S6K), which is a downstream effector of phosphatidylinositol 3-kinase/Akt, is frequently constitutively active in ovarian carcinoma. Here we identify p70S6K as a vital regulator of MCS formation. We also uncover a new mechanism of p70S6K function as a component of the microRNA biogenesis machinery in this process. We show that p70S6K phosphorylates, and inhibits the interaction of tristetraprolin (TTP) and Dicer that promotes the expression of a subset of miRNAs, including the maturation of miR-145. Twist and Sox9 are two divergent targets of miR-145, thereby enhancing N-cadherin, but not other cadherin, expression and MCS formation. Activating miR-145 suppresses ovarian tumor growth and metastasis in an orthotopic xenograft mouse model. Meta-analysis in the Oncomine database reveals that high p70S6K and low TTP levels are associated with ovarian tumor progression. These results define a critical link between p70S6K, miRNA maturation, and MCS formation that may underlie poor clinical outcome of ovarian cancer patients for developing novel therapeutic strategies.Ovarian cancer is the leading cause of death of all gynecologic tumors, associated with widespread peritoneal dissemination and malignant ascites. Key to this is the ability to form multicellular spheroids (MCS); however, the tumor-specific factors that regulate MCS formation are unclear. p70 S6 kinase (p70 The majoplastic –8. TheseS6K), a downstream effector of PI3K/Akt, in ovarian cancer [S6K activation occurs significantly more often in ovarian tumors than in benign or borderline lesions, and that constitutive activation of p70S6K correlates with aggressive malignant phenotypes [S6K in other aspects of tumor progression, such as metastasis [S6K affects MCS formation, although this is a key mechanism of ovarian cancer metastasis. The ribosomal protein S6 was the first and most well-known substrate of p70S6K. However, despite the diverse process controlled by p70S6K, only few substrates are known.There is growing evidence supports the importance of p70 S6 kinase  are small non-coding RNAs that have recently emerged as fundamental posttranscriptional regulators of target gene expression. Although first identified in  cancers . Of equa cancers , 15. WhiS6K is a pivotal regulator of MCS formation. We also provide mechanistic insight that a unique aspect of the function of p70S6K by which it suppresses the maturation of miR-145, which controls the turnover of two critical transcripts Twist and Sox9 in the regulation of N-cadherin through tristetraprolin (TTP)/Dicer-dependent pathway.Here we show for the first time that p70S6K, which is a key intracellular signaling mediator for the effects of multiple growth factors in the malignant ascites and is frequently hyperactive in human ovarian cancer [S6K, (D3E-E389) was sufficient to robustly increase MCS formation (3.3-fold) , and the nonspecific siRNA had no effect  Figure . Using ar Figure . A secont Figure .S6K was expressed at higher levels in SKOV-3 and HEYA8 cells, which exhibit physiologically higher endogenous levels of N-cadherin . As shown in Figure 3E-E389 markedly induced an increase in Twist and Sox9 mRNA, which correlated with the upregulation of its target gene, N-cadherin  by approximately 2-fold  (4.7-fold)  Figure . These rS6K in mediating Twist and Sox9 mRNA half-lives, we were particularly interested in miRNAs that have been demonstrated to function in these processes [S6K and increased by the p70S6K siRNA as compared to their respective empty vector and nonspecific siRNA controls  of both Twist and Sox9, revealing Twist and Sox9 to be directly targeted by miR-145 Figure . ConsistS Figure . To inveS Figure . ExpressS Figure .S6K regulates miR-145 expression, we assessed different aspects of miR-145 expression in response to p70S6K. Despite the increases in mature miR-145 expression, no significant changes were observed in primary miR-145 levels following p70S6K siRNA as determined by RT-PCR , KH-type splicing regulatory protein (KSRP), to Dicer is critical for loading miRNA precursors onto the microprocessor complex and facilitating miRNA maturation from precursor to mature miRNAs [S6K activation , a kinase substrate prediction database, TTP has been found to be a putative substrate for p70S6K  to survey a large number of ovarian cancers. There was a significant inverse association between p70S6K and TTP levels, in which p70S6K was highly expressed in tumor samples whereas TTP was suppressed  and 265-289 of the Sox9 3′UTR (NG_012490.1). The simultaneous targeting of multiple genes for metastasis promotion by a single metastasis suppressor miRNA may explain the dramatic effects observed , highlighttps://clinicaltrials.gov/ct2/show/NCT01829971?term=NCT01829971&rank=1). The closed space of the peritoneal cavity may offer an advantage for gene transfer in that the therapeutic genes can be sensitive and specific to the target cells.The importance of miRNAs in cancer biology has opened avenues for drug development based on the inhibition of oncomiRs or replacement of suppressor miRNAs , 39. TheS6K signaling frequently occurs in many human cancers, and it appears to play an important role in their progression. For example, active p70S6K-expressing breast cancer has a worse prognosis [S6K overexpression in breast, colon, liver, and ovarian tumors are found to be related to aggressive malignant phenotypes [S6K in MCS that we propose in ovarian cancer may be extended to other tumor cell types and thereby p70S6K could be an attractive target for therapeutics. Despite the fact that many PI3K pathway inhibitors have been developed, the clinical trials with these drugs have not been promising due to crosstalk with multiple pathways and the presence of potent feedback loops [S6K may thus represent a potential therapeutic advantage over current PI3K inhibitors, which might prove to be more effective and specific while minimizing toxicity. Moreover, the addiction of many tumors to the pathway provides a rationale for PI3K/Akt target, particularly p70S6K, exploitation.Deregulation of p70rognosis . Furtherenotypes , 42–44, ck loops . TargetiS6K and miRNA biogenesis that not only highlights a new mechanism through which p70S6K functions to establish its pro-tumor regimen but also defines the MCS properties. These findings introduce a new dimension to our existing knowledge of the regulation of miRNA machinery and signaling molecules that remains to be explored by which can be exploited as more precise targets for therapy. The prominent role of p70S6K in a broad range of human cancers suggests that its targeting may have key roles, not only in ovarian cancer, but also in other tumors.In summary, we have established a novel regulatory link between p702 incubator at 37°C before experiments. MCS were generated by liquid overlay method [S6K siRNA, Twist siRNA, Sox9 siRNA were acquired from Dharmacon . miRNA or siRNA duplex were transfected into cells by using Lipofectamine 2000 according to the manufacturer's protocol .The human epithelial ovarian cancer cell lines SKOV-3, CaOV-3, and A2780 were gifts from Dr. N. Auersperg . HEYA8 was a gift from Dr. J. Liu . Cells were routinely grown in medium 199:MCDB 105 (1:1)  with 10% fetal bovine serum  and 1% penicillin-streptomycin mixture  in a humidified 95% air, 5% COy method . BrieflyThe half-life of Twist and Sox9 mRNAs were determined using actinomycin D chase experiments, following a standard protocol described elsewhere . BrieflyTotal RNA was isolated by Trizol . For detecting miR-145 levels, RNA was reversed transcribed to cDNA by TaqMan miRNA assays  using specific stem-loop reverse transcription primer (5′-GTTGGCTCTGGTGCAGGGTCCGAGGTATTCGCACCAGAGCCAACAGGAT-3). The expression levels of mature miR-145 was quantified by PCR using sequence specific primers . Endogenous U6 small nuclear RNA levels were determined simultaneously for normalization.http://mirnamap.mbc.nctu.edu.tw/) which employs 3 publicly available algorithms, miRanda (http://www.microrna.org/microrna/home.do), TargetScan (http://www. targetscan.org/), and RNA hybrid (http://bibiserv.techfak.uni-bielefeld.de/rnahybrid).To define potential upstream regulators of Twist and Sox9, we matched candidate miRNAs that were predicted by using miRNAMap 2.0 (available at S6K (Thr389)  , anti-Twist  (Santa Cruz), anti-Sox9  (Abcam), anti-N-cadherin  (Zymed), anti-E-cadherin  (BD Transduction Laboratories) in phosphate-buffered saline containing 0.1% Tween 20 rotating at 4°C overnight. After washing, the membrane was further incubated with secondary antibodies coupled to horseradish peroxidase at room temperature for 1 h and developed with an enhanced chemiluminescent detection kit (Amersham).Cell lysates (20 μg protein each) were separated by 7.5% SDS-polyacrylamide gel electrophoresis and transblotted onto nitrocellulose membrane (Bio-Rad). The membrane was blocked with 5% nonfat dry milk and incubated with anti-phospho-p70The putative miR-145 binding sites at the 3′UTR of Twist and Sox9 were cloned downstream of a cytomegalovirus promoter-driven firefly luciferase cassette in a vector. The β-galactosidase plasmid was cotransfected as internal control in the presence of either synthetic miR-145 or mock control. Luciferase reporter assay  was performed 72 hr after transfection.5 cells) stably transduced with nonspecific miRNA or pre-miR-145 were injected intraperitoneally into 6- to 8-week-old female NOD/SCID mice  . Mice were assessed weekly for signs of pain and discomfort, weight change, and ascites formation. At the time of sacrifice, the number of all visible (> 0.1 cm diameter) tumor nodules in the peritoneal cavity and the volume of ascites were assessed as evidence of metastases.All animal care and experimental procedures were carried out according to the Committee for the Use of Laboratory Animals of the University of Hong Kong approved protocols. SKOV-3 MCS  to determine differences between the experimental groups and the control groups. A


To distinguish between excitatory and inhibitory interneurons, we used transgenic mice expressing enhanced green fluorescent protein driven by the GAD67 promoter.NMDA receptors expressed by spinal cord neurons in the superficial dorsal horn are involved in the development of chronic pain associated with inflammation and nerve injury. The superficial dorsal horn has a complex and still poorly understood circuitry that is mainly populated by inhibitory and excitatory interneurons. Little is known about how NMDA receptor subunit composition, and therefore pharmacology and voltage dependence, varies with neuronal cell type. NMDA receptors are typically composed of two NR1 subunits and two of four NR2 subunits, NR2A-2D. We took advantage of the differences in Mg2+ sensitive receptors and the NR2C/D containing NMDA receptors with less Mg2+ sensitivity. In contrast, excitatory lamina II interneurons express primarily NR2A/B containing receptors. Despite this clear difference in NMDA receptor subunit expression in the two neuronal populations, focally stimulated synaptic input is mediated exclusively by NR2A and 2B containing receptors in both neuronal populations.Analysis of conductance ratio and selective antagonists showed that lamina II GABAergic interneurons express both the NR2A/B containing MgStronger expression of NMDA receptors with NR2C/D subunits by inhibitory interneurons compared to excitatory interneurons may provide a mechanism to selectively increase activity of inhibitory neurons during intense excitatory drive that can provide inhibitory feedback. A receptor antagonist, bicuculline, or the glycine receptor antagonist strychnine [The large majority of neurons in the superficial dorsal horn are local circuitry interneurons, including both excitatory and inhibitory interneurons . Inhibitrychnine -5. Thesein vitro strongly enhances low-threshold (Aβ fiber) driven polysynaptic input onto lamina I projection neurons relayed by excitatory interneurons [In vivo studies also show that D-APV prevents disinhibition-induced allodynia [NMDA receptors are expressed at glutamatergic synapses throughout the superficial dorsal horn and have been implicated in driving the new excitatory activity that accompanies disinhibition. For example, disinhibition rneurons ,6,7. In rneurons ,6,8. In llodynia ,5. These2+ sensitivity [2+ sensitivity at negative membrane potentials than those with NR2C or NR2D subunits. Projection neurons in lamina I expressing NK1 receptors (NK1R+) express heterogeneous NMDA receptors including those that are NR2A/B like and NR2C/D like [Most commonly, NMDA receptors consist of two NR1 subunits and two NR2 subunits of which there are four types: NR2A, NR2B, NR2C,NR2D . NMDA resitivity . ReceptoC/D like . Lamina C/D like . However2+ sensitivities expressed by inhibitory and excitatory interneurons in lamina II.In this study, we took advantage of a recently developed technique  to functEssentially all of the neurons in lamina II of the dorsal horn are interneurons. Lamina II can be distinguished from other laminae based on the translucent appearance of that cell layer under transmitted light. Seventy percent of the interneurons are excitatory and 30% are inhibitory . To iden2+ sensitivity of these receptors, triangle voltage ramps from -100 mV to +50 mV and back to -100 mV were applied at 20 second intervals before, during and after NMDA application [NMDA currents were recorded under voltage clamp control from EGFP+ and EGFP- interneurons in spinal cord slices taken from postnatal day 14 (P14) to P21 mice. Bath application of NMDA (15 μM) generated inward currents at a holding potential of -70 mV. To evaluate the voltage-dependent Mglication . The con2+ inherent in NMDA receptors composed of different NR2 subunits. The voltage sensitivity of NMDA currents depends predominantly on the voltage dependence of Mg2+ block of the receptors [2+ sensitivity at negative membrane potentials than those with NR2C or NR2D [2+ sensitivity among receptor types is enhanced when extracellular Mg2+ concentration is low, 100 μM extracellular Mg2+ was used throughout the current experiments.Previously, we demonstrated a new method to identify subunit-associated characteristics of NMDA receptors expressed by neurons, including lamina I NK1R+ neurons in the spinal cord . We ideneceptors ,18. NMDA or NR2D ,19. BecaFrom the I-V curves recorded repeatedly during agonist application, we determined the voltage dependence of NMDA currents in a manner independent of absolute current amplitude . The curFigure Using pharmacological analysis, we demonstrated that conductance ratio and VMIC are good indicators of NMDA receptor subtypes expressed by dorsal horn interneurons. When NMDA was co-applied with UBP141(25 μM), a mildly selective NR2C/D antagonist , to an EWhen NMDA was co-applied with EAB318 (100 nM), an NR2A/B selective blocker , to an EThe data shown in Figure A and glycine receptor antagonists . Extracellular Mg2+ concentration was kept at 100 μM to keep conditions comparable to those used in the experiments with exogenous NMDA application.NMDA application activates receptors at both synaptic and extrasynaptic sites. To identify synaptic NMDA receptors, we used focal synaptic stimulation in the presence of AMPA, GABA2+ sensitivity, suggesting that mainly NR2A/B, not NR2C/D subunits, are synaptically expressed on both EGFP+  and EGFP- neurons .NMDA receptor-mediated, monosynaptic EPSCs were studied following addition of NBQX (see Methods) after first identifying monosynaptic AMPA EPSCs associated with focal stimulation at 10 Hz. In order to obtain the current voltage relationships for these NMDA EPSCs, we stepped the membrane to different holding potentials from -90 mV to +50 mV at 20 mV increments and then focally stimulated. After subtracting baseline membrane currents to isolate the NMDA EPSCs Figure , we measWe further tested NR2 subunit composition of synaptic NMDA receptors for both EGFP+ and EGFP- neurons using a pharmacological approach. With the membrane potential held at +50 mV, focal stimulation was applied at 0.06 Hz in the presence of NBQX, SR95531 and strychnine. As shown in Figure Ifenprodil (3 μM), a selective NR2B antagonist , and CPPWe have identified NMDA receptor subtypes expressed by two different populations of lamina II interneurons in the spinal cord of GAD67 EGFP knock-in mice. The GABAergic EGFP+ neurons express both NR2A/B and NR2C/D subunit containing NMDA receptors. The mostly excitatory, EGFP- neurons mainly express NR2A/B containing receptors with minimal evidence for NR2C/D subunit expression. Furthermore, we found evidence that only NR2A and NR2B subunits are expressed as synaptic NMDA receptors in nearly identical proportions on both inhibitory and excitatory interneurons.2+ sensitivity of the NMDA response by defining two electrophysiological measures, conductance ratio (g(-90 mV)/g(MIC)) and VMIC, and compared our values to those from heterologous expression data [Without antagonists that are perfectly subunit selective, it is often difficult to use pharmacology to identify NMDA receptor subunit composition by quantifying drug-induced changes in current amplitude. For example, UBP-141 is only mildly selective for NR2C/D subunit mediated responses and can also block NR2A/B subunit mediated responses. Therefore a depression of current amplitude does not provide information about the subunit contribution to the receptors mediating NMDA evoked current responses. Instead, we quantified the voltage dependent Mgion data ,19. BothAs is the case for NMDA currents studied using whole cell agonist application, synaptic currents mediated by NMDA receptors are difficult to analyze for subunit composition due to lack of highly subunit specific antagonists. Predictions made from focally stimulated NMDA EPSCs for unidentified lamina I and II neurons in neonatal rats indicated that synapses co-expressing AMPA and NMDA receptors have both NR2A and NR2B subunits while silent synapses have mainly NR2A subunits . These pTo probe more specifically for NR2A and B subunit contributions to synaptic receptors, we tested the sensitivity of NMDA EPSCs to three antagonists; the NR2A/B antagonist EAB318, the NR2A preferring antagonist, CPP and the NR2B antagonist, ifenprodil. CPP and ifenprodil both produced partial blocks of NMDA EPSCs from both populations of neurons. These effects indicate that both NR2A and NR2B are expressed at these synaptic sites in lamina II. EAB318 used at concentrations that block both NR2A and B decreased the amplitude of NMDA EPSCs by a little over 80%, indicating NR2A/B expression dominates postsynaptically. This also confirmed our earlier observations on unidentified rat lamina I and II neonatal rat neurons . HoweverIt is also interesting to consider why our observations differ from earlier studies  concludiIn our study, bath application of NMDA activates synaptic and extrasynaptic receptors on the inhibitory and excitatory neurons under study. While the dominant synaptic receptors are NR2A/B type receptors, our bath application data indicate that NR2C/D receptors as well as NR2A/B receptors are expressed by inhibitory neurons and these may be extrasynaptic. We did not see significant evidence for NR2C/D subunit expression by excitatory interneurons. In situ hybridization studies report the distinct existence of NR2A and NR2B subunit mRNA in rodent lamina II -28. mRNAThe expression pattern of NR2 subunits observed for inhibitory neurons, which includes both NR2A/B and NR2D, is similar to that observed for the larger NK1R+, excitatory projection neurons in lamina I . In contDisinhibition-induced recruitment of polysynaptic Aβ input onto lamina I projection neurons is relayed by excitatory interneurons. This recruitment is blocked by the non-selective NMDA receptor antagonist D-APV . Here we2+ block [2+ influx. Accumulation of ambient glutamate occurs due to spillover associated with high neuronal activity, glial release [Our results suggest that NR2D containing NMDA receptors are likely to be expressed at extrasynaptic regions on inhibitory interneurons. NR2D containing receptors have a much higher affinity for glutamate , are les2+ block ,19 and h2+ block . Thus, i release ,31 and d release ,33. Unde release . Selecti release -37 altho release . Thus, t2+ sensitivity and NR2D containing NMDA receptors with low Mg2+ sensitive are both expressed on GABAergic lamina II interneurons. Lamina II excitatory interneurons express predominantly NMDA receptors with NR2A/B subunits. The differential expression of NR2D containing NMDA receptors on lamina II inhibitory interneurons compared to excitatory interneurons suggests a role for NR2D containing NMDA receptors in modulating the activity of inhibitory interneurons.In conclusion, we demonstrated that NR2A and NR2B containing receptors with high MgAll experiments were conducted with the approval of the Columbia University Institutional Animal Care and Use Committee and in accord with the Guide for the Care and Use of Laboratory Animals. Lumbar spinal cords were obtained from mice of postnatal day 14 (P14) to P21. Animals were first anesthetized with isoflurane and then decapitated.gad1 gene promoter. This gene encodes for GAD67 [Mice used in this study were homozygous for a transgene that has enhanced green fluorescent protein (EGFP) expression driven by the mouse or GAD67 , a 67 kDor GAD67 ,41. Breeor GAD67 ,16,39.3, 1.25 NaH2PO4H2O, 6 MgCl2, 1.5 CaCl2, 25 glucose, 50 sucrose, 1 kynurenic acid), saturated with 95%O2/5%CO2. After removal of the dura mater and arachnoid membranes, all dorsal and ventral roots were cut close to the cord and the spinal cord embedded in low melting agarose (Invitrogen Life Technologies) for slicing. Transverse slices (400 μm) were obtained using a Leica VT1200S vibrating blade microtome. Slices were then incubated in 95%O2/5%CO2 saturated recovery Krebs  at 37°C for 1 h and then at room temperature until use.Spinal cords were excised and placed in ice-cold dissection Krebs solution  and fluorescence. Whole-cell patch-clamp recordings were made from GABAergic EGFP+ and mostly excitatory EGFP- neurons. EGFP+ neurons were initially identified under epi-fluorescence then approached with a pipette using DIC optics. EGFP- neurons were indentified as non-fluorescent under epi-fluoresence then patched under DIC. Intracellular solution used for most of these experiments had the following composition (in mM): Cs-methylsulfonate 130, Na-methylsulfonate 10, EGTA 10, CaCl2+ influx through activated NMDA receptors, we replaced 95-98% of the extracellular Ca2+ with 3 mM Ba2+ for experiments in which NMDA was bath applied. The barium Krebs comprised: NaCl 125, KCl 2.5, NaH2PO41.25, NaHCO3 26, glucose 25, MgCl2 0.1, CaCl2 0.1, BaCl2 3 and pH 7.4. TTX (0.5 μM), SR95531 (10 μM) and strychnine (1 μM) were included in the extracellular solutions to eliminate action potential generation and involvement of inhibitory circuits. Krebs solution for synaptic studies comprised NaCl 125, KCl 2.5, NaH2PO41.25, NaHCO3 26, glucose 25, MgCl2 0.1, CaCl2 2 and pH 7.4. SR95531(10 μM) and Strychnine (1 μM) were included in the extracellular solutions to block inhibitory circuits.For the extracellular bath, modified Krebs solutions were used. To prevent possible neurotoxicity associated with CaTo obtain the current-voltage relationships of NMDA receptor-mediated currents, NMDA (15 μM) was superfused onto pre-identified, lamina II neurons for 4-5 minutes following several minutes of baseline, whole-cell recording. Triangle voltage ramp commands (the ramp up and ramp down were 0.9 sec duration each) were applied continuously at low frequency (0.05 Hz). Digital sampling frequency was 10 KHz. NMDA applications were repeated 2 -3 times before NMDA co-application with antagonists. Current responses to triangle voltage ramps before and after recovery from NMDA application were averaged as a control current then subtracted from each triangle ramp made during NMDA induced currents. The resulting NMDA current ramps were plotted as a function of membrane potential and further analyzed. The current responses to voltage ramps before and after the first NMDA application were generally changing as a function of time. By the time of the second NMDA application, current responses to control ramps before and after NMDA application were similar. Thus the data for the first NMDA application were not included in this report. To minimize noise for measuring the following parameters, NMDA current ramps were subjected to a rolling average procedure over a 100 msec time frame (not shown).For each voltage ramp during NMDA applications, the membrane current at -90 mV (I(-90 mV)), the maximal inward current (MIC), and the membrane potential for the MIC (VMIC) were measured. The current measured at -90 mV holding potential was determined as I(-90 mV). The MIC was initially determined as the most negative current value in the rolling average. The VMIC was then determined as the voltage corresponding to the MIC. Because each ramp had an up and a down phase, each parameter from a ramp current had a pair of values and they were averaged for subsequent analysis. The conductance at -90 mV and MIC (g(-90 mV) and g(MIC) respectively) as well as conductance ratio were then calculated based on the formulae:To compare NMDA receptor g(-90 mV)/g(MIC) ratios, conductance ratios, under different pharmacological conditions, we averaged three ratio values calculated for each NMDA application near the peak NMDA response at -70 mV. The ratios under different pharmacological conditions or represented by different neuron populations were then compared.Similar to the previous paper , only ceRecorded cells were held at -70 mV under voltage clamp control unless otherwise specified. Focal synaptic stimulation was applied through a theta pipette filled with bath solution. The pipette was placed about 50 μm from the recorded cell. Stimulus intensities ranged from 30 V to 80 V and each stimulus was presented for 100 μs. Evoked EPSCs (eEPSCs) were tested at 10 Hz to ensure that each excitatory response included in the analysis was monosynaptic. The time from the stimulus artifact to the peak of the monosynaptic AMPA receptor mediated EPSCs (AMPA EPSCs) was measured from events recorded at 10 Hz stimulation. Subsequently, 5 μM NBQX, 1 μM strychnine, and 10 μM SR95531 was added to the bath and NMDA synaptic currents were stimulated at 0.06 Hz while recording at different membrane potentials from -90 mV to +50 mV using 20 mV steps. The amplitudes of monosynaptic NMDA receptor mediated EPSCs (NMDA EPSCs) were measured 10 ms after the peak of the monosynaptic AMPA EPSC to ensure minimal contamination by polysynaptic NMDA receptors .i of UBP141 for NMDA receptors with NR2A - NR2D are 14, 19, 4 and 2.7 μM respectively [SR 95531 hydrobromide were purchased from Tocris Cookson . QX-314·Cl was purchased from Alomone labs . Strychnine was obtained from Sigma-Aldrich . NBQX and Ifenprodil were purchased from Ascent . EAB318 was provided by Wyeth Research. EAB318 has IC50 of 20, 80 and 3500 nM for NMDA receptors with NR2A, NR2B and NR2C respectively . UBP141 The authors declare that they have no competing interests.All of the authors contributed to conception and design of the project, analysis and interpretation of data, drafting the article, revising it critically for important intellectual content, and approval of the final version. HS prepared and recorded from the spinal cord slices. All experiments were performed at Columbia University Medical Center, New York, NY, USA.Representative traces showing inhibition of focally evoked NMDA EPSCs by EAB-318 (200 nM), ifenprodil (3 μM) and CPP (200 nM) followed by recovery of NMDA EPSC amplitudes during antagonist washout. The upper and the bottom data are from different EGFP+ neurons. Middle traces are from an EGFP- neuron.Click here for file


The influenza-like illness that began in the Unites States and Mexico was first reported by the World Health Organization (WHO) on 24 April, 2009, and declared a phase 6 pandemic on 11 June. As of 6 July 2009, over 90 000 cases and more than 400 deaths in some 120 countries had been confirmed . ImportaThe pandemic virus 2009 H1N1 was a triple reassortant of human-, swine- and avian-derived influenza A virus segments and the HA gene was classified as being of swine-origin , a clinical isolate of A/H1N1 (A/Osaka/16/2008), a classical swine isolate of A/H1N1 (A/Swine/Hokkaido/2/1981) and a pandemic influenza isolate of A/H1N1 (A/Osaka/168/2009 H1N1 pdm) were used in this study. Three lots  of IVIG derived from pooled plasma collected in Japan and manufactured in 2008  were also used. In addition, two lots of IVIG that were manufactured in 1999, derived from plasma pooled collected in Japan and the USA , were used.The viruses were propagated in Madin-Darby canine kidney (MDCK) cells or in the allantoic cavity of chicken embryonated eggs. The culture media and the allantoic fluids were stored at −80°C prior to use. Infectivity, as infectious focus-forming units (FFU) per ml, was titrated in MDCK cells using peroxidase and an anti-peroxidase (PAP) staining technique (Intravenous human immunoglobulins were manufactured using plasma pooled from over 10 000 healthy donors. The HI and VN activities of IVIGs were titrated against pandemic, seasonal human and swine influenza A viruses . Of note


Rasmussen syndrome is an intractable epilepsy with a putative causal relation with cellular and humoral autoimmunity. Almost half of the patients have some preceding causative factors, with infections found in 38.2%, vaccinations in 5.9% and head trauma in 8.9% of Japanese patients. In a patient with seizure onset after influenza A infections, cross-reaction of the patient's lymphocytes with GluRε2 and influenza vaccine components was demonstrated by lymphocyte stimulation test. Database analyses revealed that influenza A virus hemagglutinin and GluRε2 molecules contain peptides with the patient's HLA class I binding motif (HLA − A*0201). The relative risks of HLA class I genotypes for Rasmussen syndrome are 6.1 (A*2402), 6.4 (A*0201), 6.3 (A*2601) and 11.4 (B*4601). The relative risks of HLA class I-A and B haplotypes are infinity (A*2601+B*5401), 21.1 (A*2402+B*1501), 13.3 (A*2402+B*4801) and 5.1 (A*2402+B*5201). Some alleles and haplotypes of HLA class I may be the risk factors in Japanese patients. Cross-reactivity of cytotoxic T lymphocytes may contribute to the processes leading from infection to the involvement of CNS.


In animal models of amyotrophic lateral sclerosis (ALS), astrocytes expressing superoxide dismutase 1 (SOD1G93A) mutations display a neuroinflammatory phenotype and contribute to disease progression and motor neuron death. Here we studied the role of extracellular ATP acting through P2X7 receptors as an initiator of a neurotoxic phenotype that leads to astrocyte-mediated motor neuron death in non-transgenic and SOD1G93A astrocytes.During pathology of the nervous system, increased extracellular ATP acts both as a cytotoxic factor and pro-inflammatory mediator through P2XG93A or non-transgenic astrocytes pretreated with agents known to modulate ATP release or P2X7 receptor. We also characterized astrocyte proliferation and extracellular ATP degradation.We evaluated motor neuron survival after co-culture with SOD17-selective agonist BzATP caused astrocytes to become neurotoxic, inducing death of motor neurons. Involvement of P2X7 receptor was further confirmed by Brilliant blue G inhibition of ATP and BzATP effects. In SOD1G93A astrocyte cultures, pharmacological inhibition of P2X7 receptor or increased extracellular ATP degradation with the enzyme apyrase was sufficient to completely abolish their toxicity towards motor neurons. SOD1G93A astrocytes also displayed increased ATP-dependent proliferation and a basal increase in extracellular ATP degradation.Repeated stimulation by ATP or the P2X7 receptor activation in spinal cord astrocytes initiated a neurotoxic phenotype that leads to motor neuron death. Remarkably, the neurotoxic phenotype of SOD1G93A astrocytes depended upon basal activation the P2X7 receptor. Thus, pharmacological inhibition of P2X7 receptor might reduce neuroinflammation in ALS through astrocytes.Here we found that P2X Amyotrophic lateral sclerosis (ALS) is characterized by the progressive degeneration of motor neurons in the spinal cord, brainstem and motor cortex, leading to respiratory failure and death of affected patients within a few years of diagnosis . The disG93A mutation display mitochondrial dysfunction, increased nitric oxide and superoxide production and altered cytokine liberation profile [Accumulating evidence indicates that non-neuronal cells contribute to motor neuron dysfunction and death in ALS by the maintenance of a chronic inflammatory response -12. Acti profile ,17,19-22 profile ,19,23.In the central nervous system, extracellular adenosine-5'-triphosphate (ATP) has physiological roles in neurotransmission, glial communication, neurite outgrowth and proliferation . Extrace7 receptor (P2X7r) is a ligand-gated cation channel that elicits a robust increase in intracellular calcium [7r has the highest EC50 (>100 μM) for ATP. The high extracellular concentrations of ATP needed to activate P2X7r are most likely to arise under pathological conditions. In the normal rodent brain, P2X7r expression in astrocytes is generally low, but quickly upregulated in response to brain injury or pro-inflammatory stimulation in cell culture conditions [7r activation can potentiate pro-inflammatory signaling, as it enhances IL-1β-induced activation of NF-κB and AP-1, leading to increased production of nitric oxide as well as increased production of the chemokines MCP-1 and IL-8 [Extracellular ATP effects are mediated by metabotropic (P2Y) and ionotropic (P2X) receptors, both widely expressed in the nervous system . The P2X calcium . Of all nditions -32. In aand IL-8 ,34.7r and other P2X receptors is neuroprotective in animal models of experimental autoimmune encephalomyelitis and Alzheimer's and Huntington's disease [7r mediates motor neuron death after traumatic spinal cord injury, and systemic inhibition in vivo protects motor neurons and promotes functional recovery [G93A animals, increased immunoreactivity for P2X7r has been found in spinal cord microglia [G93A microglia in culture display an increased sensitivity to ATP, and P2X7r activation drives a pro-inflammatory activation that leads to decreased survival of neuronal cell lines [Inhibition of P2X disease -37. In arecovery ,38. In Aicroglia ,40. Furtll lines .7r signaling during nervous system pathology, little is known about its effects on astrocytes or its possible role in ALS. We investigated whether ATP acting through P2X7r could trigger a neurotoxic transformation of astrocytes leading to motor neuron death. We also explored whether ATP signaling in SOD1G93A astrocytes is involved in the maintenance of their neurotoxic phenotype towards motor neurons.Despite the recognized detrimental role of extracellular ATP and P2XCell culture media and reagents, 5-bromo-2-deoxyuridine (BrdU), primary antibody against BrdU and secondary antibodies were purchased from Invitrogen (Life Technologies). The Malachite Green Phosphate Assay kit was purchased from Cayman Chemical. All other reagents were from Sigma.Procedures using laboratory animals were in accordance with the international guidelines for the use of live animals and were approved by the Institutional Animal Care Organization of the School of Medicine, Universidad de la República (Uruguay) and by the Oregon State University IACUC.4 cells/cm2 and maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, HEPES (3.6 g/L), penicillin (100 IU/mL) and streptomycin (100 μg/mL). Monolayers were >98% pure as determined by GFAP immunoreactivity and devoid of OX42-positive microglial cells. Transgenic SOD1G93A and non-transgenic astrocytes were prepared in parallel using littermate pups previously genotyped by PCR.Astrocytes were prepared from spinal cords of 1 day old rat pups as previously described . AstrocyNTR expressing motor neurons by immunoaffinity selection with the IgG 192 monoclonal antibody. For co-culture experiments, astrocyte monolayers were washed twice with phosphate buffered saline (PBS) after experimental treatments and then non-transgenic motor neurons were plated on top at a density of 350 cells/cm2. Co-cultures were maintained for 48 hours in L15 medium supplemented with 0.63 mg/ml sodium bicarbonate, 5 μg/ml insulin, 0.1 mg/ml conalbumin, 0.1 mM putrescine, 30 nM sodium selenite, 20 nM progesterone, 20 mM glucose, 100 IU/ml penicillin, 100 μg/ml streptomycin, and 2% horse serum [Motor neurons were prepared from embryonic day 15 rat spinal cords as previously described ,43. Briese serum ,43. Purese serum ,43. SurvAll astrocyte treatments were performed in DMEM 2% FBS for 48 hours unless otherwise stated. Stock solutions were prepared as 100× and added directly to the well after media change. Inhibitors were added 1 hour prior to subsequent treatment. As noted in Figure To produce conditioned media, astrocytes were treated with ATP 3 times during the course of 5 days. Twenty-four hours after the last treatment, monolayers were washed 3 times with PBS and then incubated for 48 hours with Neurobasal media supplemented with 2% horse serum. Conditioned media was centrifuged to remove debris, aliquoted and stored at -80°C until use. Pure motor neuron cultures were exposed to astrocyte conditioned media 3 hours after plating by replacing 50% of their complete media with conditioned media. GDNF was then added to a final concentration of 1 ng/ml.2 in 24-well plates. In pure cultures, motor neurons were counted under phase contrast. In co-cultures cells were fixed, immunostained for p75NTR  was incubated overnight at 4°C. After washing, cultures were incubated for 1 hour at room temperature with Alexa Fluor 488-conjugated goat anti-mouse antibody (1:500). Nuclei were stained with DAPI (1 μg/mL).Confluent astrocyte monolayers were treated with apyrase for 48 hours in DMEM 2% FBS. At the end of the first 24 hours, BrdU (10 μg/mL) was added. BrdU immunofluorescence was performed as described for GFAP with the addition of a DNA denaturalization step with 1 M hydrochloric acid (30 min at room temperature) after permeabilization. Percentage of BrdU nuclei was calculated as the number of coincident BrdU and DAPI stained nuclei over the total number of DAPI-stained nuclei.2, 120 mM NaCl, 5 mM KCl, 10 mM glucose, 20 mM Hepes, pH 7.4) and incubated in 500 μl with 3 mM ATP as described [To determine extracellular ATP hydrolysis, extracellular phosphate production was measured with the Malachite Green Phosphate Assay kit. After the treatment, astrocyte cultures in 24 well plates were washed 3 times with a phosphate free buffer .Exposure to extracellular ATP caused a neurotoxic activation of spinal cord astrocytes, which lead to death of co-cultured motor neurons in a time dependent-manner. Because ATP is quickly hydrolyzed in the extracellular media and to mimic pathological conditions with persistent ATP stimuli, we treated astrocytes repeatedly as shown in diagram in Figure To confirm that the effects seen on astrocytes were caused by ATP and not its degradation products ADP, AMP or adenosine (ADO), we treated astrocytes with ATP in combination with the enzyme apyrase (5 U/mL), which rapidly degrades ATP to AMP and phosphate. In this condition, the death of co-cultured motor neurons was completely prevented. Moreover, motor neuron survival increased above controls to 134 ± 8% Figure . Pretrea7r as an initiator of astrocyte-mediated motor neuron death, we used the preferential P2X7r agonist 2',3'-O-(4-benzoylbenzoyl)ATP (BzATP). Figure 7r antagonist BBG (1 μM), suggesting that P2X7r activation was required to induce the astrocyte neurotoxic phenotype , the superoxide scavenger MnTBAP (Manganese (III) tetrakis (4-benzoic acid) porphyrin, 100 μM) and urate (200 μM)  Figure . Urate eG93A rats induced death of 37 ± 8% of co-cultured motor neurons. Remarkably, pre-incubation of SOD1G93A astrocytes with apyrase to degrade endogenous extracellular ATP for 48 hours before co-culture completely prevented motor neuron death (Figure 7r inhibitor BBG also restored motor neuron survival to non-transgenic levels (Figure 7r could be basally activated in SOD1G93A astrocytes in an autocrine/paracrine manner, resulting in neurotoxicity to motor neurons.Consistent with previous reports , spinal G93A astrocytes. Cultured SOD1G93A astrocytes showed a 4- to 5-fold increased proliferation rate as compared with non-transgenic astrocytes (Figure G93A astrocytes was decreased in half by apyrase to the same level as apyrase-treated non-transgenic astrocytes (Figure Because purinergic signaling plays a key role in modulating astrocyte proliferation in pathological conditions ,49, we as Figure . Prolifes Figure . The smaG93A astrocytes did not result from decreased extracellular degradation. On the contrary, ATP hydrolysis was 11% greater in SOD1G93A astrocytes (Figure G93A astrocytes, these agents did not induce further ATP degradation.The increase in ATP signaling observed in SOD1s Figure . SimilarG93A astrocytes in basal conditions and contributes to the maintenance of their neurotoxic phenotype.Extracellular ATP has become increasingly recognized to have a major role in neurodegenerative processes, but its role in astrocyte-mediated neuronal death has not been explored. Here, we found that spinal cord astrocytes assume a neurotoxic phenotype in response to extracellular ATP, leading to the induction of motor neuron death in co-cultures. Furthermore, evidence indicates that endogenous ATP stimulates SOD17r-selective agonist BzATP was sufficient to activate astrocytes to induce the same extent of motor neuron death. BzATP is most potent as an agonist for P2X7r, but it is also a weaker agonist of P2X1r and P2X3r [7r was further implicated in the activation of astrocyte neurotoxicity by the antagonist BBG, as it completely inhibited the action of ATP and BzATP. BBG is a selective antagonist for both P2X7r and P2X5r. [7r appears to be the most likely receptor responsible for inducing the neurotoxic phenotype in astrocytes.Non-transgenic astrocytes required multiple stimuli with ATP over several days to induce the neurotoxic phenotype, while a single stimulus with the P2Xnd P2X3r -53. The d P2X5r. -53. Thus7r activation in microglia stimulated peroxynitrite production and led to death of co-cultured neurons [7r signaling in microglia and astrocytes could lead to the generation of an adverse environment for vulnerable neurons during neurodegenerative processes.We have previously shown that oxidative stress induced by superoxide and nitric oxide forming peroxynitrite in non-transgenic astrocytes leads to a neurotoxic phenotype ,42. Here neurons . Thus, aG93A astrocytes in culture display a neurotoxic phenotype that is maintained by chronic oxidative stress and autocrine pro-inflammatory signaling [G93A astrocytes display basally augmented extracellular ATP signaling as evidenced by an ATP-dependent neurotoxic phenotype, increased ATP-dependent proliferation, and increased extracellular ATP metabolism. Thus, ATP emerges as an extracellular factor that could chronically maintain the SOD1G93A astrocyte aberrant phenotype in an autocrine/paracrine manner.Because SOD1ignaling ,19,20,22G93A astrocytes degraded ATP faster than non-transgenic astrocytes, ruling out that their basal alteration in ATP signaling could be caused by a decrease in its extracellular degradation, thereby allowing ATP to accumulate near receptors. An increase in ATP degradation could also be induced in non-transgenic astrocytes exposed to BzATP or LPS. We have previously shown that LPS induces a neurotoxic phenotype in astrocytes, leading to motor neuron death [We found that SOD1on death . Increason death ,56. ThisDegradation of ATP by ectonucleotidases cannot only terminate deleterious ATP signaling, but also initiates ADP and adenosine signaling through P2Y and P1 receptors. To our surprise, in non-transgenic astrocytes, ATP degraded with apyrase, ADP, AMP, or adenosine led to ~35% more motor neuron attachment and survival compared to untreated controls. Because survival is determined 48 hours after plating of the motor neurons freshly isolated from spinal cords, any treatment that increases attachment of motor neurons will result in an increase of motor neuron survival above the untreated control. These results illustrate how the astrocyte phenotype can be modulated from toxic to highly trophic by changing the balance between ATP, ADP and adenosine signaling through P2X, P2Y or adenosine receptors.G93A astrocytes with apyrase or BBG blocked their neurotoxic phenotype, completely preventing astrocyte-mediated death of motor neurons. A role for ATP and P2X7r in the SOD1G93A model was recently proposed by D'Ambrosi et al [G93A microglia are sensitized to BzATP activation. A combination of aberrant ATP signaling in astrocytes and microglia could generate a positive feedback loop driving a sustained inflammatory response in the spinal cord. The results presented here and the findings in SOD1G93A microglia [7r inhibition in ALS could slow disease progression by decreasing astrocyte and microglial activation.In animal models of ALS, proliferative activated astrocytes interact with microglia to accelerate disease progression . Remarkasi et al , who shoicroglia  suggest 7r causes astrocytes to develop a neurotoxic phenotype. In SOD1G93A astrocytes evidence suggests that P2X7r is basally activated and contribute to their toxicity towards motor neurons. Thus, modulation of astrocyte P2X7r during disease could lead to decreased oxidative stress and inflammatory signaling and in turn the switch to a more trophic phenotype towards neurons. A better understanding of ATP and P2X7r signaling in astrocytes could contribute to the development of novel protective therapies in ALS and other neurodegenerative diseases where astrocytes are involved.Taken together, the present work supports the idea that extracellular ATP acting through P2XThe authors declare that they have no competing interests.MG, PC, LB participated in the design of the study. MG, HP and PC prepared astrocyte and motor neuron cultures and co-cultures. MG collected the co-culture data and carried out all other experiments. All authors reviewed the data and contributed to the preparation of the manuscript. All authors have read and approved the final manuscript.


An alanine-substituted decapeptide from the variable region of this Ab displayed increased cytotoxicity in vitro and/or therapeutic effects in vivo against various bacteria, fungi, protozoa and viruses. The possibility that isolated CDRs, represented by short synthetic peptides, may display antimicrobial, antiviral and antitumor activities irrespective of Ab specificity for a given antigen is addressed here.Complementarity-determining regions (CDRs) are immunoglobulin (Ig) hypervariable domains that determine specific antibody (Ab) binding. We have shown that synthetic CDR-related peptides and many decapeptides spanning the variable region of a recombinant yeast killer toxin-like antiidiotypic Ab are candidacidal Candida albicans cell wall stress mannoprotein; b) a synthetic peptide containing well-characterized B-cell and T-cell epitopes; c) a carbohydrate blood group A substance, showed differential inhibitory activities in vitro, ex vivo and/or in vivo against C. albicans, HIV-1 and B16F10-Nex2 melanoma cells, conceivably involving different mechanisms of action. Antitumor activities involved peptide-induced caspase-dependent apoptosis. Engineered peptides, obtained by alanine substitution of Ig CDR sequences, and used as surrogates of natural point mutations, showed further differential increased/unaltered/decreased antimicrobial, antiviral and/or antitumor activities. The inhibitory effects observed were largely independent of the specificity of the native Ab and involved chiefly germline encoded CDR1 and CDR2 of light and heavy chains.CDR-based synthetic peptides of murine and human monoclonal Abs directed to: a) a protein epitope of The high frequency of bioactive peptides based on CDRs suggests that Ig molecules are sources of an unlimited number of sequences potentially active against infectious agents and tumor cells. The easy production and low cost of small sized synthetic peptides representing Ig CDRs and the possibility of peptide engineering and chemical optimization associated to new delivery mechanisms are expected to give rise to a new generation of therapeutic agents. L) and heavy chains (VH) with background variability on each side of the CDRs. Antibodies (Abs) of different specificities can assemble identical VL domains with different VH domains. The framework sequences between CDRs can be similar or identical.Immunoglobulins are composed of polymorphic heavy and light chains. The idiotypic variability is related to the diversity of the antigen binding site and in particular to the hypervariable domains called complementarity-determining regions (CDRs). There are 6 CDRs in both variable regions of light , that neutralizes the wide-spectrum antimicrobial activity of a yeast killer toxin (KT) against eukaryotic and prokaryotic microorganisms presenting specific cell wall receptors (KTR), elicited the production of a special sub-set of antiidiotypic Abs (KT-antiId) characterized by Candida albicans in vitroin vitro and in vivo active fragment (P6), including seven amino acids of the framework region and the first three residues of the light chain CDR1 of KT-scFv, was well represented among the sequences of many unrelated Abs. A killer decapeptide (KP) generated by alanine substitution of the first aminoacid of P6 had increased candidacidal activity in vitro. Significantly, KP exerted therapeutic effect in well established murine models of vaginal and systemic candidiasis, cryptococcosis and paracoccidioidomycosis in vitro including pathogenic bacteria and protozoa in vitro and ex vivo replication of HIV-1 The peptides corresponding to the CDRs of KT-scFv, as well as a series of two-residue displaced overlapping decapeptides spanning the variable region were synthesized. All the synthetic CDRs and most of the related decapeptides showed a fungicidal effect against C. albicans cell wall stress mannoprotein, a major target of human secretory IgA in the course of oral and vaginal candidiasis, has been recently described C. albicans Als3 and enolase, as well as with the nuclear pore complex Nup88 C. albicans, such as inhibition of germination and adhesion and a direct fungicidal effect which extended to C. lusitaniae, Cryptococcus neoformans, Aspergillus fumigatus, and Scedosporium prolificans, suggesting a pleiotropic mode of action A mAb (C7), raised against Plasmodium falciparum, sharing H1 and H2 with mAb C7 As a proof of concept of the extrinsic potential of Ab fragments, we now report on the differential antimicrobial, antiviral and antitumor activities of synthetic peptides with sequences identical to CDRs of the light chain  and heavy chain  of: a) mAb C7; b) mouse mAb pc42 (IgM), directed to a synthetic peptide containing the surface antigen of hepatitis B virus and the T-helper-cell epitope from the circumsporozoite protein of in vitro microbicidal activity of mAb C7, mAb pc42 and mAb HuA CDR-based synthetic peptides at 100 µg/mL, and the EC50 of CDRs that exhibited a significant activity against C. albicans UP10 are shown in C. albicans NCPF 3153 (data not shown). Most H2 alanine-substituted derivatives (asd) had activities similar to H2, but some showed modulating effects  by mAb C7/pc42 H1, and even more (about 90%) by mAb pc42 L1. In addition, a derivative of mAb C7/pc42 H1, H1 Y3A, had enhanced inhibitory effect on HIV-1, while G1A and H5A lost their activity, showing that substitution of one residue can influence the antiviral property. In experimental conditions allowing the infection of healthy PHA-activated PBMCs, the peptide-mediated effect was dependent on the HIV-1 phenotype. As shown in The All CDR peptides from all three mAbs were assayed for cytotoxicity in B16F10-Nex2 murine melanoma cells. Although most of them were inactive, C7/pc42 H2 inhibited 50% of tumor cell growth at 0.05 mM; mAb HuA L1 was also effective but 5–10-fold less inhibitory than C7/pc42 H2 . MAb C7/C7/pc42 H2 peptide is predicted to contain a beta amphipathic (Eisenberg) region corresponding to SYNQKFK C-terminal sequence. Alanine substitutions in this region, rather than on the N-terminal (YISCYN) and turn region (GAT) reduced the cytotoxicity of H2 .In vivo growth of tumor cells depends strictly on angiogenesis, therefore CDR peptides were also tested for endothelial cell cytotoxicity. The mAb C7 CDRs inhibited endothelial cell sprouting as tested in vitro with HUVEC cells grown on Matrigel™. CDRs H2, and also L1, L2 and L3 at higher concentrations, inhibited endothelial cell sprouting antibodies C. albicans antigen, and also CDRs from mouse mAb pc42, sharing H1 and H2 with mAb C7, and human mAb HuA, sharing no CDR either with mAb C7 or mAb pc42, showed in vitro, ex vivo and/or in vivo differential antimicrobial, antiviral and/or antitumor activities.Here we show that, independent of the specificity of the native Ab, CDRs other than H3 may display, with high frequency, antimicrobial, antiviral and antitumor activities in a way reminiscent of molecules of early innate immunity in vivo antitumor activity of mAb C7/pc42 H2 and mAb HuA L1, the protection conferred by mAb C7/pc42 H1, H2 and mAb C7 L1 against invasive candidiasis, as well as ex vivo inhibitory activity of mAb pc42 L1 against HIV-1 are examples of hypervariable Ab sequences with biological activity. Synthetic peptides representing Ig CDRs are linear sequences with specific binding properties. The high frequency of peptide binding in Ig CDRs may reflect their increased diversity by somatic mutation and clonal selection by antigens. Whether there may occur a proteolytic release of active fragments from immunoglobulins is a debatable hypothesis, that would be reminiscent of the extrinsic activity of Hb33-61 from bovine haemoglobin that displays antimicrobial activity against Gram-positive bacteria and fungi at µM concentrations, as well as the carboxy-terminal tripeptide  of α-MSH that inhibits Staphylococcus aureus, C. albicans and HIV-1 at picomolar concentrations The A wide variety of organisms, from single-cell microorganisms, insects and other invertebrates, plants, amphibians, birds, fishes, and mammals, man included, produce antimicrobial, antiparasitic and antiviral peptides as part of their first line of defense Here, it is clear that C7/pc42 H2 and HuA L1 are directly cytotoxic to tumor cells causing caspase-dependent apoptosis. Apparently, the pro-apoptotic activity of C7/pc42 H2 on melanoma cells depended on peptide binding to a surface receptor that specifically recognizes the C-terminal sequence. The C-terminal SYNQKFK peptide which is not apoptotic by itself competed with and inhibited the cytotoxicity of mAb C7/pc42 H2 in melanoma cells (data not shown). Amino acids K14 and F15 are crucial for the receptor-mediated mAb C7/pc42 H2 tumor cell cytotoxicity. C7 H3 but not C7/pc42 H2 competed with mAb C7 for binding to phosphatidylcholine, the probable ligand of polyreactive C7 on melanoma cells (unpublished results).in vivo antitumor effect could also involve inhibition of angiogenesis. Presently we show that both peptides in the more stable C-terminal amidated form were active against lung colonization by melanoma cells injected i.v. using a protocol of i.p. administration every-other-day for 11 days. A direct effect of peptides on tumor cells was needed for protection since the suspension of treatment resulted in a delayed death curve similar to the untreated control.Apoptosis was characterized in both melanoma and HL-60 leukemia cells. H2 also inhibited endothelial cell growth on Matrigel™ suggesting that its http://www.ebi.ac.uk/cgi-bin/clustalw) of the viral protein with the CDR sequences with high antiviral activity showed that important peptide motifs of HuA L3 (QRSNWPR) and of pc42 L1 (SKSVSTSG) were present in reverse transcriptase (RT) and Rev proteins, respectively. For HIV-1 inhibition, it has already been reported that many peptides could be competitive or non-competitive inhibitors of enzymatic activities by structure mimicry or by hindering the formation of enzyme-DNA/RNA complexes CDRs interference on HIV-1 replication was studied in PBMCs exogenously infected with different viral strains and in a more physiologic model of endogenously infected mononuclear cells. Presently, we show that some CDR synthetic peptides were able to inhibit R5 HIV-1 expression in naturally and exogenously infected PBMCs. We speculate that the interaction between CDR molecules and HIV-1 co-receptors CCR5 and CXCR4 in our culture systems were, probably, the limiting factor for HIV-1 replication. An alignment analysis . In the case of C7 H2, an N-terminal sequence in ALS3 is hypothesized to be a target based on its functional similarity with a peptide deduced by hydropathic complementarity to C7 H2 with codons read in the 3′–5′ direction Possible molecular targets in in vitro to the inhibitory activity of mAb C7, pc42 and HuA CDRs and engineered derivatives (unpublished data). They include eukaryotic and prokaryotic microorganisms such as C. neoformans, A. fumigatus, S. prolificans, Pseudomonas aeruginosa, methicillin-resistant S. aureus, viruses such as influenza A Ulster 73 (H7N1) and human NWS (neurotropic H1N1) and tumor cell lines HeLa (human cervix epitheloid carcinoma), CEM (human leukemia), Hs294T (human melanoma).Additional pathogenic agents and tumor cell lines obtained from type culture collections or isolated from clinical samples were also sensitive The easy production and the low cost of small sized synthetic peptides representing Ig CDRs may offer significant advantages compared to recombinant Abs or Ab fragments for the rational identification and design of novel antitumor, antimicrobial and antiviral biologically active and therapeutic compounds. As we demonstrated with KP and its D-isomeric form, such peptides can be easily engineered and stabilized against proteolysis by incorporation of non-natural amino acids or other modifications without affecting, or even enhancing, their activity Mouse mAb C7 (IgM), was produced as described elsewhere P. falciparum (CT3) The mouse mAb pc42 (IgM), was selected from the Basic Local Alignment Search Tool (BLAST) of the National Center for Biotechnology Information data base, searching for short, nearly exact matches to the CDRs of mAb C7, as both mAbs shared the same CDRs H1 and H2. MAb pc42 is directed to the dominant epitope within a synthetic peptide model antigen (PS1CT3) including residues 28–42 of the large protein of the surface antigen of hepatitis B virus containing a B cell epitope (PS1) and the known T-helper lymphocyte epitope derived from the circumsporozoite protein of the malaria parasite H and VL chain cDNAs of HuA had been sequenced Human mAb HuA (IgM) was secreted by a human-mouse trioma cell line which was a cloned Epstein Barr virus-transformed B cell line stabilized by fusion with the human-mouse fusion partner SBC-H20. The Vin vitro or ex vivo were engineered by alanine-scanning. The alanine substituted derivatives (asd), defined according to the position held by the alanine-substituted aminoacid, were tested in the same biological assays to critically establish the functional relevance of each residue In vitro microbicidal activity of CDR peptides and selected asd against C. albicans UP 10  and C. albicans NCPF 3153  were used for the in vitro experiments.The CDRs shared by mAb C7 and mAb pc42 (H1 and H2) showing antimicrobial, antiviral and/or antitumor activity C. albicans strains was preliminarily evaluated by testing 100 µg/mL of each synthetic peptide by colony forming unit (CFU) assays as previously described 4/mL germinating yeast cells were added to 90 µL of distilled water containing the synthetic peptide to be tested. Yeast suspensions were incubated at 37°C for 6 h. After incubation, the cell suspensions were plated on Sabouraud dextrose agar, incubated at 30°C and observed for CFU determination after 48–72 hours. Each assay was carried out in triplicate. MAb CDR synthetic peptides exhibiting candidacidal activity at 100 µg/mL, and asd of mAb C7/pc42 H2, selected for the highest candidacidal activity, were further tested to determine EC50 values. EC50 was calculated by nonlinear regression analysis using Graph Pad Prism 4.01 software, San Diego, CA, USA.The microbicidal activity of synthetic CDRs against 5C. albicans NCPF 3153 yeast cells suspended in 0.1 mL saline. Mice were treated i.p. with 200 µg of mAb C7 L1 and mAb C7/pc42 H1 and H2 for 3 days starting on day 0, and 4 h after the fungal challenge, and at 24 and 48 h thereafter. Control mice were injected with saline (same treatment schedule as those treated with the CDRs). Protection was evaluated by monitoring animal survival for 20 days. The mean survival time and numbers of CFU of C. albicans in infected tissues were calculated as reported previously Animal experiments were approved by the Institutional Review Board of the School of Medicine and Odontology at the University of the Basque Country, Spain. Female BALB/c mice, 8-weeks old , were infected intravenously with 5×10ex vivo antiviral activity of synthetic CDRs and asd of mAb C7 H1, selected for the highest antiviral activity in HIV-1 endogenous infection was performed as previously described 6 cells/mL in RPMI 1640 medium with 10% fetal calf serum (FCS) and 20 U/mL of rIL-2. Exogenous rIL-2 was added every 3–4 days. Cultures were treated with synthetic mAb CDRs and mAb C7/pc42 H1 asd at 10 µg/mL during the time of culture. Virus production on days 5 and 10 was assayed in the supernatants using ultrasensitive Alliance® HIV-1 p24 ELISA kit (Perkin Elmer); p24 Ag in the supernatants of untreated cultures corresponded to 100% of viral production. For exogenous infection assays, PHA-stimulated PBMCs obtained from healthy individuals were infected with BaL (R5) or IIIB (X4) HIV-1 strain at a median tissue culture infective dose (TCID50) of 500 TCID50/ml. After 2 h of adsorption, the cells were washed, suspended at 2×105 cells/ml in medium and cultured in 96-well cultures plates. At time zero of infection, synthetic peptides were added to the cultures at 10 µg/ml and were maintained throughout the experiment. All assays were performed in triplicate. Virus production was assayed 10 days after infection in the supernatants of HIV-1-infected PBMCs by p24 assay.Assessment of 2, in RPMI-1640 medium with 10 mM N-2-hydroxyethylpiperazine-N2 ethanesulphonic acid (HEPES), 24 mM sodium bicarbonate, 40 mg/L gentamycin, pH 7.2 and 10% FCS.Murine B16F10-Nex2 melanoma cells , and human SKmel28 and SKmel25 human melanoma cell lines originally obtained from the Memorial Sloan Kettering Cancer Center, New York, were cultured at 37°C, under humid atmosphere and 5% COasd of mAb C7/pc42 H2, selected for the highest cytotoxic activity, were diluted from 1 mM to 0.05 mM in RPMI with 10% FCS and incubated with B16F10-Nex2, SKmel28 and SKmel25 cells (5×103 cells/well) in 100 µL per well for 12 h at 37°C. Each peptide was tested in triplicate. After 12 h, the cytotoxic activities of the peptides were determined by measuring cell viability by Trypan Blue exclusion. A 50% inhibition of cell growth was taken as a comparative index of cytotoxicity (EC50).Synthetic CDRs, scrambled peptides (controls), and 5cells/well), and then were further incubated for 12 h at 37°C with mAb C7/pc42 H2 (0.3 mM) and mAb HuA L1 (0.6 mM). Cells were then collected in phosphate-buffered saline-EDTA (PBS-0.02% EDTA), and centrifuged at 1,500 rpm. The pellets were suspended in 500 µL of TELT buffer B16F10 cells were grown for 24 h in 12-well plates (103cells/well) were added alone or mixed with mAb C7 CDR peptides in 100 µL of RPMI medium supplemented with 0.2% of FCS in each well. All peptides were used at 1 mM, with the exception of mAb C7/pc42 H2 that was used at 50 µM. The plates were incubated at 37°C for 18 h and images were captured at 8× magnification with a Sony Cyber-shot camera coupled to a light microscope. The number of pro-angiogenic structures  was counted from 3 different wells, and the average value determined in each system Matrigel™ Matrix  was thawed on ice, added (10 µL/well) to coat 96-well plates, and allowed to polymerize for 1 h at 37°C. Human umbilical vein endothelial cells (HUVEC) (5×105 syngeneic B16F10-Nex2 viable cells in 0.1 mL for each mouse. For protection experiments three groups of 10 animals received on days 1, 3, 5, 7, 9, 11 after tumor cell challenge, i.p. doses of 250 µg of mAb C7/pc42 H2 and of mAb HuA L1, or PBS, at the same time periods, respectively. After 23 days, the lungs were collected from half of the animals of each group and inspected for metastatic colonization and the melanotic nodules were counted at 2× magnification. The survivals of remaining peptide-treated mice and controls were recorded till 40 days after i.v. challenge of tumor cells.All animal experiments were carried out using protocols approved by the Ethics Committee for animal experimentation of Federal University of São Paulo, Brazil. Thirty male C57BL/6 mice seven-to eight-week-old were injected i.v. with 5×10t test. p values of 0.05 were considered significant.The Kaplan-Meier log rank test was applied to survival data. Data on CFU in infected tissues were analyzed by Student's


Helicobacter pylori (H. pylori) infection is known to be associated with a spectrum of gastroduodenal diseases. We studied the association of H. pylori virulence markers cytotoxin-associated gene (cagA) and vacuolating associated cytotoxin gene (vacA) alleles in patients with non ulcer dyspepsia (NUD), gastric ulcer (GU), gastric carcinoma (GC) and duodenal ulcer (DU).H. pylori infection established by both rapid urease test and histology were studied. The cagA and vacA allelic status was determined by polymerase chain reaction (PCR). Sequencing of vacA i1 and i2 PCR product was carried out.cagA was associated with GU in 20(63%) (p = 0.04), DU in 23(72%) (p = 0.003) and GC in 29(73%) (p = 0.001) compared to NUD in 51(42%). VacA s1am1 was associated with GU in 23(72%) (p = 0.001), DU in 17(53%) (p < 0.001) and GC in 23(58%) (p = 0.003) compared to NUD in 38(32%) while vacA s1bm1 was also associated with GU in 9(28%) (p = 0.001), DU in 12(37%) (p < 0.001) and GC 11(28%) (p < 0.001) compared to NUD in 13(11%), respectively. The diagnoses of GU in 21(66%), DU in 16(50%), GC in 20(50%) and NUD in 50(42%) were associated with moderately active chronic inflammation. CagA in 55(45%) (p = 0.037), vacA s1am1 in 51(51%) (P < 0.001), s1bm1 in 25(56%) (p = 0.002), s1am2 32(30%) (p < 0.001) and s1bm2 29(69%) (p = 0.004) were also associated with moderately active chronic inflammation.Two hundred and twenty-four patients were included, 141 (63%) were males with a mean age of 45 ± 16, range 16-83 years. The virulence marker CagA was negative in majority of NUD patients with H. pylori infection. However, cagA was associated with peptic ulcer and GC. VacA allele's s1am1 and s1bm1 were associated with H. pylori associated diseases and inflammation. Helicobacter pylori (H. pylori) infection leads to the development of chronic gastritis and may lead to the development of duodenal and gastric ulcers, gastric adenocarcinoma and lymphoma  Helicobacter pylori 26695, ref [NC-000921.1] Helicobacter pylori J99, ref [NC-008086.1] Helicobacter pylori HPAG1. We randomly selected three vacAi1 and two vacAi2 PCR products for sequencing. Of the three vacAi1 two were from GC  and one from a DU patient (GenBank ACN25131). The sequences of i region were almost identical to the vacA gene of strain 60190(GenBank U05676) except for seven mutations leading to amino acid substitution. Among these sequences, there were five conserved substitutions of N84K in cluster B (GTYNLSGL), G121S and S137L in cluster C (GANRTTTRVDFNAKNISID), D171S and K194N in cluster D (TLQASEGITSDKNAEISLYDGATLNLASNSVKLK). In these strains from GC patients G -to-S, S-to-L and D-to-S substitutions were found. In strain from NUD patient with gastritis (GenBank ACN25133) in cluster A, K-to-Q; in cluster B R-to-I while in cluster C, A-to-T while S remained S and L remained L.We sequenced PCR product of VacA ACN25129, ACN25130, ACN25131). The strain from NUD patient with gastritis having substitution similar to GC and DU was deposited in the GenBank (accession no. ACN25132). The strain from NUD patient with gastritis for which the S remained S and L remained L was deposited in the GenBank (accession no. ACN25133).The sequences of two strains from GC and one from DU patient with an S-to-G substitution was deposited in GenBank  and s1am1 in (19.7%) [cagA was 42% and vacA genotype s1am1 exceeded s1bm1 in patients with NUD. In keeping with previous study, only 10(5%) were detected with genotypes vacAs2m2 and no s2m1 was detected. Also, in our NUD patients, cagA was not significantly associated with s1am1 21(41%) (p = 0.201) and majority of vacA genotypes with s1bm1, s1am2 and s1bm2 were cagA negative. This higher prevalence of cagA may be explained by not only an increase number of patients in our study but also that our hospital is a tertiary care referral center with a large number of patients coming across from the different parts of our country.The study of patients . The majm1 Table . On histm1 Table . VacA i in 24.2% . The com (19.7%) . This isH. pylori virulence pattern in patients with peptic ulcer and GC were similar to the one described in Iran, India and Bangladesh [cagA prevalence of 76% was found in Iranian isolates [cag pathogenicity island (PAI) and potentially toxigenic vacAs1 alleles of the vacA, independent of disease status [cagA was present in 75% of the strains from patients with peptic ulcer (PU) compared to 55% in patients with NUD, and 80% from PU carried potentially toxigenic vacAs1 alleles compared to 60% in isolates from patients with NUD [vacA m region and the 5' end of the cagA gene indicated that Bangladeshi isolates were more closely related to H. pylori isolates from India and were different from isolates from East Asia [i1 and GC while Hussein et al finding no such relationship found i1 associated with GU in Iraq isolates [i1 not only with GC but also with DU in Italian isolates [i1 association with GC, GU and DU.This ngladesh -25. A caisolates . PCR tese status . In Bangisolates ,23. Receisolates . Our rescagA positive H. pylori infection is low, 56% compared to over 70% seen in neighboring countries, Iran, India and Bangladesh. It may possibly be explained by the lack of genetic predisposition and/or low frequency of particularly virulent cagA positive strain in this area. Karachi is a metropolitan city with people from different racial descent migrating to this part of the country may have resulted in the dilution of particularly virulent strains.In our patients, the overall prevalence of The authors declare that they have no competing interests.JY conceived and designed the study, JY, SA, ZA, WJ coordinated the study, JY did the work, JY, ZA, RA, analyzed the data, JY and MI performed the statistical analysis. JY wrote the manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-230X/9/87/prepub