Sunali Goonesekera

BACKGROUND: Multiple studies of tuberculosis treatment have indicated that patients with diabetes mellitus may experience poor outcomes.We performed a systematic review and meta-analysis to quantitatively summarize evidence for the impact of diabetes on tuberculosis outcomes. METHODS: We searched PubMed, EMBASE and the World Health Organization Regional Indexes from 1 January 1980 to 31 December 2010 and references of relevant articles for reports of observational studies that included people with diabetes treated for tuberculosis. We reviewed the full text of 742 papers and included 33 studies of which 9 reported culture conversion at two to three months, 12 reported the combined outcome of failure and death, 23 reported death, 4 reported death adjusted for age and other potential confounding factors, 5 reported relapse, and 4 reported drug resistant recurrent tuberculosis. RESULTS: Diabetes is associated with an increased risk of failure and death during tuberculosis treatment. Patients with diabetes have a risk ratio (RR) for the combined outcome of failure and death of 1.69 (95% CI, 1.36 to 2.12). The RR of death during tuberculosis treatment among the 23 unadjusted studies is 1.89 (95% CI, 1.52 to 2.36), and this increased to an effect estimate of 4.95 (95% CI, 2.69 to 9.10) among the 4 studies that adjusted for age and other potential confounding factors. Diabetes is also associated with an increased risk of relapse (RR, 3.89; 95% CI, 2.43 to 6.23). We did not find evidence for an increased risk of tuberculosis recurrence with drug resistant strains among people with diabetes. The studies assessing sputum culture conversion after two to three months of tuberculosis therapy were heterogeneous with relative risks that ranged from 0.79 to 3.25. CONCLUSIONS: Diabetes increases the risk of failure and death combined, death, and relapse among patients with tuberculosis. This study highlights a need for increased attention to treatment of tuberculosis in people with diabetes, which may include testing for suspected diabetes, improved glucose control, and increased clinical and therapeutic monitoring.

Objective To assess the yield of finding additional TB or diabetes mellitus (DM) cases through systematic screening and to determine the effectiveness of preventive TB therapy in people with DM. Methods We systematically reviewed studies that had screened for active TB or implemented preventive therapy for TB among people with DM, and those that screened for DM among patients with TB. We searched published literature through PubMed and EMBASE and included studies that reported the number of TB cases identified among people with DM; the number of DM cases identified among patients with TB, or the relative incidence of TB between people with DM who received a TB prophylaxis and those who did not. We assessed the yield of screening by estimating the prevalence of TB or DM in each study, the prevalence ratio and difference where comparison populations were available, and the number of persons to screen to detect an additional case of TB or DM. Results Twelve studies on screening for TB in people with DM and 18 studies on screening for DM in patients with TB met our inclusion criteria. Screening for TB in persons with DM demonstrated that TB prevalence in this population is high, ranging from 1.7% to 36%, and increasing with rising TB prevalence in the underlying population as well as with DM severity. Screening patients with TB for DM also yielded high prevalences of DM ranging from 1.9% to 35%. Two studies examining the role of TB preventive therapy in people with DM did not provide sufficient details for clear evidence of the effectiveness. Conclusion Active screening leads to the detection of more TB and DM with varying yield. This review highlights the need for further research in screening and preventive therapy. Dépistage bidirectionnel de la tuberculose et du diabète: une revue systématique Objectif: Evaluer le rendement du dépistage systématique pour la détection de cas supplémentaires de TB ou de diabète sucré (DS) et déterminer l’efficacité du traitement préventif de la TB chez les personnes atteintes de DS. Méthodes: Nous avons passé en revue systématiquement les études portant sur le dépistage actif de la TB ou l’implémentation d’un traitement préventif de la TB chez les personnes atteintes de DS et celles portant sur un dépistage du DS chez les patients TB. Nous avons recherché la documentation publiée sur PubMed et EMBASE et comprenant des études portant sur le nombre de cas de TB identifiés chez les personnes atteintes de DS, le nombre de cas de DS identifiés chez les patients TB, ou l’incidence relative de la TB chez les personnes atteintes de DS qui ont reçu une prophylaxie contre la TB et ceux qui n’en ont pas reçu. Nous avons évalué le rendement du dépistage en estimant la prévalence de la TB ou du DS dans chaque étude, le rapport et la différence de prévalence là où des comparaisons de populations étaient disponibles et le nombre de personnes à tester afin de détecter un cas supplémentaire de TB ou de DM. Résultats: 12 études sur le dépistage de TB chez les personnes atteintes de DS et 18 études sur le dépistage du DS chez les patients TB ont rempli les critères d’inclusion. Le dépistage de la TB chez les personnes atteintes de diabète a démontré que la prévalence de TB dans cette population est élevée, variant de 1,7%à 36% et augmentant avec la prévalence croissante de TB dans la population générale ainsi qu’avec la sévérité du DS. Le dépistage du DS chez les patients TB a également révélé des prévalences élevées de DS variant de 1,9%à 35%. Deux études portant sur le rôle du traitement préventif de la TB chez les personnes atteintes de DS n’ont pas fourni suffisamment de détails pour une preuve concrète de son efficacité. Conclusion: Le dépistage actif conduit à la détection de plus de TB et de DS avec un rendement variable. Cette revue met en évidence la nécessité de recherches supplémentaires dans le dépistage et le traitement préventif. Tamizaje Bidireccional para tuberculosis y diabetes: una revisión sistemática Objetivo: Evaluar el rendimiento de encontrar casos adicionales de TB o diabetes mellitus (DM) mediante el tamizaje sistemático, y determinar la efectividad de la terapia preventiva de la TB en personas con DM. Métodos: Hemos realizado una revisión sistemática de estudios en los que se tamizó para TB activa o se implementó una terapia preventiva para TB entre personas con DM, y aquellos que tamizaron para DM entre pacientes con TB. Se realizó una búsqueda de literatura a través de PubMed y EMBASE y se incluyeron los estudios que reportaban el número de casos de TB identificados en personas con DM; el número de casos de DM identificados entre pacientes con TB, o la incidencia relativa de TB entre personas con DM que recibieron una profilaxis para TB y aquellos que no la recibieron. Evaluamos el rendimiento del tamizaje estimando la prevalencia de TB o DM en cada estudio, la tasa de prevalencia y la diferencia donde había poblaciones disponibles para hacer la comparación, y el número de personas que han de ser tamizadas para detectar un caso adicional de TB o DM. Resultados: 12 estudios con tamizaje para TB en personas con DM y 18 estudios con tamizaje para DM en pacientes con TB cumplían con los criterios de inclusión. El tamizaje para TB en personas con diabetes demostró que la prevalencia de TB en esta población es alta, con rangos de 1.7% a 36%, y que aumenta con el aumento de la prevalencia de TB en la población subyacente así como con la severidad de la DM. El tamizaje de pacientes con TB para DM también arrojó una alta prevalencia de DM con un rango de 1.9% a 35%. Dos estudios que examinaban el papel de la terapia preventiva de TB en personas con DM no proveyó suficientes detalles que pudiese aportar una evidencia clara de la efectividad. Conclusión: El tamizaje activo lleva a la detección de más casos de TB y DM con un rendimiento variable. Esta revisión subraya la necesidad de más investigaciones sobre el tamizaje y la terapia preventiva. Currently, an estimated 285 million people live with diabetes mellitus (DM), a number which is expected to grow to at least 439 million by the year 2030 (IDF, 2010). At the same time, 9.6–13.6 million people live with tuberculosis [TB] disease and 1.1–1.7 million people die from the disease every year (WHO, 2009). Previous studies have demonstrated that DM not only increases the risk of active tuberculosis but also puts co-affected patients at increased risk for poor outcomes (Alisjahbana et al. 2007; Stevenson et al. 2007; Wu et al. 2007; Jeon & Murray 2008; Leung et al. 2008; Dooley et al. 2009). The potential for ‘syndemics’ of DM and TB in countries with high burdens of both diseases raises the question of how best to integrate management of these diseases (Restrepo 2007; Dooley & Chaisson 2009; Harries et al. 2009). Over the past two decades, TB control efforts have focused on the strategy of Directly Observed Therapy Short-Course (DOTS), a package of interventions, including diagnosis by sputum smear microscopy and supervised chemotherapy. While this strategy has improved both the detection rate of smear-positive cases and the outcomes of treatment, its impact on TB prevalence is less clear (Dye et al. 2005). Experts suggest that TB control would be further improved by intervening patients with known determinants of TB, including those with diabetes (Lonnroth et al. 2009, 2010). This entails both TB prevention through actions to diminish the prevalence of risk factors and targeted diagnostic and treatment interventions in risk groups, such as people with DM. Screening for active TB in people with DM could hasten case detection, which could lead to earlier therapy and prevention of transmission; the administration of preventive TB therapy in TB-infected people with DM could avert progression to TB. Conversely, screening for DM in patients with TB could improve case detection, early treatment and tertiary prevention of DM, and indirectly lead to better TB-specific treatment outcomes. Recognizing the opportunities offered by screening and preventive therapy, we systematically reviewed studies that had implemented screening or preventive therapy for TB among people with DM and those that screened for DM among patients with TB to assess the yield of finding additional TB or DM cases through active screening and to determine the effectiveness of preventive TB therapy in patients with diabetes. We conducted this systematic review to address three distinct aims: (1) to assess the yield of screening people with DM for the detection of TB in various settings, (2) to assess the yield of screening the patients with TB for the detection of DM in various settings and (3) to measure the effectiveness of TB preventive therapy in people with DM. For these aims, we conducted a literature search in PubMed from 1965 to May 2009 and in EMBASE from 1974 to May 2009 using the search strategy outlined below with no language restriction. For the PubMed search, we used the MeSH Terms: 1. ‘diabetes mellitus’ and 2. ‘tuberculosis’ [majr] and the Text terms 3. ‘prevent*’ OR ‘isoniazid*’ OR ‘chemoproph*’; 4. ‘detect*’ OR ‘screen*’ OR ‘diagnos*’ with the search string: 1 AND 2 AND (3 OR 4 in abstract). For the EMBASE search, we used the subject terms 1. ‘diabetes mellitus’ and 2. ‘tuberculosis’ [majr] and the text terms 3. ‘prevent*’ OR ‘isoniazid*’ OR ‘chemoproph*’; 4. ‘detect*’ OR ‘screen*’ OR ‘diagnos*’, with the search string: 1 AND 2 AND (3 OR 4 in abstract). We chose the root terms ‘detect*’, ‘screen*’ and ‘diagnos*’ to find articles that described a screening programme for people with DM. We also employed the root terms ‘prevent*’, ‘isoniazid*’ and ‘chemoproph*’ to find articles that described preventive therapy against TB among people with DM. We specifically included ‘isoniazid*’ as one of the search terms, given that isoniazid is the most commonly used preventive therapeutic drug against TB. We also searched the bibliographies of relevant literature and the abstracts of World Lung Conferences held in 2007 and 2008, limiting the search to these years because we expected high-quality studies reported in prior years to be published by May 2009. Among the retrieved citations, we examined the full texts of articles with abstracts that described or mentioned (1) the screening of people with DM for TB, (2) the screening of patients with TB for DM or glucose intolerance, (3) the effectiveness of preventive therapy in people with DM or (4) ‘diabetes’ and ‘tuberculosis’ but did not provide enough detail on the methods to determine if screening or preventive therapy were implemented. Our initial aim was to identify studies that assessed the effectiveness of TB screening in people with DM for preventing TB-related morbidity or mortality, but because of the lack of such studies, we focused on assessing the yield of screening for TB disease among people with DM. For this aim, we included studies that screened people with DM for TB using any of the following methods of identification: X-ray consistent with TB, positive sputum smear microscopy, positive mycobacterial culture and clinical evaluation. We excluded studies that did not allow computation of TB prevalence or TB incidence in the screened population and those that did not describe the age distribution of the screened population. Because our focus was on summarizing the yield of screening for TB in people with DM, we included studies even if they did not provide a non-diabetic control group; in the latter case, we searched for published estimates of TB prevalence in a comparable population based on surveys conducted within ±5 years in the same population that gave rise to the screened DM patients and conducted with the same screening methods as the screening study. To assess the yield of screening for DM among patients diagnosed with active TB, we included studies that screened for DM using the following types of blood glucose tests: random blood glucose, fasting blood glucose and oral glucose tolerance test. We excluded studies that did not describe the age distribution of the screened population and studies that only diagnosed impaired glucose tolerance. Here again, we did not require that studies include a control group but used an external estimation of the population prevalence of DM as a comparator if the prevalence was estimated from a survey conducted within ±5 years in the same population that gave rise to the screened TB cases and employed the same definition of DM as the study. To assess the efficacy of TB preventive therapy in people with DM, we included studies that had compared the incidence of TB in a diabetic population receiving any form of preventive therapy to a control population with DM. We included studies whether or not TB infection was confirmed to consider evidence on the potential effect of preventive therapy in people with DM even in settings where TB infection is not a prerequisite for preventive therapy. Two investigators (CYJ, SG) independently extracted data from the studies using standardized extraction forms. Evaluation and data extraction of non-English papers was carried out in conjunction with translators fluent in the language of the paper. For all studies, we extracted information on the study population, location, study period and method of recruitment of the screened population. For the studies on screening for TB among people with DM, we extracted information on the proportion of people with DM that were insulin dependent, the method of TB diagnosis, the sample size, the number of patients with TB identified in the screened populations (and control population, if available), as well as the age and sex distribution of the screened DM patients and identified TB patients. We noted whether studies included prevalent or incident TB cases and if incident TB, we extracted information on the period during which patients were followed for TB. For the studies on screening for DM among patients with TB, we extracted information on the method of DM diagnosis, the timing of DM diagnosis relative to the onset of TB treatment, the sample size, the number of people with DM identified in the studied TB population (and control population, if available), as well as the age and sex distribution of the screened TB patients and DM patients identified through screening. For studies on preventive therapy, we extracted information on the duration and type of regimen, the follow-up period, the incidence of TB in the intervention group and control groups, and the criteria by which intervention was assigned. Differences in the extracted information were by between the two data for non-English any information on extracted data was to be reviewed by the same We studies on screening for TB among people with DM those that assessed TB prevalence and those that assessed TB incidence through We the prevalence or the incidence of TB in people with DM and where data were For follow-up studies, we the TB by the incidence by the number of years of For studies that the number of TB cases diagnosed by X-ray from those diagnosed by we used the latter definition to TB We prevalence or incidence and prevalence or incidence to assess the relative and in yield of finding TB cases between the screened diabetic populations and the comparison populations where we the number of people to screen to detect one additional case of TB in a diabetic population by the of the prevalence or incidence difference for each study. To the potential yield in screening for TB in people with DM in various settings, we how patients with DM would need to be screened to detect one additional case of TB, TB prevalence ranging from 1 to for prevalence estimated from the screened populations and Because DM be in patients with TB a at the of treatment, we studies on screening for DM among people with TB (1) those that screening TB treatment (2) those that had screened the population prior to TB treatment and (3) those in which the timing of screening relative to the of TB treatment was not We the DM prevalence for the screened diabetic populations and We the prevalence and to assess the relative and between the screened TB populations and the comparison populations where We also the number of people to screen to detect one additional case of DM in a TB population by the of the prevalence or incidence difference for each study. To the potential yield in screening for DM in patients with TB in various settings, we how patients with TB would need to be screened to detect one additional case of DM, DM prevalence ranging from to for prevalence estimated from the screened populations and The PubMed and EMBASE search yielded we examined the full text of articles and excluded studies as they did not describe a screening study. of the bibliographies yielded additional studies for full text review of of we included 12 studies that reported the of TB among people with of these reported TB prevalence et al. et al. et al. et al. et al. et al. et al. & et al. et al. and 2 TB incidence et al. and the methods of screening in 1. The prevalence of active TB among people with DM from 1.7% in in to 36% in in the incidence from people with DM in to people with DM in of the 12 studies screened a non-diabetic control group or an of the TB prevalence in the population that gave rise to the study group within years of the study. prevalence from in and to in Among the studies that were by the of TB was more in those with insulin compared to those with diabetes with prevalence from to 2 the number of people with DM that would need to be screened to detect one additional case of TB based on prevalence from 2 to for varying of TB settings in which TB prevalence is at least people with DM would need to be screened to find a additional case of TB. in with TB such as where TB prevalence is estimated at screening people with DM would yield one or more cases of TB. of people with diabetes to screen to detect one additional case of tuberculosis by varying tuberculosis given prevalence in screening We identified 18 studies that met our inclusion criteria on screening for DM among patients with studies conducted screening TB treatment et al. et al. & et al. et al. et al. with of these screening for DM at during TB therapy et al. & et al. screened for DM only treatment & et al. et al. et al. and did not screening had & & et al. et al. et al. et al. 2009). and methods of screening in 4. of the 18 studies also reported the of screening for DM in a population TB. we were to find DM prevalence estimates for a comparison population based on prevalence surveys using the same method of screening for three of the studies that did not a control group et al. et al. et al. DM prevalence reported in studies that screened TB treatment from 1.9% in in to in in The prevalence of DM was in patients with TB in with prevalence of and from studies with a control population that had a for people with DM Among studies that conducted screening prior to TB treatment, the prevalence of DM from in to in Among those that did not the timing of DM screening relative to TB treatment, estimates from in to in these latter two of studies, diabetes prevalence was in patients with TB in the where available, with prevalence ranging from in to in from the studies that screened for DM at in the of TB treatment in et al. & et al. the prevalence of the of TB in in patients with tuberculosis tuberculosis 4 the number of TB patients that would need to be screened to detect one additional case of DM, varying DM prevalence and prevalence settings with a DM prevalence of (IDF, screening as as patients with TB would lead to the detection of at least one additional case of DM. of patients with tuberculosis to screen to detect one additional case of diabetes by varying diabetes given prevalence in screening We identified two studies in which people with DM had for prevention of TB. a study conducted in & in the patients with diabetes who had a of treatment for active TB were with isoniazid for a comparison group of people with DM treatment for active TB was not While the comparison group 18 cases of TB a of years of follow-up time, the intervention group no TB years of follow-up a study conducted in in the & investigators a of to patients with diabetes and compared to who were not patients were reported to incidence of TB compared to who did not any during a follow-up period of The study did not of TB whether the follow-up period during intervention or the for not in the control this systematic we the published of screening for TB in people with DM and screening for DM in patients with TB a and We that the prevalence of TB in screened people with DM was high, with estimates for populations in which active case finding is such as & et al. and in countries et al. 2009). The yield of screening for TB in people with DM increases with the prevalence of TB in the as well as with the of as by insulin that screened for DM among patients with TB also reported a of DM prevalence ranging from 1.9% to as high as with the reported for in which DM prevalence is For in where the DM prevalence is high at of the screened TB patients were to have DM et al. The studies reviewed on how to screening efforts in people with DM and patients with they were in of the studies followed an population to measure the effectiveness of screening in preventing TB or in disease outcomes. studies did not a control group and an This the computation of prevalence to a the studies did not provide details on the of the screened population, which the of finding TB and DM. would be to factors populations to screening. DM have in studies that for glucose prior to the of TB treatment, as TB disease an that be as DM. DM prevalence estimates were in studies that had screened prior to TB treatment or did not screening had that glucose screening for DM diagnosis be more TB treatment has the methods of screening for TB and diabetes to the TB or DM prevalence the studies used methods to TB, which have lead to to the of TB prevalence estimates was by more such as culture or smear microscopy those studies that reported from both X-ray and prevalence as assessed by was by culture et al. et al. TB screening studies in people with DM focus on TB TB preventive therapy the incidence of TB in two the lack of details of intervention method evidence for TB preventive therapy in people with DM. This systematic review a that research screening be a high on the research for both DM and TB et al. 2010). studies need to be conducted in diabetes with a focus on by of glucose patients with TB, research is to determine the and best methods for DM, on and by type of TB The question of TB preventive therapy only be through a which be and to Screening and to better DM control be a more of preventing TB and DM This review was by the and Lung the World and the World and by the World

By manufacturing a single-particle system in two particulate forms (i.e., micrometer size and nanometer size), we have designed a bacterial vaccine form that exhibits improved efficacy of immunization. Microstructural properties are adapted to alter dispersive and aerosol properties independently. Dried "nanomicroparticle" vaccines possess two axes of nanoscale dimensions and a third axis of micrometer dimension; the last one permits effective micrometer-like physical dispersion, and the former provides alignment of the principal nanodimension particle axes with the direction of airflow. Particles formed with this combination of nano- and micrometer-scale dimensions possess a greater ability to aerosolize than particles of standard spherical isotropic shape and of similar geometric diameter. Here, we demonstrate effective application of this biomaterial by using the live attenuated tuberculosis vaccine bacille Calmette-Guérin (BCG). Prepared as a spray-dried nanomicroparticle aerosol, BCG vaccine exhibited high-efficiency delivery and peripheral lung targeting capacity from a low-cost and technically simple delivery system. Aerosol delivery of the BCG nanomicroparticle to normal guinea pigs subsequently challenged with virulent Mycobacterium tuberculosis significantly reduced bacterial burden and lung pathology both relative to untreated animals and to control animals immunized with the standard parenteral BCG.

The bacterial surface protein flagellin is widely distributed and well conserved among distant bacterial species. We and other investigators have reported recently that purified flagellin from Salmonella dublin or recombinant flagellin of Salmonella muenchen origin binds to the eukaryotic toll receptor TLR5 and activates the nuclear translocation of NF-κB and mitogen-activated protein kinase, resulting in the release of a host of pro-inflammatory mediators in vitro and in vivo. The amino acid sequence alignment of flagellins from various Gram-negative bacteria shows that the C and N termini are well conserved. It is possible that sequences within the N and C termini or both may regulate the pro-inflammatory activity of flagellin. Here we set out to map more precisely the regions in both termini that are required for TLR5 activation and pro-inflammatory signaling. Systematic deletion of amino acids from either terminus progressively reduced eukaryotic pro-inflammatory activation. However, deletion of amino acids 95–108 (motif N) in the N terminus and 441–449 (motif C) in the C terminus abolished pro-inflammatory activity completely. Site-directed mutagenesis analysis provided further evidence for the importance of motifs N and C. We also present evidence for the functional role of motifs N and C with the TLR5 receptor using a reporter assay system. Taken together, our results demonstrate that the pro-inflammatory activity of flagellin results from the interaction of motif N with the TLR5 receptor on the cell surface. The bacterial surface protein flagellin is widely distributed and well conserved among distant bacterial species. We and other investigators have reported recently that purified flagellin from Salmonella dublin or recombinant flagellin of Salmonella muenchen origin binds to the eukaryotic toll receptor TLR5 and activates the nuclear translocation of NF-κB and mitogen-activated protein kinase, resulting in the release of a host of pro-inflammatory mediators in vitro and in vivo. The amino acid sequence alignment of flagellins from various Gram-negative bacteria shows that the C and N termini are well conserved. It is possible that sequences within the N and C termini or both may regulate the pro-inflammatory activity of flagellin. Here we set out to map more precisely the regions in both termini that are required for TLR5 activation and pro-inflammatory signaling. Systematic deletion of amino acids from either terminus progressively reduced eukaryotic pro-inflammatory activation. However, deletion of amino acids 95–108 (motif N) in the N terminus and 441–449 (motif C) in the C terminus abolished pro-inflammatory activity completely. Site-directed mutagenesis analysis provided further evidence for the importance of motifs N and C. We also present evidence for the functional role of motifs N and C with the TLR5 receptor using a reporter assay system. Taken together, our results demonstrate that the pro-inflammatory activity of flagellin results from the interaction of motif N with the TLR5 receptor on the cell surface. The whiplike flagellum of Gram-negative bacterium is implicated in microbial pathogenicity, serving as a means of propulsion and attachment and invasion of host epithelium. Recently it has been shown that the monomeric constituent of flagella, the 55-kDa protein flagellin, is a potent activator of pro-inflammatory eukaryotic cell signaling via its interaction with membrane-bound TLR5. Flagellin-TLR5 binding induces NF-κB nuclear translocation and activation of mitogen-activated protein kinase, resulting in the up-regulated expression of cytokines, such as tumor necrosis factor-α and IL 1The abbreviations used are: ILinterleukinNOnitric oxidePAMPSpathogen-associated molecular patternsTLRToll-like receptorsDMEMDulbecco's modified Eagle's mediumFBSfetal bovine serumIFN-γinterferon-γELISAenzyme-linked immunosorbent assayGFPgreen fluorescent proteinPBSphosphate-buffered salinePMSFphenylmethylsulfonyl fluorideDTTdithiothreitolEMSAelectrophoretic mobility shift assaysCHOChinese hamster ovaryLPSlipopolysaccharide.1The abbreviations used are: ILinterleukinNOnitric oxidePAMPSpathogen-associated molecular patternsTLRToll-like receptorsDMEMDulbecco's modified Eagle's mediumFBSfetal bovine serumIFN-γinterferon-γELISAenzyme-linked immunosorbent assayGFPgreen fluorescent proteinPBSphosphate-buffered salinePMSFphenylmethylsulfonyl fluorideDTTdithiothreitolEMSAelectrophoretic mobility shift assaysCHOChinese hamster ovaryLPSlipopolysaccharide.-6, chemokines, including IL-8, and pro-inflammatory free radical synthesizing enzymes, such as the inducible nitric-oxide synthase (1Feldman M. Bryan R. Rajan S. Scheffler L. Brunnert S. Tang H. Prince A. Infect. Immun. 1998; 66: 43-51Crossref PubMed Google Scholar, 2McDermott P.F. Ciacci-Woolwine F. Snipes J.A. Mizel S.B. Infect. Immun. 2000; 68: 5525-5529Crossref PubMed Scopus (128) Google Scholar, 3Ciacci-Woolwine F. McDermott P.F. Mizel S.B. Infect. Immun. 1999; 67: 5176-5185Crossref PubMed Google Scholar, 4Steiner T.S. Nataro J.P. Poteet-Smith C.E. Smith J.A. Guerrant R.L. J. Clin. Investig. 2000; 105: 1769-1777Crossref PubMed Scopus (218) Google Scholar, 5Eaves-Pyles T. Murthy K. Liaudet L. Virag L. Ross G. Soriano F.G. Szabo C. Salzman A.L. J. Immunol. 2001; 166: 1248-1260Crossref PubMed Scopus (241) Google Scholar, 6Eaves-Pyles T.D. Wong H.R. Odoms K. Pyles R.B. J. Immunol. 2001; 167: 7009-7016Crossref PubMed Scopus (121) Google Scholar). Because many Gram-negative pathogens express flagellin, it is conceivable that flagella contribute to the host inflammatory response to infection. Evidence in support of this role comes from experimental studies in which we have shown that administration of flagellin engenders systemic tissue injury, characterized by exudative lung inflammation, liver necrosis, and circulatory shock. Therefore, flagellin presents all the features of pathogen-associated molecular patterns (PAMPs) and can be regarded as another PAMP. interleukin nitric oxide pathogen-associated molecular patterns Toll-like receptors Dulbecco's modified Eagle's medium fetal bovine serum interferon-γ enzyme-linked immunosorbent assay green fluorescent protein phosphate-buffered saline phenylmethylsulfonyl fluoride dithiothreitol electrophoretic mobility shift assays Chinese hamster ovary lipopolysaccharide. interleukin nitric oxide pathogen-associated molecular patterns Toll-like receptors Dulbecco's modified Eagle's medium fetal bovine serum interferon-γ enzyme-linked immunosorbent assay green fluorescent protein phosphate-buffered saline phenylmethylsulfonyl fluoride dithiothreitol electrophoretic mobility shift assays Chinese hamster ovary lipopolysaccharide. Therapeutic opportunities to interrupt the pathologic effects of flagellin will be based upon a precise delineation of its interaction with TLR5 that induces pro-inflammatory signaling pathways. Biochemical analysis of Salmonella flagellin reveals that both conserved domains within both termini are important in inducing pro-inflammatory responses in cultured intestinal epithelial cells (6Eaves-Pyles T.D. Wong H.R. Odoms K. Pyles R.B. J. Immunol. 2001; 167: 7009-7016Crossref PubMed Scopus (121) Google Scholar). Recent studies also suggest that the hypervariable domain is not involved in pro-inflammatory activation. A recombinantly expressed flagellin mutant, in which the central hypervariable domain was deleted, was shown not to detract from its ability to induce NF-κB signaling, suggesting that the highly conserved N and C termini are sufficient for TLR5 activation (6Eaves-Pyles T.D. Wong H.R. Odoms K. Pyles R.B. J. Immunol. 2001; 167: 7009-7016Crossref PubMed Scopus (121) Google Scholar). Further definition of the pro-inflammatory flagellin sequences has been provided by evidence that neutralization of the N-terminal “RINSA” domain (amino acid 31–52) by monoclonal antibodies blocks the proinflammatory activity of recombinant flagellins of different bacterial origin (5Eaves-Pyles T. Murthy K. Liaudet L. Virag L. Ross G. Soriano F.G. Szabo C. Salzman A.L. J. Immunol. 2001; 166: 1248-1260Crossref PubMed Scopus (241) Google Scholar). Recent studies (7Mizel S.B. West A.P. Hantgan R.R. J. Biol. Chem. 2003; 278: 23624-23629Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar, 8Jacchieri S.G. Torquato R. Brentani R.R. J. Bacteriol. 2003; 185: 4243-4247Crossref PubMed Scopus (60) Google Scholar) have also delineated the extramembranous region of TLR5 responsible for flagellin binding. The crystal structure of flagellin resembles an aircraft, with two wings and a central rod-shaped body. The core of the latter is derived from the α-helices of both the N and C termini, whereas the outer surface of the rod is constituted by the hypervariable domain (9Samatey F.A. Imada K. S. F. T. M. K. 2001; PubMed Scopus Google Scholar). has been on Salmonella flagellin and its role in the of host pro-inflammatory recombinant flagellin or flagella from other Gram-negative such as and pro-inflammatory recombinant flagellin or flagella from Salmonella are the potent P.F. Ciacci-Woolwine F. Snipes J.A. Mizel S.B. Infect. Immun. 2000; 68: 5525-5529Crossref PubMed Scopus (128) Google Scholar, 3Ciacci-Woolwine F. McDermott P.F. Mizel S.B. Infect. Immun. 1999; 67: 5176-5185Crossref PubMed Google Scholar, 5Eaves-Pyles T. Murthy K. Liaudet L. Virag L. Ross G. Soriano F.G. Szabo C. Salzman A.L. J. Immunol. 2001; 166: 1248-1260Crossref PubMed Scopus (241) Google Scholar, F. A. J.P. S. A. 2001; PubMed Scopus Google Scholar). and flagellin induce pro-inflammatory flagellin is more potent and induces in L. Murthy Soriano F.G. Salzman A.L. Szabo C. Infect. Immun. PubMed Scopus Google Scholar). it was that pro-inflammatory activity of flagellin be to studies have the pro-inflammatory of flagellin. It is that Toll-like receptors are involved in the of and are an conserved of receptors that in of an domain with a or two and a region the receptor The of signaling is to that of the receptor both receptor receptor it has been shown that TLR5 signaling in cells F. Smith A. S. A. 2001; PubMed Scopus Google Scholar). of expression in the TLR5 a signaling that and F. Smith A. S. A. 2001; PubMed Scopus Google Scholar, L. Mizel S.B. Infect. Immun. 2001; PubMed Scopus Google Scholar, S.B. Snipes J.A. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). Further studies also that TLR5 is expressed on the surface of epithelial and flagellin epithelial from the domain to the domain J. Clin. Investig. 2001; PubMed Scopus Google Scholar, S. J. Immunol. 2001; 167: PubMed Scopus Google Scholar). a it was that TLR5 with as well as and that are involved in the of nitric-oxide synthase expression by flagellin S.B. Smith West A.P. J. Immunol. 2003; PubMed Scopus Google Scholar). The of the present was to further the regions in the N and C termini that contribute to the eukaryotic pro-inflammatory activity of flagellin. using deletion and we have the to a region in the and evidence is provided on the region of flagellin responsible for functional activation of and of Chinese hamster ovary cells in with and and cells in in Dulbecco's modified Eagle's medium with and and a of in and to to medium was the with the of flagellin in and cells with flagellin for The of and the of in the was by the as A. M. Szabo C. J. Google Scholar). epithelial cells an cell with the in in medium cells with recombinant flagellin for The medium was and for the of by of and of a bacterial expression S. muenchen sequences has been (5Eaves-Pyles T. Murthy K. Liaudet L. Virag L. Ross G. Soriano F.G. Szabo C. Salzman A.L. J. Immunol. 2001; 166: 1248-1260Crossref PubMed Scopus (241) Google Scholar). an N-terminal of a and was used as the for and with used to flagellin was on of in a using and of for of for for and for with enzymes, and a the been with and and its deletion and the hypervariable two of and domains an expression in the flagellin by mutagenesis The recombinant by and in the of by sequence and of TLR5 was from a fetal lung A expression sequences to the N-terminal of TLR5 was by TLR5 sequences a eukaryotic expression of TLR5 protein in cells by by with and sequences by using a recombinant from of as the The with was with and the of the reporter of of the was in to an of and for with bacteria and with phosphate-buffered saline in and the by the was to a with binding and with binding The purified and protein by the The by and with to protein and cells and the with of using to the of medium was and cells with medium and to for a in and to in a medium and the medium was a for to and in medium the with the and in the of and The by for of and cell or nuclear as PubMed Scopus Google Scholar). cell cells from the by and in in and and on for the by and in nuclear cell in of A and of of was and the for by for and the in of and The and for protein by for and of the cell with of protein and on a a with bovine serum in and for and with or for and with The by binding assays using of NF-κB was from the of nuclear with binding and of and for in with as the was the the of the to and to antibodies and from to the as and for an the functional of the conserved regions of flagellin, both domains as and S. muenchen flagellin sequences in a used as a to sequences by with with enzymes, an expression with expressed in and purified to in to the of other bacterial and that with recombinant flagellin, by using as shown in various for activity by with for the of and of in was we recombinantly expressed with in the N terminus or C terminus or hypervariable region recombinant different a pro-inflammatory response in cells a of a response shown in recombinant flagellin either region to induce However, the recombinant the hypervariable domain a of with that of recombinant flagellin. we that the was in to the recombinant was also or in the activity of the protein sequences of to that of the flagellin hypervariable region the N and C domains not Taken together, results that the conserved N and C domains are important for pro-inflammatory signaling in eukaryotic cells and that the hypervariable in is not to this The of the hypervariable domain was this region in and sequence from to species. that both domains of flagellin are required to a pro-inflammatory we to the regions within domain that are required for pro-inflammatory of amino acids within the N terminus and or C terminus and recombinant for ability to as by the of in of flagellin proinflammatory in to the of the to a deletion of amino acids in the N terminus and and amino acids in the C terminus and A and Further in either the N terminus or the C terminus abolished of by flagellin. Taken together, results suggest that both termini of flagellin contribute to its pro-inflammatory activity and that involved in this are to a acid region in the N terminus (motif and a acid region in the C terminus (motif the highly conserved region of flagellins Gram-negative the region in the N was not for the of in activity of flagellin is by not also by the epithelial of pro-inflammatory cytokines, including tumor necrosis IL-8, and it has been shown that epithelial cells in response to flagellin T.S. Nataro J.P. Poteet-Smith C.E. Smith J.A. Guerrant R.L. J. Clin. Investig. 2000; 105: 1769-1777Crossref PubMed Scopus (218) Google Scholar, L. Szabo C. Murthy Virag L. A. Soriano F.G. Salzman A.L. 2003; PubMed Scopus Google Scholar, C.E. M. M. S. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, J.A. J.A. Infect. Immun. 2003; PubMed Scopus Google Scholar). The of eukaryotic pro-inflammatory expression in response to flagellin to cell intestinal epithelial cells are with lung epithelial which have been reported to expression in response to of flagellin as as L. Szabo C. Murthy Virag L. A. Soriano F.G. Salzman A.L. 2003; PubMed Scopus Google Scholar). and recombinant S. muenchen in with cells for the and release was by The results with cells in cells and that the N-terminal motif and the motif are both for proinflammatory regions in the conserved N and C termini also to expression in cells not for pro-inflammatory Because the the that within the N terminus and C terminus have a on the of pro-inflammatory signaling in eukaryotic we and a recombinant both of regions shown in in a of pro-inflammatory is either in the N terminus or C terminus have on flagellin We in an in which the hypervariable region was this we of the N terminus C terminus or both recombinant and in inducing in we recombinant two with in the N terminus and two with in the C terminus deletion of amino acids from the and a on pro-inflammatory whereas deletion from the C terminus and in a in pro-inflammatory results demonstrate that the N and C motifs are important for pro-inflammatory activity and suggest that the sequences in both domains in The results to further the of recombinant flagellins in which motif N and motif C the and the in pro-inflammatory activity of and may have from a of flagellin structure by deletion of a of conserved and this we in which all amino acids in motif N and motif C to different amino acids by The amino acid and sequences of and S. muenchen flagellin are shown in the and of motifs N and C to in the deletion of the the we the motif N or motif C or shown in or flagellin in motif C also reduced pro-inflammatory activity whereas deletion of the motif C abolished pro-inflammatory results suggest that flagellin two widely of which is to induce a proinflammatory with this or in motifs N and C abolished pro-inflammatory that sequences in both motifs are to an sequences in the N-terminal domain that are important for the pro-inflammatory activity of flagellin, we to the amino acids to induce a pro-inflammatory We in motif N acid was to or and was to two and based upon the crystal structure of flagellin, which reveals possible and and and of the and of S. flagellin, (9Samatey F.A. Imada K. S. F. T. M. K. 2001; PubMed Scopus Google Scholar). The inducing activity of recombinant flagellins with is shown in the pro-inflammatory activity of all was to that of flagellin. Because NF-κB is to of many the nitric-oxide synthase we used electrophoretic mobility shift assays to flagellin with from cells with or flagellin and for NF-κB shown in the of flagellin or the a shift for whereas and activity to that of the flagellin or of NF-κB binding was reduced to a not with results of or with and activity in and However, with recombinant flagellin and NF-κB binding. the analysis antibodies the and the as of a well However, the of a monoclonal on the of the NF-κB support our that the pro-inflammatory activity of flagellin is in motifs N and C. the we evidence for possible of motifs N and C in the pro-inflammatory activity of flagellin. However, the not that motifs in flagellin the eukaryotic receptor for flagellin, TLR5. It is well that the pro-inflammatory activity of flagellin is via the TLR5 receptor (7Mizel S.B. West A.P. Hantgan R.R. J. Biol. Chem. 2003; 278: 23624-23629Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar, F. Smith A. S. A. 2001; PubMed Scopus Google Scholar, S. J. Immunol. 2001; 167: PubMed Scopus Google Scholar). cells TLR5 and a reporter have been to for by this TLR5 not to of the to such as cell or is by flagellin. binds to and the interaction induces NF-κB which is required for the of many pro-inflammatory F. Smith A. S. A. 2001; PubMed Scopus Google Scholar). a of studies have shown the importance of TLR5 in is the domains of flagellin and TLR5. We have the to functional activation of TLR5 by flagellin. We cell TLR5 protein and reporter the of an cells express of TLR5 expression of TLR5 in cells in NF-κB activity in response to with flagellin of cells to purified S. muenchen flagellin a of to the region of flagellin for its activation of we a of in and for the expression of in assays studies that the in and on NF-κB activation and However, further deletion of the N and C terminus sequences of flagellin and with and in which all the amino acids in motifs N and C are also to the reporter such that expression was with that of Taken together, results that the regions in flagellin required for pro-inflammatory activity and NF-κB activation are also required for its functional activation of the TLR5 is the monomeric protein of flagella, a as a means of propulsion or attachment to host is recently implicated as a of eukaryotic proinflammatory flagellin activates eukaryotic such as and intestinal and epithelial to release a of pro-inflammatory mediators in vitro and in including tumor necrosis as well as inducible P.F. Ciacci-Woolwine F. Snipes J.A. Mizel S.B. Infect. Immun. 2000; 68: 5525-5529Crossref PubMed Scopus (128) Google Scholar, 3Ciacci-Woolwine F. McDermott P.F. Mizel S.B. Infect. Immun. 1999; 67: 5176-5185Crossref PubMed Google Scholar, 4Steiner T.S. Nataro J.P. Poteet-Smith C.E. Smith J.A. Guerrant R.L. J. Clin. Investig. 2000; 105: 1769-1777Crossref PubMed Scopus (218) Google Scholar, 5Eaves-Pyles T. Murthy K. Liaudet L. Virag L. Ross G. Soriano F.G. Szabo C. Salzman A.L. J. Immunol. 2001; 166: 1248-1260Crossref PubMed Scopus (241) Google Scholar, F. Smith A. S. A. 2001; PubMed Scopus Google Scholar, J. Clin. Investig. 2001; PubMed Scopus Google Scholar, S. J. Immunol. 2001; 167: PubMed Scopus Google Scholar, PubMed Scopus Google Scholar, Immunol. PubMed Scopus Google Scholar, F. Mizel S.B. Infect. Immun. 1998; 66: PubMed Google Scholar). The of the present was to the regions and the amino acid within flagellin that contribute to its activation of pro-inflammatory was in response to from other that a central hypervariable domain is for the of pro-inflammatory activity P.F. Ciacci-Woolwine F. Snipes J.A. Mizel S.B. Infect. Immun. 2000; 68: 5525-5529Crossref PubMed Scopus (128) Google Scholar) or that pro-inflammatory activation was upon the highly conserved regions (6Eaves-Pyles T.D. Wong H.R. Odoms K. Pyles R.B. J. Immunol. 2001; 167: 7009-7016Crossref PubMed Scopus (121) Google Scholar, T.S. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). The precise within the N and C termini that the pro-inflammatory host response to flagellin are this we have a analysis of recombinant flagellin in to that are for pro-inflammatory eukaryotic with the crystal structure of flagellin, our results a functional that a by which flagellin activates its host TLR5. of the structure of flagellin is based upon crystal structure analysis by (9Samatey F.A. Imada K. S. F. T. M. K. 2001; PubMed Scopus Google Scholar). has reported that the N-terminal domain of flagellin two α-helices by a which is to our motif The in the N-terminal is the in the The domain in to the in the N-terminal domain and to α-helices are and the central whereas the hypervariable region the outer surface (9Samatey F.A. Imada K. S. F. T. M. K. 2001; PubMed Scopus Google Scholar). upon our functional and the crystal structure from (9Samatey F.A. Imada K. S. F. T. M. K. 2001; PubMed Scopus Google we have a to for the of various on the structure of monomeric flagellin and to its functional activation of TLR5 regions of flagellin that are for activation of TLR5. we have a acid region within the N-terminal domain that a of amino acids in the N-terminal domain on proinflammatory activity by flagellin. However, deletion of motif N pro-inflammatory mutagenesis of all amino acids abolished pro-inflammatory of suggesting the of attachment within the motif are required for functional activation of TLR5. The of is by a S.G. Torquato R. Brentani R.R. J. Bacteriol. 2003; 185: 4243-4247Crossref PubMed Scopus (60) Google based on that that the amino acids of flagellin are involved in binding with amino acids of the TLR5 our binding sequences (motif N) with we have a acid region motif within the conserved C is for functional activation of TLR5. or of all amino acids in motif C reduced the pro-inflammatory not completely. we have sequences within the domains and C) that may be involved in and are for flagellin We that in either the N terminus or the C terminus have on pro-inflammatory activity by flagellin. However, pro-inflammatory Because are many of attachment the N and C which are in to our results suggest that in either the N or C termini not this and in both termini, are of the alignment of the two termini, resulting in a of pro-inflammatory the role of the hypervariable domain in the functional activation of flagellin has been a of in the this we have recombinant of flagellin in which the hypervariable domain has been deleted, the N and C termini We have in with a (6Eaves-Pyles T.D. Wong H.R. Odoms K. Pyles R.B. J. Immunol. 2001; 167: 7009-7016Crossref PubMed Scopus (121) Google that flagellin the N or C termini pro-inflammatory the the hypervariable domain in cells with that of flagellin results that the conserved N and C domains are required for pro-inflammatory and the hypervariable domain is and to the of pro-inflammatory expression (6Eaves-Pyles T.D. Wong H.R. Odoms K. Pyles R.B. J. Immunol. 2001; 167: 7009-7016Crossref PubMed Scopus (121) Google Scholar). the flagellin the rod-shaped structure in our in the of the hypervariable Because the hypervariable region the N and C termini, it to that it the structure of flagellin, the the hypervariable pro-inflammatory to this we the activity of or in the We that and in recombinant pro-inflammatory activity the in flagellin. Taken together, results suggest that the hypervariable domain a role in a of the N and C a was not N-terminal in the that the C terminus may be more for the N The of various in the regions of flagellin on pro-inflammatory activation and is a and further will be required to the role of Because we have functional activation of TLR5 as a for the of flagellin our are we have not crystal of the various recombinant and can we that have the A to this is from electrophoretic studies we have that recombinant flagellins of on G. K. C. and A. L. and flagellin are in and amino acid flagellin by was and molecular of other flagellin in a further that the functional in pro-inflammatory activity involved in the which a in an Recent have our of the by which flagellin induces pro-inflammatory it has been that the TLR5 receptor bacterial flagellin derived from both and Gram-negative The interaction in the activation of which is required for the of many pro-inflammatory to be an of TLR5 for signaling (7Mizel S.B. West A.P. Hantgan R.R. J. Biol. Chem. 2003; 278: 23624-23629Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar, F. Smith A. S. A. 2001; PubMed Scopus Google Scholar, S. J. Immunol. 2001; 167: PubMed Scopus Google Scholar, S.B. Smith West A.P. J. Immunol. 2003; PubMed Scopus Google Scholar, H. S. A. J. 2003; PubMed Scopus Google Scholar, A. G. S. K. J. Biol. Chem. 2003; 278: Full Text Full Text PDF PubMed Scopus Google Scholar, F. Smith A. J. Immunol. 2003; PubMed Scopus Google Scholar). in and cells not that all of the effects with various TLR5. we used cells with TLR5 and reporter the with flagellin that to induce a pro-inflammatory response in epithelial cells also to expression in our on the ability of the flagellin to induce or the ability of the to induce TLR5 activation. are with the that TLR5 is the receptor for and inflammatory Recent studies have the role of the in intestinal J. Clin. Investig. 2001; PubMed Scopus Google Scholar, S. J. Immunol. 2001; 167: PubMed Scopus Google Scholar, C.E. M. M. S. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, K. A. T. K. T. A. H. S. H. M. T. H. H. J. T. J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus Google in systemic and (5Eaves-Pyles T. Murthy K. Liaudet L. Virag L. Ross G. Soriano F.G. Szabo C. Salzman A.L. J. Immunol. 2001; 166: 1248-1260Crossref PubMed Scopus (241) Google Scholar, L. Murthy Soriano F.G. Salzman A.L. Szabo C. Infect. Immun. PubMed Scopus Google and in various of inflammation, and (1Feldman M. Bryan R. Rajan S. Scheffler L. Brunnert S. Tang H. Prince A. Infect. Immun. 1998; 66: 43-51Crossref PubMed Google Scholar, L. Szabo C. Murthy Virag L. A. Soriano F.G. Salzman A.L. 2003; PubMed Scopus Google Scholar). or flagellin may pro-inflammatory by and may and in of Gram-negative The of the proinflammatory activity of flagellin in the may studies in this

We have recently shown that the neuronal exchange factor p140 Ras-GRF becomes activated in vivo in response to elevated calcium levels [C. L. Farnsworth, N. W. Freshney, L. B. Rosen, A. Ghosh, M. E. Greenberg, and L. A. Feig, Nature (London) 376:524-527, 1995]. Activation is mediated by calcium-induced calmodulin binding to an IQ domain near the N terminus of Ras-GRF. Here we show that the adjacent N-terminal pleckstrin homology (PH), coiled-coil, and IQ domains function cooperatively to allow Ras-GRF activation. Deletion of the N-terminal PH domain redistributes a large percentage of Ras-GRF from the particulate to the cytosolic fraction of cells and renders the protein insensitive to calcium stimulation. A similar cellular distribution and biological activity are observed when only the core catalytic domain is expressed. Although the PH domain is necessary for particulate association of Ras-GRF, it is not sufficient for targeting the core catalytic domain to this cellular location. This requires the PH domain and the adjacent coiled-coil and IQ sequences. Remarkably, this form of Ras-GRF is constitutively activated. The PH and coiled-coil domains must also perform an additional function, since targeting to the particulate fraction of cells is not sufficient to allow Ras-GRF activation by calcium. A Ras-GRF mutant containing the PH domain from Ras-GTPase-activating protein in place of its own N-terminal PH domain localizes to the particulate fraction of cells but does not respond to calcium. Similar phenotypes are seen with mutant Ras-GRFs containing point mutations in either the PH or coiled-coil domain. These findings argue that the N-terminal PH, coiled-coil, and IQ domains of Ras-GRF function together to connect Ras-GRF to multiple components in the particulate fractions of cells that are required for responsiveness of the protein to calcium signaling.