Background In yeast and  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.
Background
In yeast and  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.Caenorhabditis elegansLeishmaniaLeishmaniaLeishmaniaLeishmaniaLeishmaniaResults 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  Leihmania  infection.
Results
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  Leihmania  infection.LeihmaniaConclusions LmSIR2 is able to substantially interfere with the host cell physiology. Thus, it is tempting to speculate that these modifications could help  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.
Conclusions
LmSIR2 is able to substantially interfere with the host cell physiology. Thus, it is tempting to speculate that these modifications could help  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.Leishmaniain vivo

Background 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 [ 1 , 2 ].  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 [ 3 ]. In a previous paper we have characterized a  Leishmania major  gene encoding a protein carrying extensive homology to SIR2 of yeast that we consequently termed LmSIR2rp [ 4 ]. It belongs to a large family of closely related NAD-dependent deacetylase named Hst proteins (Homologous of Sir two) or sirTuins, present in both prokaryotic and eukaryotic species [ 5 ]. Post-translational modifications of histones, like acetylation by histone acetylase (HAT) and deacetylation by histone deacetylase (HDAC), within the context of chromatin has been shown to regulate gene expression [ 6 , 7 ]. To date, three classes of HDACs are characterized in eukaryotes, the class I and II HDACs, and the class III defined by the sir2 family. In eukaryotic species some members of the Sir2 family are much more closely related to the core domain of the yHst2p protein than to the core domain of ySir2p itself [ 8 ]. These are: the SIR2 family members from  Schizosaccharomyces pombe ,  C albicans ,  Leishmania , chicken, human (hSirT2p, hSirT3p) and the closely related mouse protein (MmSirT2p and MmSirT3p) [ 8 ]. This group has been designated Hst2-like, as it forms an independent branch within the Sir2 family tree. Five members of this group (LmSIR2rp), like it yeast (yHst2) human (SIR2L) and mouse (MmSIR2L2 and MmSIR2L3), are cytoplasmic [ 4 , 9 , 8 , 11 ]. The implication of SIR2 proteins bearing nuclear localization, in aging process has been extensively studied. In yeast, the deletion of SIR2 shortens lifespan and over expression extends lifespan [ 12 ]. This phenotype of life extension has been also observed in  Caenorhabditis elegans  carrying extra copies of the SIR2 orthologue, Sir-2.1 [review in [ 13 ]]. In mammalian cell, SIRT1 promote cell survival [ 14 ]. SIRT1 is able to deacetylate FOXO3 and/or FOXO4, thus attenuating FOXO-induced apoptosis and potentiating FOXO-induced cell-cycle arrest. Thus SIRT1 might increase longevity by shifting FOXO dependent responses away from cell death and toward cell survival [ 15 ]. Also, the unusual NAD-requirement for the Sir2 deacetylase may link metabolic rate to silencing and life span [ 16 ]. We have shown that the cytosolic LmSIR2rp protein, when over expressed in  Leishmania  amastigotes, was able to promote parasite survival in  in vitro  culture systems [ 9 ]. Further, we also observed that LmSIR2rp can be detected in the excreted/secreted material of  Leishmania  parasites when using radiolabelled immunoprecipitation technique, suggesting therefore that fraction of LmSIR2 is actively excreted by parasite (personal observations). 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.
Background
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 [ 1 , 2 ].  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 [ 3 ].LeishmaniaLeishmaniaLeishmaniaLeishmaniaIn a previous paper we have characterized a  Leishmania major  gene encoding a protein carrying extensive homology to SIR2 of yeast that we consequently termed LmSIR2rp [ 4 ]. It belongs to a large family of closely related NAD-dependent deacetylase named Hst proteins (Homologous of Sir two) or sirTuins, present in both prokaryotic and eukaryotic species [ 5 ]. Post-translational modifications of histones, like acetylation by histone acetylase (HAT) and deacetylation by histone deacetylase (HDAC), within the context of chromatin has been shown to regulate gene expression [ 6 , 7 ]. To date, three classes of HDACs are characterized in eukaryotes, the class I and II HDACs, and the class III defined by the sir2 family. In eukaryotic species some members of the Sir2 family are much more closely related to the core domain of the yHst2p protein than to the core domain of ySir2p itself [ 8 ]. These are: the SIR2 family members from  Schizosaccharomyces pombe ,  C albicans ,  Leishmania , chicken, human (hSirT2p, hSirT3p) and the closely related mouse protein (MmSirT2p and MmSirT3p) [ 8 ]. This group has been designated Hst2-like, as it forms an independent branch within the Sir2 family tree. Five members of this group (LmSIR2rp), like it yeast (yHst2) human (SIR2L) and mouse (MmSIR2L2 and MmSIR2L3), are cytoplasmic [ 4 , 9 , 8 , 11 ]. The implication of SIR2 proteins bearing nuclear localization, in aging process has been extensively studied. In yeast, the deletion of SIR2 shortens lifespan and over expression extends lifespan [ 12 ]. This phenotype of life extension has been also observed in  Caenorhabditis elegans  carrying extra copies of the SIR2 orthologue, Sir-2.1 [review in [ 13 ]]. In mammalian cell, SIRT1 promote cell survival [ 14 ]. SIRT1 is able to deacetylate FOXO3 and/or FOXO4, thus attenuating FOXO-induced apoptosis and potentiating FOXO-induced cell-cycle arrest. Thus SIRT1 might increase longevity by shifting FOXO dependent responses away from cell death and toward cell survival [ 15 ]. Also, the unusual NAD-requirement for the Sir2 deacetylase may link metabolic rate to silencing and life span [ 16 ].Leishmania majorSchizosaccharomyces pombeC albicansLeishmaniaCaenorhabditis elegansWe have shown that the cytosolic LmSIR2rp protein, when over expressed in  Leishmania  amastigotes, was able to promote parasite survival in  in vitro  culture systems [ 9 ]. Further, we also observed that LmSIR2rp can be detected in the excreted/secreted material of  Leishmania  parasites when using radiolabelled immunoprecipitation technique, suggesting therefore that fraction of LmSIR2 is actively excreted by parasite (personal observations). 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.Leishmaniain vitroLeishmanialeishmaniaLeishmaniaLeishmaniaMaterials and Methods Molecular construct and transfection procedure LmSIR2  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  Eco RI and inserted into the  Eco RI digested and dephosphorylated pcDNA3.1 shuttle vector (Amersham). Restriction analysis of the recombinant plasmids using  Eco RI and  Xho I 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. Production of monoclonal antibodies (mAb) against the fusion protein 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 [ 17 ]. Mice of BALB/c strain were injected intraperitoneally with 50 mg of hisLmSIR2 in saline solution emulsified with 0.1 ml of Freud's Complete Adjuvant. After 2 weeks, a booster injection of the same preparation was given. The antibody response of the animals was tested 1 month later. The same dose of antigen was injected intravenously 3 days before the hybridization experiment. The fusion procedure and the screening of mAb were carried out as previously described [ 18 ]. The class of mAb was determined by agarose double diffusion of supernatants against antisera specific for immunoglobulin subclasses obtained from cliniscience (Montrouge, France). Several hybridoma clones producing mAb reacting in an ELISA test with LmSIR2 protein were obtained; only one mAb of the IgG1 isotype (IIIG4) reacted in Western blot against the fusion as well as the native LmSIR2 protein. Growth kinetic and  3 H-thymidine incorporation L929 cells were inoculated at 400 cells/cm 2 , 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  3 H-thymidine incorporation, cells were seeded in 96 wells plates at 1000 cells/cm2, after various periods of incubation 1 μCi of  3 H-thymidine was added to each well. Cells were further incubated for 4 hours before reading the level of incorporated radioactivity. Giemsa staining and Immunofluorescence analysis L929 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 (final dilution in PBS: 1: 10.000) and mounted in glycerol PBS (1:1). Giemsa staining of the cell culture was performed as follows: cells were inoculated at 400 cells/cm 2  in 25 cm 2  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). RT-PCR analysis 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) 12–18 ] 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. β-galactosidase pH6 analysis Acidic (pH 6.0) β-galactosidase was assayed according to Dimri et al. [ 19 ]. Briefly, stationary-phase cells were washed three times in phosphate buffered saline (0.1 M PBS, pH 7.4), fixed in 2% formaldehyde/0,2% glutaraldehyde for 5 min at room temperature, washed three times with PBS, then incubated at 37°C without CO 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 (X-Gal) 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.
Materials and Methods
Molecular construct and transfection procedure LmSIR2  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  Eco RI and inserted into the  Eco RI digested and dephosphorylated pcDNA3.1 shuttle vector (Amersham). Restriction analysis of the recombinant plasmids using  Eco RI and  Xho I 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.
Molecular construct and transfection procedure
LmSIR2  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  Eco RI and inserted into the  Eco RI digested and dephosphorylated pcDNA3.1 shuttle vector (Amersham). Restriction analysis of the recombinant plasmids using  Eco RI and  Xho I 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.LmSIR2EcoEcoEcoXhoLmSIR2LmSIR2Production of monoclonal antibodies (mAb) against the fusion protein 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 [ 17 ]. Mice of BALB/c strain were injected intraperitoneally with 50 mg of hisLmSIR2 in saline solution emulsified with 0.1 ml of Freud's Complete Adjuvant. After 2 weeks, a booster injection of the same preparation was given. The antibody response of the animals was tested 1 month later. The same dose of antigen was injected intravenously 3 days before the hybridization experiment. The fusion procedure and the screening of mAb were carried out as previously described [ 18 ]. The class of mAb was determined by agarose double diffusion of supernatants against antisera specific for immunoglobulin subclasses obtained from cliniscience (Montrouge, France). Several hybridoma clones producing mAb reacting in an ELISA test with LmSIR2 protein were obtained; only one mAb of the IgG1 isotype (IIIG4) reacted in Western blot against the fusion as well as the native LmSIR2 protein.
Production of monoclonal antibodies (mAb) against the fusion protein
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 [ 17 ]. Mice of BALB/c strain were injected intraperitoneally with 50 mg of hisLmSIR2 in saline solution emulsified with 0.1 ml of Freud's Complete Adjuvant. After 2 weeks, a booster injection of the same preparation was given. The antibody response of the animals was tested 1 month later. The same dose of antigen was injected intravenously 3 days before the hybridization experiment.The fusion procedure and the screening of mAb were carried out as previously described [ 18 ]. The class of mAb was determined by agarose double diffusion of supernatants against antisera specific for immunoglobulin subclasses obtained from cliniscience (Montrouge, France). Several hybridoma clones producing mAb reacting in an ELISA test with LmSIR2 protein were obtained; only one mAb of the IgG1 isotype (IIIG4) reacted in Western blot against the fusion as well as the native LmSIR2 protein.Growth kinetic and  3 H-thymidine incorporation L929 cells were inoculated at 400 cells/cm 2 , 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  3 H-thymidine incorporation, cells were seeded in 96 wells plates at 1000 cells/cm2, after various periods of incubation 1 μCi of  3 H-thymidine was added to each well. Cells were further incubated for 4 hours before reading the level of incorporated radioactivity.
Growth kinetic and  3 H-thymidine incorporation
L929 cells were inoculated at 400 cells/cm 2 , 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  3 H-thymidine incorporation, cells were seeded in 96 wells plates at 1000 cells/cm2, after various periods of incubation 1 μCi of  3 H-thymidine was added to each well. Cells were further incubated for 4 hours before reading the level of incorporated radioactivity.Giemsa staining and Immunofluorescence analysis L929 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 (final dilution in PBS: 1: 10.000) and mounted in glycerol PBS (1:1). Giemsa staining of the cell culture was performed as follows: cells were inoculated at 400 cells/cm 2  in 25 cm 2  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 and Immunofluorescence analysis
L929 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 (final dilution in PBS: 1: 10.000) and mounted in glycerol PBS (1:1).Giemsa staining of the cell culture was performed as follows: cells were inoculated at 400 cells/cm 2  in 25 cm 2  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).RT-PCR analysis 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) 12–18 ] 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.
RT-PCR analysis
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) 12–18 ] 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.LmSIR2β-galactosidase pH6 analysis Acidic (pH 6.0) β-galactosidase was assayed according to Dimri et al. [ 19 ]. Briefly, stationary-phase cells were washed three times in phosphate buffered saline (0.1 M PBS, pH 7.4), fixed in 2% formaldehyde/0,2% glutaraldehyde for 5 min at room temperature, washed three times with PBS, then incubated at 37°C without CO 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 (X-Gal) 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.
β-galactosidase pH6 analysis
Acidic (pH 6.0) β-galactosidase was assayed according to Dimri et al. [ 19 ]. Briefly, stationary-phase cells were washed three times in phosphate buffered saline (0.1 M PBS, pH 7.4), fixed in 2% formaldehyde/0,2% glutaraldehyde for 5 min at room temperature, washed three times with PBS, then incubated at 37°C without CO 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 (X-Gal) 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.Results and Discussion Expression of LmSIR2 by L929 fibroblasts The Level of the LmSIR2 gene transcription was evaluated by RT-PCR analysis. As shown in Figure  1 , an amplification product of around 1.2 Kb was observed only in recombinant L929 cells transformed with the pcDNA carrying LmSIR2 gene (Fig  1  lane 2), No amplification could be detected in Wild-type fibroblasts or in fibroblasts carrying the empty pcDNA vector (Lane 1 and 3 respectively). Accordingly, Western blot analysis of cellular extracts derived from the clones 2 and 11 reacted with with the mAb IIIG4 showed a polypeptide of 50 kDa (Fig  2  lanes 3 and 5), which is slightly higher than the molecular size of the pcDNA encoded protein (≈ 43 kDa). However, it is noteworthy that this molecular size is similar to that of the parasite native protein shown in previous studies [ 9 ]. It is therefore likely that the transfected gene product is submitted to posttranslational modifications that occur during its processing. No cross reactivity was observed on cell extract derived from Wild-type cell or fibroblast carrying the empty vector (Fig  2  Lane 1 and 2). Thus, these results confirm that mouse L929 fibroblast cell line properly expressed the  LmSIR2  gene. Figure 1 Expression of LmSIR2 gene in L-929 cells.  RT-PCR analysis of total RNA from various L-929 clones was carried out using LmSIR2 or GAPDH primers. The lanes correspond to the following samples: standard size marker (PM); WT L929 cells (1); L929 clone 2 (Cl2) (2); L929 carrying an empty pcDNA plasmid (3). Figure 2 Detection of the LmSIR2 protein in L929 cell extracts.  The lanes correspond to the following samples: WT L929 cells (1); L929 carrying an empty pcDNA plasmid (2), L929 Cl2 (3); Molecular weight markers (4); L-929 Clone 9 (5). The  LmSIR2  gene was originally isolated via immunoscreening of a cDNA library, using polyclonal antibodies raised against excreted factors which could bind to glutathione [ 4 ]. The presence of such protein in the  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 (Figure  3B ) react with the monoclonal antibody as compared to cells carrying the empty pcDNA vector (Figure  3A ). Interestingly, high number of cells positive for LmSIR2 bears abnormal morphology i.e. giant multinucleated cells (Figure  3B ). In these cells, the protein is localized both in the cytosol and the nucleus. Functional studies of several mammalian SIR2 related proteins have been carried out including those who are mainly localized in the cytosol. Nevertheless, similar cellular localization pattern has never been reported nor such modification of cell physiology. Our observations suggest that LmSIR2 possesses unrelated specificity that can strongly affect the biological properties of the host cell. Interestingly, LmSIR2 protein presents a NES (the nuclear export signal or NES)-like motif LX 3 L X LX 3 L at its carboxy-terminal region, where L represents Leucine and X any amino acid [ 20 ]. The intracellular localization of key regulatory proteins tagged with a short Leucine-rich motif (nuclear export signal) is controlled by CRM1/exportin1, which is involved in the export of these proteins from the nucleus [reviewed in [ 21 ]]. However, the ability of the  Leishmania  SIR2 to shuttle between the cytoplasm and nucleus of the host cell through the NES putative sequence, await further investigations. Figure 3 Indirect immunofluorescence analysis and morphology of transgenic cells . Localisation of LmSIR2 using immunofluorescence analysis (A) L929 cells carrying an empty pcDNA vector (B) L929 (Cl2) expressing LmSIR2 protein. Morphology of cells in culture: (C) L929 cells carrying an empty pcDNA vector and (D) L929 Cl2. Note the reduced cell density and the presence of cells bearing abnormal morphology (arrow). When L929 cell culture, of either clone 1, clone 2, clone 9 or clone 10, were stained with Giemsa, a high rate of cells with unusual shape and size associated with the presence of multiple nuclei was revealed (Figure  3 D ) as compared to cells carrying the empty pcDNA vector (Figure  3C ). The rate of multinucleated cells was determined by counting the number of cells bearing more than 2 nucleus per 10 champs at 10-× magnification. In fact all the clone cultures present a 10 to 15-fold increase in giant multinucleated cells as compared to cultures of L-929 fibroblast transformed with the empty vector (Figure  3 C ). Moreover, majority of the cells if not all, present an enlarged and flattened morphology (Figure  3 D ). Growth kinetic parameters of L929 cell 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  4A ). The reduction observed in this experiment was as high as 40% as compared to cells carrying the empty pcDNA vector. These preliminary observations were further confirmed using  3 H thymidine. Indeed, as shown in figure ( 4B ), expression of LmSIR2 strongly reduced the thymidine incorporation over the time course of the experiment. The most dramatic reduction of 3H-thymidine incorporation was observed a day 7 which correspond to the maximal cell density observed for both Wild type control and the four clones studied (reduction in  3 H-thymidine incorporation of about 61% for clone 2, 60% for clone 9, 52% for clone 10 and 42% for clone 11). Figure 4 Growth kinetics analysis.  Cell proliferation was determined using two methods: cell counting (A), the results are given as a mean value of a triplicate experiment ; and  3 H-thymidine incorporation (B), the results are given as mean value of a sextuplate experiment Expression of senescence biomarker Parameters of senescence include increased cell size, reduced saturation density as well as altered expression of some gene products that have been proposed to serve as biomarkers of aging. Reduction in the final cell yield or saturation density is known to occur in human diploid fibroblast senescence and is considered as a criterion of the onset of senescence [ 22 ]. With respect to biomarkers, pH 6 β-galactosidase is particularly useful, since this activity has been found to distinguish senescent from quiescent cells both  in vivo  and  in vitro  [ 19 ]. We found that L929 cells expressing LmSIR2 exhibit reduced saturation densities (40% to 60%) and altered cell morphology. We have thus evaluated the occurrence of the pH 6 β-glactosidase activity. As shown on Figure  5  (B and C), a strong pH-6 β-glactosidase was detected in L929 cells expressing the  LmSIR2  gene but was not detected in wild-type cells or cells transformed with the empty pcDNA vector (Figure  5A ). Figure 5 Expression of a senescence biomarker.  (pH6.0) β-galactosidase activity detected in L929 cells expressing LmSIR2 (Cl2) (B and C) but not in L929 carrying an empty pcDNA plasmid (A). Cells were seeded at 40 cells/mm 2  in 25 cm 2  flasks and after 13 days of growth, the presence of pH6-galactosidase activity (Arrow) was determined as described in the Materials and Methods section. N nucleus, note the perinuclear galactosidase activity. Permissivity of fibroblast expressing LmSIR2 towards  Leishmania amazoensis Fibroblasts at the lymph are used as safe target for long lasting residual parasite population [ 23 ]. However, only few reports deal with  in vitro  infection of fibroblast. In 1978, Chang [ 24 ] demonstrated that promastigotes of  L. amazonsensis  but not  L. donovani  were able to infect human skin fibroblasts. However, once differentiated into amastigotes the parasites were unable to multiply inside the cell. Dedet and co-workers further confirmed these observations [ 25 ]. Thus, we decided to test the capacity of  L. amazonensis  to infect L929 cell lines. When  L. amazonensis  promastigotes were incubated with Wild-type fibroblasts the proportion of infected cells reached a peak of about 50% after 24 hours of contact, after what the proportion of infected fibroblasts decreased quickly. On day 2, only few parasites could be seen and after 3 days of incubation less that 1% of fibroblasts remain infected (data not shown). As shown in Figure  6 , fibroblasts expressing LmSIR2 were more permissive toward  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). Figure 6 Permissivity of L929 cells toward  Leishmania amazonensis  infection.  L929 cells were seeded in 25 cm2 flasks and treated with mitomycin. They were then infected, at different parasite/fibroblast ratio, with freshly differentiated stationary phase  L. amazonensis  promastigotes. After 24 hours of contact cells were washed and the percentage of fibroblats bearing attached or internalized parasites was determined. Results are expressed as a mean value of a triplicate experiment.
Results and Discussion
Expression of LmSIR2 by L929 fibroblasts The Level of the LmSIR2 gene transcription was evaluated by RT-PCR analysis. As shown in Figure  1 , an amplification product of around 1.2 Kb was observed only in recombinant L929 cells transformed with the pcDNA carrying LmSIR2 gene (Fig  1  lane 2), No amplification could be detected in Wild-type fibroblasts or in fibroblasts carrying the empty pcDNA vector (Lane 1 and 3 respectively). Accordingly, Western blot analysis of cellular extracts derived from the clones 2 and 11 reacted with with the mAb IIIG4 showed a polypeptide of 50 kDa (Fig  2  lanes 3 and 5), which is slightly higher than the molecular size of the pcDNA encoded protein (≈ 43 kDa). However, it is noteworthy that this molecular size is similar to that of the parasite native protein shown in previous studies [ 9 ]. It is therefore likely that the transfected gene product is submitted to posttranslational modifications that occur during its processing. No cross reactivity was observed on cell extract derived from Wild-type cell or fibroblast carrying the empty vector (Fig  2  Lane 1 and 2). Thus, these results confirm that mouse L929 fibroblast cell line properly expressed the  LmSIR2  gene. Figure 1 Expression of LmSIR2 gene in L-929 cells.  RT-PCR analysis of total RNA from various L-929 clones was carried out using LmSIR2 or GAPDH primers. The lanes correspond to the following samples: standard size marker (PM); WT L929 cells (1); L929 clone 2 (Cl2) (2); L929 carrying an empty pcDNA plasmid (3). Figure 2 Detection of the LmSIR2 protein in L929 cell extracts.  The lanes correspond to the following samples: WT L929 cells (1); L929 carrying an empty pcDNA plasmid (2), L929 Cl2 (3); Molecular weight markers (4); L-929 Clone 9 (5). The  LmSIR2  gene was originally isolated via immunoscreening of a cDNA library, using polyclonal antibodies raised against excreted factors which could bind to glutathione [ 4 ]. The presence of such protein in the  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 (Figure  3B ) react with the monoclonal antibody as compared to cells carrying the empty pcDNA vector (Figure  3A ). Interestingly, high number of cells positive for LmSIR2 bears abnormal morphology i.e. giant multinucleated cells (Figure  3B ). In these cells, the protein is localized both in the cytosol and the nucleus. Functional studies of several mammalian SIR2 related proteins have been carried out including those who are mainly localized in the cytosol. Nevertheless, similar cellular localization pattern has never been reported nor such modification of cell physiology. Our observations suggest that LmSIR2 possesses unrelated specificity that can strongly affect the biological properties of the host cell. Interestingly, LmSIR2 protein presents a NES (the nuclear export signal or NES)-like motif LX 3 L X LX 3 L at its carboxy-terminal region, where L represents Leucine and X any amino acid [ 20 ]. The intracellular localization of key regulatory proteins tagged with a short Leucine-rich motif (nuclear export signal) is controlled by CRM1/exportin1, which is involved in the export of these proteins from the nucleus [reviewed in [ 21 ]]. However, the ability of the  Leishmania  SIR2 to shuttle between the cytoplasm and nucleus of the host cell through the NES putative sequence, await further investigations. Figure 3 Indirect immunofluorescence analysis and morphology of transgenic cells . Localisation of LmSIR2 using immunofluorescence analysis (A) L929 cells carrying an empty pcDNA vector (B) L929 (Cl2) expressing LmSIR2 protein. Morphology of cells in culture: (C) L929 cells carrying an empty pcDNA vector and (D) L929 Cl2. Note the reduced cell density and the presence of cells bearing abnormal morphology (arrow). When L929 cell culture, of either clone 1, clone 2, clone 9 or clone 10, were stained with Giemsa, a high rate of cells with unusual shape and size associated with the presence of multiple nuclei was revealed (Figure  3 D ) as compared to cells carrying the empty pcDNA vector (Figure  3C ). The rate of multinucleated cells was determined by counting the number of cells bearing more than 2 nucleus per 10 champs at 10-× magnification. In fact all the clone cultures present a 10 to 15-fold increase in giant multinucleated cells as compared to cultures of L-929 fibroblast transformed with the empty vector (Figure  3 C ). Moreover, majority of the cells if not all, present an enlarged and flattened morphology (Figure  3 D ).
Expression of LmSIR2 by L929 fibroblasts
The Level of the LmSIR2 gene transcription was evaluated by RT-PCR analysis. As shown in Figure  1 , an amplification product of around 1.2 Kb was observed only in recombinant L929 cells transformed with the pcDNA carrying LmSIR2 gene (Fig  1  lane 2), No amplification could be detected in Wild-type fibroblasts or in fibroblasts carrying the empty pcDNA vector (Lane 1 and 3 respectively). Accordingly, Western blot analysis of cellular extracts derived from the clones 2 and 11 reacted with with the mAb IIIG4 showed a polypeptide of 50 kDa (Fig  2  lanes 3 and 5), which is slightly higher than the molecular size of the pcDNA encoded protein (≈ 43 kDa). However, it is noteworthy that this molecular size is similar to that of the parasite native protein shown in previous studies [ 9 ]. It is therefore likely that the transfected gene product is submitted to posttranslational modifications that occur during its processing. No cross reactivity was observed on cell extract derived from Wild-type cell or fibroblast carrying the empty vector (Fig  2  Lane 1 and 2). Thus, these results confirm that mouse L929 fibroblast cell line properly expressed the  LmSIR2  gene.LmSIR2The  LmSIR2  gene was originally isolated via immunoscreening of a cDNA library, using polyclonal antibodies raised against excreted factors which could bind to glutathione [ 4 ]. The presence of such protein in the  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 (Figure  3B ) react with the monoclonal antibody as compared to cells carrying the empty pcDNA vector (Figure  3A ). Interestingly, high number of cells positive for LmSIR2 bears abnormal morphology i.e. giant multinucleated cells (Figure  3B ). In these cells, the protein is localized both in the cytosol and the nucleus. Functional studies of several mammalian SIR2 related proteins have been carried out including those who are mainly localized in the cytosol. Nevertheless, similar cellular localization pattern has never been reported nor such modification of cell physiology. Our observations suggest that LmSIR2 possesses unrelated specificity that can strongly affect the biological properties of the host cell. Interestingly, LmSIR2 protein presents a NES (the nuclear export signal or NES)-like motif LX 3 L X LX 3 L at its carboxy-terminal region, where L represents Leucine and X any amino acid [ 20 ]. The intracellular localization of key regulatory proteins tagged with a short Leucine-rich motif (nuclear export signal) is controlled by CRM1/exportin1, which is involved in the export of these proteins from the nucleus [reviewed in [ 21 ]]. However, the ability of the  Leishmania  SIR2 to shuttle between the cytoplasm and nucleus of the host cell through the NES putative sequence, await further investigations.LmSIR2leishmaniaLeishmaniaWhen L929 cell culture, of either clone 1, clone 2, clone 9 or clone 10, were stained with Giemsa, a high rate of cells with unusual shape and size associated with the presence of multiple nuclei was revealed (Figure  3 D ) as compared to cells carrying the empty pcDNA vector (Figure  3C ). The rate of multinucleated cells was determined by counting the number of cells bearing more than 2 nucleus per 10 champs at 10-× magnification. In fact all the clone cultures present a 10 to 15-fold increase in giant multinucleated cells as compared to cultures of L-929 fibroblast transformed with the empty vector (Figure  3 C ). Moreover, majority of the cells if not all, present an enlarged and flattened morphology (Figure  3 D ).Growth kinetic parameters of L929 cell 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  4A ). The reduction observed in this experiment was as high as 40% as compared to cells carrying the empty pcDNA vector. These preliminary observations were further confirmed using  3 H thymidine. Indeed, as shown in figure ( 4B ), expression of LmSIR2 strongly reduced the thymidine incorporation over the time course of the experiment. The most dramatic reduction of 3H-thymidine incorporation was observed a day 7 which correspond to the maximal cell density observed for both Wild type control and the four clones studied (reduction in  3 H-thymidine incorporation of about 61% for clone 2, 60% for clone 9, 52% for clone 10 and 42% for clone 11). Figure 4 Growth kinetics analysis.  Cell proliferation was determined using two methods: cell counting (A), the results are given as a mean value of a triplicate experiment ; and  3 H-thymidine incorporation (B), the results are given as mean value of a sextuplate experiment
Growth kinetic parameters of L929 cell 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  4A ). The reduction observed in this experiment was as high as 40% as compared to cells carrying the empty pcDNA vector. These preliminary observations were further confirmed using  3 H thymidine. Indeed, as shown in figure ( 4B ), expression of LmSIR2 strongly reduced the thymidine incorporation over the time course of the experiment. The most dramatic reduction of 3H-thymidine incorporation was observed a day 7 which correspond to the maximal cell density observed for both Wild type control and the four clones studied (reduction in  3 H-thymidine incorporation of about 61% for clone 2, 60% for clone 9, 52% for clone 10 and 42% for clone 11).Expression of senescence biomarker Parameters of senescence include increased cell size, reduced saturation density as well as altered expression of some gene products that have been proposed to serve as biomarkers of aging. Reduction in the final cell yield or saturation density is known to occur in human diploid fibroblast senescence and is considered as a criterion of the onset of senescence [ 22 ]. With respect to biomarkers, pH 6 β-galactosidase is particularly useful, since this activity has been found to distinguish senescent from quiescent cells both  in vivo  and  in vitro  [ 19 ]. We found that L929 cells expressing LmSIR2 exhibit reduced saturation densities (40% to 60%) and altered cell morphology. We have thus evaluated the occurrence of the pH 6 β-glactosidase activity. As shown on Figure  5  (B and C), a strong pH-6 β-glactosidase was detected in L929 cells expressing the  LmSIR2  gene but was not detected in wild-type cells or cells transformed with the empty pcDNA vector (Figure  5A ). Figure 5 Expression of a senescence biomarker.  (pH6.0) β-galactosidase activity detected in L929 cells expressing LmSIR2 (Cl2) (B and C) but not in L929 carrying an empty pcDNA plasmid (A). Cells were seeded at 40 cells/mm 2  in 25 cm 2  flasks and after 13 days of growth, the presence of pH6-galactosidase activity (Arrow) was determined as described in the Materials and Methods section. N nucleus, note the perinuclear galactosidase activity.
Expression of senescence biomarker
Parameters of senescence include increased cell size, reduced saturation density as well as altered expression of some gene products that have been proposed to serve as biomarkers of aging. Reduction in the final cell yield or saturation density is known to occur in human diploid fibroblast senescence and is considered as a criterion of the onset of senescence [ 22 ]. With respect to biomarkers, pH 6 β-galactosidase is particularly useful, since this activity has been found to distinguish senescent from quiescent cells both  in vivo  and  in vitro  [ 19 ]. We found that L929 cells expressing LmSIR2 exhibit reduced saturation densities (40% to 60%) and altered cell morphology. We have thus evaluated the occurrence of the pH 6 β-glactosidase activity. As shown on Figure  5  (B and C), a strong pH-6 β-glactosidase was detected in L929 cells expressing the  LmSIR2  gene but was not detected in wild-type cells or cells transformed with the empty pcDNA vector (Figure  5A ).in vivoin vitroLmSIR2Permissivity of fibroblast expressing LmSIR2 towards  Leishmania amazoensis Fibroblasts at the lymph are used as safe target for long lasting residual parasite population [ 23 ]. However, only few reports deal with  in vitro  infection of fibroblast. In 1978, Chang [ 24 ] demonstrated that promastigotes of  L. amazonsensis  but not  L. donovani  were able to infect human skin fibroblasts. However, once differentiated into amastigotes the parasites were unable to multiply inside the cell. Dedet and co-workers further confirmed these observations [ 25 ]. Thus, we decided to test the capacity of  L. amazonensis  to infect L929 cell lines. When  L. amazonensis  promastigotes were incubated with Wild-type fibroblasts the proportion of infected cells reached a peak of about 50% after 24 hours of contact, after what the proportion of infected fibroblasts decreased quickly. On day 2, only few parasites could be seen and after 3 days of incubation less that 1% of fibroblasts remain infected (data not shown). As shown in Figure  6 , fibroblasts expressing LmSIR2 were more permissive toward  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). Figure 6 Permissivity of L929 cells toward  Leishmania amazonensis  infection.  L929 cells were seeded in 25 cm2 flasks and treated with mitomycin. They were then infected, at different parasite/fibroblast ratio, with freshly differentiated stationary phase  L. amazonensis  promastigotes. After 24 hours of contact cells were washed and the percentage of fibroblats bearing attached or internalized parasites was determined. Results are expressed as a mean value of a triplicate experiment.
Permissivity of fibroblast expressing LmSIR2 towards  Leishmania amazoensis
Leishmania amazoensisFibroblasts at the lymph are used as safe target for long lasting residual parasite population [ 23 ]. However, only few reports deal with  in vitro  infection of fibroblast. In 1978, Chang [ 24 ] demonstrated that promastigotes of  L. amazonsensis  but not  L. donovani  were able to infect human skin fibroblasts. However, once differentiated into amastigotes the parasites were unable to multiply inside the cell. Dedet and co-workers further confirmed these observations [ 25 ]. Thus, we decided to test the capacity of  L. amazonensis  to infect L929 cell lines. When  L. amazonensis  promastigotes were incubated with Wild-type fibroblasts the proportion of infected cells reached a peak of about 50% after 24 hours of contact, after what the proportion of infected fibroblasts decreased quickly. On day 2, only few parasites could be seen and after 3 days of incubation less that 1% of fibroblasts remain infected (data not shown). As shown in Figure  6 , fibroblasts expressing LmSIR2 were more permissive toward  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).in vitroL. amazonsensisL. donovaniL. amazonensisL. amazonensisLeishmaniaConclusions 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  Leishmania  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 [ 26 ]. In this view, it will be of interest to determine if fibroblasts that express LmSIR2 were more resistant to apoptotic stimuli. It is known that infected macrophages are usually more resistant to apoptosis, however it is not know if such mechanism operate in infected fibroblast. Thus, the implication of LmSIR2 in a general mechanism leading to apoptosis resistance of both macrophages and fibroblasts await further investigations. The  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 [ 27 ]; (2) the ultrastructural examinations of skin biopsies obtained from patients with cutaneous leishmaniasis showed that intracellular amastigotes could be frequently identified in vacuoles and in the cytosol of the host cell [ 28 ]. In 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  in vivo  infection process awaits further investigations
Conclusions
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  Leishmania  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 [ 26 ]. In this view, it will be of interest to determine if fibroblasts that express LmSIR2 were more resistant to apoptotic stimuli. It is known that infected macrophages are usually more resistant to apoptosis, however it is not know if such mechanism operate in infected fibroblast. Thus, the implication of LmSIR2 in a general mechanism leading to apoptosis resistance of both macrophages and fibroblasts await further investigations. The  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 [ 27 ]; (2) the ultrastructural examinations of skin biopsies obtained from patients with cutaneous leishmaniasis showed that intracellular amastigotes could be frequently identified in vacuoles and in the cytosol of the host cell [ 28 ].LeishmaniaLeishmaniaLeishmaniain vivoin vivoLeishmaniaIn 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  in vivo  infection process awaits further investigationsin vivo


Background 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.
Background
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.Method 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.
Method
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.Results 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 H 2 0/Lps from an average nasal resistance of 5.5 cm H 2 0/Lps.
Results
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 H 2 0/Lps from an average nasal resistance of 5.5 cm H 2 0/Lps.Conclusions Nasal strips reduce nasal resistance when measured with the APD. The effect is equal during exhalation and during inhalation.
Conclusions
Nasal strips reduce nasal resistance when measured with the APD. The effect is equal during exhalation and during inhalation.

Background 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 [ 1 ]. By lowering nasal resistance, they reduce the work of breathing and the supply of oxygen into the body could increase [ 2 , 3 ]. The 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 [ 3 ]. 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 [ 2 , 4 - 9 ], which contradict results found by others [ 1 , 3 , 10 - 14 ]. Various techniques were used to assess NDS effectiveness. Some measured the amount of airflow, others the area of the nostrils, and still others the nasal airflow resistance. The Airflow Perturbation Device (APD) is a small, light weight, and easy to use instrument that measures respiratory resistance [ 15 ]. A segmented rotating wheel in the air flow path changes air flow and mouth pressure as the wheel momentarily partially obstructs the flow passage (Figure  1 ). The magnitude of these perturbations depends on the resistance of the wheel and respiratory resistance. Measurement of wheel resistance allows respiratory resistance to be calculated directly (Figure  2 ). Resistance values appear on a computer screen within a minute from starting the measurement. Thereafter they are updated as they occur. Figure 1 The APD Sensor consists of a rotating wheel in the air path, a pneumotach, and pressure transducers [15] Figure 2 The APD system consists of pressure and flow transducers, analog-to-digital conversion, and a computer display of results [1]. People 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.
Background
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 [ 1 ]. By lowering nasal resistance, they reduce the work of breathing and the supply of oxygen into the body could increase [ 2 , 3 ].The 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 [ 3 ].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 [ 2 , 4 - 9 ], which contradict results found by others [ 1 , 3 , 10 - 14 ]. Various techniques were used to assess NDS effectiveness. Some measured the amount of airflow, others the area of the nostrils, and still others the nasal airflow resistance.The Airflow Perturbation Device (APD) is a small, light weight, and easy to use instrument that measures respiratory resistance [ 15 ]. A segmented rotating wheel in the air flow path changes air flow and mouth pressure as the wheel momentarily partially obstructs the flow passage (Figure  1 ). The magnitude of these perturbations depends on the resistance of the wheel and respiratory resistance. Measurement of wheel resistance allows respiratory resistance to be calculated directly (Figure  2 ). Resistance values appear on a computer screen within a minute from starting the measurement. Thereafter they are updated as they occur.People 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.Methods This study tested forty-seven volunteers (14 men, 33 women; age 17–51 yr; height 147–188 cm; weight 38–105 kg). 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" (CNS, Inc., Minneapolis, MN, clear, medium/large nasal strips). 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. 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 (Adult Mask 4–5 + , 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. The second phase of this study consisted of a test to measure nasal breathing resistance. The subject was instructed to breathe normally through the nose with the mouth closed and with no NDS. For the third phase, the subject placed a NDS across the nasal valve on his/her nose as shown on the instructions provided by the manufacturer. In both of these tests, the oronasal mask was used. Air flow perturbations with the APD occur at a rate of about 10 per second [ 15 ]. Measurements were obtained in these experiments over approximately 100 perturbations. It has been previously found that measurements made over that time are relatively stable and reproducible [ 15 ]. Several time-averaged resistance values are displayed: resistance during inhalation, 2) resistance during exhalation, and 3) the average of inhalation and exhalation resistances. All three of these have been found to be useful. Primary comparisons for this study were made using the average respiratory resistance. Secondary comparisons in the second phase of this study investigated the effects of NDS on inhalation and exhalation respiratory resistances. Statistical comparisons were made using a paired mean t-test with significance at the p = 0.05 level.
Methods
This study tested forty-seven volunteers (14 men, 33 women; age 17–51 yr; height 147–188 cm; weight 38–105 kg). 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" (CNS, Inc., Minneapolis, MN, clear, medium/large nasal strips). 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.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 (Adult Mask 4–5 + , 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.The second phase of this study consisted of a test to measure nasal breathing resistance. The subject was instructed to breathe normally through the nose with the mouth closed and with no NDS. For the third phase, the subject placed a NDS across the nasal valve on his/her nose as shown on the instructions provided by the manufacturer. In both of these tests, the oronasal mask was used.Air flow perturbations with the APD occur at a rate of about 10 per second [ 15 ]. Measurements were obtained in these experiments over approximately 100 perturbations. It has been previously found that measurements made over that time are relatively stable and reproducible [ 15 ]. Several time-averaged resistance values are displayed: resistance during inhalation, 2) resistance during exhalation, and 3) the average of inhalation and exhalation resistances. All three of these have been found to be useful.Primary comparisons for this study were made using the average respiratory resistance. Secondary comparisons in the second phase of this study investigated the effects of NDS on inhalation and exhalation respiratory resistances. Statistical comparisons were made using a paired mean t-test with significance at the p = 0.05 level.Results Subject data appear in Table  1 . Average resistance measured during mouth breathing with mouthpiece ranges from 1.90 to 5.03 cm H 2 O/Lps. In the past, average respiratory resistances for healthy adults have generally fallen in the range of 2.5 - 3.5 cm H 2 O/Lps, and such is the case here. Also, as expected, most respiratory resistance values during exhalation exceed those measured during inhalation. Table 1 Subject data for APD Measurements of Respiratory Resistance when Measured Through the Mouth and Nose. Resistances are given in cm H 2 O/Lps. Subject No. Sex Mouth Piece Mask Mouth Mask Nose Mask NDS Inh Avg Exh Inh Avg Exh Inh Avg Exh Inh Avg Exh 1 F 3.15 3.06 2.97 2.86 3.08 3.30 4.65 5.13 5.61 4.08 4.13 4.18 2 F 3.00 3.26 3.52 2.98 3.29 3.59 5.84 5.72 5.59 3.64 3.47 3.30 3 F 2.69 3.19 3.68 2.84 3.38 3.92 4.94 5.73 6.52 4.55 5.26 5.97 4 F 4.00 4.62 5.25 3.97 4.46 4.95 5.40 5.87 6.31 5.11 5.45 5.79 5 F 3.13 4.04 4.95 3.58 4.11 4.63 5.33 5.59 5.85 4.62 5.30 5.98 6 M 2.14 2.41 2.69 2.10 2.34 2.58 3.76 4.15 4.53 3.71 4.16 4.61 7 F 2.73 3.15 3.85 2.43 3.11 3.79 3.92 4.16 4.39 3.54 3.62 3.69 8 M 2.03 2.33 2.64 2.12 2.24 2.36 4.32 4.50 4.69 3.65 3.85 4.06 9 M 1.87 1.96 2.06 1.83 1.82 1.80 3.16 3.53 3.91 3.26 3.47 3.68 10 M 3.49 3.95 4.41 3.27 3.90 4.52 5.96 6.27 6.57 6.13 6.20 6.28 11 F 2.89 2.97 3.04 2.76 2.91 3.07 7.59 7.33 7.07 7.48 7.05 6.61 12 F 3.67 4.24 4.82 4.18 4.38 4.58 6.36 7.14 7.92 6.75 6.82 6.90 13 M 2.20 2.54 2.89 2.13 2.49 2.84 5.27 5.50 5.73 4.80 5.04 5.27 14 F 3.08 2.98 2.88 2.98 3.07 3.17 5.12 5.25 5.38 5.34 5.36 5.38 15 M 2.37 2.45 2.53 2.64 2.77 2.90 5.44 5.63 5.82 5.33 5.57 5.81 16 M 3.48 3.58 3.68 3.16 3.47 3.77 5.71 5.30 4.90 4.71 4.88 5.04 17 M 2.40 2.85 3.30 2.22 2.74 3.26 4.54 4.90 5.25 4.38 4.70 5.07 18 F 2.96 2.96 2.95 2.95 3.04 3.12 6.84 7.19 7.54 6.71 6.89 7.07 19 M 2.00 2.30 2.59 2.54 2.10 1.67 3.82 4.25 4.68 3.20 3.39 3.57 20 F 2.40 2.85 3.30 2.81 2.77 2.73 4.12 4.16 4.20 3.61 3.73 3.85 21 F 4.22 4.20 4.17 4.34 4.42 4.49 6.76 6.97 7.10 6.36 6.75 7.14 22 F 2.73 3.12 3.50 3.02 3.12 3.22 6.60 6.74 6.89 5.90 6.08 6.26 23 F 4.87 5.03 5.19 4.49 4.96 5.42 5.96 6.23 6.70 5.06 5.30 5.54 24 F 4.38 4.79 5.20 4.41 4.64 4.87 6.35 6.68 7.01 5.70 6.05 6.39 25 F 2.61 3.02 3.44 2.51 2.94 3.36 3.98 4.03 4.08 3.56 3.42 3.28 26 M 2.12 2.42 2.71 1.80 2.26 2.71 5.87 5.84 5.81 5.31 4.38 5.44 27 F 3.22 3.77 4.31 3.48 3.58 3.68 4.51 4.76 5.01 4.24 4.58 4.93 28 F 2.85 3.01 3.17 2.74 2.94 3.14 4.71 4.64 4.56 3.57 4.17 4.76 29 F 2.84 3.26 3.67 3.04 3.39 3.75 4.94 5.56 6.18 4.40 5.04 5.68 30 F 2.76 3.08 3.40 2.81 3.05 3.29 6.24 6.40 6.56 5.57 5.76 5.94 31 F 2.90 3.21 3.51 2.68 2.88 3.09 4.71 4.88 5.04 5.03 5.16 5.29 32 M 1.76 1.90 2.03 1.54 1.87 2.20 4.02 4.49 4.97 4.00 4.18 4.35 33 F 3.65 3.94 4.23 3.76 3.92 4.09 4.90 5.30 5.71 4.90 5.26 5.61 34 M 1.94 2.15 2.36 1.97 2.17 2.36 5.19 4.90 4.61 4.37 4.36 4.36 35 F 2.78 2.93 3.09 2.81 3.05 3.29 3.81 4.45 5.10 3.75 4.20 4.65 36 F 5.21 5.09 4.97 4.46 4.81 5.71 4.81 5.19 5.56 4.00 4.04 4.08 37 F 3.18 3.44 3.69 3.02 3.34 3.66 5.49 5.70 5.91 5.50 5.58 5.65 38 F 2.77 3.48 4.20 2.62 3.31 4.00 6.70 6.60 6.51 5.97 6.12 6.26 39 F 3.66 3.57 3.48 3.53 3.54 3.55 7.62 7.73 7.83 6.94 7.33 7.72 40 F 2.57 3.05 3.52 2.58 2.97 3.36 3.64 4.13 4.63 3.58 3.78 3.98 41 F 3.76 3.74 3.71 3.17 3.68 4.18 4.70 5.11 5.51 4.13 4.54 4.95 42 M 3.27 3.39 3.58 3.43 3.61 3.80 6.08 6.44 6.79 5.90 6.25 6.60 43 F 3.27 3.65 4.03 3.89 3.68 4.47 4.47 4.78 5.09 3.69 3.98 4.27 44 M 2.23 2.51 2.79 2.17 2.34 2.51 6.48 6.42 6.35 5.87 6.01 6.15 45 F 2.09 2.95 3.02 2.98 3.07 3.16 6.10 6.65 7.20 5.86 6.20 6.55 46 F 2.98 3.12 3.26 3.15 3.23 3.30 5.70 5.65 5.60 4.57 4.68 4.78 47 F 2.67 2.64 2.61 2.56 2.56 2.57 4.35 4.74 5.12 3.29 3.64 3.99 Average 2.96 3.24 3.51 2.96 3.21 3.48 5.24 5.50 5.74 4.80 5.00 5.25 Std dev 0.76 0.75 0.82 0.73 0.76 0.88 1.09 1.02 1.03 1.11 1.10 1.12 Breathing through the mouth into the oronasal mask yielded almost the same values. Means of values with the mouthpieces and oronasal mask are 3.24 and 3.21, respectively. The difference was not statistically significant using a paired-t test at p = 0.05. Figure  3  shows the graph of average respiratory resistance of mask vs. mouthpiece while breathing through the mouth. The graph has a slope of nearly 1.0 and an intercept of nearly 0.0, indicating a nearly perfect correspondence between the two methods of measurement. Both slope and intercept were tested statistically and the line was found to be identical to y = x at the p = 0.05 level. Comparison of inhalation resistance between mouthpiece and oronasal mask yielded the following equation: Figure 3 Average oral respiratory resistance measured with a mouthpiece and an oronasal mask. There is almost perfect agreement between the two methods. y = 0.9624 + 0.1041 R 2  = 0.8435    (1) where y = mask value of resistance and x = mouthpiece value of resistance This 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: y = 0.874bx + 0.4594 R 2  = 0.8828    (2) This 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. Figure  4  shows the relationship of average respiratory resistance when breathing through the nose measured with and without the nasal strips. The NDS data have a slope of nearly 1.0 and a y-intercept is approximately -0.4. This signifies a reduction of nasal breathing resistance using the nasal strip. Nasal resistances with no nasal strip range from 3.53 to 7.73 cm H 2 O/Lps, while nasal resistance with the NDS ranges from 3.39 to 7.33 cm H 2 O/Lps. This demonstrates the expected resistance reduction with NDS. Figure 4 Average respiratory resistance while breathing through the nose in 47 subjects. Nasal strips showed a decrease of nasal resistance of 0.43 cm H 2 O/Lps. 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. The effect of NDS on resistance during exhalation was also statistically highly significant. There was an average reduction of 0.45 cm H 2 O/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 inhalation tested to be statistically highly significant, as well. The average resistance reduction was 0.49 cm H 2 0/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. Resistance 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.
Results
Subject data appear in Table  1 . Average resistance measured during mouth breathing with mouthpiece ranges from 1.90 to 5.03 cm H 2 O/Lps. In the past, average respiratory resistances for healthy adults have generally fallen in the range of 2.5 - 3.5 cm H 2 O/Lps, and such is the case here. Also, as expected, most respiratory resistance values during exhalation exceed those measured during inhalation.Subject data for APD Measurements of Respiratory Resistance when Measured Through the Mouth and Nose. Resistances are given in cm H 2 O/Lps.Subject No.SexMouth PieceMask MouthMask NoseMask NDSBreathing through the mouth into the oronasal mask yielded almost the same values. Means of values with the mouthpieces and oronasal mask are 3.24 and 3.21, respectively. The difference was not statistically significant using a paired-t test at p = 0.05.Figure  3  shows the graph of average respiratory resistance of mask vs. mouthpiece while breathing through the mouth. The graph has a slope of nearly 1.0 and an intercept of nearly 0.0, indicating a nearly perfect correspondence between the two methods of measurement. Both slope and intercept were tested statistically and the line was found to be identical to y = x at the p = 0.05 level. Comparison of inhalation resistance between mouthpiece and oronasal mask yielded the following equation:y = 0.9624 + 0.1041 R 2  = 0.8435    (1)where 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:y = 0.874bx + 0.4594 R 2  = 0.8828    (2)This 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.Figure  4  shows the relationship of average respiratory resistance when breathing through the nose measured with and without the nasal strips. The NDS data have a slope of nearly 1.0 and a y-intercept is approximately -0.4. This signifies a reduction of nasal breathing resistance using the nasal strip. Nasal resistances with no nasal strip range from 3.53 to 7.73 cm H 2 O/Lps, while nasal resistance with the NDS ranges from 3.39 to 7.33 cm H 2 O/Lps. This demonstrates the expected resistance reduction with NDS.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.The effect of NDS on resistance during exhalation was also statistically highly significant. There was an average reduction of 0.45 cm H 2 O/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 inhalation tested to be statistically highly significant, as well. The average resistance reduction was 0.49 cm H 2 0/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.Resistance 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.Discussion 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 [ 15 , 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 [ 17 , 18 ] have concluded that the minimum detectable external resistance is about a constant 25–30% proportion of the resistance already present. The resistance change measured in this study is about 10% of the baseline resistance. If the use of NDS does result in a detectable change, then it may be that a different detection mechanism is operating. It is possible that the subject could detect nasal resistance only, rather than total respiratory resistance. Based on that supposition, NDS reduce nasal resistance by about 17%. 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 [ 1 - 11 ]. The fact that the APD directly measures respiratory resistance, and is not an indirect measurement may be one reason for this consistency. Then, again, our subject population exhibited some homogeneity in age, social class, and racial makeup.
Discussion
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 [ 15 , 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 [ 17 , 18 ] have concluded that the minimum detectable external resistance is about a constant 25–30% proportion of the resistance already present. The resistance change measured in this study is about 10% of the baseline resistance. If the use of NDS does result in a detectable change, then it may be that a different detection mechanism is operating. It is possible that the subject could detect nasal resistance only, rather than total respiratory resistance. Based on that supposition, NDS reduce nasal resistance by about 17%.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 [ 1 - 11 ]. The fact that the APD directly measures respiratory resistance, and is not an indirect measurement may be one reason for this consistency. Then, again, our subject population exhibited some homogeneity in age, social class, and racial makeup.Conclusions Nasal strips reduce nasal resistance by about 0.5 cm H 2 O/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. The 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.
Conclusions
Nasal strips reduce nasal resistance by about 0.5 cm H 2 O/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.The 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.Authors' Contributions LW conducted the testing as an undergraduate student. ATJ provided the APD and mentored LW. All authors have read and approved this manuscript.
Authors' Contributions
LW conducted the testing as an undergraduate student. ATJ provided the APD and mentored LW. All authors have read and approved this manuscript.