral Genetics, CO, USA
Neuronal nicotinic acetylcholine receptors (nAChRs) are pentameric assemblies made up of different combinations of alpha- (ligand binding) and beta- (structural) subunits. The alpha4beta2 nAChR is the most widely distributed subtype in the central nervous system. Recently it has been demonstrated that the presynaptic nAChRs in the brain dopaminergic neuron terminals consist of at least five different functional nAChR subtypes, two alpha4beta2-type nAChRs (alpha4beta2 and alpha4alpha5beta2) and three alpha6-type nAChRs (alpha6alpha4beta2beta3, alpha6beta2beta3 and alpha6beta2)(reviewed in Grady et al. 2007). The alpha6-type nAChRs have raised considerable interest because of their differing pharmacological properties compared with alpha4-type nAChRs. The distribution of alpha6 nAChRs is fairly restricted to the dopaminergic nerve terminals and the optical tract in rodent brain. One of the alpha6 nAChRs (alpha6alpha4beta2beta3) has the highest sensitivity to nicotine of any native nAChR that has been studied, to date (Salminen et al. 2004, 2007). This implies that this particular alpha6-type of nAChR is the first receptor to be activated following the initial puffs of tobacco. Functional and binding studies have yielded readily measured differences in sensitivity to nicotinic agonists and antagonists among these five nAChR subtypes. Several research groups have demonstrated that chronic nicotine treatment induces the downregulation of the alpha6 nAChRs while it is well established that other nAChRs (alpha4beta2 and alpha7) are upregulated following chronic nicotine administration. Therefore, there is a reason to suggest that while alpha6 nAChRs differ in many ways from the other neural nAChRs it could be possible to develop subtype selective compounds that would allow therapeutic manipulation of nAChRs in number of conditions such as parkinsonism or smoking cessation.
Phage Display: Opportunities for Development of Personalized Anti-Cancer
Therapeutics SAMOYLOVA TI, COX NR
Scott-Ritchey Research Center, College of Veterinary Medicine, Auburn University, Auburn, AL 36830, USA
A personalized therapeutics approach is particularly critical for cancer patients since they have limited time to experiment with different chemotherapies in the attempt to find the most effective and the least toxic. Additionally, cancer is a very heterogeneous disease with significant molecular differences in patients with the same tumor type and grade. Designing therapies becomes even more complex as the disease progresses due to further accumulation of mutations. Finally, cancer therapies frequently are extremely toxic to normal tissues and organs causing severe side effects rarely seen in patients with other diseases. To reduce adverse reactions and to drastically improve the outcome of cancer treatments, cytotoxic therapies have to be targeted and each patient needs to be profiled for the presence of cancer targets before the therapy is put into practice. Phage display technology that uses combinatorial libraries of peptides and proteins displayed on phage particles is an essential tool widely used for identification of cancer-specific targeting molecules. Applications for such molecules include: targeted delivery of cytotoxic agents to cancer cells, affinity isolation of specific biomarkers expressed on the cancer cell surface, profiling of cancer specimens from individual patients, and the design of peptidebased anti-cancer therapeutics. Our research group is exploring these and additional novel avenues for development of customized therapeutics for cancer patients. Here, we discuss the current status, present our recent data, and highlight the potentials of phage display in development of personalized approaches for solid tumors.
Calreticulin, a novel B-cell molecular target in gastrointestinal malignancies SÁNCHEZ D1, PEKÁRIKOVÁ A1, PALOVÁ-JELÍNKOVÁ L1, BENEŠ Z2,HOFFMANOVÁ I3, MOTHES T4, TLASKALOVÁ-HOGENOVÁ H1, TUČKOVÁ L1 1Department of Immunology, Institute of Microbiology, Czech Academy of Sciences, Prague, Czech Republic; 2Thomayer´s Faculty Hospital, Prague, Czech Republic; 33rd Medical Faculty of Charles University, Prague, Czech Republic; 4Institute of Laboratory Medicine, Clinical Chemistry and Molecular Diagnostics of the University Hospital, Leipzig, Germany
Background: Calreticulin (CRT), a calcium binding protein and cellular chaperon is suggested to play a key role in the recognition of dying tumor cells by the immune system. Calreticulin is recognized by antibodies (Ab) of some patients with autoimmune mediated hepatic and intestinal diseases. In an attempt to analyze the immune reaction to CRT during carcinogenesis, we 1) quantified serum levels of anti-CRT Ab in newly diagnosed patients with primary hepatocellular carcinoma (PHC, n=41), pancreatic tumors (PT, n=55), colorectal carcinoma (CRC, n=30) and gall bladder tumor (GBT, n=27), and patients with high risk for PHC – patients with viral hepatitis C (VHC, n=18), and for PT – patients with chronic pancreatitis (PI, n=16), 2) analyzed B-cell epitopes of CRT.
Methods: ELISA assays with human recombinant CRT or its peptides as an antigen were used for quantification of serum anti-CRT Ab. Pepscan method employing synthetic decapeptides of CRT was used for determination of immunodominant epitopes of CRT.
Results: Statistically significantly elevated levels of anti-CRT Ab were found in patients with PHC and PT (IgA, IgG, P<0.001), CRC (IgA, P<0.001) and GBT (IgG, P<0.001) when compared with healthy controls. Interestingly, statistically significantly (P<0.001) higher levels of IgA anti-CRT Ab were found in sera of patients suffering form PHC in comparison with VHC patients. Significantly higher levels of IgA Ab against CRT peptide KGEWKPRQIDNP (frequently recognized by IgA Ab of oncological patients tested in Pepscan experiments) quantified by ELISA confirmed immunodominance of this peptide for PHC and PT.
Conclusions: 1) we revealed a developed immune reaction against CRT including affinity maturation and isotype switching in patients with gastrointestinal malignancies, 2) IgA and IgG anti-CRT Ab were predominantly detected in newly diagnosed patients with PHC, while 3) patients with VHC – representing a risk group for development of PHC – produced only low levels of anti-CRT Ab.
New Approaches in the Treatment of Lymphoma Patients, R-Chop: Rapid Infusion Rituximab SANCHEZ-RUIZ A, CANTOS B, MAXIMIANO C, MENDEZ M, GALAN L, HUELVES M, LOPEZ A, PROVENCIO M
Medical Oncology Service. University Hospital Puerta de Hierro. Madrid, Spain.
Background: Rituximab is a well tolerated treatment, but a cautious administration schedule is recommended to avoid infusion reactions. Rapid infusion over 90 minutes is feasible and safe. Since Rituximab is usually linked to other drugs with known myocardial toxicity, such as adriamycin, we decided to check the safety of a rapid infusion regimen and the presence of clinical or sub-clinical cardiac toxicity related to this method of administration.
Methods: We treated 42 patients with non-Hodgkin lymphoma with rapid infusion (over 60 minutes) Rituximab-based chemotherapy (CHOP-R), with average of 6.5 infusions of Rituximab per patient. Infusion-related events were analysed. We measured basal left ventricular ejection function (LVEF), every six months after chemotherapy, and the incidence of adverse cardiac events to assess cardiac toxicity.
Results: We had no grade 3 or 4 infusion-related events and no increased incidence or minor reactions during rapid infusions. None of the patients experienced cardiac events nor symptoms of cardiac failure during the whole observational period. Decreased in the post-treatment LVEF of over 10% was observed in 13 patients, and those with drop > 10% recovered normally. Patients with a LVEF decreased by 15% did not recover their normal level.
Conclusions: Rapid infusion Rituximab is safe and well tolerated and when add to chemotherapy does not cause clinical cardiac toxicity although some subclinical decreased in LVEF can be observed.
Construction -Dependent DNA Liposome Efectiveness on Tuberculosis Vaccination GAZIOLA DE LA TORRE L2; ROSADA RS1; COELHO-CASTELO AAM1 ; SILVA CL1, SANTANA MHA2 * 1Núcleo de Pesquisas em Tuberculose, Faculdade de Medicina de Ribeirão Preto, Universidade de São Paulo, Brazil; 2 Departamento de Processos Biotecnológicos, Faculdade de Engenharia Química, Universidade Estadual de Campinas, Brazil
Nowadays, non-viral adjuvants have been successful tested in DNA vaccination. Delivery systems such as multicomponent cationic liposomes have demonstrated transfection efficiency and elicited immune responses useful for vaccination. However, a great challenge is to create single - dose vaccines. Furthermore , the administration by a non-invasive route is comfortable and easier to reach populations in distant areas. Like ?magic bullets?, cationic liposomes composed by EPC/DOPE/DOTAP produce low citotoxicity and efficient transfection imparted by the gradual release of DNA as well as by the functionality of their components: structural (EPC) reducing citotoxicity, electrostatic (DOTAP) promoting interaction with DNA and cells and phase transition (DOPE) destabilizating the endosomal membrane for DNA delivery . In spite of that, the effects of DNA localization and partial complexation with the cationic lipid on the effectiveness of vaccination were not investigated. Using the functional EPC/DOPE/DOTAP liposomes, we produced constructions with internal and external DNA localization. In the first structure the DNA was entrapped and totally complexed with DOTAP in a sandwich like configuration. Secondly, DNA was partially complexed close to the surface of liposomes. Both contructions had the same DNA loading and charge ratio between DNA and DOTAP. The rigid control of the processing condition assured similar vesicle sizes (1-2µm) as well as reproductibility of the structures in small and pilot scale. The DNA-hsp65 was used in the liposomes due to its known successful prophylatic action against tuberculosis when administered as naked DNA. The effectiveness of vaccination was evaluated by intranasal route through CFU reduction in the lungs of immunized mice previously infected by mycobacterium tuberculosis. The results shown that when we used the external DNA-liposome construction, in a single dose with only 25 µg, a higher protection was observed with significant reduction of 1.97±0.23 log in the bacterial load compared to the saline group. No significant reduction on CFU was obtained by the internal DNA-liposome construction. This CFU reduction was the same observed when we used four doses (400 mg of total DNA) of naked hsp65 immunization by intramuscular route and also similar to BCG immunization. Besides, four doses of naked hsp65 by intranasal route did not induce reduction of bacilli number in the lungs. These results suggest an important structure ?function relationship: the effectiveness of cationic liposomes on vaccination depends on the rate of DNA delivery, which is not sustained by the structural stability only, but also it depends on the combination of DNA localization and partial complexation.
Effectiveness of Nystatin in Polysymptomatic Patients
A randomized, double-blind study in 116 individuals selected by a 7-item questionnaire (FRDQ-7) SANTELMANN H1, LAERUM E2, ROENNEVIG J3, FAGERTUN H4 1Holistic Center, Oslo, Norway; 2University of Oslo, Oslo, Norway; 3National Hospital, Oslo, Norway; 4Medstat Research, Stroemmen, Norway
Background: Antifungal therapy has been claimed to be effectivein polysymptomatic patients with diffuse symptoms from multiplebody systems and even well defined diseases, traditionally notrelated to fungi. Hypersensitivity to fungus proteins and mycotoxinshas been proposed as the cause..
Methods: We conducted a 4-week randomised, double-blind, placebo-controlledstudy in 116 individuals selected by a 7-item questionnaireto determine whether the antifungal agent nystatin given orallywas superior to placebo. At the onset of the study, the patientswere free to select either their regular diet or a sugar- andyeast-free diet, which resulted in four different subgroups:nystatin + diet (ND); placebo + diet (PD); nystatin (N); andplacebo (P).
Results:Nystatin was significantly better than placebo in reductionof the overall symptom score (P < 0.003). In six of the 45individually recorded symptoms, the improvement was significant(P < 0.01). All three active treatment groups reduced theiroverall symptom scores significantly (P < 0.0001), whilethe placebo regimen had no effect (P = 0.83). The benefit ofdiet was significant within both the nystatin (ND > N) andthe placebo groups (PD > P).
Conclusions:Nystatin is superior to placebo in reducing localisedand systemic symptoms in individuals with presumed fungus hypersensitivityas selected by the 7-item questionnaire FRDQ-7. This superiority isenhanced even further by a sugar- and yeast-free diet.
Telomerase as a Possible new Target for Cancer Treatment SARETZKI GC Newcastle University, Newcastle upon Tyne, UK
Background: Telomerase is a unique reverse transcriptase. Its main role is the protection and maintenance of telomeres, nucleoprotein structures at the end of linear chromosomes.
Most human somatic cells that do not or express low amounts of the enzyme and enter replicative senescence ones their telomeres are short and signal a DNA damage response. In contrast, tumour cells and around 85% of cancer express high levels of telomerase activity constitutively. This contributes to unlimited proliferation and cellular immortality. The broad expression of the enzyme in many cancer entities makes it an attractive target for the development of new cancer-therapeutic drugs.
