Injection of APL resulted in positive selection of CD8 T cells expressing the relevant viral specificity and in the export of those virus-specific CTL to lymph nodes without inducing T cell proliferation. Thus, exogenous APL administration offers the potential of expanding repertoires in vivo in a manner useful to the organism. To efficiently peripheralize antigen-specific T cells, concomitant enhancement of mechanisms promoting thymocyte migration appears to be required. This commentary describes the rationale for thymic vaccination and addresses the potential prophylactic and therapeutic applications of this approach for treatment of infectious diseases and cancer. Thymic vaccination-induced peptide-specific T cells might generate effective immune protection against disease-causing agents, including those for which no effective natural protection exists.Conventional vaccines afford protection against infectious diseases by expanding existing pathogen-specific peripheral lymphocytes, both CD8 cytotoxic effector (CTL) and CD4 helper T cells. The latter induce B cell maturation and antibody production. As a consequence, lymphocytes within the memory pool are poised to rapidly proliferate at the time of a subsequent infection. The "thymic vaccination" concept offers a novel way to alter the primary T cell repertoire through exposure of thymocytes to altered peptide ligands (APL) with reduced T cell receptor (TCR) affinity relative to cognate antigens recognized by those same TCRs. Thymocyte maturation (i.e. positive selection) is enhanced by low affinity interaction between a TCR and an MHC-bound peptide in the thymus and subsequent emigration of mature cells into the peripheral T lymphocyte pool follows. In principal, such variants of antigens derived from infectious agents could be utilized for peptide-driven maturation of thymocytes bearing pathogen-specific TCRs. To test this idea, APLs of gp Vaccination has improved healthcare by providing the most cost effective means to prevent disease on a global basis ,2. SinceTo date, the fundamental principles of vaccination have remained unchanged. The overriding concept for each vaccine has been the establishment of protective immunity largely due to antigen-specific T cell expansion, facilitating subsequent proliferation and differentiation of CD8 cytotoxic effector T cells and CD4 helper T cells capable of producing antiviral cytokines and chemokines Fig. . CD4 T cHowever, conventional vaccines have their pitfalls. Microorganisms including HIV and malaria, among others, may alter their antigenic proteins through rapid mutagenesis, thereby hindering cytotoxic T lymphocyte (CTL)-based immunity, exploiting holes in the T cell repertoire, and/or misdirecting both cellular and humoral responses away from key cell-binding receptors to pathogen components which cannot provide epitopes for neutralizing antibodies -13.in vivo, controlling the generation of naive T cells and hence, subsequent memory development within the peripheral lymphoid compartment. Through repertoire manipulation, it should be possible to sculpt the specificity and diversity of disease-fighting cells. This thymic vaccination approach aims to deliver, by parenteral administration, positively selecting APL of cognate antigens into the thymus, eliciting maturation of thymocytes with desired TCR specificities at the level of thymic repertoire development . APL derived from infectious agents or tumor antigens, with low affinity to the TCR could, in principle, mediate positive selection and export of specific T cells from the thymus . As suchent Fig. . Note hoExpanding T cell repertoires has enormous potential in aiding the organism's fight against infections or in affording tumor immunity. This strategy is principally different from conventional "peripheral" vaccination, which leads to proliferation of pre-existing mature T cells but does not alter the repertoire through creation of T lymphocytes with new T cell specificities. Thymic vaccination, by contrast, will alter the thymic repertoire to create desired T cell specificities. Moreover, a key feature of thymic vaccination is that it should be capable of directing the immune response towards those non-mutable components of proteins derived from infectious agents and tumors and away from misguiding cues that are part of pathogen or cancer chicanery. The rationale for this approach and the advantages over traditional vaccines are described below.-CD8- double negative) → DP (CD4+CD8+ double positive → SP (CD4+CD8-, CD4-CD8+ single positive)] is depicted in Fig. T cells bearing a highly diverse αβ T cell receptor (TCR) repertoire develop in the thymus from stem cells originating in the hemopoietic tissues -17. On eThe avidity of pMHC/TCR interactions plays a major role in T cell recognition . Crystal52–59 (VSV8), VSV8 triggers negative selection of DP thymocytes in the context of H-2Kb. In contrast, a weak agonist peptide variant, identical to the VSV8 peptide except for substitution of leucine for valine at the p4 peptide residue, termed L4, induces positive selection [b-restricted immunodominant LCMV epitope gp33–41 [33–41 peptide causes negative selection due to high affinity pMHC/TCR interactions. However, certain mutations of amino acid residues in the gp33–41 peptide affects the fate of thymocyte development in fetal thymic organ culture (FTOC) leading to positive selection P14-bearing thymocytes [366–379 in the context of H-2Db, a peptide antagonist mediates positive selection in FTOC [TCR-transgenic mice provide useful tools for studies of peptide-based thymocyte selection. For example, in N15 transgenic mice carrying a TCR specific for the vesicular stomatitis virus nucleoprotein octapeptide Nelection . Similar gp33–41 , the cogymocytes . In yet  in FTOC ,36, wher33–41 and VSV8 peptides with substitutions at residues interacting with the TCR aimed at reducing TCR-pMHC affinity via diminution of the number of atomic contacts between the peptide and the TCR. Subsequently, we examined the effects of these APL on thymocyte maturation and emigration in vivo in two well-defined TCR-transgenic mouse systems. In the case of gp33–41 cognate peptide, amino acids at the peptide positions p4 (Tyr, Y) and p6 (Phe, F) were modified to Ser (S) and Ala (A), respectively, based on the crystal structure of the gp33–41/H-2Db complex showing exposure of the side chains of these amino acid residues to the solvent and hence, TCR accessibility [b, thus ensuring proper peptide presentation in the context of MHC. In the other less extreme peptide variant of gp33–41, Ala (A) at p7 was substituted with Glu (E). For the VSV8 peptide, the weak L4 agonist with the substitution of Leu (L) for Val (V) at the p4 peptide residue has been employed. The crystal structure of the N15 TCR-VSV8/Kb complex as well as the space-filling models of Kb in complex with VSV8 and L4 peptides are shown in Fig. b as compared to L4/Kb, these focal changes (p4 and Lys 66 on the α1 helix of H-2Kb) determine the outcome of thymic selection [b/ gp33–41 vs. APL in which amino acid residues at p4 and p6 are altered, differentially affect development of thymocytes expressing the P14 TCR (data not shown). Experimental data using these APL (Y4S/F6A and A7E) for studies of thymocyte selection and emigration as applied to the thymic vaccination approach are summarized below .-/- lymph node and spleen showed a response to the high TCR affinity cognate peptide gp33–41, but not to the Y4S/F6A variant peptide. In support of this observation, tetramers of H-2Db in complex with the Y4S/F6A peptide did not bind to SP CD8 thymocytes from P14 Rag2-/- mice at any tetramer dilution, as judged by immunofluorescence analysis, whereas high fluorescence intensity staining was detected using tetramers of H-2Db in complex with the gp33–41 peptide. The A7E/H-2Db tetramer gave intermediate staining. These data suggested that the Y4S/F6A mutant must interact with the P14 TCR with extremely weak affinity, if at all.The binding of the APL to the MHC class I molecules using RMA-S cells confirmed that amino acid substitutions at peptide residues interacting with the TCR did not affect peptide binding and, by extension, peptide presentation to T cells. A series of experiments was next performed to evaluate the functional potential of the APL to stimulate peripheral T cells in mice injected with the variant peptides. Results of both proliferation and cytokine secretion assays using mature T cells from P14Rag233–41 and VSV8 leads to negative selection of P14- and N15TCR-bearing thymocytes, respectively, due to relatively high affinity pMHC/TCR interactions [In vivo administration of gp33–41 in P14Rag2-/- mice and VSV8 injection into N15Rag2-/- mice resulted in pronounced depletion of DP thymocytes. Surprisingly, injection of P14 Rag2-/- mice with the Y4S/F6A peptide mutant resulted in a significant increase in the total number of thymocytes as well as the DP thymocyte subpopulation, while the A7E variant had no effect on thymocyte counts. As with Y4S/F6A in the P14Rag2-/- system, injection of L4 in the N15Rag2-/- mouse preserved the DP thymocytes and led to an increase in total thymocyte counts. The unusual increase in the number of DP thymocytes following exposure to Y4S/F6A peptide was not due to cellular proliferation and attendant DNA synthesis as examined by in vivo BrdU incorporation assay. Rather, Y4S/F6A peptide administration prevented apoptosis as confirmed by staining of thymocytes with anti-Annexin V mAb. To more directly test this hypothesis, we injected P14 Rag2-/- mice with mixtures of the negatively selecting cognate peptide gp33–41 plus the Y4S/F6A APL. Increasing the amount of Y4S/F6A peptide in the injection mixture resulted in a higher number of total and DP thymocytes. Thus, we infer that the Y4S/F6A variant may compete with other endogenous negatively-selecting peptides for binding to H-2Db molecules expressed on thymic stroma either by binding to "empty" surface MHC class I molecules or, perhaps, by a cross-presentation mechanism [b/A7E tetramer binding assays suggest that this APL does not reduce TCR binding affinity sufficiently to stimulate positive selection.Injection of the cognate viral peptides gpractions ,34. In v-/- mice (donor) were injected into irradiated congenic B6 mice (recipient) and the development of donor-type cells was monitored weekly by immunofluorescence staining and multicolor FACS analysis [The numerically small population of antigen-specific recent thymic emigrants (RTE) makes thymic selection/emigration studies difficult even with the use of TCR transgenic mice. To resolve this issue, we employed irradiation chimeras of congenic mouse strains (expressing the CD45.1 marker in B6 and CD45.2 in P14 and N15 transgenic mice) to determine whether interactions between the low affinity ligands, Y4S/F6A and L4, and their specific TCRs would result in thymic positive selection and subsequent emigration from the thymus. For this purpose, lineage-minus BM precursors of P14 – or N15- TCR transgenic Rag2analysis . Followi+ thymocytes in irradiation chimeras injected with Y4S/F6A were greatest, suggesting that this ligand mediated positive selection of P14 Rag2-/--specific T cells. Similarly, in N15 Rag2-/--B6 irradiation chimeras injected with L4, the numbers of both DP and SP CD8 donor thymocytes were highest, consistent with positive selection. In contrast, injection of either gp33–41 or VSV8 cognate viral peptides into irradiation chimeras led to thymocyte depletion by negative selection. Analysis of the peripheral lymphoid organs in these chimeras by triple color immunofluorescence with anti-CD45.2, anti-CD8α and anti-TCR-specific mAbs showed the greatest number of donor-type CD45.2+CD8+Vα2+ T cells in the lymph nodes of Y4S/F6A -injected chimeras (2–3 fold over PBS-injected control mice), suggesting that donor-type thymocytes expressing the P14 TCR had developed in the presence of Y4S/F6A, matured and emigrated to the lymph nodes. A similar increase in the donor cell numbers were observed up to 9 weeks after injection of Y4S/F6A peptide. Positive selection was also evident in N15 Rag2-/- -B6 irradiation chimeras injected with the L4 variant. In this case, higher CD8+Vβ5.2+ N15 TCR transgene donor-type T cell numbers were observed both in lymph nodes and spleens. The functional analysis of donor-type CD8+ lymph node T cells in irradiation chimeras injected with the positively selecting Y4S/F6A or L4 peptides showed approximately two-fold higher proliferation levels in response to the cognate peptides gp33–41 and VSV8, respectively, in vitro, compared to cells from PBS control-injected chimeric mice, reflecting the two-fold difference in the number of donor-type CD8+ T cells in lymph nodes of chimeras injected with the APL. However, importantly, these mature donor-type T cells did not proliferate in response to Y4S/F6A or L4 variant peptides in vivo. Injection of the viral peptides and their APLs in vivo led to reduction of CFSE+ staining in the case of gp33–41 and VSV8, suggestive of proliferation and/or activation-induced cell death (AICD). In contrast, no change in CFSE+ staining was observed upon injection of Y4S/F6A or L4 peptides, implying that these APLs do not facilitate T cell expansion per se. In sum, the cognate peptide ligands gp33–41 and VSV8 which interact with the TCR with relatively high affinity compared to their respective APL, induce activation of peripheral T cells, whereas peptide variants Y4S/F6A and L4, which bind TCR with low affinity and mediate positive selection, do not stimulate mature T cell divisions.The numbers of donor DP and SP CD8in vivo. In addition, we show that thymocyte emigration is dependent on the affinity/avidity of pMHC/TCR interactions. These results suggest that although the low affinity pMHC/TCR interactions are insufficient to trigger cell divisions in mature cells, differentiation of immature thymocytes nevertheless follows. Affinity measurements support the idea that positively selecting peptide ligand affinities are lower than those of negatively selecting ligands for TCRs, but additionally linked to their MHC binding/stability properties [The data described above and previously  represenoperties . Our repoperties . Two recoperties ,44.in vivo in a manner useful to the organism. Whether these peptide-specific T cells generate stronger defense mechanisms to fight viral infection or tumors in normal, non-transgenic mice remains to be investigated. The magnitude of the APL-driven increase in thymocytes and subsequent egress is only 2–3 fold, however. This level of change likely reflects the tightly regulated thymocyte egress process. In this respect, exploring peptide-based means of enhancing differentiation of thymocytes bearing desired TCRs together with the modulation of mechanisms controlling thymocyte emigration to the periphery would be of a great importance. To this end, various pathways regulating egress from the thymus are described below and should be considered as potential targets for such manipulation in conjunction with APL administration. Although not discussed further here, thymic vaccination followed by conventional cognate antigen immunization may be the best way to insure a robust memory T cell response.Collectively, our data show that cognate peptides can be modified at key TCR recognition positions to create variants that result in selection, directly or indirectly, of desired TCR specificities at the level of thymic development. This exogenous peptide administration offers a potential of expanding repertoire generation Lymphocyte migration plays an important role in regulating the localization and orchestration of immune responses. As thymocytes progress through the developmental stages, they migrate from the cortico-medullary junction, the site of entry of T cell progenitors from the BM, to the subcapsular region of the thymus, then to the cortex and to the medulla ,19. Finaiα protein-coupled seven-transmembrane receptors (GPCR), leading to activation of intracellular secondary mediators which control directional cell migration. To date, 43 human chemokines have been identified, acting via binding to 19 different GPCR.Chemokines are basic polypeptides of about 100 amino acids, usually containing four Cys residues linked by disulphide bonds, which are produced by certain thymic stroma cells and are abundantly expressed in the thymus. Specifically, thymic epithelial, medullary epithelial and dendritic cells have been shown to secrete various chemokines. Growing evidence suggests that chemokines and their receptors, expressed differentially on thymocytes during discrete maturational stages, control homing of T cell progenitors to the thymus, their intrathymic migration, and exit to the periphery. Chemokines deliver signals for lymphocyte proliferation and survival, and regulate thymocyte trafficking by functioning in concert with other adhesion molecules such as selectins and integrins. Chemokines stimulate responding cells by activating pertussis toxin-sensitive GSome chemokine receptors are expressed in DP and SP thymocytes, e.g. CCR9, with its ligand CCL25 secreted by TEC and DC. Others, e.g. CCR5 and CCR8, expressed on mature SP thymocytes, have been suggested to play a role in mediating thymocyte emigration. In particular, CCR7 has been demonstrated to mediate homing of naïve T cells to peripheral lymphoid organs via ligands CCL19 and CCL21.+ macrophages in the thymus. Other ECM proteins including nidogen, associated with laminin, and galectins -1, -3, and -5 as well as glycosaminoglycans are produced by thymic epithelium. ECM proteins form molecular bridges between thymocytes and the thymic microenvironment, mediating adhesion of thymocytes via their ECM receptors VLA-4, -5 and -6, and their disassembly from the cell complexes. In the absence of ECM proteins, normal thymocyte development and migration are severely perturbed, both in in vitro cultures of TEC and in in vivo knockout mouse models, suggesting a crucial role of the ECM protein network in the thymic function.Extracellular matrix (ECM) proteins laminin and fibronectin are produced by TECs, fibroblasts and MHC class II1–5/Edg) with up to nanomolar affinity, triggering diverse effects, including proliferation, survival, migration, morphogenesis, adhesion molecule expression, and cytoskeletal changes. S1P receptors are widely expressed during embryonic development and in the adult. The tissue distribution shows that lymphoid organs express high levels of S1P1 and S1P4. Thus, these receptors may be potential targets for pharmacological drug design aimed at effecting thymocyte migration.Sphingosine 1-phosphate (S1P), a member of sphingolipid family, is an important signaling molecule present in high concentrations in body fluids. SIP binds to members of a family of G protein-coupled receptors .Thymic vaccination might offer promising clinical applications as a way of immunization against these infectious diseases and cancers, enabling "designer" thymic development to produce suitable and long-lasting protective T cell immune specificities. In a converse role, deletion of unwanted T cell specificities in the case of autoimmunity by agonist administration early in life could be considered. Assuming thymic vaccination proves clinically viable, immune responses against invariant components of infectious agents, such as HIV and malaria, which otherwise utilize their intrinsic mutational capacity to evade human immune recognition, can be targeted. Design of novel vaccines based on the thymic vaccination approaches will benefit from the information gained in the recently completed Human Genome Project, particularly as genetic polymorphism associated with high risk of developing certain diseases later in life including cancers, infectious disease susceptibility and autoimmunity are uncovered. For example, the ability to manipulate the T cell repertoire to elicit anti-tumor responses early in life may prevent clinical disease evolution later. Powerful bioinformatic tools such as computer-based identification of HLA-allele specific binding epitopes and structural insight into TCR-pMHC interactions will aid in the epitope-based APL design process .in vivo mouse models using irradiation chimeras reconstituted with bone marrow progenitors from TCR-transgenic animals. Increasing emigration of antigen-specific T cells from the thymus to the periphery is a challenging goal. In the future, a combined approach of exposing the subject to a positively selecting APL plus a thymic export-enhancing agent might generate practical and efficient protective repertoire manipulations. Potential applications may include design and administration of APL against cancer, infectious and autoimmune diseases.A thymic vaccination strategy has been conceived based on the current knowledge of thymocyte differentiation and repertoire generation. This approach differs substantially from conventional vaccination since it aims to shape T cell responses through thymic repertoire manipulation, exposing developing thymocytes to positively selecting APL derived from infectious agents or tumors. Experimental data to date suggest that this strategy is possible in APL, altered peptide ligand; BM, bone marrow; CMJ, cortico-medullary junction; DC, dendritic cells; DN, double negative; DP, double positive; GPCR, G-protein-coupled receptors; HEV, high endothelial venule; MMP, matrix metalloproteinases; RTE, recent thymic emigrants; SP, single positive; S1P, sphingosine 1-phosphate; TCR, T cell receptor; TEC, thymic epithelial cells.The author(s) declare that they have no competing interests.MFH carried out the study, including experimental design and data acquisition, drafted and revised the manuscript. ELR conceived of the study, participated in its design and coordination, and helped to draft and revise the manuscript. The authors read and approved the final manuscript.


Recent evidence suggests that endogenous arginase activity potentiates airway responsiveness to methacholine by attenuation of agonist-induced nitric oxide (NO) production, presumably by competition with epithelial constitutive NO synthase for the common substrate, L-arginine. Using guinea pig tracheal open-ring preparations, we now investigated the involvement of arginase in the modulation of neuronal nitric oxide synthase (nNOS)-mediated relaxation induced by inhibitory nonadrenergic noncholinergic (iNANC) nerve stimulation.Electrical field stimulation -induced relaxation was measured in tracheal preparations precontracted to 30% with histamine, in the presence of 1 μM atropine and 3 μM indomethacin. The contribution of NO to the EFS-induced relaxation was assessed by the nonselective NOS inhibitor L-NNA (0.1 mM), while the involvement of arginase activity in the regulation of EFS-induced NO production and relaxation was investigated by the effect of the specific arginase inhibitor nor-NOHA (10 μM). Furthermore, the role of substrate availability to nNOS in EFS-induced relaxation was measured in the presence of various concentrations of exogenous L-arginine.EFS induced a frequency-dependent relaxation, ranging from 6.6 ± 0.8% at 0.5 Hz to 74.6 ± 1.2% at 16 Hz, which was inhibited with the NOS inhibitor L-NNA by 78.0 ± 10.5% at 0.5 Hz to 26.7 ± 7.7% at 8 Hz . In contrast, the arginase inhibitor nor-NOHA increased EFS-induced relaxation by 3.3 ± 1.2-fold at 0.5 Hz to 1.2 ± 0.1-fold at 4 Hz , which was reversed by L-NNA to the level of control airways in the presence of L-NNA . Similar to nor-NOHA, exogenous L-arginine increased EFS-induced airway relaxation .The results indicate that endogenous arginase activity attenuates iNANC nerve-mediated airway relaxation by inhibition of NO generation, presumably by limiting L-arginine availability to nNOS. The inhibitory nonadrenergic noncholinergic (iNANC) nervous system is the most effective bronchodilating neural pathway of the airways. Inhibition of nitric oxide synthase (NOS) markedly reduces the iNANC relaxation of both guinea pigs -3 and huNO is generated by a family of NOS isoforms that utilize the semi-essential amino acid L-arginine, oxygen and NADPH as substrates to produce NO and L-citrulline . Three iAnother L-arginine metabolizing enzyme is arginase, which hydrolyzes L-arginine to L-ornithine and urea. Arginase is classically considered to be an enzyme of the urea cycle in the liver, but also occurs in extrahepatic tissues, including the lung ,13. Two A role for arginase in the iNANC system has been found in internal anal sphincter  and peniThe role of endogenous arginase in the regulation of iNANC-derived NO generation in the airways has not yet been investigated. In the present study, we demonstrated that endogenous arginase activity and L-arginine availability are importantly involved in the modulation of iNANC nerve-mediated NO-production and relaxation of guinea pig tracheal smooth muscle.ad libitum, while a 12-h on/12-h off light cycle was maintained.Male specific pathogen free HsdPoc:Dunkin Hartley guinea pigs (Harlan Heathfield, UK), weighing 500 – 800 g, were used in this study. The animals were group-housed in individual cages in climate-controlled animal quarters and given water and food All protocols described in this study were approved by the University of Groningen Committee for Animal Experimentation.2 and 5% CO2. The composition of the KH-solution in mM was: NaCl 117.50; KCl 5.60; MgSO4 1.18; CaCl2 2.50; NaH2PO4 1.28; NaHCO3 25.0 and D-glucose 5.50; pH 7.4. The trachea was prepared free of serosal connective tissue. Twelve single proximal tracheal open-ring preparations were mounted for isotonic recording (0.3 g preload) between two parallel platinum point-electrodes in water-jacketed (37°C) organ baths containing 20.0 ml of gassed KH-solution and indomethacin (3 μM), which remained present during the whole experiment to eliminate any influence of prostanoids.The guinea pigs were sacrificed by a sharp blow on the head. After exsanguination, the trachea was removed from the larynx to the bronchi and rapidly placed in a Krebs-Henseleit (KH) buffer solution of 37°C, gassed with 95% Oω-nitro-L-arginine , the specific arginase inhibitor Nω-hydroxy-nor-L-arginine , a combination of both inhibitors, or L-arginine (0.3, 1.0 or 5.0 mM) were applied 30 min prior to histamine-addition. In line with previous observations [After a 30 min equilibration period, tracheal preparations were relaxed with isoprenaline (0.1 μM) to establish basal tone. After a washout period of 30 min with three washes with fresh KH solution, maximal contraction of the tracheal preparations to histamine was determined with cumulative additions of the agonist (0.1, 1, 10 and 100 μM). After washout (30 min), the tracheal preparations were precontracted with histamine to 30% of the maximal histamine-induced tone in the presence of atropine (1 μM) to prevent EFS-induced cholinergic airway contraction. On the plateau, biphasic EFS (150 mA, 4 ms, 4 s, 0.5 – 16 Hz) was applied and frequency response curves (0.5 – 16 Hz in doubling steps) were recorded. Per preparation, one frequency response curve was performed. When used, the nonselective NOS inhibitor Nrvations , neitherAll individual relaxations elicited by EFS were estimated as peak height of the EFS-induced response, and were expressed as a percentage of maximal relaxation as established in the presence of isoprenaline. The contribution of NO to the EFS-induced relaxation was determined by the effect of the NOS inhibitor L-NNA. Similarly, the role of arginase activity in the modulation of EFS-induced airway relaxation was determined by the effect of the arginase inhibitor nor-NOHA. The role of substrate availability in EFS-induced airway relaxation was assessed by measuring the responses in the presence of various concentrations of exogenous L-arginine.P < 0.05.All data are expressed as means ± s.e.m. Statistical significance of differences was evaluated using a paired or unpaired two-tailed Student's t-test as appropriate, and significance was accepted when ω-nitro-L-arginine, (-)-isoprenaline hydrochloride and L-arginine hydrochloride were obtained from Sigma Chemical Co. (St. Louis, MO, USA). Nω-hydroxy-nor-L-arginine was kindly provided by Dr J.-L. Boucher (Université Paris V).Histamine dihydrochloride, indomethacin, atropine sulphate, NP < 0.01 all; Fig. In guinea pig tracheal open-ring preparations, EFS induced a frequency-dependent relaxation of histamine-induced tone ranging from 6.6 ± 0.8% at 0.5 Hz to 74.6 ± 1.2% at 16 Hz. Incubation with the NOS inhibitor L-NNA caused a significant inhibition of the EFS-induced relaxation at 0.5 to 8 Hz, particularly at the lower frequencies. The effect of L-NNA ranged from 78.0 ± 10.5% inhibition at 0.5 Hz to 26.7 ± 7.7% inhibition at 8 Hz , to the level of control preparations in the presence of L-NNA alone . In the presence of 5.0 mM L-arginine, a significant increase in EFS-induced relaxation was observed at all frequencies compared to untreated preparations (Using perfused tracheal preparations, we have previously demonstrated that endogenous arginase activity is involved in the regulation of agonist-induced airway constriction by inhibition of NO production, presumably by competition with cNOS for L-arginine . In the In line with previous observations , it was The observation that exogenous L-arginine significantly enhanced the EFS-induced airway smooth muscle relaxation confirms that L-arginine is indeed a limiting factor in EFS-induced, NO-mediated airway smooth muscle relaxation under basal conditions. Remarkably, the effect of nor-NOHA was similar to that observed in the presence of the maximally effective L-arginine concentration, indicating that endogenous arginase activity is a major factor in regulating the neural NO-mediated airway smooth muscle relaxation.Aspergillus fumigatus [Recently, we discovered that increased arginase activity is importantly involved in the pathophysiology of asthma by contributing to the allergen-induced NO-deficiency and subsequent airway hyperresponsiveness to methacholine after the early asthmatic reaction, by limiting the availability of L-arginine for cNOS to produce bronchodilating NO . Arginasumigatus . Moreoveumigatus , while iumigatus . In guinumigatus . Thus, iA role for arginase by restricting the L-arginine availability for nNOS in iNANC nerves has also been proposed in the pathophysiology of erectile dysfunction . In suppThis is the first demonstration that endogenous arginase activity is functionally involved in iNANC nerve activity in the airways, by attenuating the generation of nNOS-derived NO. Disturbance of this novel regulation mechanism of airway responsiveness might be involved in the pathophysiology of allergic asthma.ω-nitro-L-arginine; NADPH, nicotinamide adenine dinucleotide phosphate; nNOS, neuronal nitric oxide synthase; nor-NOHA, Nω-hydroxy-nor-L-arginine; VIP, vasoactive intestinal polypeptidecNOS, constitutive nitric oxide synthase; EFS, electrical field stimulation; eNOS, endothelial nitric oxide synthase; iNANC, inhibitory nonadrenergic noncholinergic; iNOS, inducible nitric oxide synthase; KH, Krebs-Henseleit; L-NNA, NThe authors declare that they have no competing interests.HMa designed and coordinated the study, performed a major part of the experiments, performed the statistical analysis and drafted the manuscript. MAT assisted substantially in performing the experiments. JZ participated in the design of the study, interpretation of results and final revision of the manuscript. HMe conceived of the study, participated in its design and direction, as well as in preparing the manuscript. All authors read and approved the final manuscript.


Obstetric palsy is the injury of the brachial plexus during delivery. Although many infants with plexopathy recover with minor or no residual functional deficits, some children don't regain sufficient limb function because of functional limitations, bony deformities and joint contractures. Shoulder is the most frequently affected joint with internal rotation contracture causing limitation of abduction, external rotation. The treatment comprises muscle release procedures such as posterior subscapularis sliding or anterior subscapularis tendon lengtening and muscle transfers to restore the missing external rotation and abduction function.We evaluated whether the preoperative abduction degree affects functional outcome. Between 1998 and 2002, 46 children were operated on to restore shoulder abduction and external rotation. The average age at surgery was 7.6 years and average follow up was 40.8 months. We compared the postoperative results of the patients who had preoperative abduction less than 90° (Group I: n = 37) with the patients who had preoperative abduction greater than 90° (Group II: n = 9), in terms of abduction and external rotation function with angle measurements and Mallet classification. We inquired whether patients in Group I needed another muscle transfer along with latissimus dorsi and teres major transfers.In Group I the average abduction improved from 62.5° to 131.4° (a 68.9° ± 22.9°gain) and the average external rotation improved from 21.4° to 82.6° (a 61.1° ± 23°gain). In Group II the average abduction improved from 99.4°to 140°(a40.5° ± 16°gain) and the average external rotation improved from 33.2°to 82.7° (a 49.5° ± 23.9° gain). Although there was a significant difference between Group I and II for preoperative abduction (p = 0.000) and abduction gain in degrees (p = 0.001), the difference between postoperative values of both groups was not significant (p = 0.268). There was also no significant difference between the two groups in the preoperative external rotation, the external rotation gain and the postoperative external rotation (p = 0.163, p = 0.181 and p = 0.803, respectively).Obstetric palsy patients with shoulder sequela who had a preoperative abduction less than 90°hadas good functional results using latissimus dorsi, teres major muscle transfer and subscapularis muscle release as the patients who hada preoperative abduction greater than 90°. Although the extent and severity of the deformity in obstetrical palsy may vary from patient to patient, shoulder is the most frequently affected joint. Many clinical and radiological classification systems had been described to address the problem and tailor the solution at the shoulder -5. AmongUsually release and transfer procedures are seperated depending on the age of the patient and condition of the shoulder but in older age group (>4 years), it is preferred to combine the shoulder release and muscle transfer simultaneously -8.Some authors perform other muscle transfers like levator scapulae and trapezius along with latissimus dorsi and teres major only for total flail shoulders , others In this study, we compared the results of combined release and tendon transfer operations performed at the same stage because of the late presentation of the cases, for shoulder abduction and external rotation in two groups of patients who did not have any surgical treatment before. In one group preoperative abduction degree was less than 90°, while in the other group it was equal or more than 90°. We compared the gains of abduction and external rotation in two groups and evaluated whether the preoperative abduction degree affects the functional outcome and in patients with preoperative abduction degree <90° and also another muscle transfer is needed along with latissimus dorsi and teres major transfers.Between 1998 and 2002, 46 children with obstetrical brachial plexus palsy who had no surgical treatment before  were operated to restore shoulder abduction and external rotation in our clinic. The average age at surgery was 7.6 years (3–16). The patients had an average follow up of 40.8 months (range 24 to 60 months).Involvement of right side was seen in 30 patients and the left side in 16 patients. No patient had bilateral involvement. All of the patients were vaginally delivered with vertex presentations. Obstetrical history revealed that most mothers were multiparous and 8 of the patients were delivered with the help of forceps or vacuum. The mean birth weight of the patients was 4.5 kg (3–6.6 kg).The pool of the patients consisted 12 of patients with C5–C6 spinal root involvement, while 11 of the patients had additional C7 involvement. Finally 23 of the patients had total brachial plexus roots involvement. Accompanying birth complications were fracture of clavicle in one case, injury of sternocleidomastoideus muscle in one case and Horner's Syndrome in 2 cases.Table 1 see  summarizp < 0.05.We compared the postoperative results of the patients who had preoperative abduction less than 90° [Group I: n = 37, mean abduction 62.5° (20°–85°) and mean external rotation 21.4° (0–80°)] with the patients who had preoperative abduction equal or more than 90° [Group II: n = 9, mean abduction 99.4° (90–110) and mean external rotation 33.2° (0–65)], in terms of abduction and external rotation function with angle measurements and Mallet classification. The statistical analysis was performed with analysis of variance (ANOVA) and Student's t-test. Statistical significance was presumed at Preoperative and postoperative active and passive range of motion degrees of abduction and external rotation were measured, videos were recorded during shoulder abduction and external rotation and Mallet scores Figure  were notRadiography of the shoulder in adduction and 90° abduction and axial magnetic resonance imaging of the shoulder was performed. Shoulder deformity was classified according to Waters-Peljovich grading system, Table Most of the times, preoperative physical examination revealed weakness of the deltoid muscle and external rotators as well as co-contraction of pectoralis major, latissimus dorsi and teres major muscles at the anterior and posterior margin of the axillary fossa during shoulder elevation specially in Group I patients.® Plus (Johnson & Johnson) which had size 2 green ethibond polyster sutures on it, was applied at the insertion point of infraspinatus muscle on the rotator cuff  to 131.4° (90°–165°) and the average external rotation degree improved from 21.4° (0°–80°) to 82.6° (30°–95°) in Group I. We obtained 68.9° ± 22.9° (109%) gain for abduction and 61.1° ± 23° (285%) gain for external rotation. The difference between preoperative and postoperative values of abduction and external rotation was significant (ANOVA, F = 265 p = 0.000, F = 201 p = 0.000, respectively).The average abduction degree improved from 99.4° (90°–110°) to 140° (110°–170°) and the average external rotation degree improved from 33.2° (0°–65°) to 82.7° (45°–90°) in Group II. We obtained 40.5° ± 16° (40%) gain for abduction and 49.5° ± 23.9° (149%) gain for external rotation. The difference between preoperative and postoperative values of abduction and external rotation was significant (ANOVA, F = 25 p = 0.000 and F = 32.3 p = 0.000, respectively). The results were summarized in Table Although difference between preoperative abduction and abduction gain values in terms of degrees of Group I and Group II were significant (ANOVA, F= 43.1 p = 0.000 and F = 12 p = 0.001, respectively), the difference between postoperative values of both groups was insignificant (ANOVA, F = 1.257 p = 0.268).There was also no significant difference between the preoperative external rotation, the external rotation gain and the postoperative external rotation for both groups (ANOVA, F = 2.017 p = 0.163, F = 1.848 p = 0.181 and F = 0.063 p = 0.803, respectively).The results of both groups were shown in graphics in Figure Mean Mallet scores increased from 2.8 to 3.9 for global abduction, from 2.5 to 3.9 for global external rotation, from 2.1 to 3.6 for hand to head and from 2.5 to 3.5 for hand to mouth for Group I. Hand to back Mallet score decreased from 2.5 to 2.2. All the differences between preoperative and postoperative values were found significant according to the Student's t-test .Mean Mallet scores increased from 3.5 to 4 for global abduction, from 2.8 to 4 for global external rotation, from 2.7 to 4 for hand to head and from 3.2 to 3.5 for hand to mouth for Group II. Hand to back Mallet score decreased from 2.8 to 2.1. All the differences, apart from hand to mouth score, between preoperative and postoperative values were found significant according to the Student's t-test . The difference between preoperative and postoperative hand to mouth scores was found insignificant (t = -2, p = 0.081).While the difference between preoperative Mallet scores of Group I and Group II concerning abduction, hand to head and hand to mouth was significant (ANOVA, F = 23.211 p = 0.000, F = 11.407 p = 0.002 and F = 7.692 p = 0.008, respectively), the difference was insignificant for external rotation and hand to back scores (ANOVA, F = 1.393 p = 0.244 and F = 2.475 p = 0.123, respectively). The difference between postoperative values of both groups was insignificant .The results were summarized in Table Example cases from each group can be seen in Figure Internal rotation contracture is the most frequent and important secondary deformity of the shoulder in birth palsy. The problem is sometimes addressed by muscle release procedures such as the posterior subscapular slide or an anterior subscapularis tendon lenghtening operations. Once passive external rotation is improved, the child is later assessed for muscle transfers to reconstruct active external rotation if necessary .According to Chang et al  there arHowever, there are still many controversies concerning donor muscle choice for transfer, timing and operative tecniques of palliative surgical theraphy for the shoulder deformity.The infraspinatus muscle works to center the humeral head in the glenoid throughout elevation. External rotation of the shoulder allows greater arm elevation by clearing the greater tuberosity from impingement by the coracoacromial arch. External rotation of the humerus also positions the long head biceps centrally to aid in its function as humeral stabilizer and loosens the inferior glenohumeral ligaments, thereby allowing greater arm elevation. Hence the infraspinatus muscle plays a key role in shoulder elevation as a humeral head stabilizer, an active abductor, and an external rotator of the shoulder .The importance of transferring the teres major and latissimus dorsi as one conjoined tendon and anchoring into the posterior aspect of the greater tuberosity at the insertion of the infraspinatus similar to Hoffer method is augmentation of the weakened infraspinatus. Transfer with this technique instead of rerouting around humeral neck enables a stronger external rotator power because of the increased mechanical advantage at its insertion in the humeral head as opposed to the humeral shaft. The reason for the dramatic improvement of shoulder abduction after latissimus muscle transfer is probably because the transfer enhances the stabilizing effect of the rotator cuff which enables the deltoid to act more effectively, this phenomenon was called "force couple" effect by Phipps and Hoffer .In many centers, muscle release procedures are performed before the age of two years, however for older children tendon transfer to restore abduction and external rotation is added . It is aGilbert  suggesteMuhlig et al  describeAs all of our patients were older than 2 years of age, we performed latissimus dorsi and teres major transfer at the same session with subscapularis and pectoralis major release.In total flail shoulders, despite a certain degree of innervation, the functional results of shoulder corresponds to zero with the absence of a strong latissimus dorsi. In that condition, the levator scapulae muscle is utilised as an intrinsic stabilizer of glenohumeral joint and trapezius muscle is used as a prime mover for shoulder abduction with or without latissimus dorsi and teres major transfers .Gilbert in his series of 44 patients with transfer of latissimus dorsi, the improvement of abduction was satisfactory in the shoulders which preoperatively coded as Grade III  or more, but not in those coded as Grade II  or less. Hence he thought it may be necessary to add a concomitant transfer of the trapezius to the patients whose abduction of the shoulder was weak or absent .Chen et al  assertedAl-Qattan  performeIt is our opinion that in our Group I patients, the co-contraction between shoulder abductors (supraspinatus, infraspinatus and deltoid) and adductors  and also subscapularis muscle tightness cause limitation of shoulder elevation. If antagonistic muscles (teres major and latissimus dorsi) are transferred for the paretic muscles (infraspinatus) and the pectoralis major and subscapularis muscles are released with preserving their shoulder stability function, these children can have as succesfull postoperative shoulder abduction and external rotation as the children in Group II, who has less cross innervation hence better preoperative abduction value. Group I and II patient had almost similar postoperative mean abduction (131.4° & 140°, respectively) and external rotation values (82.6° & 82.7°, respectively).Extensive dissection of latissimus dorsi and teres major muscles from the surrounding structures gave us the opportunity to utilize both muscles for transfer, without any difficulty during the passage of the conjoined tendon through the tunnel which was prepared between long head of triceps and deltoid muscle, and also during reinsertion to the humerus.Several authors reported recurrences of the deformity in terms of reduction of external rotation and abduction gain. Two of the 12 children in Al-Qattan series  and threWe believe that in cases who has congruent glenohumeral joint (Type I-III Waters-Peljovich grading system), and deltoid muscle strength of M3–M4  but weak or absent external rotation, if the latissimus dorsi and teres major muscles have sufficient strength (M3 or more), the ideal procedure is transfer of latissimus dorsi and /or teres major onto the posterior aspect of the greater tuberosity of humerus, at the insertion of the infraspinatus. So we are not totally convinced about adding trapezius muscle transfer concomitantly with the latissimus dorsi + teres major transfer session. We rather preserve this muscle for the patients that could not achieve enough shoulder abduction after the first operation.Since our study was not randomised to treatment, the groups did not comprise equal number of patients, the assessments were not performed by independent observers but by a physiotherapy group of our team at postoperative 24 – 60 months (not at the same time for everyone), we would like to interpret our conclusions cautiously.Almost near normal shoulder function can still be reached in children who could not receive primary early neural reconstruction, by combined muscle release and muscle transfer operations which are performed before the severe glenohumeral deformities occur.We found out that the patients with obstetric palsy shoulder sequela who had a preoperative abduction value less than 90° could have good functional results as the patients who had preoperative abduction values equal or more than 90°, with latissimus dorsi, teres major muscle transfer and subscapularis muscle release.None declared.All authors participated in the design of the study, operations and drafting the manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:Preoperative and postoperative range motion degrees and Mallet scores of the patients. Patients with bold numbers are in Group II with preoperative abduction values ≥ 90° and the others are in Group I with preoperative abduction values < 90° . Passive range of motion degrees are in parenthesis.Click here for file


Hyblaea puera) is a pest moth of teak woodlands in India and other tropical regions (e.g. Thailand) and is of major economic significance. This pest is of major concern as it is involved in complete defoliation of trees during the early part of the growing season. Defoliation does not kill teak trees, but it results in huge amount of timber loss. Teak defoliator outbreaks are a regular annual feature in most teak plantations in India and it is extremely difficult to predict the exact time and place of occurrence of these outbreaks. Evidence from the study of the population dynamics of H. puera indicated habitual, short range movements of emerging moth populations, suggesting that these populations have spread to larger areas, generation after generation, affecting the entire teak plantations. We were therefore interested in investigating the temporal and spatial relationship among various population groups in Nilambur, Kerala (India) and address the cause of outbreak at the landscape level.The Teak defoliator . We have used this method extensively to evaluate the species specificity, reproducibility and to discriminate among the three different characterised populations of teak defoliator.H. puera do not play a major role in contributing to large-scale infestations. With respect to the hypotheses put forward regarding the origin of outbreaks of the moth, this study confirms the role of migration in outbreak causation, while negating the belief that endemic populations aggregate to cause an epidemic.This method also allowed us to comment with some certainty that the endemic teak defoliator,  Tectona grandis L.) is a very valuable timber species, and is a member of the moist deciduous and dry deciduous forest types. Teak plantations are threatened by two major pests: the Teak Defoliator (Hyblaea puera Cramer) Lepidoptera: Hyblaeidae and the Teak Skeletonizer  syn.). H. puera is less widely distributed in the tropics: in Oriental and Australian regions ; in the West Indies; and in South and parts of East Africa [3/ha compared with 3.7 m3/ha for unprotected trees, a gain of 3 m3/ha per annum [Teak , have proved very useful for genetic fingerprinting and for facilitating positional cloning of genes. This class of markers are particularly important for less studied species, for which genome sequence information is generally not known. These technologies include randomly amplified polymorphic DNA (RAPDs) ,14, DNA The nuclear and mitochondrial gene specific primers chosen did not produce any amplification product when used in combination with the corresponding primers as described in the UBC primer set kit . This reRAGEP markers were first tested for polymorphisms, species-specificity and repeatability. Similar fingerprinting pattern were observed in subsequent PCRs for the same individual using the same primers Fig , which dH. puera fall in two clusters, which are further divided into two major sub clusters. Average similarity between the two major clusters was 20%, while that between the two sub clusters was 34%. In one of the major clusters, we observed all the endemic insects clustering together with some of the populations from the epicenter insects; however, both populations fall in two distinct sub-clusters  tree. While evaluating the similarity matrix based on the Dice coefficient for all nuclear specific RAGEP markers and whilst constructing a UPGMA tree, it was observed that the various population groups of Using the mitochondrial RAGEP markers, the average numbers of bands scored for each primer ranged from 6–15. All bands scored were of size range 300 bp to 1600 kb. The maximum numbers of bands detected was found using primer SR-J-14233, the minimum numbers using marker N4-N-8924. Among mitochondrial markers, an average of 1–2 monomorphic bands were observed. The maximum number of monomorphic bands was observed using marker CB-N-10920.Two distinct clusters were observed in the UPGMA dendrogram for mitochondrial markers. Similarity between the two clusters was only 20%. One of these clusters comprised the majority of the endemic samples with a few samples from epicenter insects, whilst the other cluster was comparatively larger and had the two major sub clusters. Both these sub-clusters have insects from epicenter and epidemic populations Fig . From thH. puera and the nature of its damage was published in 1898 [H. puera outbreaks have been reported to begin in small epicenters and later spread to larger areas. It was then suspected that population build-up in the early outbreak epicenters might account for the subsequent widespread epidemic. However, a study using the time lapse  between two epidemics to determine whether an earlier epidemic was responsible for causing the subsequent outbreak showed that all subsequent outbreaks could not be attributed to previous outbreaks, thereby indicating the possibility of migrant populations being involved [The first teak plantation in India was started as early as 1842 in Nilambur, Kerala State, India. Preliminary information on the life history of involved .H. puera infesting teak plantations in South India. It can also be used to discriminate taxonomically various closely – related moths to the species level. Mitochondrial RAGEP fingerprints are derived from the randomness of RAGEP-PCR. It is difficult to predict with certainty that the bands are diagnostic feature of the mitochondrial genome, but since RAGEP PCR uses gene specific primers, the PCR products could therefore be a result of amplification of homologous genes or pseudogenes which could represent nuclear mitochondrial DNA (NUMTs). Mitochondrial DNA sequences are frequently transferred to the nucleus-giving rise to NUMTs, which are considered to be common in eukaryotes [Podisma pedestris (L.)[Several technical advancements on the DNA fingerprinting methodologies have been established to resolve the taxonomic uncertainties and address the issue on species variability and migration -16,20,21karyotes . Very hitris (L.).Sitobion avenae (F.) populations across agricultural ecosystems using DNA fingerprinting [(GATA)4] and RAPDs, it was possible to discriminate the micro- and macro geographical heterogeneity [S. avenae on wheat and cocksfoot grass, Dactylis glomerata (L.) were observed during the early months of infestation, which declined as the season progressed, largely as a result of genetic drift and local movement between adjacent host species [Bemisia tabaci (Gennadius) populations studied throughout the world [Solenopsis invicta (Buren) using different classes of markers, it was confirmed that both mitochondrial and nuclear markers display the same hierarchical structure [Age groups, sexes, life history variants, etc. and the processes including birth, death, immigration and emigration as different phenotypic classes have been very well defined . While sogeneity . Highly  species . Monophyhe world . While etructure . Distinctructure .The present study using RAGEP-PCR provides a tool for a logical continuation of the earlier work to trace the relationship of endemic, epicenter and epidemic populations of the teak defoliator. The dendrogram produced from nuclear RAGEP clearly indicates that the endemic insects are not involved in causing the epidemic; however, they are apparently involved in the localized spread by building up small epicenter populations. Similarly, while evaluating the observation based on mitochondrial RAGEP's, it is further apparent that endemic populations were not involved in causing the epidemic. This suggests that all the epidemic insects, which are spatially distinct, but temporally co-occurring, share the same gene pool.Aubeonymus mariafranciscae (Roudier), which had diverged recently [Listronotus bonariensis (Kuschel) in New Zealand, was traced to the eastern coast of South America [Oryzaephilus surinamensis (L.) [Lymantria dispar (L.) [S. avenae [Simulium spp. [Drosophila subobscura (Collin) [Drosophila athabasca Sturtevant and Dobzhansky, and Drosophila azteca Sturtevant and Dobzhansky has been extensively studied by allozymes, mitochondrial DNA (mtDNA) and RAPD markers.Randomness of genome amplification methods have been efficiently used in constructing the phylogenetic history in the weevil, recently , whilst  America . Use of sis (L.) , charactpar (L.) , host ba. avenae , and gen. avenae , has beeium spp. , whilst (Collin)  in the wH. puera coexist during the outbreak season. The degree of variability observed for RAGEPs also argues that this technique could be useful for a variety of questions, including individual identification, strain identification and phylogenetic analysis.With the Teak defoliator, earlier studies based on temporal and spatial distribution of the larvae indicated that the epicenters were not constant over the years and did not represent highly favourable local environments . The preH. puera epicenter populations would help prevent large-scale outbreaks of the teak defoliator in teak plantations. Therefore, appropriate strategies should be adopted to control the epicenter populations, which occurs in a smaller area. This appears to be a more practical and economical approach for teak defoliator management when compared with management of the pest in the total plantation area covering thousands of hectares. Thus the molecular markers detected using RAGEP-PCR can enhance the understanding of insect population dynamics and aid in tracing the spread and cause of epidemics.The present results appear to validate the hypothesis, that control of H. puera. Larval samples were collected from the infestation sites. Whenever fifth instar larvae were available, ten larvae were preserved in 70 % alcohol and stored deep frozen at -20°C. If only lower stages were available, i.e., third or fourth instar, they were reared up to 5th instar in the laboratory. Ten 5th instar larvae were preserved for DNA isolation from each sample site, whilst the remaining larvae were reared into the next generation. Each sample was assigned a code number containing the details of Year / Month / Date / Block / Grid / Generation for further reference. Using the duration of each instar , the temporal data on outbreaks were examined to see whether each subsequent epidemic could be explained on the basis of a previous outbreak. The details of location of pest incidence and extent of infestation were later transferred to the field map in order to understand the spatial pattern of infestation , 0.25 mM of dNTPs (Amersham) and 12 pM of primer per reaction. Primers were initially screened for polymorphism and repeatability. Amplifications were performed in similar cycling conditions in a Thermocycler (Biorad) programmed as follows: initial denaturation at 95°C for 5 min., followed by 45 cycles of cycle denaturation at 94°C for 1 min., annealing at 36°C for 1 min., extension at 72°C for 2 min. and final extension at 72°C for 5 mins. The amplification products were separated using 1.2% agarose gel in 0.5 × TBE buffer with ethidium bromide staining to visualize the product separation using a Bio-Rad's Fluor S imager. The molecular weight of each band was estimated by comparing with a co-migrating 100-bp ladder (Amersham). RAGEP fingerprints of each sample from different regions were then interpreted using Fingerprint type module of Bionumerics software (Applied Maths Kortrijk Belgium, ver.2.0).Both nuclear and mitochondrial DNA RAGEP amplifications were performed in a total volume of 30 μl. Each reaction consisted of 1x Taq buffer with 1.5mM MgClH. puera with those from the Teak skeletonizer, E. machaeralis , Leaf roller, Sylepta derogata (F.), Leaf folder, Cnaphalocrocis medinalis (Guenée), and the Silkworm, Bombyx mori (L.).A preliminary screening with 50 nuclear RAGEPs and 37 mitochondrial RAGEP primers were evaluated for polymorphism and repeatability. Only 11 nuclear Table- from eacThe polymorphic content for nuclear and mitochondrial primers were analyzed using Bionumerics software . Band seCN and MB performed the molecular studies and are responsible for the interpretation of molecular data whilst TV and VVS performed the field data collection and are responsible for spatial and temporal data interpretation. All authors read and approved the final manuscript. MB and VVS conceived and designed the study.


RNA-mediated gene silencing has emerged in recent years as an important mechanism for regulating gene expression. Some of the key discoveries have been made in plants Although the word ‘revolution’ should not be used lightly in science, there is no other way to describe the recent explosion in our awareness and understanding of RNA-mediated gene silencing pathways. The central player in RNA-mediated gene silencing is a double-stranded RNA (dsRNA) that is chopped into tiny RNAs by the enzyme Dicer. The tiny RNAs associate with various silencing effector complexes and attach to homologous target sequences (RNA or DNA) by basepairing. Depending on the protein composition of the effector complex and the nature of the target sequence, the outcome can be either mRNA degradation, translational repression, or genome modification, all of which silence gene expression . PresentCaenorhabditis elegans . Unexpectedly, the first transgene complex, which was stably active on its own, often became silenced in the presence of the second . The proNeurospora crassa , sequence-specific DNA methylation that triggered TGS, and RNA-directed DNA methylation. They had also proposed models for PTGS involving dsRNA , small RNeurospora identified common components in the respective silencing pathways (RNAi was discovered in experiments designed to compare the silencing activity of single-stranded RNAs (ssRNAs) (antisense or sense) with their dsRNA hybrid. While only marginal silencing of a target gene was achieved after injecting worms with the individual strands, injection of a sense–antisense mixture resulted in potent and specific silencing . This unpathways . This inpathways . PTGS isDrosophila systems demonstrated that 21–23 nucleotide ‘short interfering'RNAs (siRNAs), derived from cutting longer dsRNA, can guide mRNA cleavage  RNAs corresponding to silenced target genes in transgenic plants . They prcleavage ; identifcleavage ; and idecleavage   moves from cell to cell and through the vascular system to induce silencing at distant sites, was initially detected in plants in 1997  and late in 1998 . One of  in 1998 . A recen in 1998 .Arabidopsis has four Dicer-like (DCL) proteins, in contrast to mammals and worms, whose genomes encode only one Dicer protein (Arabidopsis to include at least three active members. An important goal has been to determine the functions of individual family members. Previous studies in Arabidopsis have shown that DCL1 is needed for processing miRNA precursors important for plant development (PLoS Biology delineates distinct functions for DCL2, DCL3, and RDR2. Nuclear-localized DCL3 acts with RDR2 to generate short RNAs that elicit DNA and histone modifications; DCL2 produces short RNAs active in antiviral defense in the cytoplasm of cells. This study illustrates nicely how RNA silencing components have diversified in plants to carry out specialized functions.Although RNA-mediated gene silencing pathways are evolutionarily conserved, there are various elaborations in different organisms. For example, the plant  protein . The RDRelopment , but notelopment . The papBy identifying small RNAs as agents of gene silencing that act at multiple levels throughout the cell, molecular biologists have created a new paradigm for eukaryotic gene regulation. Plant scientists have figured prominently in RNA-mediated silencing research. Instrumental to their success was the early ability to produce large numbers of transgenic plants, which displayed a rich variety of gene silencing phenomena that were amenable to analysis. The agricultural biotechnology industry provided incentives to find ways to stabilize transgene expression and use transgenic approaches to modulate plant gene expression and to genetically engineer virus resistance. As exemplified by the petunia cosuppression experiments, nonessential plant pigments provide conspicuous visual markers that vividly reveal gene silencing. The history of gene silencing research shows once again that plants offer outstanding experimental systems for elucidating general biological principles.


Why three non-governmental organizations are launching an alternative to the World Health Report Mobilising civil society around an alternative World Health Report At the World Health Assembly in May 2003, three civil society groups—the People's Health Movement, the Global Equity Gauge Alliance, and Medact—discussed the need for civil society to produce its own alternative to the World Health Organisation's World Health Report. We felt strongly that we needed to produce a global health report that had equity and the right to health at its heart. We also needed a way to monitor the performance of global health institutions themselves. The idea of an alternative to the World Health Report has developed into an initiative called the Global Health Watch, which we are launching next year.http://www.medact.org) is a United Kingdom–based global health charity, undertaking education, research, and advocacy on conflict, poverty, and the environment.Medact (http://www.gega.org.za) was created to participate in and support an active approach to monitoring health inequalities and promoting equity within and between societies. The Alliance currently includes 11 member-teams, called Equity Gauges, located in ten countries in the Americas, Africa, and Asia.The Global Equity Gauge Alliance (http://www.phmovement.org) is a global network of activists, organisations, and social movements. Its goal is to re-establish health and equitable development as top priorities in local, national, and international policy-making, with comprehensive primary health care as the strategy to achieve these priorities.The People's Health Movement Monitoring of international promises on aid and debt reliefSection F: Summary and Strategies for ActionThis is how the Watch will be alternative: it will present options for health policy-makers that question the dominant reform agenda that emphasises market-driven and diseased-based approaches to health care. A policy bias against government action and a lack of creative thinking about how governments can shape health care markets to work in favour of equity and social inclusion are unfortunate features of global health debates. More recently, the emphasis has been placed once again on campaigns against specific diseases such as HIV/AIDS and tuberculosis, despite the universally acknowledged importance of building and maintaining health systems that can respond to the broader needs of patients.We hope the Watch will present some alternative and imaginative thinking about how health services can respond creatively to the many challenges they face, with a strong focus on basic principles of equity and universality and avoiding top-down disease-focussed programmes that neglect the broader determinants of health. We have invited some of the most interesting and innovative thinkers in health policy—from both developing and developed countries and from academia and civil society—to help us achieve these objectives.The Watch will also be ‘alternative’ in another sense—it will act as a regular monitor of the policies, governance, and funding of the institutions affecting global health, including the World Health Organisation and World Bank, something no other health report undertakes. We hope to offer proposals for reform, as well as to stimulate further action by civil society to make these institutions more accountable and responsive to the needs of the poor and vulnerable.It is important to say that the three networks and organisations that have convened the Watch are really just its initiators. In the end we hope the Watch will be backed by as many individuals, organisations, and social movements as possible, strengthening the links between civil society organisations across countries and across health-related sectors, and increasing the power and influence of the report itself. Already, many have expressed their interest in the project, and their willingness to contribute: through writing chapters, contributing case studies, and launching the Watch and promoting it in their country when it is finished. Groups from India and Brazil are planning parallel national Watches.We plan to launch the Watch at the second People's Health Assembly, which will be held in Ecuador in July 2005. We don't want this report to be addressed just to health activists or health policy-makers or academics. If we are going to create change we need to capture the imagination of the broader health professional community and the public at large. That is why we encourage readers to get involved and tell others about the Watch and to use it to throw down a challenge to those who call the shots at national and international levels.


Pseudomonas aeruginosa entrapped in agar beads in the mouse lung leads to chronic lung infection in susceptible mouse strains. As the infection generates a strong inflammatory response with some lung edema, we tested if it could modulate the expression of genes involved in lung liquid clearance, such as the α, β and γ subunits of the epithelial sodium channel (ENaC) and the catalytic subunit of Na+-K+-ATPase.The intratracheal instillation of Pseudomonas aeruginosa entrapped in agar beads were instilled in the lung of resistant  and susceptible (DBA/2, C57BL/6 and A/J) mouse strains. The mRNA expression of ENaC and Na+-K+-ATPase subunits was tested in the lung by Northern blot following a 3 hours to 14 days infection.1Na+-K+-ATPase mRNA, the catalytic subunit of the sodium pump, was recorded. The distinctive expression profiles of the three subunits were not different, between the susceptible and resistant mouse strains.The infection of the different mouse strains evoked regulation of α and β ENaC mRNA. Following Pseudomonas instillation, the expression of αENaC mRNA decreased to a median of 43% on days 3 and 7 after infection and was still decreased to a median of 45% 14 days after infection (p < 0.05). The relative expression of βENaC mRNA was transiently increased to a median of 241%, 24 h post-infection before decreasing to a median of 43% and 54% of control on days 3 and 7 post-infection (p < 0.05). No significant modulation of γENaC mRNA was detected although the general pattern of expression of the subunit was similar to α and β subunits. No modulation of αPseudomonas infection, by modulating ENaC subunit expression, could influence edema formation and clearance in infected lungs.These results show that  Xenopus laevis oocytes [+ transport from the alveoli to the interstitium is the main force that drives water out of the alveoli [+ transport is also important in adults for lung liquid clearance [The epithelial sodium channel (ENaC) is expressed in epithelial cells of several tissues involved in salt and water reabsorption. The channel is composed of three related subunits (α, β, γ) that are able to reconstitute a functional channel when expressed in  oocytes ,2. ENaC  oocytes ,3-5, dis oocytes ,3,5, lun oocytes -8, ear e oocytes ,10, papi oocytes -13, eyes oocytes , chondro oocytes  and diff oocytes . ENaC sy oocytes ,18. In t alveoli ,20. This alveoli . Its imp alveoli . Na+ tralearance ,23.in vitro, following stimulation with steroids, β-agonists, catecholamines, and agents that increase cAMP concentration [+ transport compared to normal cells [Increased ENaC expression has been detected in the lung and in alveolar epithelial cells ntration -27. αENantration ,6,27. Itntration ,29 and dntration ,31. Seventration -34. Althal cells ,36. Thisal cells ,38.Pseudomonas aeruginosa is a bacterium occuring naturally in a wide range of environments such as in soil, fresh and seawater, plants and decomposing organic matter [Pseudomonas can promote nosocomial lung infection after artificial ventilation [Pseudomonas aeruginosa is the main source of chronic lung infection in CF patients [c matter . Althougc matter . Pseudomtilation  and is atilation . Chronictilation  where Pspatients .Pseudomonas aeruginosa in the lung of anaesthetised rabbits has been reported to promote acute pneumonia, resulting in alveolar epithelial injury, loss of epithelial barrier integrity, lung edema, pleural empyema and pleural effusions within 8 h of infection [P. aeruginosa entrapped in agar beads. In this model, the lungs of susceptible mouse strains develop severe lung infection with a strong inflammatory response and some lung edema [Pseudomonas by itself has been shown to inhibit active sodium absorption in cultured airway epithelial cells [+-K+-ATPase.Instillation of nfection . A more ng edema ,45. Pseual cells . Here, wPseudomonas entrapped in agar beads was instilled in the lung of resistant  and susceptible (DBA/2, C57BL/6 and A/J) mouse strains, and the expression of α, β, γENaC and α1 Na+-K+-ATPase mRNA was studied by Northern blotting in lungs infected between 3 hours and 14 days.P. aeruginosa  was entrapped in agar beads, and 50-μl suspensions containing 2 × 105 to 1 × 106 CFU/ml were instilled intratracheally in male mice of resistant (BALB/c) or susceptible (DBA/2, C57BL/6 and A/J) strains as described previously [Clinical strain 508 of eviously ,45.2 inhalation at different time points after P. aeruginosa instillation in the lungs. The trachea was cannulated, and the lungs were washed seven times with 1 ml PBS. Total cell counts were conducted in a hemacytometer. Differential cell counts were made by Diff-Quick staining (American Scientific Products) of Cytospin preparations. Number of animals: day 1, n = 6; day 4, n = 16; day 6, n = 6; day 14, n = 3.BAL were performed as described elsewhere with a few modifications . The inf1Na+-K+-ATPase, glyceraldehyde-3-phosphate dehydrogenase (GADPH) or 18S rRNA). To detect αENaC mRNA, the blots were hybridized with 764-bp mouse αENaC cDNA (His-445 to stop codon) [1Na+-K+-ATPase probe was a gift from Dr. J. Orlowski  and consisted of a NarI-StuI 332-bp fragment coding from nucleotide 89 to 421 (from the 5'UTR to Arg-61) of the rat kidney and brain α isoform [The lungs from infected mice were harvested between 3 h to 14 days after infection, homogenized in 5 ml of 4 M guanidine isothiocyanate, and centrifuged on a cesium chloride gradient . Fifteenp codon) . The α1N isoform . For qua isoform . The bloBetween each round of hybridization, the membranes were stripped by treatment with 0.1 X SSC, 1% SDS and 2.5 mM EDTA at 95°C. The blots were allowed to cool gradually with agitation for 30 min at room temperature. The membranes were then rinsed with 5 X SSC and rehybridized. Number of animals: n = between 6 and 8 animals for each time point and each mRNA studied.+, K+-ATPase mRNA expresion, the comparisons between groups were analyzed by Wilcoxon signed rank non-parametric test using Statsview software (SAS Institute, Inc., Cary, NC, USA). Probability p values < 0.05 were considered to be significant.For the BAL cell count, the data are presented as means ± SE (standard error). For ENaC and NaPseudomonas instillation in the lung of C57BL/6 mice was monitored by studying the number of total cells in BAL at different times after infection. As shown in Figure The inflammation process evoked by Pseudomonas embedded in agar beads was administered intratracheally in resistant (BALB/c) and susceptible strains of mice (DBA/2, C57BL/6, A/J) as described previously [Pseudomonas infection [1 Na+-K+-ATPase mRNA coding for the catalytic domain of the sodium pump, but could not find any significant change during infection  and -susceptible (DBA/2, C57BL/6, A/J) mouse strains. Modulation of ENaC expression is therefore most likely not a genetic marker linked to the susceptibility of mouse strains to establishment of a chronic infection with Pseudomonas.The instillation of ammation ,45 and lammation  secondar strains ,45. The g injury , and becg injury , we testithelium ,23. The Pseudomonas infection affected ENaC mRNA with a pattern consisting of increased expression at 24 h, followed by a marked decrease on day 3 post-infection. The change in ENaC mRNA was related to bacterial infection, since agarose beads alone failed to evoke any modulation of these RNA. The three ENaC subunits were modulated with a similar profile, with some noticeable differences, however. Although αENaC mRNA expression tends to increase by day 1, the most noticeable feature brought by Pseudomonas infection to αENaC mRNA was the significant decreases after 3 days, 7 days and 14 days post-infection. To the best of our knowledge, this is the first report demonstrating that bacterial infection in vivo can lead to modulation of ENaC mRNA expression. Recently, αENaC mRNA expression was found to be downregulated in the mouse lung after 7 and 14 days of adenoviral infection [nfection . Furthernfection  reportednfection . All thePseudomonas infection. There was a significant increase in the mRNA expression on day 1 post-infection, followed, as for αENaC, by a decreased expression on day 3 and day 7 post-infection. Different stoichiometries have been proposed for ENaC. One model suggests a 2α, β, γ ratio [Xenopus laevis oocytes increases amiloride-sensitive Na+ current by 100% compared to αENaC alone [+ transport across the airway epithelium and a reduced height of the airway surface liquid [Pseudomonas infection, could certainly influence amiloride-sensitive current and lung liquid clearance as in αENaC KO mice rescued by transgenic expression of αENaC that has a lower expression of ENaC in the lung [The β ENaC subunit was also modulated by  γ ratio ,54 where γ ratio  or nonam γ ratio ,56. AlthaC alone ,58. GeneaC alone . Lung liaC alone , but is aC alone . Increase liquid . For allthe lung ,63. In sthe lung .Pseudomonas infection. Acute bacterial pneumonia in rats has been shown to increase alveolar epithelial fluid clearance [Pseudomonas infection could be of potential clinical significance to understand the slow improvement in some ARDS patients.The general biphasic modulation of ENaC mRNA expression with an increase at 24 h followed by a decrease thereafter is an interesting finding that could explain some contradictory reports concerning ENaC expression in lung following learance ,66 when 1 Na+-K+-ATPase mRNA was unaffected in the course of lung infection. This is similar to what has been reported during adenovirus lung infection [1 Na+-K+-ATPase. In ischemia reperfusion injury, there was also no modulation of α1 Na+-K+-ATPase expression despite significant ENaC downregulation [+-K+-ATPase is an important element in lung liquid clearance, however, by being one of the key generator of membrane potential, the enzyme also affects other channels and ion transport process. It is possible that by modulating ENaC expression and not α1 Na+-K+-ATPase, Pseudomonas infection alters the Na+ transport system but does not change other important cell functions meditated by Na+-K+-ATPase. Furthermore, despite a similar mRNA expression level, there could be a fall in protein content or activity of the sodium pump. Additional experiments are necessary to answer this question.In contrast to α and β ENaC, αnfection  where αEgulation . These rSeveral studies report that in lung epithelial cells, viral infection ,68, mycoPseudomonas infection modulates ENaC mRNA expression. This modulation is independent of mouse strain susceptibility to establishment of chronic infection with Pseudomonas. Although there is an elevation of ENaC expression after 24 h, the most important feature is probably the long-lasting decrease of αENaC transcripts on days 3 and 7 post-infection. The lung inflammation induced by Pseudomonas infection therefore seems to favour a reduction in the expression of an essential element involved in lung liquid clearance as well as the regulation of airway surface liquid volume.We have shown in this report that Pseudomonas instillation, RNA extraction and Northern blotting of RNA sample. The BAL recovery as well as PMN and macrophage counting was performed by CG. YB and DR designed and co-ordinated the study. All authors read and approved the final manuscript.AD performed the hybridization, the statistical analysis of the blots and wrote the manuscript. DG performed the


We tested the hypothesis that the tube cuff is inadequately inflated when manometers are not used.Cuff pressure in endotracheal (ET) tubes should be in the range of 20–30 cm H2O.With IRB approval, we studied 93 patients under general anesthesia with an ET tube in place in one teaching and two private hospitals. Anesthetists were blinded to study purpose. Cuff pressure in tube sizes 7.0 to 8.5 mm was evaluated 60 min after induction of general anesthesia using a manometer connected to the cuff pilot balloon. Nitrous oxide was disallowed. After deflating the cuff, we reinflated it in 0.5-ml increments until pressure was 20 cmH2O). Only 27% of pressures were within 20–30 cmH2O; 27% exceeded 40 cmH2O. Although it varied considerably, the amount of air required to achieve a cuff pressure of 20 cmH2O was similar with each tube size.Neither patient morphometrics, institution, experience of anesthesia provider, nor tube size influenced measured cuff pressure (35.3 ± 21.6 cmHWe recommend that ET cuff pressure be set and monitored with a manometer. However, complications have been associated with insufficient cuff inflation. Consequences of micro-aspiration of oropharyngeal secretions include nosocomial pulmonary infections . Conventfficient . In cont2O may significantly reduce tracheal blood flow with normal blood pressure and critically reduces it during severe hypotension [2O for just four hours produces serious ciliary damage that persists for at least three days [Catastrophic consequences of endotracheal tube cuff over-inflation such as rupture of the trachea -6, trachotension . Similarree days . Thus, a2O as a safe minimum cuff pressure to prevent aspiration and leaks past the cuff [2O (20 mmHg) [in vitro study and recommended cuff inflation pressure not exceed 30 cm H2O [2OLomholt et al. recommended selecting a cuff pressure of 25 cmHthe cuff ; Bernharthe cuff . On the 20 mmHg) . Seegobi2O in 40-to-90% of tested patients [2O, when inflated using the palpation method. Because cuff inflation practices are likely to differ among clinical environments, we evaluated cuff pressure in three different practice settings: an academic university hospital and two private hospitals. It is also likely that cuff inflation practices differ among providers. We therefore also evaluated cuff pressure during anesthesia provided by certified registered nurse anesthetists (CRNAs), anesthesia residents, and anesthesia faculty.Adequacy of cuff inflation is conventionally determined by palpation of the external balloon. Previous studies suggest that this approach is unreliable ,22. One 2O for various sizes of endotracheal tubes. Taking another approach to the same question, we also determined compliance of the cuff-trachea system in vivo by plotting measured cuff pressure against cuff volume.Cuff pressure can be easily measured with a small aneroid manometer , but thiWith approval of the University of Louisville Human Studies Committee and informed consent, we recruited 93 patients (42 men and 51 women) undergoing elective surgery with general endotracheal anesthesia from three hospitals in Louisville, Kentucky: 41 patients from University Hospital (an academic centre), 32 from Jewish Hospital , and 20 from Norton Hospital .Patients with emergency intubations, difficult intubations, or intubation performed by non-anesthesiology staff; pregnant women; patients with higher risk for aspiration (e.g., full stomach, history of reflux, etc.); and patients with known anatomical laryngeo-tracheal abnormalities were excluded from this study. The Human Studies Committee did not require consent from participating anesthesia providers. However, no data were recorded that would link the study results to specific providers.Independent anesthesia groups at the three participating hospitals provided anesthesia to the participating patients. Because one purpose of our study was to measure pressure in the endotracheal tube cuff during routine practice, anesthesia providers were blinded to the nature of the study. They were only informed about the second purpose of the study: determining the relationship between cuff volume and pressure. Ninety-three patients were randomly assigned to the study. The groups were not equal for the three different types of practitioners; however, determining differences of practice between different anesthesia providers was not the primary purpose of our study.General anesthesia was induced by intravenous bolus of induction agents, and paralysis was achieved with succinylcholine or a non-depolarizing muscle relaxant. Male patients were intubated with an 8 or 8.5 mm internal diameter endotracheal tube, and female patients were intubated with a 7 or 7.5 mm internal diameter endotracheal tube. This is a standard practice at these hospitals. Patients who were intubated with sizes other than these were excluded from the study. Anesthesia was maintained with a volatile aesthetic in a combination of air and oxygen; nitrous oxide was not used during the study period.® Tracheal Tube, Mallinckrodt, St. Louis, MO]); at Jewish Hospital, at least 10 patients each were evaluated with size 7, 7.5, and 8 mm Mallinckrodt Intermediate Hi-Lo® Tracheal Tubes; and at Norton Hospital, 10 patients each were evaluated with size 7 and 8-mm Mallinckrodt Intermediate Hi-Lo® Tracheal Tubes. Consecutive available patients were enrolled until we had recruited at least 10 patients for each endotracheal tube size at each participating hospital. All tubes had high-volume, low-pressure cuffs.At the University of Louisville Hospital, at least 10 patients were evaluated with each endotracheal tube size  that was connected to the pilot balloon of the endotracheal tube cuff via a three-way stopcock. This type of aneroid manometer is nearly as accurate as a mercury manometer, but easier to use . Pressur2O was achieved. The entire process required about a minute.A syringe attached to the third limb of the stopcock was then used to completely deflate the cuff, and the volume of air removed was recorded. The cuff was considered empty when no more air could be removed on aspiration with a syringe. The cuff was then progressively inflated by injecting air in 0.5-ml increments until a cuff pressure of 20 cmH2O as a function of tube size. Outcomes were compared by tube size, provider, and hospital with either an ANOVA  or the Kruskal-Wallis statistic . Compliance of the cuff system was evaluated by linear regression of measured cuff pressure vs. measured cuff volume. Data are presented as means (SD) or medians [interquartile ranges] unless otherwise noted; P < 0.05 was considered statistically significant.Our primary outcomes were 1) measured endotracheal tube cuff pressures as a function of tube size, provider, and hospital; and 2) the volume of air required to produce a cuff pressure of 20 cmH2O; only 27% of the patients had measured pressures within the recommended range of 20–30 cmH2O. Fifty percent of the values exceeded 30 cmH2O, and 27% of the measured pressures exceeded 40 cmH2O. Thus, 23% of the measured cuff pressures were less than 20 mmHg. Measured cuff volume averaged 4.4 ± 1.8 ml. Neither measured cuff pressure nor measured cuff volume differed among the hospitals .CRNAs (n = 72), anesthesia residents (n = 15), and anesthesia faculty (n = 6) performed the intubations. There were no statistically significant differences in measured cuff pressures among these three practitioner groups (2O) and the volume of air (ml) retrieved at complete deflation of the cuff; this showed a linear pressure-volume relationship: Pressure= 7.5. Volume+2.7, r2 = 0.39  than outside the range. It was nonetheless encouraging that we observed relatively few extremely high values, at least many fewer than reported in previous studies [Previous studies suggest that the cuff pressure is usually under-estimated by manual palpation. For example, Braz et al.  observedz et al.  observed studies . This reMeasured cuff inflation pressures were virtually identical at the three study sites: one academic center and two private hospitals. These data suggest that management of cuff pressure was similar in these two disparate settings. Interestingly, there was also no significant or important difference as a function of provider – measured cuff pressures were virtually identical whether filled by CRNAs, residents, or attending anesthesiologists. Our results thus fail to support the theory that increased training improves cuff management.We evaluated three different types of anesthesia provider in three different practice settings. Although we were unable to identify any statistically significant or clinically important differences among the sites or providers, our results apply only to the specific sites and providers we evaluated. While it is likely that these results are fairly representative, it is obvious that results would not be identical elsewhere because of regional practice differences.et al. [2O did not differ significantly as a function of endotracheal tube size. These data suggest that tube size is not an important determinant of appropriate cuff inflation volume. A caveat, though, is that tube sizes were chosen by clinicians in our study and presumably matched patient size; results may well have differed if tube size had been randomly assigned. We intentionally avoided this approach since our purpose was to evaluate cuff pressures and associated volumes in three routine clinical settings.Fernandez et al.  found thA limitation of this study is that cuff pressure was evaluated just once 60 minutes after induction of anesthesia. Because nitrous oxide was not used, it is unlikely that the cuff pressures varied much during the first hour of the study cases. We recognize that people other than the anesthesia provider who actually conducted the case often inflated the cuffs. However, a full hour was plenty of time for the provider to have checked and adjusted cuff pressure to a suitable level.2O independent of tube size for the same type of tube. However, there was considerable patient-to-patient variability in the required air volume. Measuring actual cuff pressure thus appears preferable to injecting a given volume of air.We observed a linear relationship between the measured cuff pressure and the volume of air retrieved from the cuff. The regression equation indicated that injected volumes between 2 and 4 ml usually produce cuff pressures between 20 and 30 cmHCuff pressure should be measured with a manometer and, if necessary, corrected.The author(s) declare that they have no competing interests.SP oversaw day-to-day study mechanics, collected data on many of the patients, and wrote an initial draft of manuscript.DIS contributed to study design, data analysis, and manuscript preparation.PM, SW, and AV recruited patients and performed many of the measurements.JD conceived of the study and participated in its design.AW contributed to protocol development, patient recruitment, and manuscript preparation.All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here: