Background: Hepatitis B Virus (HBV) is a unique DNA virus which replicates through an RNA intermediate, lacks proof reading ability, has high viral replication rate. This leads to random mutations with amino acid substitution in reverse transcriptase region. In clinical parlance antiviral drug resistance is defined as selection of variants bearing amino acid substitution confirming reduced susceptibility to drug those results in primary or secondary treatment failure. For the management of HBV infection in addition to interferon/Peg Interferon/Thymosin alpha. 4 oral antiviral like lamivudine, adefovir, entecavir, Telbuvidine are licensed and Tenofovir and emcetarabine are licensed for HIV & HBV coinfection. Success rate of these antiviral agents do not exceeded more than 30 to 40% in long term treatment and with prolonged therapy, menace of antiviral resistance exists. Clinical consequence of resistance are decreased HBeAg clearance, reversal of histological improvement, increased rate of disease progression, ,clinical decompensation or even death in patients with cirrhosis, risk of graft loss and death in liver transplant recipients, transmission of drug resistance strain and vaccine failure mutation.
Methods: We analyzed real life data on our patients receiving long term Lamivudine treatment and development of resistance clinical consequences and their management...
Results: Our study included 82 patients (male 66, Age range 5-85 years). Of the 82 patients 50 patients were HBeAg +ve. These patients received mean duration of Lamivudine treatment 32.44 months. 17 out of 50 (34%) developed resistance to Lamivudine, 32 patients were HBeAg –ve, Mean duration of treatment with Lamivudine was 28 months. 8 out 32 (25%) developed Lamuvidine resistance. The presentation of Lamivudine resistance was clinical decompensation 3, sero-reversion 7, flare of liver enzymes 8, and increased viral load 5. All the patients who developed resistance were treated with addition of Adefovir to Lamivudine. Mean period of 8.5 months of follow up; 2 patients died dues to decompensation, remaining patients are stable with normalized liver function
Conclusion: Antiviral drug resistance is a major problem in management of chronic HBV infection. Combining second drug with no cross resistance at appropriate time seem to be best policy currently.
The F1-V Plague Vaccine Can Activate the Innate Immune Response Through Toll-Like Receptor2 and 4 but Does Not Need Them for an Antibody Response to the Vaccine AMEMIYA K U.S. Army Medical Research Institute of Infectious Diseases, Frederick, Maryland, USA
Background: A new recombinant fusion protein F1-V is in advanced development for preventing plague caused by Yersinia pestis. F1 is a capsular protein, and V is a low calcium response (Lcr) protein or V-antigen. V-antigen has been reported to activate Toll-like receptor (TLR) 2. We hypothesized that activating the innate immune system through TLRs is required for an antibody response to the F1-V vaccine.
Methods: We evaluated the interaction of V-antigen and F1-V with human embryonic kidney (HEK)-293 cells expressing TLR2 or TLR4 (InvivoGen). Further, we examined the antibody response, the proliferative response, and cytokine expression by splenocytes from F1-V-vaccinated TLR 2, TLR 4, TLR2/4, or myeloid differentiation factor 88 ( MyD88) knock-out (KO) mice. Activation of TLR 2 and 4 in HEK-293 cells was measured as per manufacturer’s instructions. Mice were vaccinated subcutaneously twice (0 and 28 days) with F1-V (1 – 2 ug) formulated with Alhydrogel. Serum and spleens were removed from anesthetized mice 21 days after the boost. The antibody response to the vaccine was measured by end-point ELISA. Cytokine expression by antigen-stimulated splenocytes were measured by BD FACSArray analysis after 40 h. Proliferation of splenocytes was determined by the amount of 3H-thymidine incorporated after an additional 18 h incubation.
Results: TLR2 and TLR4 HEK-293 cell lines were weakly activated by V-antigen but strongly by the F1-V vaccine. These antigens activated no other TLR cell lines. Wild- type, TLR2, and TLR4 mutant mice vaccinated with the F1-V vaccine all appeared to respond similarly to the prime and boost administration of the vaccine. This included the IgG1 and IgG2a isotype response to the vaccine. Furthermore, the antibody response in MyD88 KO mice was similar to that in wild-type mice. The proliferative response and cytokine expression was partially affected in the TLR2 KO but was essentially background level in the TLR4 KO, TLR2/4 KO, and MyD88 KO mice.
Conclusion: 1. TLR2 and TLR4 mediated innate immune responses are not required for an antibody response to the plague vaccine. 2. MyD88 is also not required for an antibody response to the vaccine. 3. Cellular immune responses to the vaccine appear to be partially dependent on TLR2 but appear to be completely dependent on TLR4.
Pharmacokinetic study of rivastigmine in Iranian healthy subjects following 3 and 4.5 mg dosing using a simple and sensitive HPLC-UV method AMINI H1, AHMADIANI A2 1Golestan University of Medical Sciences, Gorgan, Iran; 2Beheshti University of Medical Sciences, Tehran, Iran.
Background: Rivastigmine is relatively new drug and the evaluation of its pharmacokinetic properties in different ethnic populations is important to optimize the dosage regimens. For the pharmacokinetic study of rivastigmine, a simple and rapid but also a highly sensitive and selective bioanalytical assay method should be available. Aims: 1) To develop and validate a sensitive and selective analytical method for rivastigmine assay in plasma 2) To perform the pharmacokinetic study in Iranian healthy subjects 3) To compare the pharmacokinetics of rivastigmine following 3 and 4.5 mg dosing.
Methods: A simple and reproducible HPLC method with spectrophotometric detection at 200 nm was developed and validated for the determination of rivastigmine in human plasma. The assay was used for pharmacokinetic study of rivastigmine capsules in healthy Iranian subjects following 3 and 4.5 mg dosing. 23 healthy and fasted volunteers participated in both groups. Food and drinks were not allowed until 3 h after ingestion of the capsules. Multiple blood samples (5 ml) were collected before and 0.25, 0.5, 0.75, 1, 1.5, 2, 2.5, 3, 3.5, 4, 5, 6 and 8 h post-dosing. A non-compartmental analysis was used in the data processing.
Results: In HPLC analysis, it was founded that in addition to reversed-phase retention, other retention mechanisms such as hydrogen-binding or ion-exchange are probably involved in the chromatogeraphic behaviour of rivastigmine. Triethylamine should not be used in the mobile phase, while an acidic mobile phase and chromatography at high temperatures can increase theoritical plates for rivastigmine. A selective extraction of rivastigmine from plasma was obtained using 1-butanol/n-hexane (2:98, v/v) and back extraction into diluted acetic acid. The newly developed HPLC-UV method had an limit of quantification (LOQ) of 0.5 ng/ml, which is comparable to LOQ of 0.2 ng/ml obtained by current LC/MS methods. The pharmacokinetic studies showed that rivastigmine has a rapid oral absorption with a large inter-subjects variations. The mean values of maximum plasma concentration (Cmax), time to Cmax (tmax), area under the plasma concentration-time curve from time 0 to 8 hours (AUC8) and from time 0 to infinity (AUC∞), and plasma half-life following administration of the rivastigmine at 3 mg dosing were 6.27 ng/ml, 0.98 h, 11.95 ngh/ml, 12.79 ngh/ml and 1.11 h, respectively, and for 4.5 mg dosing 10.74 ng/ml, 0.91 h, 21.60 ngh/ml, 22.98 ngh/ml and 1.22 h, respectively.
Conclusions: For the first time, a highly sensitive HPLC-UV method was developed and validated for rivastigmine assay in plasma. Pharmacokinetics of rivastigmine in Iranian healthy subjects were comparable with results obtained in other ethnic populations. As reported by others, the oral bioavailability of rivastigmine increased with dose.
Antibiotic resistance: what can we learn from evolution?
AMINOV RI Rowett Inst. of Nutrition and Health, Univ. Aberdeen, Aberdeen, UK
Background: The brilliant “Zauberkugeln” (Magic bullets) idea of Paul Ehrlich was a major breakthrough in treating infectious diseases. It opened the whole new era in medicine and led to the discovery of many antibiotics that saved millions of lives. Despite the considerable success, however, bacteria invented various shields thus compromising the magic power of antibiotics. The number of pathogens that learned how to dodge these bullets is increasing and the question is how they acquired these properties? Aim: reconstruction of evolutionary history of selected antibiotic resistance genes to answer this question.
Methods: Four sets of genes encoding resistance to tetracyclines, macrolides, vancomycin and fluoroquinolones were chosen for this analysis: 1) tetracycline resistance genes, encoding ribosomal protection proteins; 2) the erm genes encoding enzymes that methylate the specific adenine residue in the 23S rRNA molecule; 3) the vancomycin resistance gene cluster represented by the concatenated set of vanHAX; and 4) the qnr genes conferring resistance to fluoroquinolones. The sequences were downloaded from GenBank and aligned using ClustalX ver. 1.83. Maximum-likelihood and Bayesian inference were used to reveal the evolutionary history of these genes.
Results: Phylogenetic reconstruction suggested a long evolutionary history of diversification of antibiotic resistance genes that began well before the “antibiotic era”. There is no indication that lateral gene transfer from antibiotic-producing bacteria has played any significant role in shaping the pool of antibiotic resistance genes in pathogenic and commensal bacteria. The primary antibiotic resistance gene pool originated and diversified within the environmental bacterial communities, from which the genes were mobilized and penetrated into taxonomically and ecologically distant bacterial populations, including pathogens.
Conclusions: Enormous metabolic diversity of bacteria allows them to come up with protection mechanisms even against novel antibiotics. To preserve the magic of novel antibiotic bullets we have to pay more attention to the pool of antibiotic resistance genes in the environment and carefully monitor the possible movement of such genes into commensal and pathogenic bacteria.
Activity of Antimalarial Constituents of Spathodea campanulata AMUSAN OOG University of Swaziland, Private Bag 4, Kwaluseni, Swaziland.
Background: The search for antimalaria drugs is a continuous one because of the devastating effect of the disease. Aim: To determine the antimalarial properties of Spathodea campanulata.
Methods: This study included 165 Swiss albino mice in Fink and Kretschmar’s, and Rane in vivo tests. Five mice were used per treatment, weight range: 18-22g. In the Fink and Kretschmar’s test, each mouse was inoculated with Plasmodium berghei berghei and treated post-infection subcutaneously once daily for 4 days with plant constituents, chloroquine or blank control. The % parasitaemia was evaluated on the fifth day post-infection. In the Rane test, the mice were treated with the drugs once daily for 4 days starting 3 days post-infection. The % parasitaemia for each mouse was determined for 5 days starting from the fouth day post-infection. The active constituents of the plants were isolated by column chromatography and characterised.
Results: Antimalarial principles of stem bark were ursolic acid, tomentosolic acid, 20β-hydroxyursolic acid and caffeic acid from leaves. In Fink and Kretschmar’s test ursolic acid at 15-60 mgkg-1day-1 produced 34-97% suppresion of parasitaemia and mean survival period of 13-25 days. Tomentosolic acid at 10-80 mgkg-1day-1 produced 35-82% suppresion of parasitaemia and mean survival period of 10-19 days. 20β-hydroxyursolic acid at 20-80mgkg-1day-1 produced 11-53% suppresion of parasitaemia and mean survival period of 8-13 days. The aqueous leaf extract at 50-400 mgkg-1day-1 produced 0-74% suppresion of parasitaemia. Chloroquine at 10 mgkg-1day-1 produced 98% suppresion of parasitaemia and mean survival period of 26 days.
In Rane test the aqueus leaf extract at 50-400 mgkg-1day-1 produced mean survival time of 11-15 days. Ursolic acid at 15-60 mgkg-1day-1 produced mean survival period of 9-24 days. 20β-hydroxyursolic acid at 20-80 mgkg-1day-1 produced mean survival period of 6-16 days. Tomentosolic acid at 5-40 mgkg-1day-1 produced mean survival period of 9-18 days. Blank control gave mean survival period of 7 days in both tests.
Conclusions: 1) The antimalarial principles of Spathodea campanulata demonstrated significant schizontocidal properties, the activity of chloroquine was however superior to any of them. 2) The activity of the antimalarial principles provides the scientific basis for the use of Spathodea campanulata in the management of malaria in traditional medicine.
Metalloantibiotics: Synthesis and Antibacterial Activity of Metal(II) Complexes Containing Cephalosporin and Sulfathiazole ANACONA JR Universidad de Oriente.Departamento de Quimica, Cumana. Venezuela
Background: The interaction of antibiotics with main and transition metal ions has attracted our attention and compelled us to combine their chemistry in order to improve the stability and efficiency of antibiotics. Thus, designing a new area of research in the synthesis of stable metalloantibiotic compounds that may be used as active drugs working effectively against antibiotic resistance species.
Methods: The metal(II) complexes were prepared by mixing clear solutions of the appropriate cephalosporin sodium salt (1 mmol) and NiCl2.6H2O or CuCl2 metal salts (1 mmol) in distilled water (10 mL) and sulfathiazole (1 mmol) in EtOH (10 mL). The reaction mixture was then stirred at room temperature for 12 h. and green precipitates formed. The precipitated complexes were filtered off, washed with water, MeOH and ether and dried under reduced pressure at room temperature. Yield 55-65%. No attempts to use different molar ratios to prepare the complexes were made. Antibacterial activities were tested using the paper disc diffusion method. The chosen strains were Staphylococcus aureus ATCC 25923, Escherichia coli ATCC 11775, Pseudomonas aeruginosa ATCC 27853, Klebsiellapneumoniae ATCC 23357, Salmonella enteritidis CDC 64 and Bacillus subtilis ATCC 6051.
Results: Nickel(II) and copper(II) react with cephalosporins plus sulfathiazole (Hstz) to form the following mixed ligand complexes: [M(cefazol)(stz)(H2O)], [M(cephalot)(stz)(H2O)2], [M(cefotax)(stz)], [M(ceftria)(Hstz)] and [M(cefepime)(stz)]Cl (Hcefazol = cefazolin, Hcephalot = cephalothin, Hcefotax = cefotaxime, H2ceftria = ceftriaxone) which were characterized by physicochemical and spectroscopic methods. Their spectra indicated that most the cephalosporins are acting as a monoanionic multidentate chelating agents, the exception being ceftriaxone which is dianionic. The complexes are insoluble in water and common organic solvents and probably have polymeric structures. They have been screened for antibacterial activity in DMSO solutions against several bacteria, and the results are compared with the activity of cephalosporins. The [M(cefepime)(stz)]Cl complexes showed better activity than free cefepime against all bacteria strains, including against P. aeruginosa and S. aureus where cefepime is inactive.
Conclusions: The synthesized compounds showed antibacterial properties. In comparison, the copper(II) and nickel(II) complexes containing cefepime plus stz showed better activity against several bacterial strains than the cefepime, thus introducing a novel class of metal-based bactericidal agents.
Production, isolation, partial characterization and antimicrobial spectrum of a novel bacteriocin produced by a Lactobacillus plantarum strain in fermentation ANASTASIADOU S*, PAPAGIANNI M, AMBROSIADIS I, KOIDIS P
Department of Hygiene and Technology of Food of Animal Origin, School of Veterinary Medicine, Aristotle University of Thessaloniki. Thessaloniki 54006, Greece
Background: Fermentation broths (in MRS) of an isolated Lactobacillus plantarum strain exhibited strong antimicrobial activity against common food spoilage and also food born pathogens. Antimicrobial activity was assessed in the agar diffusion assay using a total of 9 indicator food-grade bacteria. The antimicrobial activity appeared to be higher against closely related species and Lactobacillus curvatus was chosen as the most suitable among the tested microorganisms to serve as indicator for the quantification of the produced bacteriocin.The exhibited antimicrobial activity was due to the production of a proteinaceus compound, a bacterocin, most of which appears to be cell-associated.
Methods: A number of mechanical and physicochemical treatments were applied to washed cells in attempting to solubilize the bacteriocin. The most convenient method for extraction was centrifugation at 20200 rcf for 10 minutes at 4oC . Repeated tricine - SDS - PAGE electrophoresis of samples taken at various fermentation time-points showed that bonds as ~ 30kDa were of interest.The positon was finally determined by overlying the gel with MRS agar in which L. curvatus was embedded.
Results: The M.W. of the bacteriocin was estimated at 30kDa.The isolated bacteriocin lost its activity after treatment with lipase and ?-chymothrypsin, but retained activity following proteolytic treatment. The stability of bacteriocin was studied over a range of pHs,T and mechanical stresses.
Conclusions: It appeared to be heat labile, a characteristic indicative, along with M.W. and the sensibility to lipase and ?-chymothrypsin, of a certain category of antimicrobial peptides, the class IV of bacteriocins produced by lactic acid bacteria. Fermentation kinetics studies performed in a stirred tank bioreactor showed that the production of the bacteriocin was not associated to growth, but it was rather formed as a secondary metabolite.
Systems-directed targeted therapies in metastatic tumors: Equitable to reductionist therapy approaches? REICHLE A, VOGELHUBER M, VOGT T, BERAND A, BROSS K, WIEST R, KLEBL F, KULLMANN F, ROGENHOFER R, WALTER B, HAU P, ANDREESEN R University Hospital of Regensburg, Regensburg, Germany
Introduction: As we consider the exchange of information between tumor, adjacent stroma cells, and cells of the involved organ from a systems perspective, we may disregard operational prerequisites that a combination of activities triggered by specific action systems must be intended by single participating pathophysiological mechanisms, such as inflammation, angiogenesis, Warburg effect, immune response, extracellular matrix remodeling, proliferation, apoptosis, coagulation. Activities that seem to be operationally induced by the division of function present itself from a systems perspective as an enhancement of complexity. We hypothesized, that tumor systems-directed therapies might have the capability to use aggregated action effects, as adjustable sizes to therapeutically modulate the tumor systems’ stability, homeostasis, and robustness.
Methods: We performed a retrospective analysis of recently published data on 278 patients with advanced and heavily pre-treated (10% to 63%) vascular sarcoma, melanoma, renal clear cell, cholangiocellular, mucoepidermoid, and hepatocellular carcinoma, hormone-refractory prostate cancer, glioblastoma, and multivisceral Langerhans’ cell histiocytosis enrolled in nine multi-center phase II trials (13 centers). Each patient received a multi-targeted systems-directed therapy that consisted of metronomic low-dose chemotherapy, a COX-2 inhibitor, combined with one or two transcription modulators, pioglitazone +/- dexamethason or IFN-alpha.
Results: These treatment schedules may attenuate the metastatic potential, tumor-associated inflammation, may exert site-specific activities, and induce long-term disease stabilization followed by prolonged objective response (3% to 48%) despite poor monoactivity of the respective drugs. Progression-free survival (PFS) data are comparable with those of reductionist-designed standard first-line therapies targeting preferably tumor cell-specific pathways.
Conclusions: Differential response patterns indicate the therapies’ systems biological activity. Structured systems-directed therapies in metastatic cancer, targeting amongst others inflammation and neoangiogenesis, may break through the barrier of complexity of tumor-stroma-interactions, and get a source for detecting topologies of tumor-associated aggregated action effects as adjustable sizes for targeted biomodulatory therapies. Biomodulation of systems biological processes facilitates comparatively high efficacy at moderate toxicity.
A diarylquinoline targeting the energy supply of M. tuberculosis
ANDRIES K, KOUL A, LOUNIS N, GUILLEMONT J*, JARLIER V**
Tibotec NV, Beerse, Belgium, *Tibotec NV, Val de Reuil, France, **Pitié-Salpêtrière School of Medicine, Paris, France
We discovered a diarylquinoline (TMC207 or R207910) with potent bactericidal activity against drug-sensitive and drug-resistant Mycobacterium tuberculosis. Whole genome sequencing of resistant mutants suggested that the drug targets the energy supply of mycobacteria by inhibition of the ATP synthase. The oligomeric subunit c (AtpE) of ATP synthase was validated as the target by genetic, biochemical and binding assays. Unlike other TB drugs, TMC207 is equally active against growing and dormant TB bacilli, making it a good candidate for shortening TB therapy.
In mice, four weeks of TMC207 monotherapy exceeds the bactericidalactivities of isoniazid and rifampin by at least 1 log unit.Substitution of rifampin, isoniazid,or pyrazinamide (the World Health Organization'sfirst-line treatment regimen) with TMC207 accelerated bactericidal activity,leading to complete culture conversion after 2 months of treatmentin some combinations, against 5 months for the standard regimen. Four months of treatment with rifampin + pyrazinamide + TMC207 yielded the same relapse rate as six months of the standard regimen. Similar improvements were observed when TMC207 was combined with drugs to treat MDR-TB, suggesting that use of TMC207 may also significantly reduce the duration of treatment of MDR-TB.
The bactericidal activity of TMC207 was confirmed in patients in a one week early bactericidal activity trial and the drug is now being investigated in a phase 2 trial in MDR TB patients
The Chemical Interaction of the Anticancer Drugs Irinotecan-HCl and Epirubicin-HCl in the Same Infusion Solution ÖZDEMIR FA1, ANILANMERT B2, PEKIN M1 1 Marmara Univ. Faculty of Pharmacy, Istanbul, Turkey; 2 Erciyes Univ. Faculty of Pharmacy, Kayseri, Turkey
Background: Because the administration of infusions sometimes needs a long period of time in combination chemotherapy, it would appear that the best and easiest method is to give more than one drug in the same infusion solution. Because of this, investigation of the chemical compatibilities of chemotherapeutic drug combinations given in the same infusion solution before clinical studies is quite important. A new combination epirubicin and irinotecan were chosen for investigation of chemical incompatibility, in order to search the applicability of these two drugs in the same infusion solution in a combination chemotherapy.
Methods: Visual compatibility was assessed by means of effervescence, colour change, precipitation and pH change after the drugs had been injected into same infusion solution. Chemical interaction was further investigated quantitatively using a spectrophotometric method in infusion solution in clinical concentrations. The molar ratio for the reaction to proceed was also determined. All the interaction study was repeated in pharmaceutical forms, imitating a real application.
Results: No sign of incompatibility was observed upon visual examination. But a chemical incompatibility was observed in the UV spectra of the drug mixtures. The molar ratio of epirubicin-HCl/irinotecan-HCl at which the interaction reached a maximum was found to be 2:1. The chemical interaction occurred immediately after admixing and no visual or spectral change was noticed for 24 h after the interaction had occurred.
Conclusions: These drugs are chemically incompatible. The positive or negative contribution of such chemical interactions to the pharmacological effect of the combination might be of importance and while the applicability of these two drugs in combination is investigated in further pharmacological studies, their chemical interaction should also be a consideration.
A Potential Tumor Cell-Penetrating Peptide, CRGDCF(K[H-]KKK)6, for the Delivery of Antisense and siRNA Oligonucleotides AOKI Y1,2, NISHIO S2, MIYAMOTO T2, KUMAGAI M1, HASHIZUME K2 1Department of Internal Medicine, Matsumoto National Hospital, Matsumoto, Japan; 2Department of Aging Medicine and Geriatrics, Institute on Aging and Adaptation, Shinshu University Graduate School, Matsumoto, Japan
Background: We have previously developed a tumor cell-penetrating peptide (cRGD-hK) of 36 amino acid residues, which comprises a cyclic RGD motif for tumor homing and cell internalization, a DNA- or RNA-binding oligolysine, and histidyl residues to facilitate the delivery into the cytosol. The length of antisense or siRNA oligonucleotides is thought to match that of the oligolysine, presuming that both form complexes electrostatically in a 1:1 molar ratio (Figure). Then, we have been assessing the possibility of such complexes as antineoplastic agents.
Methods: 1) With luciferase expression plasmids as a reporter, we tested the functional potency of elements of cRGD-hK in cancer cells. 2) The effects of the complexes of cRGD-hK with antisense phosphorothioate DNA and siRNA corresponding to the luciferase gene were compared in vitro. 3) Using siRNA corresponding to the c-raf gene, we assessed the effects of cRGD-hK/siRNA complexes on intracellular c-Raf protein levels of pancreatic cancer cells and on the tumor growth in nude mice.
Results: 1) The three elements of cRDG-hK were indicated to function as expected, by using bafilomycin A1 and cycloRGDfV. 2) The gene-silencing effect of the complexes with siRNAs was greater than that with antisense phosphorothioate DNA. 3) The peptide/siRNA complexes lowered intracellular c-Raf protein levels of cultured cells at less than 500 nM for 48h incubation. When tumor-bearing nude mice were intraperitoneally administered with the complexes (5 μg RNAs three times a week for 4 weeks), the tumor growth was found to be significantly (P<0.05) inhibited in the third and fourth week.
Conclusions: It is suggested that the cRGD-hK could function as a tumor cell-penetrating peptide for the delivery of siRNA and antisense oligonucleotides, but further studies are needed.
Fig. Schematic drawing of a putative complex of the tumor cell-penetrating peptide (cRGD-hK) and small interfering RNA duplex (siRNA).
Preoperative use of Analgesia in Appendicites
ARAM FO Hadramout University, Mukalla, Yemen.
Background: Appendicitis is a common cause of acute abdominal pain, early analgesia was considered previously as it could mask physical signs and hence delay the diagnosis and surgical intervention, now this has been challenged
Methods: A prospective, experimental study had been carried out in Ibn Sinna general Hospital from November 2006 to March 2007, our aim was to determine the influence of Diclofenac sodium DS(voltaren) on masking the diagnosis of acute appendicitis.
The data collected by using well designed questionnaire with observation during the period of admission before the operation.
Results: The study includes 80 patients (40 as cases and 40 as controls) and The result revealed that most of the symptoms (fever, anorexia, nausea and vomiting) and sings (tenderness, obturator and Psoas signs, local guarding and rigidity), in addition to the rate of perforation and the vital sign were not hidden by DS(Voltaren) with P value >0.05.while other symptoms (pain) and signs (rebound tenderness, Rovsing and pointing) had been hindered by the use of DS (voltaren). The most common presenting symptom in placebo and DS group was pain (100%) which showed a marked decrease in severity in those who received voltaren as analgesia (72.2%)
Conclusion: Some of the symptoms and signs of acute appendicitis were masked by the use of analgesia; while others were not .and overall Diclfenac sodium did not influence the decision of diagnosis or the management of acute appendicitis.
Single Chain Antibodies (ScFvs) and Immunoconjugates: Computational and Functional Approaches ARCANGELI C1, PUGNALI M2, SPERANDEI M2, CANTALE C2, BUCHER M3, GIANESE G4, ROSATO V1,4, GALEFFI P2 1ENEA, Dept. FIM-CAMO Rome, Italy; 2ENEA, Dept. BAS-BIOTEC Rome, Italy; 3Univ. Cologne Cologne, Germany; 4Ylichron S.r.l. c/o ENEA, Rome, Italy.
Background: ScFvs in which the variable heavy and light chains are connected by a peptide linker maintain the binding specificity and affinity of the parental antibody IgG. ScFvs coupled to highly toxic molecules (immunoconjugates) are currently being developed for cancer therapy. Aims: 1) To assess the effects of specific mutations on the stability, structure and dynamics of the scFv antigen binding site. 2) To develop an in silico procedure for evaluating physicochemical properties of two tumor-targeting anti-HER2 immunoconjugates. 3) To propose plant-based expression systems for high-level scFv production.
Methods: This study included four scFvs (scFv(F8), scFv(ADDLs), scFv(FR5), scFv(800E6)) and two tumor-targeting anti-HER2 immunoconjugates (scFv(FR5)-ETA, scFv(800E6)-ETA). For the computational procedures all the antibody structures were derived by homology modeling and assessed by molecular dynamics (MD) simulations. As regards the experimental section tobacco plants were transformed for stable and transient expressions. Specific expression vectors containing the gene encoding for the scFvs of interest were used. Transgenic plants were cultivated also in hydroponic and aeroponic systems. DNA, RNA and protein analyses were performed in leaves, roots and root exudations.
Results: Structural and MD analysis indicated a strong correspondence between structurally–determined flexibility of the binding site with the different functional behaviors proved by the wild-type and its mutants. Computational analysis of anti-HER2 immunoconjugates showed that the presence of a toxin does not significantly affect the major physicochemical parameters and their structure. The highest level of scFvs expression was observed in roots.
Conclusions: 1) The computational approaches represented a good tool for structure-based design of antibody-binding site, for analyzing physicochemical properties of immunoconjugates and for predicting the effects of the linked toxin on structure, dynamics and functionality of the antibodies. 2) The proposed plant-based expression system seems to represent a promising tool for a large-scale scFv production.
Exercise as a Modality to Identify Therapeutic Molecules for Treatment & Prevention of Cancer-Associated Cachexia: Possible Enhancement of Anti-inflammatory Cytokines Through an Intermittant Activation of the Stress-Response Pathways ARDIES, CM Northeastern Illinois University, Chicago, USA.
Background: Repeated exercise is well-known to reduce risk for cancer, cardiovascular disease, type II diabetes, and a variety of neurological disorders; a magic bullet if there ever was one! Cachexia affects approximately 50% of all cancer patients and is characterized by weakness, fatigue, anorexia, adipose and skeletal muscle atrophy, insulin resistance, and impaired immune function; a condition desparately in need of a magic bullet! Cachexia appears to be associated with elevated levels of PIF, TNF-α, IL-1, IL-6, and Interferon-γ. Exercise induces the production of sTNFr, IL-1ra, and IL-10 (anti-inflammatory cytokines). Aim: To test the hypothesis that activation of stress-response pathways may be involved in the protective effect of exercise.
Methods: Rats were familiarized with a rodent treadmill on four separate days over a period of two weeks and then forced to run for 60 minutes at a speed of 27 m/min (a moderately hard workload for untrained rats). Three animals were killed at each time point: 0, 15, 30, 60, 90, 120, 180, 240, and 300 minutes after the start of the exercise. Lungs from 3 animals at each time point were pooled and nuclei prepared by Dounce homogenization and differential centrifugation. Nuclear proteins were analyzed by western blot using anti cJun/AP1 monoclonal antibody (Ab-3, Oncogene Research Products).
Results: Jun binding was appearent at 4 hours and one hour later was undetectable. This indicates that a 60 minute bout of running exercise is sufficient to enhance jun content but that this effect is of a relatively short duration.
Conclusions: Based on these preliminary results the possibility exists that a transient exercise/stress-mediated activation of the MAPK and/or JNK-MAPK pathways (possibly through enhanced Ca++ and ROS) is responsible, in part, for the anti-inflammatory effects of exercise as well as the enhanced activity of antioxidant enzymes and phase II enzymes previously observed by this lab. Because skeletal muscle is far more metabolically active than lung during exercise, one might expect that the degree of an exercise-induced activation of AP-1 in muscle would be greater than that of lung. Development of drugs which mimic this activation profile should produce similar benefits.
Magic Bullets in Removing Enterococcus faecalis Biofilms. Arias-Moliz MT1, Ferrer-Luque CM1, Espigares-Rodríguez E2, Pérez-Heredia M1, Baca-García P1 1School of Dentistry, University of Granada, Spain; 2School of Pharmacy, University of Granada, Spain.
Background:Enterococcus faecalis is the most common and, occasionally, the only single isolated bacterium from root canals of teeth with persistent periapical periodontitis. Its inherent antimicrobial resistance and ability to adapt to harsh environmental changes make E. faecalis responsible for many endodontic failures; moreover, such adverse conditions may favour the growth of this bacterium as a biofilm in root canal walls.
The elimination and/or control of E. faecalis biofilms is a goal in root canal therapy. Several irrigating solutions are used during the endodontic treatment; some of them have been widely tested against planktonic bacteria. Because of the high resistance of the biofilms to the endodontic irrigants, the aim of our study was to evaluate the effectiveness of four irrigating solutions used in root canal teeth against E. faecalis biofilms.
Methods: Four irrigants - sodium hypochlorite, chlorhexidine, ethylene-diaminetetraacetic acid (EDTA) and citric acid - were tested at 1, 5 and 10 min of exposure to biofilms of E. faecalis ATCC 29212. The biofilms were grown aerobically in the MBECTM high-throughput device for 24h at 37ºC. They were exposed to ten serial twofold dilutions of each irrigating solution. The antibacterial activity of the root canal irrigants was evaluated by determining the viable cell counts and log killing of E. faecalis biofilm cultures. A concentration of an irrigant was considered to be effective when it produced a reduction of ≥ 5 logarithmic units.
Results: Sodium hypochlorite was the most effective solution at any dilution and time of exposure tested. Chlorhexidine and citric acid solutions showed less antibacterial activity and needed more time to kill E. faecalis biofilms. EDTA solution lacks antibacterial activity against E. faecalis biofilms even after 10 min contact time.
Conclusions: Sodium hypochlorite should be elected as the best irrigating solution as it showed the highest effectiveness against E. faecalis biofilms.
Vaccination with Recombinant MHV68 Producing IFN Effectively Protects Mice Against Infection With Wild Type MHV-68 and Dramatically Reduces the Establishment of Long-term Spleen Latency. ARICO’ E1, MONQUE DM1, MOSCHELLA F1, D’AGOSTINO G1, VENDITTI M1, KALINKE U2, ALLEN D3, NASH A3, BELARDELLI F1, FERRANTINI M1.
1Istituto Superiore di Sanità, Rome, Italy; 2Paul Ehrlich Institute, Langen, Germany; 3University of Edinburgh, Edinburgh, UK.
Background: Human gammaherpesviruses such as Epstein-Barr Virus cause lifelong infections and associated diseases, by virtue of their ability to establish latent infection.
Mice infected with murine herpesvirus 68 (MHV-68) represent a versatile experimental setting to study the biology of gammaherpesviruses and to test vaccination strategies against them.
We recently observed that a clone of recombinant MHV-68 carrying the mouse IFN1 gene (MHV-68mIFN1) shows a significant in vivo attenuation, which is mediated by the cytokine released during the course of the infection, and affects both the acute replication and spleen latency.
Methods: C57 BL/6 mice received two intranasal (i.n.) administrations of 105 earstwhile pfu of psoralen-UV-inactivated wt or recombinant IFN1-producing MHV68. A group of mice received i.n. 105 pfu of live-attenuated MHV68m IFN1 as a vaccine. Four weeks later, vaccinated and a group of unvaccinated mice were infected (i.n.) with 4 x 105 PFU of MHV-68.
Virus titres in the lungs and spleen latency were measured by plaque and infectious centre assay respectively. Virus non-specific B-cell activation was assessed by FACS. Molecular analysis of the viral genomes harboured in mice spleens cells was performed by real time PCR. An ELISA was used to quantify the anti-MHV68 humoral immune response.
Results: Mice vaccinated with live-attenuated or partially inactivated MHV-68mIFN1 were protected against the challenge with wt MHV-68 in terms of acute replication and long-term latency. This protection was associated with a significant virus-specific IgG antibody response.
Conclusions: IFN1, produced at the site of the infection by the vaccinating virus, acted as an adjuvant in stimulating an anti-MHV68 immune response effective in eliciting protection against all phases of MHV68 infection. We believe that the ability of MHV-68mIFN1 to produce IFN1, thus efficiently stimulating the immune system whenever it reactivates from latency, makes this recombinant virus a safer live-attenuated vaccine as compared to the latency-deficient clones previously described by others.
Remifentanyl: How it Relives Human and Earthly Pain, and New Perspectives. ARIZA F1; TORRES G2 1Transplantation Anesthesiology,Fundación Valle del Lili, Cali, Colombia; 2Cardiovascular Anesthesiology, Fundación Valle del Lili, Cali, Colombia.
Background: Remifentanyl use has become a standard analgesic therapy during and after low-high complexity surgeries. Its indications have extended beyond surgery rooms and today, it is one of the drugs that has importantly changed the way of administrating anesthesia for a considerable number of procedures specially for those that are ambulatory (a-day surgery). However it is unknown what it´s impact will be over the emission of halogenated anesthetics which may be the origin of organic-halogenated pollutants (OHP) that are released into the atmosphere, resulting in ozone layer damage and more over these compounds are recognized as neurotoxic and endocrine modulators. Around the world there are many institutions where it is not possible to administer total endovenous anesthesia (TIVA) due to economical implications. Our objective was to analyze the impact of the use of remifentanyl during surgery over the quantity of halogenated anesthetics, using intermediate infusions of remifentanyl (0,15-0.25 mcg/kg/min).
Methods: Over a period of one year an initial prospective study was undertaken to identify mean plasma concentrations of remifentanyl for a target biespectral index (BIS) of 40-50, in a hispanic population at a level 4 hospital, based on the pharmacokinetic model described by Minto et al. The second stage of the study was to identify the total amount of halogenated gases consumed during this period comparing it to the consumption from the previous year. Variables are reported as number of patients, medians and ranges, means and SD. Nominal data were analyzed using the exact Fisher test. Ordinal and continual non-normally distributed variables were analyzed using Mann-Whitney Rank summ test. Continuous variables were compared using Student t-test.
Results: 12.532 surgical procedures were done. There were no significant differences in comparing demographic variables between studied groups. Remifentanyl mean plasma concentration in this population was 4,76 (±0,45) ng/dl for an average consumption of 0,17 (± 0,13) mcg/kg/min. being significantly high for those patients whom had undergone abdominal surgeries (p<0,025). Desflurane, sevoflurane and isorane consumptions were significantly reduced when remifentanyl was used [32 (±5), 23(±2), 15 ((±1) vs. 13 (±3); 9 (±1); 6(±1) ml/hr. (p<0,001)]. The total estimated amount of halogenated gases per month was reduced to approximately 55% (40.192 vs. 17.099 ml).
Conclusions: 1.Standardized use of remifentanyl in surgical units which use balanced inhaled anesthesia techniques, significantly reduces the usage of volatile halogenated anesthetics, and protects environment. 2.Implications derived from these observations could be useful for those institutions in various developing countries where TIVA is not feasible for economical or infrastructural reasons.
3. New associations between remifentanyl and other drugs like ketamine or midazolam, based on pharmacological interaction mechanisms could lower even more halogenated gas consumption during surgery. It is a necessity to continue searching new anesthetic technics that are friendly with our planet and at the same time giving economic benefit especially to those in poorer populations.
Differently Directed Changes in Interferon-γ Production Depending on Radioadaptive Response. ARKHIPOVA EN, ALCHINOVA IB, KARGANOV MYu
Institute of General Pathology and Pathophysiology, Russian Academy of Medical Sciences, Moscow, Russia
Background: Interferon-γ (IFN-γ) is a pleiotropic cytokine with antiproliferative and immunomodulatory activities that are crucial for the regulation of immune responses.
Methods: We examined a group of military pilots. The examinees were divided into 3 subgroups: ground personnel (9 persons, control group), 17 pilots with <1000 h flight time, and 12 pilots with >1000 h flight time. The quality of reparation is in many respects genetically determined; therefore, we used peripheral blood lymphocytes from pilots for in vitro detection of a radioadaptive response (RAR), which was evaluated by the number of chromosome aberrations.
Results: No differences in IFN-α serum content after induction by NDV virus were detected. The adaptive response was observed in 7 individuals of the control group (78%), in 10 pilots who had <1000 flight hours (59%), and in 4 pilots having >1000 flight hours (33%). The examined individuals were divided into 2 groups depending on the presence of RAR, and IFN-γ production after radiation was measured. It was shown that at doses 0.05 Gy or 0.5 Gy no differences between groups were detected. Exposure with these doses sequentially in 48 h interval resulted to differently directed changes: lymphocytes of individuals with RAR produced more IFN-γ than before while cells of persons without RAR made it less.
Conclusions: The quality of adaptive mechanists evaluated by RAR may be useful for estimation of individual sensitivity to radiation during radiotherapy in oncology and in prediction of professional risk.
Conscious drug selection and dosing by genotyping and phenotyping of alleles with mutations, deletion and/or duplication of the CYP2D6 gene ARNETH B1, SHAMS M2, HIEMKE C2, and HÄRTTER S2 Departments of Clinical Chemistry1 and Psychiatry2, University of Mainz, Germany
Background: Polymorphisms of Cytochrome p450 2D6 (CYP2D6) have a significant effect on the pharmacokinetics of most antidepressants. Individuals are classified as poor metabolizers (PMs) due to inheritance of two mutant CYP2D6 alleles and develop higher plasma drug concentrations causing an increased risk of side effects and toxicity when subjected to standard recommended doses of CYP2D6 substrate drugs. In contrast individuals are classified as ultrarapid metabolizers (UMs) due to inheritance of alleles with duplicated or multiduplicated active genes and will thus under circumstances not reach therapeutic plasma levels of CYP2D6 substrates, leading drug resistance and false accusation of non compliance. Our study aimed to develop a rapid and reliable procedure used for early detection of PMs- associated mutations and/or deletions (CYP2D6 *4, *3, *6, *9 and *5) and UMs- associated CYP2D6 gene duplication or multiplication.
Methods: EDTA blood was drawn from twenty five pre-selected depressed patients [(14 men and 11 women), mean age ±SD (49.8 ±12.7)] treated with the antidepressant venlafaxine and having unusual resulting plasma concentrations of venlafaxine were chosen for genotyping analysis. Real-time PCR reaction with subsequent fluorometric melting point analysis of the PCR product was used. Gene deletion (*5) and gene duplication or multiplication were investigated based on the measurement of the fluorescence intensity quotient (N) of the CYP2D6 gene relative to the albumin gene as an internal standard gene using a quantitative PCR technique.
Results: One homozygous (*6/*6), and three heterozygous (two with*4/*5 and one with*4/*6) were detected. Five individuals were heterozygous (*4/non detectable allele) and only one patient had heterozygote gene duplication [*4/gene duplication (2x*1)]. Six individuals had gene duplication. Melting curves were verified using DNA samples of known genotypes and by sequencing the PCR products.
Conclusions: Our new genotyping procedure was evaluated against the ratio between the O-demethylated (ODV/V) metabolite of the CYP2D6 substrate Venlafaxine (V) and determined by HPLC serving as phenotype of the genotyped patients. This genotyping procedure was regarded as fast and reliable for clinical routine.
Cardiac tamponade from slingshot metal darts in Chuuk: a retrospective review of cases. ARSENAL JC, REMIT K, YICHIRO O.
C/- Chuuk State Hospital, Federated States of Micronesia. jcars3@yahoo.com
We determined the immediate cause of death of patients with penetrating cardiac injuries from slingshot metal darts. This retrospective review of cases focused on those 7 patients with penetrating cardiac injuries from the period July 1999 to July 2005. There were 6 patients who underwent emergency thoracotomy regardless of the type of operative approach. Five of the 6 patients who were operated underwent left Lateral Thoracotomy and 1 patient underwent Median Sternotomy. There were 11 patients who sustained cardiac injuries out of the 240 cases reviewed. The patient's with cardiac injuries had a higher mortality (27.3%) than those who have penetrating thoracic injuries (3.5%) without associated cardiac injury. Of the seven patients who had penetrating cardiac injuries, 5 patients underwent left Lateral thoracotomy and 1 patient underwent median sternotomy. All 6 patients had chest tube thoracotomy insertion prior to surgery. There were 2 deaths in this review of penetrating cardiac injuries. The other patient with 7 multiple slingshot injuries died of cardiac tamponade with hypovolemic shock.
The Long Term Follow up Results of Hydatid Cysts after Albendazole and Mebandazole Tratment ARSLAN A1, ÖDEV K2, PAKSOY Y2, ARIKOGLU H2, and ŞAHİN M2 1Gaziantep University, Gaziantep, Turkey, 2Selçuk University, Meram Campus, Konya, Turkey
Background: Our experiences gained over the years of study on the treatment of hydatid cyst disease showed that women are more susceptible to hydatid cyst disease then men. Anthelminthic drugs such as albendazole or mebendazole are more effective in lung cysts than the liver cysts when prescribed orally. Initially we undertook experiments to explain this phenomenon.
Methods: Cystic liver and lungs were collected freshly from the municipal slaughter house and from the patients. Each cystic fluids collected and viable scoleces were pooled and used for analysis. Scolicidal agents such as hypertonic saline solution, 0.5% silver nitrate, 25% ethanol, and 10% albendazole suspension were used at a time to determine the effect of scolicidal agents kinetically on the viability of scoleces from minutes to 72 hours assessed by eosin Y dye solution exclusion and inclusion.
Clinical use of in vitro findings: Initially, sheeps having hydatid cyst disease were selected by ultrasonic examination of the liver and lung of the animals brought to slaughter house. The animals were grouped for albendazole and mebandazole treatment. And each animal cysts were treated percutaneously with either 10% albendazole or mebandazole suspension solution. Based on sheep experiment the patients treated with saline solution by means of PAIR-PD (percutaneous puncture, aspiration, injection and irrigation, and reaspiration) and albendazole by PAI (percutaneous aspiration and injection) procedure.
Results: Among them hypertonic saline solution, ethanol, and silver nitrate treatment exerted immediate effect within half an hour while albendazole showed its effect after 48 hours. Because of these findings we used pre and post percutaneous oral albendazole or mebandazole treatment for 72 hours prior to PAIR-PD or PAI involvement. Thus, turgid cyst becomes flaccid and enables puncture without spilling the cystic content which prevents complications, dissemination and recurrence. Percutaneous drainage and irrigation application showed that protoscoleces metabolize and convert these drugs into active form to affect their viability irreversibly. Generally, patients are referred to hospital with already existing cysts. PAI of the cysts with antihelmintic drugs or PAIR-PD of scolicidal agents or saline solution cure the cyst with a minimum morbidity. The kinetic in vitro studies showed that sclerosing agents, such as ethyl alcohol, silver nitrate, saline solution kill the protoscoleces immediately and albendazole and mebandozole suspension solution starts affecting 48 hours later and kills the protoscolecess completely after 72 hours. However, sclerosing agents can cause chemical sclerosing cholangitis which limits their use.
Conclusion: The procedure described above offerred safe alternative to surgical treatment of hydatid cysts. Over 10 years follow up our procedure did not experience any mortality which is higher with surgical procedure. Complications such as anaphylactic reaction, side effects and recurrence are minimal. Hospital stay is reduced considerably and the patient can return the work the following day. Hospital cost is reduced considerably. However, the best therapy is to prevent cyst formation from the protoscoleces at early phase of infection or to prevent cyst growth by preventing the differentiation of germinative membrane. Therefore, it looks plausible to develop vaccination targeted to germinative membrane differentiation which circumvents immune system in the infected patients.
References:
1. Arslan A, Ödev K, Baykan M, Arıkoğlu H, Çataloluk O, Paksoy Y, Bulun M: Ekinokokkus granulosus’un bulaşması, organ tutulumu, tedavisi ve karapınarlı için alınması gereken tedbirler. Karapınar Sempozyumu, 26–27 Ekim 2000, Karapınar, Konya, Sempozyum kitabı s: 281-292.
2. Ödev, K., Paksoy, Y., Arslan, A., Aygün, E., Sahin, M., Karaköse, S., Baykan, M., Arikoglu, H., Aksoy, F., 2000. Sonographically guided percutaneous treatment of hepatic hydatid cysts: Long-term results. J. Clin. Ultrasound. 28, 469-478.
3. Paksoy, Y., Ödev, K., Sahin, M., Dik, M., Ergul, R., Arslan, A., 2003. Percutaneous sonographically guided treatment of hydatid cysts in sheep. Direct injection of mebandozole and albendazole. J. Ultrasound. Med. 22, 797-803.
4. Paksoy, Y., Ödev, K., Sahin, M., Arslan, A., Koc, O., 2005. Percutaneous treatment of liver hydatid cysts: comparison of direct injection of albendazole and hypotonic saline solution. AJR. AM. J. Roentgenol. 185, 727-734.
5. Arslan, A., Arikoglu, H., Paksoy, Y., Odev, K., Koc, O., 2006. Comment on percutaneous treatment of liver hydatid cysts. Reply. Am. J. Roentgenol. 186, 1199-1200.
Efficacy of amoxicillin/clavulanic acid in preventing infectious and inflammatory complications following impacted mandibular third molar extraction. ARTEAGOITIA I1, DIEZ A2, BARBIER L12,SANTAMARIA G1 SANTAMARIA J12 1University of the Basque Country. Leioa, Spain 2Cruces Hospital. Osakidetza. Spain
Background: The aim of this clinical trial was to evaluate the efficacy of amoxicillin/clavulanic acid 500/125 in the reduction of infectious and inflammatory complications after extraction of an impacted mandibular third molar. Our hypothesis is there are more infectious and inflammatory complications in patients treated with placebo than in those treated with antibiotic, with a maximum ratio difference of 0.067.
Methods: A double-blind placebo-controlled randomized clinical trial. The sample was derived from the population of subjects attending Cruces Hospital for evaluation and extraction of a impacted mandibular third molar. A maxillofacial surgeon performed the operations under local anesthesia. The surgical technique was the same in all cases, and the follow-up period was 8 weeks. Patients were treated with postoperative placebo or amoxicillin/clavulanic acid 500/125 mg 3 times a day during 4 days. The outcome variable was infectious and inflammatory complications. Sex, age, smoking, molar depth, angulation, need for sectioning, ostectomy, and operation time were recorded. Analysis was by intention to treat.
Results: A total of 490 lower third molars were surgically removed (259 antibiotic and 231 placebo), The patients' mean age was 24,15 (23,70-24,59), the frequency of postoperative infectious and inflammatory complications was 1.9% in the antibiotic and 12.9% in the placebo group (OR 7.6, 95%CI 2.9-19.9; P < .001). The number needed to treat was 10 (7-16). Unadjusted relative risk was 0.15 (0.06-0.38) (P < .001). Absolute reduction risk was 0.11(0.066-0.155)]. Therefore, the hypothesis cannot be rejected. In third molars submucous (p=0.091 and NNT 17 with IC95% 8-infinite). Multivariate analysis shows treatment with antibiotic (OR = 8.66 (3.17-23.67); P < .001) and age (OR = 1.08 (1.00-1.16); P = .029) are the only variables to be included in the logistic regression model. Possibility of infection: P(x)=1/1+e -(-3,74+0,074 EDAD-2,075 ANTIBIOTIC(1)) Severe complications occurred in one patient in placebo group
Conclusions: Amoxicillin/clavulanic acid is efficacious in reducing the incidence of postoperative infectious and inflammatory complications following third molar extraction but should not be prescribed in all cases. Preventive antibiotic treatment would not be indicated for third molars submucous. Age should be taken into account, infection risk increases 8% every year in all patients.
Manganese (Mn) Transport at the Blood-Brain Barrier: Implications for Parkinson's-Like Disease ASCHNER M1,2, BENEDETTO A1, AU C1 1Dept. Pediatrics and2Dept. Pharmacology, Vanderbilt University Medical Center, Nashville, TN, USA
Background: Though an essential trace element, exposure to high Mn levels has been implicated in a Parkinson’s-like disease, manganism. We hypothesized that symptoms associated with these two disorders reflect perturbations in shared molecular pathways. Accordingly, studies were carried out inC. elegans to test the hypotheses that (1) divalent metal transporter (DMT1) is a putative Mn transporter, and (2) Mn preferentially targets dopaminergic (DAergic) neurons.
Methods: Bristol wild-type (WT)C. elegans N2 strain was used unless otherwise indicated and was grown at 20°C in Petri dishes on nematode growth medium inoculated with OP50E. coli. For lethality assessment, live and dead worms were counted on each plate and recorded. Mn content was measured by atomic absorption spectrometry. Neurodegeneration was assessed by confocal microscopy.
Results: TheC. elegans genome encodes three DMT1 orthologues (SMF-1, SMF-2, SMF-3).C. elegans deletion-mutantssmf-1(eh5) andsmf-3(ok1035) exhibited resistance to Mn exposure compared to WT, whilesmf-2(gk1330) was more sensitive. Corroborating these observations, after exposure, Mn content insmf-2(gk1330) was greater than WT,smf-1(eh5) andsmf-3(ok1035), the latter taking up the least Mn of all mutants. SMF-1::GFP and SMF-3::GFP were found co-expressed in the gut and the major epidermis hyp7, likely accounting for most Mn uptake, while SMF-2::GFP was mainly expressed in the "mc1-3" and "vpi1-6" cells. Confocal microscopy revealed that Mn specifically targeted DAergic neurons, while sparing others (GABA, glutamate, and acetylcholine). The DA transporter knock-outdat-1(ok157) and the DA receptordop-2(vs105);dop-1(vs100);dop-3(vs106) triple mutant were hypersensitive to Mn exposure. Combined with measurements of DA content in these strains, our results established that elevated DA levels sensitize the worm to Mn toxicity.
Conclusions: (1) DMT1 in a putative blood-brain barrier Mn transporter and a target for transport inhibition into the mammalian brain under conditions of excessive Mn exposure. (2) DAergic neurons are exquisitely sensitive to Mn and extracellular DA is involved in Mn-induced neurotoxicity. (3) C. elegans is ideally suited for studies on genetic pathways involved in Mn toxicity and PD.
Supported by R01 ES10563 & DoD W81XWH-05-1-0239.
Targeting Dexamethasone-Loaded anti-E-selectin Liposomes Prevents Glomerulonephritis Progression: The Potential of Vascular Bed-Specific Drug Delivery Ásgeirsdóttir SA, Kamps J, Heeringa P, Ruiters MHJ, Molema G
University Medical Center Groningen, University of Groningen, The Netherlands, Dept of Pathology & Medical Biology, Laboratory for Endothelial Biomedicine & Vascular Drug Targeting Research
Background: Glomerulonephritis is a renal disease characterized by glomerular inflammation which is frequently treated with glucocorticoids. However, their use has limitations because of systemic side effects.
Aim: To test the hypothesis that targeted delivery of dexamethasone by immunoliposomes to glomerular endothelium decreases renal injury, while preventing systemic side effects of dexamethasone.
Methods: Glomerulonephritis was induced in C57bl/6 mice and monitored for 2 weeks. Dexamethasone-containing immunoliposomes and free dexamethasone were injected intravenously. Gene expression was quantified in renal endothelial subsets after lasermicrodissection. Disease parameters analyzed included the extent of glomerular crescent formation, albuminuria, and blood ureum nitrogen and plasma glucose levels.
Results: E-selectin was expressed selectively by glomerular endothelial cells after induction of glomerulonephritis. Consequently, accumulation of anti-E-selectin (AbEsel) liposomes was 3.6 times higher than non-targeted IgG liposomes in diseased kidney. In glomeruli dexamethasone-AbEsel liposomes co-localized with endothelial cells. Targeted delivery of dexamethasone-AbEsel liposomes reduced glomerular endothelial expression of P-selectin, E-selectin and VCAM-1 by 60 to 70%. Other renal microvasculature was not affected by targeted dexamethasone delivery and unlike administration of free dexamethasone, site selective delivery of dexamethasone-AbEsel liposomes did not increase blood glucose. Dexamethasone-AbEsel liposomes reduced albuminuria at day 7 and ameliorated renal injury at day 14 as evidenced by a reduction of blood urea nitrogen levels, decreased glomerular crescent formation, and down regulation of disease associated genes.
Conclusion: 1) E-selectin is an excellent target for selective delivery of potent anti-inflammatory drugs to glomerular endothelium. 2) Recent new application of this powerful strategy includes encapsulation of gene specific small interference RNA to knock-down genes important for disease development.
