Plasmodium falciparum and is associated with essential biological processes. It constitutes an attractive and potential target for the development of new antimalarial drugs.Sphingolipids are key molecules regulating many essential functions in eukaryotic cells and ceramide plays a central role in sphingolipid metabolism. A sphingolipid metabolism occurs in the intraerythrocytic stages of Plasmodium activity of a series of ceramide analogs containing different linkages (amide, methylene or thiourea linkages) between the fatty acid part of ceramide and the sphingoid core was investigated in culture and compared to the sphingolipid analog PPMP . This analog is known to inhibit the parasite sphingomyelin synthase activity and block parasite development by preventing the formation of the tubovesicular network that extends from the parasitophorous vacuole to the red cell membrane and delivers essential extracellular nutrients to the parasite.The anti-Plasmodium activity (IC50 in the low nanomolar range) than PPMP and their counterparts with a natural amide linkage (IC50 in the micromolar range). The methylene analogs blocked irreversibly P. falciparum development leading to parasite eradication in contrast to PPMP whose effect is cytostatic. A high sensitivity of action towards the parasite was observed when compared to their effect on the human MRC-5 cell growth. The toxicity towards parasites did not correlate with the inhibition by methylene analogs of the parasite sphingomyelin synthase activity and the tubovesicular network formation, indicating that this enzyme is not their primary target.Analogs containing methylene linkage showed a considerably higher anti-P. falciparum growth in culture. Interestingly, the nature of the linkage between the fatty acid part and the sphingoid core considerably influences the antiplasmodial activity and the selectivity of analogs when compared to their cytotoxicity on mammalian cells. By comparison with their inhibitory effect on cancer cell growth, the ceramide analogs might inhibit P. falciparum growth through modulation of the endogenous ceramide level.It has been shown that ceramide analogs were potent inhibitors of  Sphingolipids are essential components of eukaryotic cell membranes, predominantly found in the outer leaflet. Sphingosine and ceramide Figure  are the Until recently, sphingolipids were primarily considered to be structural components of membranes. However, data accumulated during the last decade have expanded the view of their biological functions. They are now also considered to be key molecules which regulate many functions essential to eukaryotic cells -5. They Ceramide plays a central role in sphingolipid metabolism . It can Moreover, ceramide can be phosphorylated by a distinct kinase and can also be produced by enzymatic hydrolysis of complex sphingolipids. In turn, ceramide can be hydrolyzed to sphingosine and fatty acid by ceramidases.Plasmodium falciparum, the causative agent of malaria, is still limited. Gerold et al. [de-novo synthesis of sphingolipids occurs in the intraerythrocytic stages of the human malaria parasite P. falciparum and can be inhibited by the well established inhibitors of de-novo ceramide biosynthesis, fumonisin B1, cyclo-serine and myriocin [P. falciparum [i) The parasite contains two distinct forms of SPM synthase, one sensitive to sphingolipid analogs, d,1-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (PDMP) or d,1-threo-1-phenyl-2-palmitoylamino-3-morpholino-1-propanol (PPMP)  These analogs blocked the parasite proliferation in culture by preventing the formation of the tubovesicular network (TVN) that extends from the parasitophorous vacuole to the red cell membrane and delivers essential extracellular nutrients to the parasite [P. falciparum [Plasmodium. Recently, an increase in the intracellular ceramide content and an activation of parasite sphingomyelinase(s) were found to be associated with the parasite death process as induced by artemisinine and mefloquine [In contrast to yeast and mammalian cells, the current understanding of sphingolipid metabolism and the biological role of sphingolipids in the development of d et al.  providedmyriocin ,16. Howelciparum ,17. The parasite -22. Neutlciparum -25, indifloquine .Plasmodium activity of non-natural analogs of ceramides was investigated on the intraerythrocytic development of P. falciparum. Interestingly, a series of analogs containing a methylene (CH2-NH) linkage between the fatty acid and the sphingoid-analog core showed considerably higher anti-Plasmodium activity than their counterparts with a natural amide (CO-NH) linkage or than PPMP. The methylene analogs irreversibly blocked parasite development in contrast to PPMP whose effect is cytostatic. Their efficiency in inhibiting parasite growth did not correlate with their potential to inhibit parasite SPM synthase activity, indicating that SPM synthase is not their primary target. Possible mechanisms of action are discussed.Given the importance of sphingolipids in many cellular functions and the central role of ceramide in sphingolipid metabolism, the anti-6-ceramide) and N- (4,4-difluoro-5, 7-dimethyl-4-bora-3a, 4a-diaza-s-indacene-3-pentanoyl) sphingosyl phosphocholine (BODIPY-FL-C5-ceramide) were obtained from Molecular Probes, Inc. (Eugene, OR). The compounds of Figure D,1-threo-1-phenyl-2-palmitoylamino-3-morpholino-1-propanol-HCl (D,1-threo-PPMP) was purchased from Matreya (Pleasant Gap, PA). 6-((N- (7-nitrobenz-2-oxa-1, 3-diazol-4-yl) amino) hexanoyl sphingosine  were maintained in continuous culture on human erythrocytes in RPMI medium containing 7% (v/v) heat-inactivated human serum under an atmosphere of 3% CO2, 6% O2, 91% N2, at 37°C, as described by Trager and Jensen [d Jensen . Parasitd Jensen .et al. [3H] hypoxanthine (Amersham, France, 1 to 5 Ci.mmol/ml) per well. After a further incubation of 24 hour, plates were frozen and thawed. Cell lysates were then collected onto glass-filter papers and counted in a liquid scintillation spectrometer. The growth inhibition for each drug concentration was determined by comparison of the radioactivity incorporated in the treated culture with that in the control culture  maintained on the same plate. The concentration causing 50% growth inhibition (IC50) and 90% growth (IC90) were obtained from the drug concentration-response curve and the results were expressed as the means ± the standard deviations determined from several independent experiments. The DMSO concentration never exceeded 0.1% (v/v) and did not inhibit the parasite growth.Drug susceptibility assays were performed using a modification of the semi automated microdilution technique of Desjardins et al. . Stock sper well in 100 μl. After 24 hours, the cells were washed and two-fold dilutions of the drug were added in 200 μl standard culture medium  and maintained for five days under 5% CO2 atmosphere. The final DMSO concentration in the culture remained below 0.1%. Untreated cultures were included as controls. The cytotoxicity was determined using the colorimetric MTT assay according to the manufacturer's recommendations (Cell proliferation kit I, Roche Applied Science, France) and scored as a percentage of reduction in absorption at 540 nm of treated cultures versus untreated control cultures. IC50 values were obtained from the drug concentration-response curve. The results were expressed as the mean ± the standard deviations determined from several independent experiments. The index of selectivity was defined as the ratio of the IC50 value on MRC-5 to that of P. falciparum.A human diploid embryonic lung cell line (MRC-5, Bio-Whittaker 72211D) was used to assess the cytotoxic effects towards eukaryotic host cells. MRC-5 cells were seeded at 5,000 cells 50 values. Aliquots were removed at the indicated times, washed three times with culture medium and maintained in culture in the absence or in the presence of a given drug. Parasite morphology was determined on Giemsa-stained smears defined according to the following criteria: the ring stage, when parasites exhibited a peripheral cytoplasm stained by Giemsa and a unstained intraparasitic vacuole; the trophozoite stage, when parasites showed a fully stained cytoplasm, haemozoin crystals and one nucleus; the schizont stage, when parasites presented several distinctive nuclei. Parasitaemias were determined by counting 3,000 cells for each sample. Controls consisted of parasites incubated with DMSO instead of drugs processed in the same way.Synchronized cultures (1–2% parasitemia) at the ring stage (0–10 hours old parasites), the trophozoite stage (25–35 hours old parasites) and the schizonte stage (40–48 hours old parasites) were maintained in the presence of drug concentrations in the vicinity of ICP. falciparum cultures at the trophozoite stage (20–30 h old parasites). 400 μl of culture (1 × 108 parasites) were incubated for 60 min at 37°C with 10 μM NBD-C6-ceramide and 0 to 500 μM PPMP or AD2646. Cells were then lysed by freezing and thawing of the culture. Lipids were extracted by a modification of the method of Bligh and Dyer [3OH/CHCl3 mixture (1:2, v:v) were added and the mixture vortexed for one min. Organic and aqueous phases were separated by centrifugation (12,000 × g, five min) and the organic phase was dried. Lipids were dissolved in 15 μl ethanol and analysed by thin layer chromatography on HPTLC plates (Silica gel 60 F254, Merck, Darmstadt, Germany) in CH3OH/CH3Cl3/NH4OH (75:25:4, v:v:v). For qualitative analyses, the fluorescent lipids were detected under UV and for quantitative analyses, the fluorescent lipid spots were scraped, eluted in one ml methanol and quantified at an excitation of 470 nm and an emission of 530 nm in a spectrofluorometer. The percentage of SPM synthase activity for each drug concentration was determined by comparison of the fluorescence quantified in the analog-treated culture with that in the control culture (without drug).SPM synthase activity was measured as described by Haldar et al. . Brieflyand Dyer . To each5-ceramide, washed three times with culture medium without serum and fixed overnight, at 4°C, in 3.7% formaldehyde/0.05% glutaraldehyde. Cells were mounted on poly-L-lysine coated slides and viewed using a Nikon Eclipse TE 300 DV inverted microscope with an 100X oil objective mounted on a piezzo electric device using appropriate fluorescence emission filters. Image acquisition (z-series) was performed with a back illuminated cooled detector (CCD EEV: NTE/CCD-1024-EB, Roper Scientific, France) using a 0.2 μm step. Data acquisition and image deconvolution process were performed with Metamorph software . The images presented correspond to the maximum intensity projection of the deconvoluted z-series.Infected erythrocytes treated with or without ceramide analogs were incubated for 30 min, at 37°C, in culture medium containing 10 μM BODIPY-FL-C2-NH) or a thiourea (CS-NH) linkages  A pan cells  and did lciparum .50 values obtained for the different compounds on the development of the chloroquine-resistant strain FcB1 of P. falciparum in culture . Interestingly, the nature of the linkage considerably influences the anti-Plasmodium activity. Analogs with amide linkage were found to inhibit parasite growth with IC50 values in the micromolar range . However, this IC50 value for PPMP differed from the previously reported value (IC50 = 0.85 μM) [50 values in the nanomolar range  . The dis0.85 μM)  and on aD-threo nitro phenyl analogs of series A, no particular increase of the inhibitory activity was observed with the increase of the N-acyl chain length , F32 (IC50 = 21 nM) and W2 (IC50 = 28 nM), suggesting that the drug is not restricted to a specific strain and acts through a conserved mechanism in malarial parasites. Furthermore, analysis of drug combination with antimalarial drugs showed that AD2646 has a non-synergistic and non-antagonistic effect with CQ on the CQ-resistant strain K1, and with mefloquine and with artemether on the FcB1 strain (data not shown). Compound AD2646  nor hydroxylation on carbon 1 (compare compounds AD2730 and AD2724). In contrast, hydroxylation of carbon 3 is important for anti-Plasmodium activity since removal of the hydroxyl group reduced the activity 13.5 times (compare compounds AD2730 and AD2729).Structure-activity relationship around AD2646 showed that the presence of a nitro group linked to the phenyl is not essential for anti-50 values in the micromolar range, from 5 to 8 μM (except for AD2619), which are similar to the IC50 value of PPMP. No major difference of toxicity was observed between D- and L-threo enantiomers (compare AD2646 and AD2645). In contrast to what was observed for P. falciparum, hydroxylation of the sphingosine carbon 3 does not seem important for cytotoxicity since similar IC50 values were measured for AD2646 and AD2729, suggesting different mechanism(s) of action for AD2646 on MRC-5 cells and P. falciparum. AD2646 and 4 derivatives show high selectivity for P. falciparum as illustrated by the high index of selectivity of these compounds ranging from 160 to 624. The index of selectivity was defined as the ratio of the IC50 value on MRC-5 cells to that on P. falciparum. It can be noted that no selectivity was observed for PPMP. A similar range of growth inhibition was measured on P. falciparum  was evaluated Table . Derivat50 values than that observed with P. falciparum.It must be emphasized that the amide linkage of ceramide analogs is not required for activating apoptosis in cancer cells . An incrTo investigate the cytostatic or cytotoxic effects of AD2646 on the parasite development, cultures at the ring stage (0–10 hours), the trophozoite stage 25–35 hours) and the schizonte stage (40–48 hours) were incubated with 30, 100 or 250 nM of AD2646 for 24.5 hours for the ring stage, for 11 hours for the trophozoite stage, and for 14 hours for the schizonte stage. Aliquots were then taken, washed and incubated in the absence or the presence of drug for a further 13 hours to 24 hours depending upon the parasite stage tested  was insensitive to this concentration of drugs ,19. A cyFigure In contrast, PPMP and AD2646 have completely different effects on the TVN formation for drug concentrations that block parasite growth. After 24 hours of incubation, ring development was totally inhibited by 5 μM PPMP and no TVN was observed as previously described  Figure . As in cPlasmodium activity of AD2646 does not correlate with its inhibitory activity on the SPM synthase. Although AD2646 and PPMP showed similar inhibitory activity on this enzymic activity in parasites in cultures, AD2646 is about 300 times more efficient in inhibiting parasite development than PPMP; 2) In contrast to PPMP which inhibits the parasite development preferentially and reversibly at the ring stage [These data do not support the hypothesis of parasite growth inhibition due to an inhibition of the parasite SPM synthase activity as was demonstrated for PPMP -22 : 1) ng stage , AD2646 ng stage -22. ThisBy their lipidic nature, these analogs might act through a detergent effect that could lead to lysis or modification of the integrity of infected-erythrocyte membranes. This apparently is not the case. No significant lysis of normal erythrocytes was observed after 48 hous of incubation with concentrations of analogs up to 10 μM (data not shown). Furthermore, no preferential lysis of infected-erythrocytes was observed on Giemsa-stained smears of infected cultures maintained 48 hours with 250 nM AD2646, a concentration inhibiting parasite growth totally.Plasmodium and are involved, at least, in the uptake of erythrocyte raft proteins and maintenance of the parasitophorous vacuole containing the parasite, inside the erythrocyte [P. falciparum growth is characterized by a setting up of new permeabilities of the infected-erythrocyte membrane [Interestingly, the absence of a fatty acyl carbonyl group (methylene linkage) in our ceramide analogs is a critical factor for the efficacy of their antiplasmodial activity. Sphingolipids preferentially interact with cholesterol in membranes, especially in detergent-resistant microdomains (DRMs or rafts). Rafts have been described in throcyte . This inthrocyte  and 2) hthrocyte . The amithrocyte . It coulmembrane . Althougmembrane ,43. The in vitro [P. falciparum. Parasites exposed to 1 μM AD2646 for up to 36 hours failed to exhibit characteristic apoptosis, as determined by terminal deoxynucleotidyl transferase DNA fragmentation assay and DNA fragmentation using both gel electrophoresis and fluorescence microscopy methods, although the nucleus appeared highly condensed (M. Dellinger, unpublished data). Apoptosis in P. falciparum is still controversial although some characteristics of apoptosis has been described in Plasmodium [Ceramide is at the parting of different ways of sphingolipid metabolism. Analogs have the potential to inhibit different ceramide-metabolizing enzymes and then might have a pleiotropic effect. Ceramide analogs in amide linkage were described as potent inhibitors of alkaline ceramidase in HL60 human myeloid leukemic cells ,45. Methin vitro . When apin vitro ,45. In cin vitro , prelimiin vitro inhibition assays on P. falciparum and MRC-5 cells. MG and MD performed the fluorescence microscopy and apoptosis investigations on P. falciparum, respectively. SE and ST carried out electrophysiological studies on the malaria-induced anion channel. AD, CW and SG participated in the design and synthesis of ceramide analogs. All authors read and approved the final manuscript.ML and PG carried out the


Caenorhabditis elegans, Silent Information Regulator (SIR2) proteins have been shown to be involved in ageing regulation. In Leishmania, the LmSIR2rp was originally isolated from the excreted/secreted material of the Leishmania parasites. Among the function(s) of this protein in Leishmania biology, we have documented its implication in parasite survival, and in particular in Leishmania amastigotes. In this paper we question the role of the excreted/secreted form of the protein. In particular we wonder if the Leishmania Sir2 homologue is involved in some aspect of its biological function(s), in various components and pathways, which could promote the host cell survival. To test this hypothesis we have mimicked an intracellular release of the protein through constitutive expression in mouse L929 fibrosarcoma cells.In yeast and Leihmania infection.Our results demonstrate that the LmSIR2 protein was properly expressed by fibroblasts and that LmSIR2 is localized both in the cytoplasm and the nucleus of all the transformed cell clones. Unexpectedly, we found that cells expressing LmSIR2 presents reduced saturation cell density ranging from 40% to 60% and expressed an acidic (pH6.0) β-galactosidase activity, which is known to be a senescence biomarker. As a consequence, we observed that LmSIR2 positive fibroblasts were more permissive towards Leishmania to survive for a long period in a cell with reduced capacity to multiply or respond to immunologic stimuli. The potential implications of our finding during the in vivo infection process are discussed.LmSIR2 is able to substantially interfere with the host cell physiology. Thus, it is tempting to speculate that these modifications could help  Leishmania is an intracellular pathogen that causes Leishmaniasis, which remain an important medical problem in several countries. Protozoan parasites of the genus Leishmania result in a spectrum of human disease that range from self-healing cutaneous ulcers to potentially fatal visceral infection, depending primarily upon the species of parasite involved [Leishmania live as either extracellular flagellated promastigotes in the digestive tracts of their sand fly vectors or as nonflagellated amastigotes within macrophages where they survive and replicate within phagolysosome. Leishmania are known to export large range of proteins and glycoconjugates including lipophosphoglycan [involved ,2. Leishhoglycan .Leishmania major gene encoding a protein carrying extensive homology to SIR2 of yeast that we consequently termed LmSIR2rp [Schizosaccharomyces pombe, C albicans, Leishmania, chicken, human (hSirT2p, hSirT3p) and the closely related mouse protein (MmSirT2p and MmSirT3p) [Caenorhabditis elegans carrying extra copies of the SIR2 orthologue, Sir-2.1 [review in [In a previous paper we have characterized a LmSIR2rp . It beloLmSIR2rp . Post-trLmSIR2rp ,7. To daLmSIR2rp . These amSirT3p) . This grmSirT3p) ,9,8,11. mSirT3p) . This pheview in ]. In mameview in . SIRT1 ieview in . Also, teview in .Leishmania amastigotes, was able to promote parasite survival in in vitro culture systems [Leishmania parasites when using radiolabelled immunoprecipitation technique, suggesting therefore that fraction of LmSIR2 is actively excreted by parasite . We then initially surmised that this protein could also be involved in some aspect of host cell-parasite interplay, particularly in pathways that could contribute to cell survival. To test this hypothesis, the leishmania LmSIR2 gene was cloned in a mammalian shuttle vector and the expression of the protein was performed in fibroblasts. Unexpectedly, expression of the Leishmania Sir2 protein leads to a cell growth arrest phenotypes, which correlates, with the expression by the cells of an acidic (pH 6.0) β-galactosidase activity, known to be a senescence biomarker. Furthermore, fibroblasts expressing LmSIR2 were more permissive towards Leishmania infection than control ones. Altogether our results may suggest that the excreted forms of LmSIR2 could participate in the perturbation observed during chronic disease.We have shown that the cytosolic LmSIR2rp protein, when over expressed in  systems . FurtherLmSIR2 was amplified using sense GAA TTC GAT ATG ACA GGG TCT CCG and antisense CTC GAG CAG TCA CCA TGT TGG CAG primers. The 1119 bp PCR amplified genomic fragment subcloned into pCR2.1 was digested with EcoRI and inserted into the EcoRI digested and dephosphorylated pcDNA3.1 shuttle vector (Amersham). Restriction analysis of the recombinant plasmids using EcoRI and XhoI allowed the identification of recombinant vector carrying the LmSIR2 gene in sense orientation. Large-scale preparations of pcDNA-LmSIR2 and pcDNA empty vectors were performed using the QIAGEN plasmid maxi kit. Transfected cells were selected in RPMI 1640 medium containing 10% FBS and 400 μg/ml G418. Each G418-resistant clone, isolated by limiting dilution, were propagated and screened by immunofluorescence. Four clones were isolated and named Cl2, Cl9, Cl10 and Cl11.LmSIR2 recombinant protein containing 6 X histidine residues at its N-terminal (hisLmSIR2) was previously obtained after having subcloned the encoding gene in the pQE-expression vector . Mice ofThe fusion procedure and the screening of mAb were carried out as previously described . The cla2, after various period of incubation they were collected by using Trypsin-EDTA (0.025%). The mean number of viable cells was determined at 100-× magnification after staining with trypan blue. In order to evaluate the rate of 3H-thymidine incorporation, cells were seeded in 96 wells plates at 1000 cells/cm2, after various periods of incubation 1 μCi of 3H-thymidine was added to each well. Cells were further incubated for 4 hours before reading the level of incorporated radioactivity.L929 cells were inoculated at 400 cells/cmL929 cells were seeded in 16-well Labteck chambers for 48 hrs. Cells were then washed with PBS and fixed for 30 minutes with 4% paraformaldehyde at 4°C. After two washes with PBS, cells were permeabilized with 0.01 M PBS containing 0.5% TritonX-100 and 2% BSA for 30 min at 4°C. Slides were then washed and incubated for 1 hour with mAb IIIG4 in 0.01 M PBS containing 2% BSA. After two washes, slides were incubated for 45 min with fluoresceine-conjugated rabbit anti-mouse IgG (Diagnostic Pasteur, Marnes la Coquette, France) diluted 1:100 in PBS containing Evan's blue  and mounted in glycerol PBS (1:1).2 in 25 cm2 flasks, after 7 days of growth they were fixed with methanol and stained with Giemsa and examined observed on inverted microscope (40× magnification) after adding a solution of PBS 0,01 M-Glycerol (1/1).Giemsa staining of the cell culture was performed as follows: cells were inoculated at 400 cells/cm12–18] using the Superscript II RNase H-reverse transcriptase. PCR amplifications were performed using 1–2 μl of reverse-transcription of each product and 20 pmol of each primer, using Taq DNA polymerase. Each PCR cycle consisted of a denaturation step (94°C, 1 min), an annealing step (55°C, 1 min) and an elongation step (72°C 1 min). For the last cycle, the elongation step was extended to 10 min at 72°C. Reactions were carried out for 25–35 cycles in a thermocycler (PTC-100™, MJ Research, Inc.). PCR products were analyzed on 1.5% agarose gel and visualized with ethidium bromide. As an internal control, a housekeeping gene, the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) transcript, was amplified. LmSIR2 primers were: Sense GAA TTC GAT ATG ACA GGG TCT CCG and antisense CTC GAG CAG TCA CCA TGT TGG CAG. GAPDH primers were: Sense GTC TTC ACC ACC ATG GAG and antisense CCA AAG TTG TCA TGG ATG ACC.Total RNA was isolated from wild type and transformed cells using the Rneasy Mini Kit following the manufacturer's instructions. One microgram of RNA was reverse transcribed to cDNA with an oligonucleotide [poly (dT)2 for 12 h with freshly prepared β-Gal staining solution pH 6.0, containing the 5-bromo-4-chloro-3-indoyl-b-D-galactosidase  chromogenic substrate which produces blue 5-bromo-4-chloro-3-indole upon hydrolysis. Cell preparation was stored at 4°C in 70% glycerol upon analysis.Acidic (pH 6.0) β-galactosidase was assayed according to Dimri et al. . BrieflyLmSIR2 gene.The Level of the LmSIR2 gene transcription was evaluated by RT-PCR analysis. As shown in Figure LmSIR2 gene was originally isolated via immunoscreening of a cDNA library, using polyclonal antibodies raised against excreted factors which could bind to glutathione [leishmania's excreted material as previously demonstrated raised the question of its function during the infection process. Thus, heterologous expression in eukaryotic cells could represent a useful approach to give some insight into the biological activity of secreted parasite material. Immunolocalization of LmSIR2 shows that cells expressing LmSIR2 .In order to investigate the potential functions of LmSIR2 protein, when delivered intracellularly in L929 fibroblasts, we have determined the growth kinetic parameters of cells via two different methodologies. Using a counting method, we observed a high reduction in the saturation density of cells expressing LmSIR2 Figure . The redn figure , expressin vivo and in vitro [LmSIR2 gene but was not detected in wild-type cells or cells transformed with the empty pcDNA vector . As shown in Figure Leishmania infection since the mean percentage of infected fibroblast reach 70% after 24 hours of contact. However, once inside fibroblasts no differences in the capacity of parasites to survive and/or multiply was observed and the intracellular amastigote population was eliminated after three days of contact (data not shown).Fibroblasts at the lymph are used as safe target for long lasting residual parasite population . However8, Chang  demonstrrvations . Thus, wLeishmania infection. These results suggest that LmSIR2 has dual function according to the cell type in which it acts: in Leishmania parasites, LmSIR2 is able to promote cell survival while in the host fibroblast it induces a senescence phenotype. This effect could be beneficial for Leishmania since senescent cells are no longer capable of dividing yet remaining metabolically active. They could thus represent safe target for parasite. It is documented that senescent fibroblasts present an inherent higher capacity to resist to apoptotic death [in vivo relevance of our observation is rather difficult to appreciate because; (1) the level of the protein produced in fibroblasts transformed with the pcDNA is certainly far higher than the level achievable in vivo, and (2) the presence of excreted parasite material inside the fibroblast cytosol has yet not been reported. However, an action of the excreted form of LmSIR2 on he host cell could no be ruled out. Two series of observations support this possibility: (1) it has been shown that the Leishmania EF1-a could be translocated from the parasitophorous vesicle into the macrophage cytosol [Our study demonstrates that LmSIR2 is able to down regulate the proliferation of fibroblasts and to induce a senescence-like state in L929 fibroblast cell line. The most interesting finding is that a parasitic protein is able to modify the physiology of the host cell making them more permissive toward ic death . In this cytosol ; (2) the cytosol .in vivo infection process awaits further investigationsIn conclusion, although the effect of the protein, when delivered extracellularly, on the host cells have not being examined in the present study, our data support the notion that the excreted form of LmSIR2 is able to interfere with the host cell physiology, when delivered intracellularly. Unexpectedly the expression of the protein is associated with the appearance of a senescence biomarker. Relevance of such finding during the


Nasal strips are used by athletes, people who snore, and asthmatics to ease the burden of breathing. Although there are some published studies that demonstrate higher flow with nasal strips, none had directly measured the effect of the strips on nasal resistance using the airflow perturbation device (APD). The APD is an inexpensive instrument that can measure respiratory resistance based on changes in mouth pressure and rate of airflow.This study tested forty-seven volunteers (14 men and 33 women), ranging in age from 17 to 51. Each volunteer was instructed to breathe normally into the APD using an oronasal mask with and without nasal strips. The APD measured respiratory resistance during inhalation, exhalation, and an average of the two.20/Lps from an average nasal resistance of 5.5 cm H20/Lps.Results of a paired mean t-test comparing nasal strip against no nasal strip were statistically significant at the p = 0.05 level. The Breathe Right™ nasal dilator strips lowered nasal resistance by an average of 0.5 cm HNasal strips reduce nasal resistance when measured with the APD. The effect is equal during exhalation and during inhalation. Nasal dilator strips (NDS) are used by athletes, people who snore, and asthmatics to ease the burden of breathing. The nasal strips are used as a mechanical means of reducing nasal airflow resistance . By loweThe size of the nostril limits the amount of air entering into the body. The NDS is placed along the nasal valve of the nose. The adhesiveness of the strip binds to the creases of the nasal valve to prevent the outer wall tissue of the nose from collapsing inward during nasal breathing. This mechanism thus dilates the nose and allows more air to flow into the nose .The primary effect of the NDS could be either to dilate the air passage of the nose or to stiffen the nasal wall. Either mechanism would reduce nasal resistance and allow higher flow of air, but they can be distinguished over a range of air flows. Stiffening the nasal wall would have its most profound effect at higher flows where the Bernoulli effect would decrease internal nasal pressures and tend to constrict nasal passage diameter. Air passage dilation would tend to decrease nasal resistance more uniformly over a range of air flows.Recent studies on the effectiveness of Breathe Right™ nasal strips tested participants under various rest and exercise conditions. Some found that the strips neither improve or diminish airflow ,4-9, whiThe Airflow Perturbation Device (APD) is a small, light weight, and easy to use instrument that measures respiratory resistance . A segmePeople breathe normally through the APD. No special breathing maneuvers are required. For this reason, the APD can be used with young children, older adults, unconscious patients, and animals. Respiratory resistance can be separated into inhalation and exhalation components, and resistance can be displayed against lung volume and air flow rate.Respiratory resistance is normally measured through the mouth, with a nose clip and hands pressed against the cheeks. An oronasal mask may be used to obtain combined mouth and nose resistance, or nose resistance by itself if the mouth is closed. The APD used with an oronasal mask should be an ideal instrument to assess the effect of NDS.This study is as much a test of the capabilities of the APD as it is a study of NDS. Objectives of this study were to: 1) determine if APD measurements of respiratory resistance measured with an oronasal mask matched those with breathing through a mouthpiece, and 2) measure the effects of NDS on respiratory resistance made with the APD. This is not a clinical study.This study tested forty-seven volunteers . Some had symptoms of nasal congestion such as asthma, allergies, and snoring. A written informed consent was obtained from each subject and the protocol was approved by the University of Maryland Institutional Review Board (IRB).The nasal strips used in this study were a commercial product called "Breathe Right" . According to the manufacturer's instructions, the nasal strips should be placed halfway down the nose along the nasal valve. The two end regions of the nasal strips should cover the left and right nasal creases.+, Laerdal Medical, Wappingers Falls, NY). The subject was instructed to press their face against the mask while he/she breathed normally. The size of the mask was large enough that it contacted only the hard tissue on the bridge of the nose and did not compress the soft nasal septum.This study contained three phases: 1) to determine the ability of the APD to measure oral resistance using either a mouthpiece or an oronasal mask, 2) to determine the ability of the APD to measure nasal resistance, and 3) to determine the effect of nasal strips on nasal resistances. Phase I consisted of two tests that measured oral breathing resistance. In the first test, the subject's nose was occluded with two layers of Durapore surgical tape (3 M, St. Paul, MN) while the subjects were sitting in an upright position. The subject was instructed to breathe normally into a cardboard mouthpiece. The second test repeated the same procedure as the first test, except the subject was breathing into an oronasal mask y = 0.9624 + 0.1041 Rwhere y = mask value of resistance and x = mouthpiece value of resistanceThis equation was tested to be statistically equivalent to y = x. This indicates that the oronasal mask had no effect on the inhalation values.A similar comparison of exhalation resistances gave the following:2 = 0.8828    (2)y = 0.874bx + 0.4594 RThis equation did not pass the statistical test for equivalence y = x. The oronasal mask may have affected the measurement of respiratory resistance in the exhalation direction.2O/Lps, while nasal resistance with the NDS ranges from 3.39 to 7.33 cm H2O/Lps. This demonstrates the expected resistance reduction with NDS.Figure All subjects except three showed a decrease in nasal resistance when breathing with the NDS. Average value of nose breathing without NDS was 5.50; with NDS it was 5.00. These means were highly statistically significantly different.2O/Lps in resistance, and only six out of 47 subjects failed to demonstrate a decrease in resistance with NDS.The effect of NDS on resistance during exhalation was also statistically highly significant. There was an average reduction of 0.45 cm H20/dps. Again, six subjects failed to demonstrate a decrease in resistance with NDS. These were not the same subjects that increased resistance in the exhalation direction.The effect of NDS on resistance during inhalation tested to be statistically highly significant, as well. The average resistance reduction was 0.49 cm HResistance differences with and without NDS in the inhalation and exhalation directions were tested to determine if NDS had a larger effect while breathing in one direction or the other. Means of the differences for inhalation and exhalation directions were tested with a paired t-test, and found to be statistically nonsignificant. It appears, therefore, that NDS affect nasal resistances equally during inhalation and exhalation.This study confirmed the results of other studies that showed a reduction of about 10% in nasal breathing resistance, as well as supported the claim of the manufacturer that the nasal strips provide nasal relief. Several subjects who had nasal congestion reported some relief in nasal breathing when using the nasal strip. Exactly which subjects these were was not recorded.There was one surprise, though, in the results. The reduction in respiratory resistance due to NDS was a constant amount and not proportional to the resistance level present without NDS. This result was not expected, and no adequate explanation for it can be given at this time. It is not clear why this should be so, but we do not doubt that measurements made with the APD are correct, based on previous studies ,16.We cannot comment on the clinical significance of the resistance reduction with NDS use. It seems likely that some benefit could be obtained from such a resistance change, but whether it is actually detectable is not clear. Other reports in the literature ,18 have The APD has been shown to be able to measure respiratory resistance with either a mouthpiece or an oronasal mask. This may be a significant advantage of the instrument, especially because respiratory resistance measurement on unconscious or uncooperative patients would be much more easily made with a mask than with a mouthpiece. Equations (1) and (2) show the close correspondence between measurements made with both techniques, although the presence of an intercept and a slope different from unity indicate that the correspondence between mask and mouthpiece is not perfect. Resistances with a mask are both higher than resistances with a mouthpiece. The reason for this seems to be different mouth positions in both cases. We have laboratory experiences (not published) that demonstrate that tongue position can influence measured resistance. Breathing through the mask is probably done with the mouth closed more than when breathing through the mouthpiece. The measured difference between inhalation and exhalation resistances could reflect the effect of a pressure difference across the distensible smaller airways, which is greater inside than outside during inhalation, but smaller inside than outside during exhalation. This would lead to a dynamic compression of the small intrathoracic airways during exhalation. Another possible explanation is natural movement of the vocal chords such that they are closer during exhalation than during inhalation.This study was a good test of the capabilities of the APD measuring device. Testing confirmed that the APD can detect resistance changes, and that measurements are easy to obtain. Results in this study are generally more consistent than other studies using other techniques -11. The 2O/Lps. Thus, nasal strips do have a measurable effect on nasal resistance. The effect of NDS appears to be equal during exhalation and during inhalation.Nasal strips reduce nasal resistance by about 0.5 cm HThe APD can be used to measure nasal resistance, and can detect resistance levels.The APD can consistently measure oral resistance with either a mask or a mouthpiece.LW conducted the testing as an undergraduate student. ATJ provided the APD and mentored LW. All authors have read and approved this manuscript.


The disparity in the scientific output between developed and developing counties is dramatic, but, as the Americas show, this grim picture is improving Kofi Annan, the Secretary-General of the United Nations, recently called attention to the clear inequalities in science between developing and developed countries and to the challenges of building bridges across these gaps that should bring the United Nations and the world scientific community closer to each other . Mr. AnnNorth America and Europe clearly dominate the number of scientific publications produced annually.It is rather obvious that richer countries are able to invest more resources in science and therefore account for the largest number of publications. It is also likely that there is a statistical bias on the part of the SCI as a bibliometric database, since it represents North American and European publications far better than those of the rest of the world . But is Using the SCI databases produced by the Institute for Scientific Information (ISI), as well as data compiled by the Red Iberoamericana de Indicadores de Ciencia y Tecnología (RICYT), we examined the differences in the number and proportion of scientific publications between the developed world and the developing world from 1990 until 2000, focusing on the Americas as a case study. Not surprisingly, there was a huge disparity in the number of publications from 1990 until 2000, with the United States contributing the lion's share (84.2%), followed by Canada (10.35%). Latin America as a whole contributed only 5.45% to the total number of scientific publications in these ten years .The total number of publications, however, is not necessarily the best measure for assessing scientific productivity or technical advances . More reOther relative indicators of scientific productivity, such as the number of publications picked up by the SCI in relation to the number of scientists in a particular country, also demonstrate that such developing regions as Latin America are making substantial contributions to science, despite the fact that the average proportion of gross domestic product (GDP) invested in science in Latin America throughout this 10-year period was only 21% of the amount invested in United States . Indeed,Among Latin American countries, there is a high degree of variability in publication rate as well as in financial investment in science and technology. Some countries have performed particularly well. For example, Uruguay, Chile, Panama, and Cuba averaged, respectively, 6.8, 5.3, 5.2, and 3.4 publications per million dollars of research and development investment in the 10 years studied, which is notoriously high compared with United States (1.5) and even Canada (3.3) . Other cWhy has the number of publications per dollar invested in research and development been increasing in Latin America while decreasing in United States and Canada?One potential explanation for the increase in scientific productivity in Latin America is that scientific development during the 1990s was particularly strong for many countries of this region. Indeed, this would explain the rapid rise in the number of publications in Latin America compared with the relatively flat increases in the United States and Canada, which were publishing just as well at the beginning of the decade. A potentially more important question, however, is why the number of publications per dollar invested in research and development has been increasing in Latin America while decreasing in the United States and Canada. This pattern could be the result of a variety of factors, none of which are mutually exclusive. It is possible that publishing in international journals as a measure of scientific productivity is becoming more important in Latin America. Increased funding to the most productive scientists from the national science development programs might have been an important stimulus. International cooperation resulting in more scientific collaborations among scientists in Latin America, Europe, and the United States may also have increased the relative number of publications in Latin America. In contrast, the decreasing trends in the number of publications per investment dollar in Canada and United States could reflect a trend towards more costly research in larger scientific programs.Nature and Science; with impact factors of 27.96 and 23.33, respectively) and in the 20 top ecological journals (with impact factors of 10.51–2.47)  between 1990 and 2002 in both the two top general science journals (51–2.47) . We credFor the top 20 ecological journals, the American subcontinents of South, Central, and North America accounted for 62% of the publications worldwide. Within the Americas, however, Latin America represented only 6%, while Canada and United States accounted, respectively, for 13% and 82% of the top 20 ecological publications. When we examined the data as contributions to the top 10 ecological journals (impact factors 10.51–3.31) versus the top 11–20 (impact factors 3.28–2.47), the Latin American countries contributed nearly twice as many publications to journals in the second category (8% in the top 11–20 compared with 4% in the top 10). These findings suggest that publications from such developing regions as Latin America are falling short of reaching the top journals. In contrast, the United States contributed somewhat more publications to the top 10 journals (84%) than the top 11–20 journals (79%). The difference in the proportion of publications contributed by the United States to the top 10 and top 20 journals was even more pronounced when we examined it in respect to worldwide publications. In this case, the United States contributed 60% of the publications to the top 10 journals and only 40% of the publications to the top 11–20 journals.Science and Nature were nearly identical to those of the top 20 ecological journals. In Science and Nature, Latin America had 7% of the publications within the Americas versus 6% in the top 20 ecological journals, whereas the United States and Canada had 81% versus 82% and 12% versus 13%, respectively. These similarities suggest that the Latin American researchers are not shying away from the two top-ranked general science journals. However, publishing in Science and Nature was not enough to gain prominence, as evidenced by the number of citations of these researchers. The latest list of the 247 most-cited researchers in ecology and environmental sciences emphasizes the overwhelming contributions of authors from North America (73%) and Europe (21%)  . No resepe (21%)  and thusAlthough there are outstanding scientific researchers in the developing world who independently are making important contributions to the international scientific community, they are the exception. Why, in general, do Latin American scientists often fail to reach the top journals or become amongst the most cited researchers in their fields? One possibility is that the main research agendas between both regions are somewhat different and that the top journals, which are published in the developed world, respond more to the scientific mainstream of the developed regions. This is not to suggest any sort of conspiracy, but rather it implies that the perception of the most important science is linked to the region and that because the major funding agencies as well as most prominent journals share a similar economic region, they also share the same perception of what science is most interesting to them. Another consideration is that more local journals from developed regions are listed by the SCI than similar journals from developing regions . ConsequThe positive trends in scientific productivity in Latin America should not be misinterpreted as a reason to be unconcerned about the existing gap highlighted by Mr. Annan. There are many compelling reasons for the push to increase scientific input from the developing world . One is Climate change and biodiversity research urgently need the scientific input from those developing regions that are so important for global processes.Although the evidence presented here demonstrates that there is a long way to go before developing countries contribute a more equitable share to the international scientific community, there are also reasons to be optimistic. The relative increase in the number of publications, especially when corrected for the amount of money available in research and development, demonstrates that many developing countries are heading in the right direction. The extremely high scientific productivity of many developing nations, corrected for and despite the rather limited availability of funds, suggests that increased funding to the sciences will be an excellent investment by developing nations in terms of publications as a measure of scientific output, particularly if these publications can target the journals that have the greatest impact. Although there may still be a long road to travel, we feel optimistic that the bridges mentioned by Mr. Annan are slowly being built.


Msx1 has been studied as an antagonist of muscle differentiation, and its expression in cultured mouse myotubes induces about 5% of the cells to undergo cellularisation and viable fragmentation, but its relevance for the endogenous programme of salamander regeneration is unknown. We dissociated muscle fibers from the limb of larval salamanders and plated them in culture. Most of the fibers were activated by dissociation to mobilise their nuclei and undergo cellularisation or breakage into viable multinucleate fragments. This was followed by microinjection of a lineage tracer into single fibers and analysis of the labelled progeny cells, as well as by time-lapse microscopy. The fibers showing morphological plasticity selectively expressed Msx1 mRNA and protein. The uptake of morpholino antisense oligonucleotides directed to Msx1 led to a specific decrease in expression of Msx1 protein in myonuclei and marked inhibition of cellularisation and fragmentation. Myofibers of the salamander respond to dissociation by activation of an endogenous programme of cellularisation and fragmentation. Lineage tracing demonstrates that cycling mononucleate progeny cells are derived from a single myofiber. The induction of Msx1 expression is required to activate this programme. Our understanding of the regulation of plasticity in postmitotic salamander cells should inform strategies to promote regeneration in other contexts.The conversion of multinucleate postmitotic muscle fibers to dividing mononucleate progeny cells (cellularisation) occurs during limb regeneration in salamanders, but the cellular events and molecular regulation underlying this remarkable process are not understood. The homeobox gene  Amphibians such as the salamander can regenerate their limbs. This paper explores how multinucleate muscle cells transform into mononuclear cells and begin to proliferate during regeneration There is currently a significant focus on strategies to promote regeneration in adult mammals and therefore a renewed interest in the mechanisms that underlie regeneration in urodele amphibians. An adult salamander such as the newt or axolotl can regenerate its limbs and tail, jaws, ocular tissues such as the lens, and small sections of the heart . A key fMsx1 in mouse myotubes , which have been used interchangeably with comparable results. After removing the epidermis, the tissue was dissociated by proteolytic digestion, filtered through a sieve to remove most of the mononucleate cells, and plated in serum-free medium. The striated myofibers readily attached to the culture dish  or 48 h (263 myofibers, three cultures), while 16% of the mononucleate cells were labelled in the latter case. It is noteworthy that the absence of S-phase entry in nuclei within multinucleate cells includes the population of myofibers that undergoes the events of cellularisation or fragmentation described below.The dissociation of viable myofibers has allowed us to evaluate their plasticity after implantation into a limb blastema, a procedure that has previously been performed only on myotubes formed in cell culture. Fibers were labelled with a cell tracker dye in suspension after dissociation, and single fibers were examined to verify the absence of any adherent mononucleate cells and were drawn into a glass micropipette A. A few After 48 h in culture, some of the striated fibers remained viable, but showed no significant change in morphology and retained the appearance of the cell shown in Approximately 10% of the total population of myofibers underwent changes in nuclear localisation within the cells such that a lobulated or ‘cauliflower’ structure formed in the middle or end of the cell A and 3B.0 nuclei in myofibers, G1 nuclei in mononucleate cells, and G2/M nuclei in mononucleate cells labelled with antibody to phosphohistone H3. The histogram of DNA content for cells in the colony is comparable to that previously observed for cycling newt mononucleate cells  showed any reaction with the probe, whereas 51% of all myofibers (n = 820) showed reactivity. After treatment with taxol, 56.3% of inhibited fibers (n = 765) were positive, whereas 63% of all myofibers (n = 901) showed expression of Msx1. It is clear, therefore, that the arrest of nuclear mobilisation does not prevent the early expression of Msx1 in activated myofibers. These data are consistent with an upstream role for Msx1 in the activation of plasticity, but direct evidence for functional activity has come from antisense perturbation.In an initial investigation of this possibility, cultures were arrested as before by treatment with taxol, followed by reaction with the In order to evaluate the uptake of morpholino-substituted oligonucleotides, larval myofibers were dissociated as usual in the presence of 10 μM biotinylated morpholinos or underivatised morpholinos. The cells were cultured for 48 h and then analysed by a detection procedure involving tyramide signal amplification see . ApproxiMsx1. The resulting cultures were analysed in parallel for the proportion of active fibers and the antisense reagent reproducibly and specifically decreased this by 60%–70% , we have observed fragmentation comparable to that described here for fibers of the larval salamander, but no cellularisation. It is possible, therefore, that there is a gradation in the degree of plasticity after dissociation, and this may be related to the ability to undergo reversal during regeneration, although more work is required to investigate these comparative issues. It is interesting that the mononucleate progeny of cellularisation were observed to reenter the cell cycle, while multinucleate fragments retained the postmitotic arrest of the parental fibers. At least half of the salamander fibers were activated after dissociation and could be scored by morphological criteria as an index of plasticity, as well as by analysis of gene expression in situ. The occurrence of cellularisation did not reflect the activation of adherent mononucleate cells since the injection of a nontransferable tracer into the fibers resulted in labelling of the mononucleate progeny, and furthermore the mobilisation of nuclear aggregates occurred without any detectable S-phase reentry. It is probable that the process of enzymatic and mechanical dissociation mimics the activation events after amputation, either in terms of mechanical factors sensed by the fibers or the release of signals from the tissue or matrix. Earlier experiments on microinjected fibers in the larval tail have explored the stimuli required to trigger cellularisation and concluded that activation apparently required both ‘clipping’ at the end of the fiber as well as tissue injury in the vicinity  or axolotl (A. mexicanum) (3–5 cm size) were removed, the epidermis was peeled off, and the tissue was rinsed in serum-free amphibian MEM (AMEM)   prior tolsewhere . After aMyofibers were dissociated as above, retained in suspension in a sterile bacteriological dish (Bibby Sterilin, Stone, United Kingdom), and incubated with 10 μM CellTracker Orange CMTMR (Molecular Probes, Eugene, Oregon, United States) for 30 min at 25 °C. The labelling was terminated by addition of 10% AMEM, and the cells were incubated for 45 min at 25 °C to permit enzymatic activation of the dye. The cell suspension was diluted several fold to allow observation of myofibers at low density. The forelimbs of axolotl larvae (7–10 cm size) were amputated at mid humerus level under tricaine (0.1%) anaesthesia 48 h before injection of labelled myofibers see A. The anMyofibers were incubated in AMEM containing 2,3-butanedionne monoxime (BDM) (4 mM) for 30 min to prevent contraction of the myofibers  and main2, and phase contrast or variable relief contrast (VAREL) (Zeiss) images were acquired using a digital camera controlled through Image-Pro Plus software (Media Cybernetics, Silver Spring, Maryland, United States). A sequence gallery was created using Image Pro-Plus and images of interest were selected, digitally enhanced, and processed in Adobe Photoshop 6.0 .To record the coordinates of the myofibers in culture, the dish was scored underneath with a scalpel, and cells in each grid square were observed daily and images were acquired with a CCD camera (Sony, Tokyo, Japan). For time-lapse microscopy, myofiber cultures were placed under an Axiovert microscope fitted with an incubation chamber maintained at 26 °C and 3% CO3H] thymidine  for 24 h, fixed in 1% glutaraldehyde, and processed for autoradiography . Baseline values for nuclear DNA content in cycling axolotl mononucleate cells were measured in parallel after incorporation of 5-deoxy-2′-bromouridine (BrdU)  was routinely used in cultures to visualize and score the myofibers. The quiescent or inactive myofiber nuclei were aligned along the fiber see  and 7D, Dissociated myofibers were plated in medium containing taxol . Parallel control cultures were incubated in vehicle (DMSO) in a similar way. The cultures were fixed at 48 h after treatment and processed for tubulin immunofluorescence or in situ hybridisation.Msx1 gene (5′-CGGTCTGCATCCTCTGCTTGCTTAG-3′) by Gene Tools Inc. . Invert control oligos of Msx1 (5′-GATTGCTTCGTCTCCTAGCTCTGGC-3′) and standard control oligos (5′-CCTCTTACCTCAGTTACAATTTATA-3′) from the supplier were used as controls. 3′-Biotin-end-labelled standard control oligos were used for evaluating the uptake of morpholino oligonucleotides by myofibers. Oligo stock solutions were prepared according to guidelines from the manufacturer and stored at 4 °C. The morpholino oligos were added at a concentration of 10 μM to the dissociation cocktail and the myofibers were dissociated as described. After purification of myofibers, fresh morpholino oligos were added to the culture medium. After sequential washes to remove any adherent morpholino  coupled with enzyme-linked immunofluorescence (ELF97, Molecular Probes) was employed to localize the uptake of morpholino oligos in cultured myofibers. Myofiber cultures were fixed at 48 h in 0.5% PFA containing 0.05% glutaraldehyde for 15 min on ice. The fixative was replaced with freshly made 0.1% NaBHMsx1 cDNA (a kind gift from H. Ide, Tohoku University, Sendai, Japan) was cloned into Bluescribe vector , and probes were generated as described elsewhere (Rad (NRad, a gift from K. Yoshizato, Hiroshima University, Hiroshima, Japan) probe was generated from a fragment of approximately 400 bp from Bluescribe vector after linearising with either HindIII (antisense) or EcoRI (sense), and riboprobes were synthesized using T3 and T7 RNA polymerase respectively (EF1α and NRad probes were used as positive controls, while neomycin (NRad sense, and Msx1 sense probes served as negative controls. For in situ hybridisation, the myofiber cultures were incubated in BDM (4 mM), fixed in chilled 1% glutaraldehyde for 15 min, postfixed in 4% PFA, and washed in 0.3% PBT. In situ hybridisation was essentially as described elsewhere  was incorporated into the fixative. After fixation, the culture was treated with freshly prepared 0.1% NaBHlsewhere . For loced cells . The samz-stacks, and composite maximum projection images were generated through Leica software. Samples stained for Msx1 protein were observed under a Zeiss Axioplan microscope and images were acquired with an Axiocam digital camera. The fluorescence intensity in myofiber nuclei was measured in Axiovision software (Zeiss), and the data were analysed by one-way analysis of variance (ANOVA) followed by multiple range test using Instat .The myofiber cultures stained for β-tubulin, MHC, or titin, or cultures injected with TR–dextran were observed under confocal laser scanning microscope (Leica). The images were acquired as Figure S1The myonuclei incorporate Syto13 live nuclear stain. The myofiber was observed with VAREL optics at 24 h after plating. Scale bar, 100 μm.(4.1 MB TIF).Click here for additional data file.Figure S2Nuclear localisation of Msx1 protein (green) was detected with a rabbit polyclonal antibody generated against the full-length mouse Msx1 homeoprotein.(5.6 MB TIF).Click here for additional data file.Video S1Time-lapse sequence was begun 6 h after plating of the myofiber on to a culture dish. The images were taken at 6 min intervals under 32× VAREL objective magnification.(110 KB AVI).Click here for additional data file.


Inappropriate use of antibiotics by individuals worried about biological agent exposures during bioterrorism events is an important public health concern. However, little is documented about the extent to which individuals with self-identified risk of anthrax exposure approached physicians for antimicrobial prophylaxis during the 2001 bioterrorism attacks in the United States.We conducted a telephone survey of randomly selected members of the Pennsylvania Chapter of the American College of Emergency Physicians to assess patients' request for and emergency physicians' prescription of antimicrobial agents during the 2001 anthrax attacks.Ninety-seven physicians completed the survey. Sixty-four (66%) respondents had received requests from patients for anthrax prophylaxis; 16 (25%) of these physicians prescribed antibiotics to a total of 23 patients. Ten physicians prescribed ciprofloxacin while 8 physicians prescribed doxycycline.During the 2001 bioterrorist attacks, the majority of the emergency physicians we surveyed encountered patients who requested anthrax prophylaxis. Public fears may lead to a high demand for antibiotic prophylaxis during bioterrorism events. Elucidation of the relationship between public health response to outbreaks and outcomes would yield insights to ease burden on frontline clinicians and guide strategies to control inappropriate antibiotic allocation during bioterrorist events. Bacillus anthracis postexposure prophylaxis and clinical management of patients with anthrax disease [The September 11, 2001 terrorism events and the ensuing anthrax attacks in the United States were associated with widespread psychological trauma [ disease . For asy disease -6.Public health officials assured the public that emergency drug supplies would be delivered to clinical settings as needed from the national antimicrobial stockpile . DespiteA total of 250 potential study subjects were randomly selected from the 2001 membership database (n = 1,060) of the Pennsylvania Chapter of the American College of Emergency Physicians (ACEP). Information in the database, such as contact telephone numbers and location of practice, is provided during enrollment or during membership renewals, and no active verification is done. Because the accuracy of the database was unknown, we used an 80% estimate based on a previous study of emergency physicians . During We used one-way frequency analyses to describe distributions of responses for all categorical items. Associations were quantified using odds ratios (ORs) with associated 95% confidence intervals (CIs). Statistical analyses were performed using SAS software (SAS Institute Inc., Cary, NC).Forty-three of the 250 physicians in the sample were excluded from the study because of insufficient contact information or because the physician was no longer practicing medicine in the state. Of the remaining 207 physicians (24% of the estimated population of 848 subjects with accurate information), 97 were interviewed (47% response rate).Sixty-four (66%) of the 97 respondents had received patient requests for antimicrobial prophylaxis against anthrax; of the physicians who received requests, 16 (25%) prescribed antibiotics. Physician setting  was not associated with either patient request for, or physician prescription of, antimicrobial prophylaxis  prescribed ciprofloxacin while 8 physicians (50%) prescribed doxycycline Figure . FourteeB. anthracis analysis. During October-December 2001, the Pennsylvania Bureau of Laboratories received approximately 1400 specimens including white powder, environmental swabs, and letters from counties in eastern and western Pennsylvania districts. Of these, 27 (or about 18 requests per 100,000 population) came from Cambria County in the western district (PA DOH: unpublished data). There were 11,063 anthrax-related telephone inquiries received from October 8 to November 11, 2001 by the Centers for Disease Control and Prevention's (CDC) emergency operations centers; queries originated from all states and one US territory. Most of these calls were from members of the public concerning anthrax vaccines (≈58%), suggesting that search for protective measures against anthrax was widespread across the United States [Following the September 11 terrorist attacks and the ensuing anthrax outbreak, the majority of the emergency physicians we surveyed in Pennsylvania had received patient requests for anthrax prophylaxis; a quarter of these physicians prescribed antibiotics for these patients. Physicians that reported patients' requests for antimicrobial prophylaxis were distributed across the state, suggesting that patients' search for protection against anthrax was widespread. Our results are consistent with data on specimens submitted to Pennsylvania public health laboratory officials for Other studies have documented an increased use of antimicrobial agents that was temporally related to the anthrax outbreak; this use could not be ascribed to that recommended by the CDC. For example, a recent national study reported that approximately 160,000 more ciprofloxacin and 96,000 more doxycycline prescriptions were written in 2001 compared to 2000 . When otB. anthracis exposure until susceptibility results were known [B. anthracis isolates recovered from patients involved in the anthrax attacks were susceptible to other antibiotics, public health officials indicated that doxycycline might be preferable over ciprofloxacin [Of the physicians reporting that they had prescribed antibiotics for anthrax prophylaxis, the majority used ciprofloxacin. This finding is consistent with other studies ,13 and ire known ,5. When floxacin . While bfloxacin , the ratUnfortunately use of antibiotics has inherent risks and costs and optimizing benefits is especially difficult in the midst of bioterrorist events. Consequences of antibiotic treatment of unexposed individuals include adverse drug reactions, increased risk of antimicrobial resistance, depletion of antibiotics, and monetary costs . FurtherPublic health response likely influences demand for and outcomes associated with antibiotics requests during bioterrorism attacks. When we asked physicians to suggest on what health departments could do to reduce the influx of patients to the emergency departments, they cited official communications to make the public "less worried." In Illinois, a surge in environmental samples received by public health officials for anthrax tests was associated with both media reports of anthrax cases in other states and a specific announcement on October 29, 2001 by the US attorney general and the FBI director. The announcement asked US citizens and law enforcement agencies to be on the "highest alert" based on "credible information" . LessonsWe acknowledge some limitations to our results. First, as in any survey, these data are subject to non-response bias. But the 47% response rate is comparable with other telephone surveys conducted among physicians in general . In addiTaken together with other recent studies of antibiotic utilization ,13, thesACEP: American College of Emergency PhysiciansCDC: Centers for Disease Control and PreventionPA DOH: Pennsylvania Department of HealthThe author(s) declare that they have no competing interests.NMM conceived of the study and its design, and coordinated revision of the manuscript drafts with all authors. KGJ participated in data collection, study design, and contributed to the study's critical review. ARK participated in the design of the study and performed statistical analyses. RCA secured funding and participated in the study's critical review. JTR participated in the study design and coordination. EL contributed to the design of the study and participated in its critical review. All authors read, commented on, and approved of the final manuscript.The pre-publication history for this paper can be accessed here:


Approaches to limiting exposure to antiretroviral therapy (ART) drugs are an active area of HIV therapy research. Here we present longitudinal follow-up of a randomized, open-label, single-center study of the immune, viral, and safety outcomes of structured therapy interruptions (TIs) in patients with chronically suppressed HIV-1 infection as compared to equal follow-up of patients on continuous therapy and including a final therapy interruption in both arms.Forty-two chronically HIV-infected patients on suppressive ART with CD4 counts higher than 400 were randomized 1:1 to either (1) three successive fixed TIs of 2, 4, and 6 wk, with intervening resumption of therapy with resuppression for 4 wk before subsequent interruption, or (2) 40 wk of continuous therapy, with a final open-ended TI in both treatment groups. Main outcome was analysis of the time to viral rebound (>5,000 copies/ml) during the open-ended TI. Secondary outcomes included study-defined safety criteria, viral resistance, therapy failure, and retention of immune reconstitution.n = 21; repeated TI, median [interquartile range] = 5 [n = 21; p = 0.36). No differences in study-related adverse events, viral set point at 12 or 20 wk of open-ended interruption, viral resistance or therapy failure, retention of CD4 T cell numbers on ART, or retention of lymphoproliferative recall antigen responses were noted between groups. Importantly, resistance detected shortly after initial viremia following the open-ended TI did not result in a lack of resuppression to less than 50 copies/ml after reinitiation of the same drug regimen.There was no difference between the groups in time to viral rebound during the open-ended TI (continuous therapy/single TI, median [interquartile range] = 4 –8 wk, n Cycles of 2- to 6-wk time-fixed TIs in patients with suppressed HIV infection failed to confer a clinically significant benefit with regard to viral suppression off ART. Also, secondary analysis showed no difference between the two strategies in terms of safety, retention of immune reconstitution, and clinical therapy failure. Based on these findings, we suggest that further clinical research on the long-term consequences of TI strategies to decrease drug exposure is warranted. Structured treatment interruptions, or "drug holidays", in HIV patients yield no clinical benefits but don't lead to short- term treatment failure either Antiretroviral therapy (ART) has been a milestone in the treatment of HIV infection. Current treatment guidelines for HIV-1 infection in the United States recommend the initiation of ART in patients with CD4 T cell counts of less than 350 cells/μl , and theWe completed a randomized, controlled trial on the outcome of repeated 2- to 6-wk TIs in patients with chronic infection in which the comparator group maintained continuous therapy and then an open-ended interruption period was applied in both treatment groups. The study addressed the potential for repeated interruptions of therapy to delay time to viral rebound as a primary outcome and analyzed secondary outcomes regarding study-defined safety criteria, viral suppression and resistance, and retention of immune reconstitution.Between August 2000 and December 2003, we enrolled 42 patients infected with HIV who were older than 18 y and on ART; eligibility criteria included CD4 counts of greater than 400 cells/μl on ART with a nadir of no less than 100 cells/μl, ART-mediated suppression (< 500 copies/ml) for more than 6 mo and less than 50 copies/ml at recruitment on any antiretroviral regimen. Approval of the study protocol was obtained from the institutional review board (IRB) of the Philadelphia Field Initiating Group for HIV Trials (Philadelphia, Pennsylvania, United States). Written informed consent was obtained from all patients. Human experimentation guidelines of the United States Department of Health and Human Services and of the authors' institutions were followed. The study protocol, including the patient consent form, the CONSORT form, and the IRB approval, can be found in Forty-two eligible patients from the Jonathan Lax Immune Disorder Clinic in Philadelphia, Pennsylvania, were randomized via sealed envelopes in a 1:1 fashion to a first phase (phase I) of either (1) three successive TIs of 2, 4, and 6 wk, respectively, or (2) maintenance of ART for 40 wk before a final interruption of therapy in both arms (phase II) subject to therapy reinitiation criteria as described below. Phase II consisted of an open-ended interruption to allow for virological and immunological comparisons between the groups off therapy. Study visits were every 2 wk for the repeated interruptions group and every 4 wk for the continuous ART group during phase I. Both groups were followed every 2 wk during phase II. We followed a study design with step-wise increases in the length of TI cycles to address potential safety concerns (resuppression was confirmed after shorter TIs before longer interruptions were initiated) and the hypothesis that sequential viral replication intervals would stimulate viral control and a delay in time to viral rebound.Phase I procedures for the repeated interruptions group included the following. (1) Interruption of therapy was individually timed to occur after two HIV RNA measurements of less than 50 copies/ml without any viral load measurements greater than 400 copies/ml in between; these interruptions increased from 2 to 4 to 6 wk sequentially. (2) If a 0.5-log or greater reduction in viral load did not occur by 6 wk of reinitiated therapy or less than 50 copies/ml was not achieved within 20 wk of reinitiated therapy, patients were withdrawn as therapy failures and a resistance test was performed. (3) Patients were also withdrawn as therapy failures if (a) the CD4 cell number declined by more than 45% of the baseline CD4 count, (b) participants developed an opportunistic infection, even if retaining required CD4 count levels, or (c) a viral load of greater than 500,000 copies/ml occurred once, with or without development of acute retroviral syndrome as defined by fever, skin lesions, and pharyngitis.Phase I procedures for the continuous therapy arm included the following: (1) patient monitoring if detected viremia was between 50 and 999 copies/ml, with the patient withdrawn if their viral load did not return to less than 50 copies/ml immediately prior to phase II, and (2) patient study withdrawal as therapy failure if during the 40-wk ART period viral load rebounded to more than 1,000 copies/ml at two consecutive time points.Phase II procedures for both arms included the following: (1) monitoring for patient study withdrawal criteria as described in phase I, (2) determining time to primary end point of a viral load greater than 5,000 copies/ml, (3) monitoring until the time of therapy reinitiation at a viral load greater than 30,000 copies/ml for three consecutive time points, and (4) after reinitiation of therapy, follow-up on therapy to confirm resuppression to less than 50 copies/ml at 6, 10, and 14 wk on therapy. Clinical and laboratory parameters  were monitored at each visit, and venous blood was collected for additional secondary outcomes during selected study visits.In both phase I and II, participants taking non-nucleoside reverse-transcriptase inhibitors (NNRTIs) were instructed to stop them a day earlier than the remaining drugs in the regimen.HIV RNA) was measured as a secondary outcome at weeks 12 and 20 of phase II based on reinitiation-of-therapy criteria outlined above.The primary outcome was time to confirmed virological rebound during phase II. Rebound was defined as first time point with greater than 5,000 copies/ml. Viral replication magnitude as defined by mean HIV-1 plasma RNA area under the curve  safety outcomes , (2) retention of ART-mediated immune reconstitution, and (3) detection of viral resistance. Retention of immune reconstitution was analyzed by (1) same-day whole blood flow-cytometry-based analysis of CD4 and CD8 T cells, including total and naïve (CD62 l/CD45RA) and memory (CD45RO) subsets as described , and (2)escribed . Viral rescribed ,19.The sample size required was calculated using PS  softwareHIV RNA up to 12 and 20 wk. In all cases, a two-sided alpha level of 0.05 was used to define statistical significance. Unless otherwise stated, results are presented as median (interquartile range) in text and tables.The primary analysis was an intent-to-treat analysis in which dropouts were assigned a week 0 rebound time (e.g., maximum failure to delay rebound). In secondary analyses, these dropouts were excluded. The log-rank test was used to test the null hypothesis of no difference between arms in the number of weeks from initiation of the open-ended TI to reaching viral rebound as defined. Patients not reaching end point at 26 wk after the beginning of the open-ended TI were censored. Wilcoxon rank sum tests were used to compare baseline and week 0 of the open-ended interruption between groups. Wilcoxon signed rank tests were used to test for no change from baseline to week 0 of phase II. Finally, Wilcoxon rank sum tests were employed to test between groups for equality of the mean AUCn = 1; patient moved away) and virological failure during continuous therapy . In the repeated interruptions arm, 18 of 21 patients reached the open-ended interruption following three TIs of 2, 4, and 6 wk duration, with median peak rises in viral loads of 136 (50–2,590), 13,651 (180–222,589), and 18,887 (3,893–96,101) copies/ml, respectively. Median time to less than 50 copies/ml after resumption of therapy was 2 (0–4), 3 (1.8–12), and 9.5 (2–12) wk, respectively, with 9, 18, and 20 wk as the maximum time needed to achieve suppression in 100% of patients before reaching the open-ended interruption. Study discontinuation in the repeated interruptions arm was due to protocol violation , loss to follow-up , and virological failure during on-therapy period .Trial patient flow is summarized in . No significant difference was found in baseline parameters between arms, with 33%–47% of patients on protease-inhibitor-containing and 61%–71% on NNRTI-containing regimens. Owing to the high participation of patients on NNRTI-based regimens and concerns about TI and safety in general, patient outcomes and treatment failure were reviewed monthly by the IRB of this study during the first 8 mo of study, quarterly for the following 4 mo, and semi-annually thereafter. The demographic and clinical characteristics of the two groups at baseline are summarized in n = 21; repeated TI, median = 5 [n = 21; p = 0.36). n = 21 per group). Exclusion of drop-outs in an as-treated analysis did not alter conclusions . Additional secondary analysis of the magnitude of viral load as shown in HIV RNA analysis at week 12  or week 20 .An intent-to-treat analysis of the time to viral rebound (>5,000 copies/ml) in the open-ended interruption showed no difference between groups (continuous therapy/single TI, median = 4 –8 wk, n dian = 5  wk, n = No patient discontinuation in either group was due to study-defined changes in CD4 cell count (reviewed further below) or due to study-associated SAEs . However, four non-study-related SAEs occurred: two patients from the continuous therapy/single interruption arm were hospitalized, one for a cholecystectomy and one for acute rectal bleeding, during the 40-wk ART period; a patient from the repeated interruptions arm died of liver cancer during week 26 of the open-ended interruption after previously reaching a viral load greater than 5,000 copies/ml yet electing to stay off ART; and a patient from the repeated interruptions arm developed a transient ileitis.C. albicans was observed, confirming the absence of significant differences in the retention of baseline immune reconstitution correlates between arms. However, a significant decrease in the abundance of CD4 cells relative to other T cell types as summarized in CD4% (but not in absolute CD4 count ) was present in the repeated TI arm, corresponding to a significant increase in CD8 T cell count. In spite of fluctuations in CD4 T cell count levels between the start and end of each monitored TI, a recovery of CD4 count levels was achieved upon resuppression following each TI in conjunction with a retention of lymphoproliferative responses against C. albicans before, during, and after each TI, as illustrated in No significant difference was observed between groups in CD4 T cell counts at the start of phase II, as illustrated in p > 0.05).An intent-to-treat analysis of the combined number of patients per arm with detected resistance mutations irrespective of therapy failure in phase I and during the final TI in phase II showed no significant difference between arms  in association with self-reported nonadherence to therapy and detection of resistance mutations in phase I, as listed in p = 0.06). A greater number of resistance mutations was detected in the repeated interruption arm, as summarized in In patients who reached phase II in the absence of therapy failure, a total of 12 patients were identified to have resistance mutations at the first viremic time point  or clinical disease progression (as indicated by CD4 count on therapy or opportunistic infections) was not observed to be different between arms. Prospective safety outcomes in our study are in accordance with reports from a retrospective analysis of 1,290 patients who interrupted treatment at least once (< 3 mo) without an increased risk of HIV-associated morbidity or mortality (with the exception of patients in Center for Disease Control and Prevention stage C during first interruption only) . In regad TI see , nor fold TI see . The latd TI see . InteresImportantly, no evidence for an increase of viral resistance in association with therapy failure was present in the repeated interruptions arm See . We did In spite of the lack of difference in the total number of patients with resistant mutations detected on therapy during phase I and off therapy in phase II (7/21 [33%] versus 10/21 [47%], respectively) in both arms, we do report in similarity to others a greater detection of resistance mutations in the TI arm when restricting analysis to the last off-drug period only ,30 as thDrug resistance that occurs during virological drug failure predicts virological responses to salvage treatment ,32,33. ITaken together, while our data show no clinically significant benefit for repeated TIs of less than 1.5 mo in patients with CD4 counts greater than 400 on therapy with regard to viral control as defined by time to rebound, secondary outcomes document no significant difference in levels of retention of immune reconstitution between arms and no increased incidence of virological failure as a consequence of TIs. While our data indicate that this TI strategy should not be pursued outside of a clinical trial setting, we argue that it will be important to collect additional data on the potential benefits of drug-sparing regimens (such as reduced long-term toxicity and reduced cost) and to define long-term outcomes in comparison with continuous therapy.clinicaltrials.gov under identifier NCT00051818.Registration of randomized trial at Protocol S1(614 KB DOC).Click here for additional data file.Protocol S2Current IRB approval for study at clinical site.(179 KB PDF).Click here for additional data file.Protocol S3IRB approval to receive study biological material at the Wistar Institute for research.(201 KB PDF).Click here for additional data file.Protocol S4(50 KB DOC).Click here for additional data file.Table S1(36 KB DOC).Click here for additional data file.Highly active antiretroviral therapy has revolutionized HIV treatment for patients who have access to the medications. But the drugs are expensive, have side effects, and can become ineffective when the virus develops resistance. Structured treatment interruptions (STIs), also known as “drug holidays” (because patients take a holiday from their drugs), have been suggested as possible alternatives to continuous therapy. Initially, there was fear that patients who went back on therapy after an interruption would not be able to control the virus again, but there was also hope that STIs might actually strengthen the immune system. In addition, STIs might alleviate some side effects, and they would certainly reduce costs. This study uses a particular design to examine the risks and benefits of STIs.The researchers studied 42 patients who received either continuous therapy for 40 weeks or three successive treatment interruptions of two, four, and six weeks, followed by a final open-ended interruption for both groups. The researchers then recorded how long patients were able to control the virus before their viral load reached a certain threshold and they had to restart therapy. They also examined CD4 counts and therapy failure, and looked for resistant viruses on and off therapy.In terms of being able to control the virus, it made no difference whether patients were on continuous therapy or had three STIs. In other words, when both groups stopped treatment at 40 weeks, the length of time that the patients could control the virus was the same in both groups. Eventually, all patients  re-initiated therapy because of a rising viral load, and the patients once on therapy all regained control over the virus. Resistant viruses were found in patients from both groups, but during the final interruption they were more common in the group that had received the three STIs.The study confirms that STIs do not help with viral control, consistent with other studies that found that STIs had no clinical benefit. On the other hand, no short-term adverse events were present, as all patients were able to regain control over the virus after they went back on treatment (without a drop in CD4 count), even after several rounds of interruptions and tests to detect of resistant viruses. There remains concern about whether recurrent cycles of viral replication and suppression might in themselves be harmful, and whether the presence of resistant virus is a signal for future treatment failure. Given these unanswered questions, STIs should only be undertaken within clinical trials.Possible risks and benefits of STIs in the management of therapy remain an active area of research. Evidence so far has not shown clinical benefits. Ongoing studies need to clarify whether there are long-term risks (and what they are), so that we can weigh these against the benefits of reducing costs and side effects.http://www.thebody.com/treat/sti.htmlThe Body information Web page on STIs: http://www.avert.org/conttrt.htmInformation on “continuing antriretroviral treatment” from AVERT, an international HIV and AIDS charity based in the United Kingdom: http://www.aidsmap.com/en/docs/7980314C-97B5–412F-93B1-AD8B64F51F73.aspInformation on STIs from NAM, a United Kingdom registered charity: http://www.niaid.nih.gov/factsheets/treat-hiv.htmFactsheet on HIV treatments from the United States National Institute for Allergy and Infectious Diseases: http://www.clinicaltrials.gov/search/term=%22Treatment+Interruption%22%5BCONDITION%5D+AND+HIV+%5BCONDITION%5DSearch results from Clinicaltrials.gov when searching for “HIV” and “treatment interruption” combined terms: