Methods: Xenograft models were derived following injectin of MCL Z-138 or JVM-2 cells into Rag-2M mice. Rituximab is a monoclonal antibody targeted against the B cell marker CD20 and treatment at doses of 1, 2.5, 5 and 10 mg/kg was given every other day for a total of 5 treatments in low tumor bearing mice (palpable) and high tumor bearing mice (200mg). Each treatment arm included groups of 6 mice and the study was done in triplicate. In vivo efficacy data was analyzed using SPSS software. The time taken for subcutaneous tumors to reach a size of 0.4 cm3 was analyzed using Kaplan-Meier curves for survival analysis. Treatment and control groups were compared using a log-rank test. Patient samples were obtained from the BC Cancer Agency and consisted of 10 MCL, 10 Follicular Lymphoma, and 10 Diffuse Large B cell Lymphoma.
Results: In contrast to rituximab-insentive JVM-2 xenografts, tumors derived following injection of Z-138 human MCL cells were sensitive to rituximab, as judged by complete tumor regressions and decreases in cell proliferation (as measured by Ki67). Staining for FasL in Z-138 xenografts demonstrates that FasL is expressed on endothelial cells lining tumor blood vessels and was confirmed in patient samples. Coincidence of the FasL signal and CD31 targeting blood vessels was confirmed by double immunofluorescence staining on frozen sections. Expression of FasL lining tumor blood vessels led to exclusion of NK cells from the tumor microenvironment unless treated with Rituximab (as measured by immunofluorescence staining and real-time PCR). Tumor-associated macrophages were predominant in the tumor milieu and were downregulated following Rituximab treatment (as measured by immunoflorescence staining and western blot analysis).
Conclusions: 1) We describe here for the first time a novel mechanism of immune evasion in MCL and other Non-Hodgkin’s Lymphomas via upregulation of FasL on endothelial cells lining tumor blood vessels.
Authors’ disclosure statement: This work is currently unpublished and we trust that the data shared herein will remain confidential.
Interactions between Cytochrome P4504A, Cyclooxygenase and Nitric Oxide Synthase during Endotoxemia: Therapeutic Implications for Inflammatory Diseases TUNCTAN B1, KORKMAZ B1, CUEZ T1, BUHARALIOGLU CK1, SAHAN-FIRAT S1, FALCK J2, MALIK KU3 1Mersin Univ., Mersin, Turkey; 2Univ. of TX, Southwestern Med. Ctr., Dallas, TX, USA; 3Univ. of TN, HSC, Memphis, TN, USA.
Background: Increased production of nitric oxide (NO) and prostaglandins (PGs) associated with a decrease in formation of 20-hydroxyeicosatetraenoic acid (20-HETE) contributes to development of hypotension during inflammatory diseases such as endotoxemia. Aim: To summarize of our recent findings indicating interactions of cytochrom P450 (CYP) 4A, cyclooxygenase (COX) and inducible NO synthase (iNOS) mediate hypotension in rats treated with endotoxin (ET).
Methods: Conscious male rats received ET (10 mg/kg) or saline (4 ml/kg) at time 0. Blood pressure (BP) was measured using a tail-cuff device. Separate groups of ET-treated rats were given 1,3-PBIT (iNOS inhibitor; 10 mg/kg), indomethacin (nonselective COX inhibitor; 5 mg/kg), piroxicam (COX-1 inhibitor; 10 mg/kg), NS398 (COX-2 inhibitor; 10 mg/kg), 5,14-HEDGE (a synthetic analogue of 20-HETE; 30 mg/kg) or 20-HEDE (a competitive antagonist of vasoconstrictor effects of 20-HETE; 30 mg/kg) 1 h after injection of saline or ET. The rats were sacrificed 4 h after ET challenge and blood, kidney, heart, thoracic aorta (TA) and superior mesenteric artery (SMA) were collected for measurement of the enzyme protein levels and/or activities.
Results: ET-induced fall in BP were associated with increases in renal iNOS protein level and nitrite, 6-keto-PGF1, PGE2 and TxB2 levels in the serum, heart, kidney, TA or SMA. ET decreased the renal CYP4A1 protein level/activity and the systemic and tissue levels of PGF2. 1,3-PBIT prevented the ET-induced decrease in BP, renal CYP4A protein level/activity and increase in systemic and renal nitrite production. Indomethacin prevented the ET-induced decrease in BP, CYP4A protein level/activity, and increase in renal iNOS protein level/activity and systemic PGE2 production. 5,14-HEDGE, piroxicam or NS398 prevented the ET-induced changes in BP and systemic and tissue nitrite and eicosanoid production. These effects of 5,14-HEDGE were abolished by 20-HEDE.
Conclusions: 1) iNOS-derived NO suppresses CYP4A expression/activity, increases the production of PGI2, PGE2 and TxA2, and decreases PGF2 levels. 2) PGI2, PGE2 and TxA2, but not PGF2, increase the iNOS protein expression/activity and decrease the CYP4A protein expression/activity. 3) 20-HETE may decrease the production of nitrite, PGI2, PGE2 and TxA2, and increase the production of PGF2. 4) Inhibition of NO and/or eicosanoid production or administration of a 20-HETE analog prevents the hypotension in endotoxemic rats.
Acknowledgement: This work was supported by USPHS-NIH Grant 19134-31, NIH Grant HLBI-19134-33A1, the Research Foundation of Mersin University (Project Code No: BAP ECZF EMB (BT) 2006-3), NIH Grant GM31278 and the Robert A. Welch Foundation.
Quinolones and Metal Ions- Friends or Enemies? TUREL I University of Ljubljana, Faculty of Chemistry and Chem. Technology, Askerceva 5, Ljubljana, Slovenia
Background: Unpredictable and never ending battle between bacteria and mankind shows that diseases considered to be controlled or even eradicated are appearing again, often in new forms that are multidrug resistant. Therefore it is extremely important to completely understand the molecular mode of action of existing drugs which could help us to exploit them even more efficiently in the future. In last years quinolones are clinically amongst the most successful synthetic antibacterial agents and one of the famous members of this large family-ciprofloxacin (cfH) is a real blockbuster drug. It is known that quinolones can in general easily interact with metal ions. In one hand metal-quinolone interactions are disturbing because the absorption of these drugs is reduced (due to the formation of sparingly soluble metal complexes). On the other hand it is believed that metal ions are needed for the biological activity of quinolones. The aim of this work was to prepare metal-quinolone complexes and study their properties.
Methods: We have isolated several novel metal ion (magnesium, copper, vanadium, bismuth, europium) -quinolone complexes and characterized them by various physico-chemical techniques (X-ray structure analysis, spectroscopy (IR, NMR, UV-VIS), etc.). Biological activity of complexes was also tested (antibacterial activity, DNA gyrase activity, DNA cleavage, insulin mimetic behavior).
Results: Crystal structure of metal-quinolone complexes confirmed that bidentate (O, O`) bonding of quinolone through ring carbonyl and carboxylate oxygen atoms is predominant mode of coordination. Results have shown that antibacterial activity of metal complexes is not increased in comparison to that of free quinolones. The most interesting biological results were found for Cu-cfH complexes that are able to cleave DNA and for V-cfH complex that exerts in vitro insulin mimetic behavior. Interesting luminescence properties were also discovered for Eu-cfH complex.
Conclusions: 1) Certain isolated metal-quinolone complexes exert different kinds of biological activities and might be considered as potential new drugs. 2) Optical properties of europium-cfH complex might be useful for staining of tissues or for analysis of quinolones in biological samples.
Interaction between simvastatin, a HMG CoA reductase inhibitor, and grapefruit juice: in vitro characterization. 1NAITCHABANE M, 1Le GOFF N, 1,2UBEAUD G 1 UMR CNRS 7175, LC1 « Biomolécules, Biotechnologie et Innovation Thérapeutique », Université Louis Pasteur, Faculté de Pharmacie, Strasbourg France
2 Pôle Pharmacie Pharmacologie, CHU Strasbourg, France
Grapefruit juice is able to modify the pharmacokinetic parameters of many drugs. In particular, it increases oral bioavailability of simvastatin, a cholesterol lowering agent that acts by competitive inhibition of 3-hydroxy3-methylglutaryl coenzyme A (HMG-CoA) reductase. Simvastatin undergoes an important first pass metabolism and this is thought to be partly responsible for its low bioavailability after oral administration. Simvastatin is a prodrug that requires metabolic activation through hydrolysis by esterases to form the active simvastatin acid. Besides, simvastatin is a substrate for cytochrome P450 enzymes. It is also an OATP (organic anion transporter peptide) carrier substrate belonging to the solute carrier (SLC) family and MDR-1 efflux carrier belonging to the ATP binding cassette (ABC) superfamily of membrane transporters.
The objectives of our works were first to identify some constituents of grapefruit juice involved in this interaction and secondly to characterize and quantify the mechanisms of this interaction in the intestine and in the liver. We have studied two groups of compounds involved in this interaction: i) the flavonoids such as the naringin and its aglycon form, the naringenin and ii) the furocoumarins (psoralens) such as bergamottin and its metabolites. We have evaluated the effects of these two compounds (naringenin and bergamottin) on the intestinal transport and the intestinal and hepatic metabolism of simvastatin by using different in vitro models.
Our works have shown that these components of the grapefruit juice were able to modify absorption and metabolism of simvastatin by inhibition of CYP-450s (in particular CYP3A) and by modulation of ABC (MDR-1, MRP2) and OATP carriers. Thus, these constituents increased the bioavailability and plasma concentrations of simvastatin, raising its potential for adverse effects.
These results should be taken into account to adjust doses in order to avoid adverse effects and risks of rhabdomyolysis when simvastatin is co-administered with grapefruit juice.
Physiology and Pharmacology of Gonadotropin-Inhibitory Hormone UBUKA T1, TSUTSUI K2, BENTLEY GE1 1Univ. of California at Berkeley, Berkeley, CA, USA; 2Waseda Univ., Shinjuku-ku,
Tokyo, Japan
Background: Gonadotropin-inhibitory hormone (GnIH) is an hypothalamicneuropeptide which modulates reproduction by inhibiting gonadotropin secretion from the anterior pituitary gland. GnIH can also directly inhibit reproductive behaviors, possibly via action within the brain. Identification of the distribution of GnIH neurons and fibers may provide us with clues to how the brain controls reproductive activities of the animal. We identified human and macaque GnIH peptides, and characterized the location and connectivity of GnIH neurons in the macaque brain.
Methods: 5 adult human (Homo sapiens) and 5 adult macaque (Macaca mulatta) hypothalami were used to identify the mature GnIH peptides by mass spectrometry combined with immunoaffinity purification. Location of GnIH neuronal cell bodies and fibers were identified by in situ hybridization and immunocytochemistry. Morphological interactions of GnIH neurons with gonadotropin-releasing hormone (GnRH)-I, GnRH-II, dopamine and β-endorphin neurons were analyzed by double labeling immunocytochemistry.
Results: We identified human GnIH (MPHSFANLPLRF-NH2 and VPNLPQRF-NH2) and macaque GnIH (SGRNMEVSLVRQVLNLPQRF-NH2) by mass spectrometry. The majority of GnIH precursor mRNA positive and GnIH-immunoreactive (GnIH-ir) cell bodies were localized in the intermediate periventricular nucleus (IPe) in the macaque hypothalamus, as determined by in situ hybridization and immunocytochemistry, respectively. Abundant GnIH-ir fibers were observed in the nucleus of stria terminalis in the telencephalon; habenular nucleus, paraventricular nucleus of thalamus, preoptic area, paraventricular nucleus of hypothalamus, IPe, arcuate nucleus of hypothalamus, median eminence and dorsal hypothalamic area in the diencephalon; medial region of superior colliculus, central gray substance of midbrain and dorsal raphe nucleus in the midbrain; and parabrachial nucleus in the pons. GnIH-ir fibers were further observed in close proximity to GnRH-I, dopamine, β-endorphin and GnRH-II neurons in the preoptic area, IPe, arcuate nucleus of hypothalamus and central gray substance of midbrain, respectively.
Conclusions: GnIH neurons may thus control reproductive activity of primates by regulating several neural systems in the brain in addition to inhibiting pituitarygonadotropin release.
A novel view of the pathophysiology of psychiatric disorders and development of pharmacotherapy based on brain energy metabolism UEHARA T, SUMIYOSHI T, MATSUOKA T, SUZUKI M
Univ. TOYAMA, Dept. Neuropsychiatry, Toyama, Japan
Background: Lactate, an energy substrate for neural activity like glucose, has been shown to be produced by astrocytes under the regulation of glutamatergic tone. The serotonin (5-HT) system, specifically 5-HT1Areceptors, is suggested to play a key role in anxiety and affective symptoms. The aims of this study were: 1) to evaluate the effect of 5-HT1A agonists on lactate production in the medial prefrontal cortex (mPFC) and basolateral amygdala (BLA); and 2) to investigate the role of glutamate transporters in the modulation of lactate production.
Methods: This study used 94 adult male Wister rats (5-7 rats per each groups, weight: 280 – 300 g). Using in vivo microdialysis technique, we measured extracellular lactate concentrations in the mPFC and BLA. Footshock stress (FS; 0.5 mA for 5 s administered every 30 s for 20 min) was administered using a plastic communication box connected to a shock-generator and a timer box.
1) Tandospirone (2.0 mg/kg), a partial agonist at 5-HT1A receptors, and WAY-100635 (0.5 mg/kg), a selective 5-HT1A antagonist, were administered intraperitoneally 10 min and 40 min respectively before the start of FS.
2) Dihydrokainate (DHK), a glutamate uptake inhibitor, was dissolved in artificial cerebrospinal fluid (CSF). Artificial CSF containing DHK was perfused at a rate of 5.0 µl/min into the dialysis probe. Twenty minutes after the start of DHK (0.1 mM) perfusion, FS was administered.
Results:
1) Tandospirone attenuated the FS stress-induced increase of eLAC in both of the brain regions, which was blocked by pretreatment with WAY-100635.
2) DHK also attenuated stress-induced increment of eLAC in the mPFC, and completely prevented it in the BLA.
Conclusions: The results of this study indicate 1) FS stress-induced increase in lactate production is partly regulated by 5-HT1A receptors both in cortical and limbic regions, 2) glutamate transporters regulate lactate availability in astrocytes, and that 3) rapid energy demand induced by glutamate contributes to local lactate production. Research into interactions between neurotransmitters and lactate metabolism may provide a novel view of the pathophysiology of some stress-related disorders, e.g., mood disorder, anxiety disorder, and post-traumatic stress disorder.
Authors’ disclosure statement:Part of this work was supported by Dainippon-Sumitomo Pharmaceutical Ltd. and Yoshitomi-yakuhin Ltd.
Antitumor potency of angiotensin II receptor blocker for prostate cancer UEMURA H, ISHIGURO H, HOSHINO K, KUBOTA Y
Department of Urology, Yokohama City University Graduate School of Medicine,
Yokohama, Japan
Since a low prevalence of cancer in hypertensive patients receiving angiotensin converting enzyme inhibitors was reported, a biological action of angiotensin II (Ang-II) on the development or progression of cancer has been drawn attention. It is known that Ang-II plays a fundamental role not only as a vasoconstrictor in controlling blood pressure and electrolyte/fluid homeostasis, but also as a mitogenic factor through the Ang-II type-1 (AT1) receptor in cardiovascular cells. In the last decade, a widespread use of AT1 receptor blockers (ARBs), antihypertensive drugs, has contributed more attractive information on the involvement of Ang-II in cancer. Interestingly, there is increasing evidence that the renin-angiotensin system (RAS) is implicated in the development of various cancers.
To date we have reported that prostatic RAS related to cell proliferation and angiogenesis has the potential especially in carcinogenesis of prostate and ARBs can suppress them by inhibiting signal transduction pathways or angiogenesis through AT1 receptor. Clinically, we demonstrated the administration of ARBs for hormone refractory prostate cancer patients induced the decline and stabilization of serum prostate specific antigen (PSA) with an improvement in performance status. Also, we confirmed that the deceleration of PSA-doubling time by ARB treatment was shown in patients with PSA failure after radical prostatectomy. Our in vitro study demonstrated that Ang-II up-regulated oxidative stress-related proteins and enhanced the production of O2·–radical in prostate cancer cells, and conversely ARBs inhibited them.
From basic and clinical data, we believe that ARB has a novel ability to decelerate PSA elevation and suppress the incidence of prostate cancer, which implies that ARB may have a chemopreventive activity for prostate cancer.
The critical role of IL-15 trans-presentation in the antitumor effects mediated by the combination therapy with Imatinib and IL-2 ULLRICH E1,2, MIGNOT G1, BONMORT M1, TEMRE M1, CHAPUT N1, ZITVOGEL L1 1INSERM U805, Institut Gustave Roussy, Villejuif, France; 2Friedrich-Alexander-Universität Erlangen-Nürnberg, Germany
Background: The synergistic antitumor effect of the combination therapy with imatinib mesylate (IM) and IL-2 has been shown to depend on NK1.1 expressing cells and led to the first description of an important effector cell population called Interferon producing killer dendritic cells (IKDC). IKDC were initially described as CD11cintB220+NK1.1+ tumor infiltrating cells that mediated the antitumor effects of combination therapy with IM and IL-2 in B16F10 melanoma-bearing mice (Taieb et al. Nat. Med. 2006). This work aims at further investigating the mechanism involved in antitumor capacity of IKDC in this combination therapy.
Methods: We used a systemic mouse B16F10 melanoma model in C57Bl/6 mice and different transgenic mice. 3 x 105 B16F10 cells were injected at day 0 into the tail vein of mice. 200µl IM (Gleevec®, Novartis) 150mg/kg was given orally twice a day combined with IL-2 (1 x 105 IU of rhIL-2, twice a day i.p.) from day 0 to day 10 after tumor inoculation. Control groups received H2O+PBS, IM or IL-2 alone. At day 12, mice were sacrified and lung metastasis were enumerated.
Results: Here, we show that the antitumor efficacy of the combination therapy was compromised in IL-15 and IFN type 1R loss-of-function mice. IL-15R was required for the proliferation of IKDC during therapy with IM and IL-2. Trans-presentation of IL-15 by IL-15R activated IKDC to express CCR2 and to respond to type 1 IFN by producing CCL2. Moreover, the antitumor effects of the combination therapy correlated with a CCL2-dependent recruitment of IKDC into tumor beds.
Conclusions: Our data indicate that the IL-15 driven peripheral expansion and intratumoral chemoattraction of IKDC is an important factor for the immunomodulatory effects of IM+IL-2.
Sinceour data suggested that the combination therapy IM+IL-2could be useful to boost natural immunosurveillance against tumors sensitive to TRAIL-dependent apoptosis, we launched the Phase I trial “IMAIL-2” that aims at targeting Imatinib-resistant and TRAIL sensitive cancers. It is conceivable that the monitoring of innate effectors in patients treated with high dosages of IL-2 combined with Imatinib might allow the identification of the human counterpart of mouse IKDC.
Designing Bone-Seeking Proteins ULUDAG H University of Alberta
An ideal therapeutic agent for bone diseases should act solely on bone tissue with no
pharmacological activity at other anatomical sites. Current therapeutic agents, however, do not usually display a preferential affinity to bones and non-specifically distribute throughout the body after administration. Attempts to design bone-specific agents have relied on engineering a desired therapeutic agent with bone-seeking molecules so that the latter delivers the therapeutic agents specifically to bones. In this presentation, we will summarize the latest attempts to engineer bone-seeking therapeutic agents based on formulating therapeutic agents with bisphosphonates, a class of compounds with high affinity to biological apatite. We first provide a relevant summary of the structure of bone mineral and bisphosphonates, highlighting the mode of interaction between these two entities. A summary of recent attempts to formulate bisphosphonates with traditional therapeutic agents to restrict their activities to bone tissues is then provided, with special emphasis on the structure-function relationships of the engineered compounds. Finally, attempts to use bisphosphonates to deliver macromolecular therapeutics (i.e., proteins) are summarized, based on recent data from the authors’ lab. The collective research into bone-seeking medicinal agents is progressively laying the foundation for next-generation ‘magic bullets’ that display desirable activities at the disease sites with no undesirable activity on other organ systems.
Chemotherapy of Duchenne’s muscular dystoropy URADE Y, ARITAKE K
Osaka Bioscience Institute, Osaka, Japan
Background: Duchenne muscular dystrophy (DMD) is a X-linked muscular abnormality caused by the loss of dystrophin and is one of the most gravely genetic disorders. We have recently found that hematopoietic prostaglandin (PG) D synthase (H-PGDS) was induced in grouped necrotic muscle fibers in DMD patients (Okinaga T. et al., Acta Neuropathol., 104, 377-384 (2002)). Cyclooxygenase-2 and phospholipase A2 were also synchronously induced in the H-PGDS-expressing necrotic muscle fibers. We developed novel H-PGDS inhibitors based on the X-ray crystallographic analysis of human H-PGDS complexed with its prototype inhibitor, HQL-79 (Aritake K. et al., J. Biol. Chem., 281, 15277-15286 (2006)). In this study, we investigated pathological significance of H-PGDS in a dystrophin null mdx mouse model and developed a novel therapy for DMD by inhibition of H-PGDS.
Methods: Localization of H-PGDS was examined in the dystrophic muscle fiber in a dystrophin null mdx mouse model by immunofluorescence staining with anti-mouse H-PGDS antibody. H-PGDS inhibitors were orally administrated to mdx mice for 5 days. The necrotic muscle in mdx mice was continuously measured by X-ray computed tomography (CT) imaging enhanced by non-ionic contrast media.
Results: H-PGDS was localized in the necrotic muscle fibers and accumulated macrophages in mdx mice. Oral administration of H-PGDS inhibitors prevented the expansion of muscular necrosis in a mdx mouse model and decreased the expression of mRNAs of pro-inflammatory cytokines. These results demonstrate that PGD2 produced by H-PGDS plays important pathological roles on the expansion of muscle necrosis. H-PGDS inhibitor also accelerated the accumulation and activation of macrophages in the necrotic area.
Conclusions: These results indicate that PGD2 produced by H-PGDS is involved in the expansion of muscle necrosis in DMD and that inhibition of H-PGDS is a novel therapy for DMD.
This study was supported by the Program for Promotion of Fundamental Studies in Health Sciences of the Nttional Institute of Biomedical Innovation (NIBIO).
Inhibition of Neutrophil Granule Exocytosis by a Novel Cell-Penetrating SNAP23 Fusion Protein: A Potential Magic Bullet? URIARTE SM1, LUERMAN GC1, LE J1, WARD RA1, MCLEISH KR1,2.
1University of Louisville, Louisville, KY, United States, 2Dept Veterans Affairs, Louisville, KY, United States.
Background: Neutrophils are the primary cellular component of innate immunity. These cells contain four granule subsets that undergo exocytosis in response to stimulation. The role of exocytosis in mediating neutrophil activation is unknown. The hypothesis that exocytosis contributes to specific neutrophil functional responses was tested.
Methods: To inhibit exocytosis, fusion proteins containing the TAT cell permeability sequence and either the amino terminus SNARE domain of SNAP-23, (TAT-SNAP-23) or an inactive amino acid sequence (TAT-Control) were expressed. Transduction of TAT-SNAP-23 into neutrophils was confirmed by confocal microscopy. Exocytosis of secretory vesicles, specific granules and azurophil granules was determined using flow cytometry to measure the expression of CD35, CD66b and CD63, respectively.
Results: TAT-SNAP-23 inhibited the fMLP-stimulated increase in CD35 and CD66b in a concentration-dependent manner, but had no effect on the increase in CD63 in the presence of latrunculin A. TAT-SNAP-23 (5 µg/ml) reduced the stimulated increase in CD35 and CD66b expression by 90% ± 7% and 75% ± 10%, respectively. Exocytosis of gelatinase granules, measured by release of gelatinase, was reduced by 60% ± 10%. TAT-Control had no effect on exocytosis of any of the four granule subsets. TAT-SNAP-23 (5 µg/ml) had no effect on fMLP-stimulated superoxide release or bacterial phagocytosis; however, phagocytosis-stimulated H2O2 production was significantly reduced by 50%. Additionally, 5 μg/ml of TAT-HA-SNAP-23 significantly reduced chemotaxis, but not chemokinesis, across a transwell membrane.
Conclusion: In conclusion, a fusion protein containing a cell penetrating peptide and a SNARE domain of SNAP-23 inhibits exocytosis of three of four neutrophil granule subsets. The data show that granule exocytosis contributes to neutrophil chemotaxis and phagosomal respiratory burst activity, but not bacterial phagocytosis or respiratory burst activity stimulated by plasma membrane receptors.
Principal parallels between Interferon type I- and small inhibitory (si)RNA- based therapies UROSEVIC N1, 2, 3, PANTELIC L1 1University of Western Australia, Microbiology & Immunology; 2School of Psychiatry and Clinical Neurosciences, Nedlands, WA and 3Edith Cowan University, Joondalup, WA, Australia.
Background: RNA interference (RNAi), a simple regulatory mechanism of gene expression and virus replication, was evolutionary replaced by the more complex interferon type I (IFN I) system. IFN I system is the major component of the innate immune response against viruses and part of the initial body defence against a range of microorganisms. However, it is less specific and it appears to be less efficient than RNAi against viruses. Here we will provide parallels between these two systems and discuss their advantages and disadvanteges for the treatment of viral infections.
Methods: In this study primary cultures of peritoneal macrophages (mΦ) isolated from congenic flavivirus susceptible or resistant mice were used as a model to study various antiviral treatments against West Nile (WN) virus. The extent of antiviral effect of treatments with IFN I, polyinosinic/polycytidylic RNA (pIC) and several off-target (random) small inhibitory (si) RNAs was quantified by virus titration while the presence/absence of viral RNA was confirmed by RT-PCR.
Results: Untreated mΦ from both susceptible and resistant mice were similarly permissive to WN virus infection although cells from resistant mice showed an initial 14% inhibition of virus growth. Following the priming with various antiviral agents, including the major antiviral cytokine IFN I, mΦ from resistant mice developed 2-4 stronger antiviral responses than cells from susceptible mice. However, a treatment with off-target siRNAs resulted in a complete eradication of WN virus replication in cells from both susceptible and resistant mice.
Treatment
Susceptible mΦ
Virus titre (log10TCID50 units/105 cells)
*Percent inhibition
(%)
Resistant mΦ
Virus titre (log10TCID50 units/105 cells)
*Percent inhibition
(%)
None
5.8
0
5.0
14
IFN I
4.9
16
2.0
66
pIC
3.2
45
0.8
86
siRNA
0
100
0
100
*Percent inhibition relative to untreated mΦ from susceptible mice
Conclusions: 1) Cells from resistant mice responded better to conventional antiviral treatments with IFN I and pIC than cells from susceptible mice. 2) Treatment with off-target siRNAs elicited the strongest antiviral effect in both cell types suggesting involvement of a resistance-unrelated mechanism. 3) Antiviral therapy based on small RNA may prove more efficient and direct than therapy based on IFN I.
WHEN SHOULD WE ADMINISTER POSTOPERATIVE INTRA-PERITONEAL MITOMYCIN THERAPY? UZUNKOY A1, BOLUKBAS C2, HOROZ M2, BOLUKBAS F2, KOCYIGIT A3 Department of Surgery1, Internal Medicine2, Biochemistry3, Harran University, Sanliurfa, Turkey
Background: There is controversy about the effect of the timing of intraperitoneal administration of chemotherapeutic agents on the healing of intestinal anastomosis. We have investigated the effect on intestinal wound healing of mitomycin-C administered at different times post-operatively.
Methods: Eighty-four Wistar-Albino female rats underwent ileal resection and end-to-end anastomosis. The rats were randomly selected for intraperitoneal administration of mitomycin-C or saline as follows: mitomycin-C group (n = 65), 2 mg/kg mitomycin-C; control group (n = 13), 10 ml saline. The former was sub-divided into 5 equal groups (A 1-5) and mitomycin-C was administered postoperatively as follows: day 0 (A1), day 3 (A2), day 5 (A3), day 7 (A4) and day 10 (A5). All the rats were sacrificed on the 14th postoperative day and anastomotic bursting pressures and tissue hydroxyproline levels were determined.
Results: Five of the animals died postoperatively: 2 (15.4%) in group A1, 2 (15.4%) in group A2 and 1(7.7%) in group A3. Non-lethal anastomotic leakage was observed in a further five animals: 1 in group A1, 2 in group A2, 1 in group A5 and 1 in the control group. Groups A1 and A2 had significantly lower anastomotic bursting pressures than the other groups (p was <0.05 for each comparison). The anastomotic bursting pressures of group A3, A4 and A5 were comparable with those of the controls (p was >0.05 for each comparison). Tissue hydroxyproline levels in group A1 and A2 were significantly lower than in the controls (p values were <0.05 for each comparison) or the other mitomycin-C sub-groups (p was <0.05 for each comparison).
Conclusıons: Intraperitoneal chemotherapy impairs intestinal wound healing when applied before the 5th postoperative day. Additional therapeutic approaches are needed to prevent this potentially lethal side effect of early intraperitoneal mitomycin-C administration.
Authors’ disclosure statement: This study has been published BMC Cancer as “the optimal starting time of postoperative intraperitoneal mitomycin-C therapy with preserved intestinal wound healing”.
