- Email: [email protected]
“Cystic fibrotics could survive cholera, choleraics could survive cystic fibrosis”; hypothesis that explores new horizons in treatment of cystic fibrosis
Medical Hypotheses 85 (2015) 715–717
Contents lists available at ScienceDirect
Medical Hypotheses journal homepage: www.elsevier.com/locate/mehy
‘‘Cystic fibrotics could survive cholera, choleraics could survive cystic fibrosis”; hypothesis that explores new horizons in treatment of cystic fibrosis Arsalan Azimi ⇑ Faculty of Medicine, Shiraz University of Medical Sciences, Zand St., Shiraz, Iran
a r t i c l e
i n f o
Article history: Received 10 July 2015 Accepted 22 October 2015
a b s t r a c t Cystic fibrosis, the most common inherited disease of white population, is a disease of CFTR channels, in which mucosal function of many organs especially respiratory tract is impaired. Decreased mucociliary clearance and accumulation of mucus in airways facilitates colonization of infectious microorganisms, followed by infection. Following chronic infection, persistent inflammation ensues, which results in airway remodeling and deterioration of mucociliary clearance and result in a vicious cycle. Here, it is hypothesized that cholera toxin (CT) could ameliorate symptoms of cystic fibrosis as CT could dilute the thickened mucus, improve mucociliary clearance and alleviate airway obstruction. CT strengthens immunity of airway mucosa and it could attenuates bacterial growth and reduce persistency of infection. CT also modulates cellular immune response and it could decrease airway inflammation, hinder airway remodeling and prevent respiratory deterioration. Thereby it is hypothesized that CT could target and ameliorate many of pathophysiologic steps of the disease and it explores new horizons in treatment of CF. Ó 2015 Elsevier Ltd. All rights reserved.
Introduction Cystic fibrosis (CF) is the most common inherited disease of white population and CF genes are disseminated in general population with an unusual prevalence [1]. Thereby focusing on strategies to approach to CF become of high importance. CF is the disease of Cystic Fibrosis Transmembrane conductance Regulator (CFTR) channels. Physiologically the channel is responsible for Cl secretion and by the disease, mucosal Cl secretion is impaired and subsequently, Na+ reabsorption is increased. Thereby the mucus becomes thickened and dehydrated and mucosal function of many organs, especially respiratory tract, becomes impaired. Accumulation of heavy, dehydrated mucus in airways and the resultant changes in the capability of the lungs to fight infections is the major problem with CF patients. A second problem is that thickening and dehydration of GI mucus could block outflow of digestive enzymes from the exocrine pancreas and cause panAbbreviations: APC, antigen presenting cells; c-AMP, Cyclic Adenosine Mono Phosphate; CF, cystic fibrosis; CT, cholera toxin; CFTR, Cystic Fibrosis Transmembrane conductance Regulator; FEV1, Forced Expiratory Volume in the first second; IL-1, interleukin 1; IL-6, interleukin 6; LCI, Lung Clearance Index; LPS, lipopolysaccharides; NO, nitric oxide; P. aeruginosa, Pseudomonas aeruginosa; RSV, respiratory syncytial virus; TLC, Total Lung Capacity; TNFa, Tumor Necrosis Factor Alpha. ⇑ Tel.: +98 9379876373. E-mail address: [email protected] http://dx.doi.org/10.1016/j.mehy.2015.10.023 0306-9877/Ó 2015 Elsevier Ltd. All rights reserved.
creatitis. Another major change (that also makes it possible to use the sweat chloride test as the standard diagnostic test for CF) is the abnormal salty sweat of CF patients. Sterility (in both males and females) and abnormal secretion in the small intestine are some other clinical manifestations of the disease [2]. Previously, it was implicated that there could be some kind of relevance between infection by Vibrio cholerae and prevalence of the genetic disease: CF [2]. Cholera is an ancient infamous diarrheal infection which was extremely fatal before development of current treatments. When cholera toxin affects intestinal mucosa, it intensifies efflux of chloride ions by CFTR and a secretory diarrhea ensues. Historically, all over the world cholera has affected human populations by devastating outbreaks [3]. On the other hand CF is the most common inherited disease of white population. It has been hypothesized that heterozygote carriers of CF genes may have benefited from them when they became contaminated by cholera and this may be the reason that these genes have achieved such high prevalence in general population [1].
Hypothesis It is hypothesized that cholera toxin, if used as an inhaled medication, could help patients who suffer from CF. In order to evaluate efficacy of such medication, animal studies and clinical trials
716
A. Azimi / Medical Hypotheses 85 (2015) 715–717
of intra-cellular c-AMP and as secretory cells of small intestinal mucosa respond to this supra-ordinary amount of secretagogues cAMP, which over-stimulates CFTR, and intensified efflux of chloride ions by the channel ensues [2]. It is hypothesized that overstimulation of defective CFTR by extraordinary amounts of c-AMP could improve impaired function of CFTRs. Dysfunction of CFTR not only impairs secretion of Cl , but it also disrupts activity of Na+/H+ and Cl /HCO3 exchanger channels [4]. Thereby CT could also improve secretion of HCO3 through the mucus [2]. CT could be of help not only in respiratory tract but also in Gastro-intestinal tract, where thickened mucus and depleted HCO3 result in GI manifestations of the disease. To evaluate the hypothesis, CT should be used as an inhaled medication, which makes it possible for the toxin to be absorbed in both GI and respiratory tract. Fig. 1. Defective ion and fluid transport due to CFTR mutation results in impaired mucosal function of many organs especially respiratory tract. Decreased mucociliary clearance and accumulation of mucus in airways facilitates infection. Following chronic infection, persistent inflammation ensues, which results in airway remodeling and deterioration of mucociliary clearance and result in a vicious cycle. Cholera toxin (CT) could dilute the thickened mucus and facilitates its clearance. CT strengthens immunity of airway mucosa and attenuates bacterial growth and development of infections. CT decreases airway inflammation and slows down remodeling of airways and respiratory deterioration. Thereby CT unsettles the vicious cycle of the disease and it could explore new horizons in treatment of Cystic Fibrosis.
should be performed. Animal studies help to define the proper dosage and concentration of the medication. In the next step, if animal studies validated the hypothesis, phase I and then phase II clinical trials could be performed. To perform phase II trials, the patients who would be enrolled in the clinical trial should be categorized in separate groups considering their age, race (e.g. Caucasian), subtype of the disease and pulmonary function. Then, each group should be divided into two subgroups, one receiving the current medication for CF and the other receiving the same medication together with nebulized CT. These two subgroups should be followed by a selected combination of: – Transport of chloride ions through CFTR channel, assessed by transepithelial nasal potential difference. – Mucocilary clearance assessed by Lung Clearance Index (LCI). – Gastrointestinal pH. – Sputum PH, inflammation and microbiology assessments. – Forced Expiratory Volume in the first second/Total Lung Capacity (FEV1/TLC). – Number of episodes of pulmonary exacerbations or respiratory tract infections. Then these subgroups should be compared to survey the difference both in short term and long term results, between these different therapeutic regimens.
CT as an immune-booster CT has been shown to have some adjuvant immuno-modulatory actions. Moreover CT is used in many transcutaneous immunization methods by simply mixing it with various antigens [4]. CT is used in vaccines to enhance presentation of antigens to the immune system by various antigen presenting cells (APCs), including macrophages, dendritic cells and B cells [5]. CT up-regulates various cell surface co-stimulatory molecules and chemokine receptors on human APCs [6,7]. CT also stimulates macrophages to secret IL-1, which enhances their own antigen presenting function [8]. As a result CT could potentiate immunogenicity of most of antigens including antigens of Pseudomonas aeruginosa, Streptococcus pneumoniae, respiratory syncytial virus (RSV) and influenza virus [9–12]. Concluding the issue, it is hypothesized that using inhaled CT in CF patients would facilitate immunization and potentiates immunity of respiratory system against most of important pathogens, particularly P. aeruginosa. This could decrease duration of airway inflammation and decelerate airway remodeling in CF patients.
CT as an immunomodulator After exposure to bacterial lipopolysaccharides (LPS), Macrophages produce and secret TNFa and IL-6, which intensify cellular immunity and tissue destruction. Administration of CT to LPS-stimulated macrophages suppresses production of TNFa, by up to 80%. Since nitric oxide (NO) synthesis is dependent on TNFa, CT simultaneously reduces the production of NO [13]. Thereby it is hypothesized that CT, if used as an inhaled medication, could modulate immune response and attenuate airway inflammation, remodeling and respiratory deterioration in CF patients.
Evaluation of the hypothesis It is hypothesized that CT could act as a mucodiluter, immunebooster and immunomodulator and if used as an inhaled medication, it could improve mucociliary clearance, decelerate rate of bacterial growth and infection, alleviate inflammation and hinder airway remodeling. As it is hypothesized, CT could unsettle the vicious cycle of airway obstruction–infection–inflammation in all of its three steps. Thereby cholera toxin (CT) could shed a new light on future treatments and approaches to CF (see Fig. 1).
Discussion and conclusion There is a vicious cycle between airway obstruction, infection and inflammation which leads to progressive respiratory disease in CF patients [14]. Thereby therapeutic strategies should focus on unsettling the vicious cycle by [15]: (1) Diluting the thickened mucus to facilitate airway clearance. (2) Diminishing bacterial growth and infection. (3) Modifying inflammation to slow down airway remodeling.
CT as a mucodiluter CT impedes inactivation of Adenylate Cyclase, resulting in constitutive action of this enzyme. Thereby CT increases concentration
As it is hypothesized, CT, if inhaled by CF patients, could unsettle the vicious cycle of airway obstruction–infection–inflammation, because:
A. Azimi / Medical Hypotheses 85 (2015) 715–717
(1) As a mucodiluter, CT could increase excretion of Cl and HCO3 in the thickened mucus which dilutes it and facilitates its clearance. (2) As an immune-booster, CT improves function of APCs and enhances presentation of antigens to the immune system which can strengthen immunity of airway mucosa and attenuate bacterial growth and development of infections [9–12]. (3) As an immunomodulator, CT modulates cellular immune response and airway inflammation as it suppresses production of TNFa which may slow down remodeling of airways and respiratory deterioration [13]. All in all cholera may be the explanation for high prevalence of CF genes in general population, but now it is hypothesized that cholera, itself can also offer a remission for CF. Conflict of interest Arsalan Azimi reports no conflict of interest. References [1] Rodman DM, Zamudio S. The cystic fibrosis heterozygote — advantage in surviving cholera? Med Hypotheses 1991;36(3):253–8. PubMed PMID: 1724059. [2] Goodman BE, Percy WH. CFTR in cystic fibrosis and cholera: from membrane transport to clinical practice. Adv Physiol Educ 2005;29(2):75–82. PubMed PMID: 15905150. [3] Juzych NS, Resnick B, Streeter R, Herbstman J, Zablotsky J, Fox M, et al. Adequacy of state capacity to address noncommunicable disease clusters in the era of environmental public health tracking. Am J Public Health 2007;97 (Suppl. 1):S163–9. http://dx.doi.org/10.2105/AJPH.2006.096453. PMCID: PMC1854996.
717
[4] Glenn GM, Rao M, Matyas GR, Alving CR. Skin immunization made possible by cholera toxin. Nature 1998;391(6670):851. [5] Lycke NY. Cholera toxin promotes B cell isotype switching by two different mechanisms. cAMP induction augments germ-line Ig H-chain RNA transcripts whereas membrane ganglioside GM1-receptor binding enhances later events in differentiation. J Immunol 1993;150(11):4810–21. [6] Gagliardi MC, De Magistris MT. Maturation of human dendritic cells induced by the adjuvant cholera toxin: role of cAMP on chemokine receptor expression. Vaccine 2003;21(9–10):856–61. [7] Eriksson K, Fredriksson M, Nordström I, Holmgren J. Cholera toxin and its B subunit promote dendritic cell vaccination with different influences on Th1 and Th2 development. Infect Immun 2003;71(4):1740–7. PubMed PMID: 12654787; PubMed Central PMCID: PMC152034. [8] Bromander A, Holmgren J, Lycke N. Cholera toxin stimulates IL-1 production and enhances antigen presentation by macrophages in vitro. J Immunol 1991;146(9):2908–14. [9] Chen KS, Strober W. Cholera holotoxin and its B subunit enhance Peyer’s patch B cell responses induced by orally administered influenza virus: disproportionate cholera toxin enhancement of the IgA B cell response. Eur J Immunol 1990;20(2):433–6. [10] Abraham E, Robinson A. Oral immunization with bacterial polysaccharide and adjuvant enhances antigen-specific pulmonary secretory antibody response and resistance to pneumonia. Vaccine 1991;9(10):757–64. [11] Walsh EE. Mucosal immunization with a subunit respiratory syncytial virus vaccine in mice. Vaccine 1993;11(11):1135–8. [12] Yamamoto M, McDaniel LS, Kawabata K, Briles DE, Jackson RJ, McGhee JR, et al. Oral immunization with PspA elicits protective humoral immunity against Streptococcus pneumoniae infection. Infect Immun 1997;65(2):640–4. PubMed Central PMCID: PMC176108. [13] Cong Y, Oliver AO, Elson CO. Effects of cholera toxin on macrophage production of co-stimulatory cytokines. Eur J Immunol 2001;31(1):64–71. PubMed PMID: 11169439. [14] Hoffman LR, Ramsey BW. Cystic fibrosis therapeutics: the road ahead. Chest 2013;143(1):207–13. http://dx.doi.org/10.1378/chest.12-1639. PubMed PMID: 23276843; PubMed Central PMCID: PMC3610617. [15] Ramsey BW, Davies J, McElvaney NG, Tullis E, Bell SC, Drˇevínek P, et al. A CFTR potentiator in patients with cystic fibrosis and the G551D mutation. N Engl J Med 2011;365(18):1663–72. http://dx.doi.org/10.1056/NEJMoa1105185. PubMed PMID: 22047557; PubMed Central PMCID: PMC3230303.
Contents lists available at ScienceDirect
Medical Hypotheses journal homepage: www.elsevier.com/locate/mehy
‘‘Cystic fibrotics could survive cholera, choleraics could survive cystic fibrosis”; hypothesis that explores new horizons in treatment of cystic fibrosis Arsalan Azimi ⇑ Faculty of Medicine, Shiraz University of Medical Sciences, Zand St., Shiraz, Iran
a r t i c l e
i n f o
Article history: Received 10 July 2015 Accepted 22 October 2015
a b s t r a c t Cystic fibrosis, the most common inherited disease of white population, is a disease of CFTR channels, in which mucosal function of many organs especially respiratory tract is impaired. Decreased mucociliary clearance and accumulation of mucus in airways facilitates colonization of infectious microorganisms, followed by infection. Following chronic infection, persistent inflammation ensues, which results in airway remodeling and deterioration of mucociliary clearance and result in a vicious cycle. Here, it is hypothesized that cholera toxin (CT) could ameliorate symptoms of cystic fibrosis as CT could dilute the thickened mucus, improve mucociliary clearance and alleviate airway obstruction. CT strengthens immunity of airway mucosa and it could attenuates bacterial growth and reduce persistency of infection. CT also modulates cellular immune response and it could decrease airway inflammation, hinder airway remodeling and prevent respiratory deterioration. Thereby it is hypothesized that CT could target and ameliorate many of pathophysiologic steps of the disease and it explores new horizons in treatment of CF. Ó 2015 Elsevier Ltd. All rights reserved.
Introduction Cystic fibrosis (CF) is the most common inherited disease of white population and CF genes are disseminated in general population with an unusual prevalence [1]. Thereby focusing on strategies to approach to CF become of high importance. CF is the disease of Cystic Fibrosis Transmembrane conductance Regulator (CFTR) channels. Physiologically the channel is responsible for Cl secretion and by the disease, mucosal Cl secretion is impaired and subsequently, Na+ reabsorption is increased. Thereby the mucus becomes thickened and dehydrated and mucosal function of many organs, especially respiratory tract, becomes impaired. Accumulation of heavy, dehydrated mucus in airways and the resultant changes in the capability of the lungs to fight infections is the major problem with CF patients. A second problem is that thickening and dehydration of GI mucus could block outflow of digestive enzymes from the exocrine pancreas and cause panAbbreviations: APC, antigen presenting cells; c-AMP, Cyclic Adenosine Mono Phosphate; CF, cystic fibrosis; CT, cholera toxin; CFTR, Cystic Fibrosis Transmembrane conductance Regulator; FEV1, Forced Expiratory Volume in the first second; IL-1, interleukin 1; IL-6, interleukin 6; LCI, Lung Clearance Index; LPS, lipopolysaccharides; NO, nitric oxide; P. aeruginosa, Pseudomonas aeruginosa; RSV, respiratory syncytial virus; TLC, Total Lung Capacity; TNFa, Tumor Necrosis Factor Alpha. ⇑ Tel.: +98 9379876373. E-mail address: [email protected] http://dx.doi.org/10.1016/j.mehy.2015.10.023 0306-9877/Ó 2015 Elsevier Ltd. All rights reserved.
creatitis. Another major change (that also makes it possible to use the sweat chloride test as the standard diagnostic test for CF) is the abnormal salty sweat of CF patients. Sterility (in both males and females) and abnormal secretion in the small intestine are some other clinical manifestations of the disease [2]. Previously, it was implicated that there could be some kind of relevance between infection by Vibrio cholerae and prevalence of the genetic disease: CF [2]. Cholera is an ancient infamous diarrheal infection which was extremely fatal before development of current treatments. When cholera toxin affects intestinal mucosa, it intensifies efflux of chloride ions by CFTR and a secretory diarrhea ensues. Historically, all over the world cholera has affected human populations by devastating outbreaks [3]. On the other hand CF is the most common inherited disease of white population. It has been hypothesized that heterozygote carriers of CF genes may have benefited from them when they became contaminated by cholera and this may be the reason that these genes have achieved such high prevalence in general population [1].
Hypothesis It is hypothesized that cholera toxin, if used as an inhaled medication, could help patients who suffer from CF. In order to evaluate efficacy of such medication, animal studies and clinical trials
716
A. Azimi / Medical Hypotheses 85 (2015) 715–717
of intra-cellular c-AMP and as secretory cells of small intestinal mucosa respond to this supra-ordinary amount of secretagogues cAMP, which over-stimulates CFTR, and intensified efflux of chloride ions by the channel ensues [2]. It is hypothesized that overstimulation of defective CFTR by extraordinary amounts of c-AMP could improve impaired function of CFTRs. Dysfunction of CFTR not only impairs secretion of Cl , but it also disrupts activity of Na+/H+ and Cl /HCO3 exchanger channels [4]. Thereby CT could also improve secretion of HCO3 through the mucus [2]. CT could be of help not only in respiratory tract but also in Gastro-intestinal tract, where thickened mucus and depleted HCO3 result in GI manifestations of the disease. To evaluate the hypothesis, CT should be used as an inhaled medication, which makes it possible for the toxin to be absorbed in both GI and respiratory tract. Fig. 1. Defective ion and fluid transport due to CFTR mutation results in impaired mucosal function of many organs especially respiratory tract. Decreased mucociliary clearance and accumulation of mucus in airways facilitates infection. Following chronic infection, persistent inflammation ensues, which results in airway remodeling and deterioration of mucociliary clearance and result in a vicious cycle. Cholera toxin (CT) could dilute the thickened mucus and facilitates its clearance. CT strengthens immunity of airway mucosa and attenuates bacterial growth and development of infections. CT decreases airway inflammation and slows down remodeling of airways and respiratory deterioration. Thereby CT unsettles the vicious cycle of the disease and it could explore new horizons in treatment of Cystic Fibrosis.
should be performed. Animal studies help to define the proper dosage and concentration of the medication. In the next step, if animal studies validated the hypothesis, phase I and then phase II clinical trials could be performed. To perform phase II trials, the patients who would be enrolled in the clinical trial should be categorized in separate groups considering their age, race (e.g. Caucasian), subtype of the disease and pulmonary function. Then, each group should be divided into two subgroups, one receiving the current medication for CF and the other receiving the same medication together with nebulized CT. These two subgroups should be followed by a selected combination of: – Transport of chloride ions through CFTR channel, assessed by transepithelial nasal potential difference. – Mucocilary clearance assessed by Lung Clearance Index (LCI). – Gastrointestinal pH. – Sputum PH, inflammation and microbiology assessments. – Forced Expiratory Volume in the first second/Total Lung Capacity (FEV1/TLC). – Number of episodes of pulmonary exacerbations or respiratory tract infections. Then these subgroups should be compared to survey the difference both in short term and long term results, between these different therapeutic regimens.
CT as an immune-booster CT has been shown to have some adjuvant immuno-modulatory actions. Moreover CT is used in many transcutaneous immunization methods by simply mixing it with various antigens [4]. CT is used in vaccines to enhance presentation of antigens to the immune system by various antigen presenting cells (APCs), including macrophages, dendritic cells and B cells [5]. CT up-regulates various cell surface co-stimulatory molecules and chemokine receptors on human APCs [6,7]. CT also stimulates macrophages to secret IL-1, which enhances their own antigen presenting function [8]. As a result CT could potentiate immunogenicity of most of antigens including antigens of Pseudomonas aeruginosa, Streptococcus pneumoniae, respiratory syncytial virus (RSV) and influenza virus [9–12]. Concluding the issue, it is hypothesized that using inhaled CT in CF patients would facilitate immunization and potentiates immunity of respiratory system against most of important pathogens, particularly P. aeruginosa. This could decrease duration of airway inflammation and decelerate airway remodeling in CF patients.
CT as an immunomodulator After exposure to bacterial lipopolysaccharides (LPS), Macrophages produce and secret TNFa and IL-6, which intensify cellular immunity and tissue destruction. Administration of CT to LPS-stimulated macrophages suppresses production of TNFa, by up to 80%. Since nitric oxide (NO) synthesis is dependent on TNFa, CT simultaneously reduces the production of NO [13]. Thereby it is hypothesized that CT, if used as an inhaled medication, could modulate immune response and attenuate airway inflammation, remodeling and respiratory deterioration in CF patients.
Evaluation of the hypothesis It is hypothesized that CT could act as a mucodiluter, immunebooster and immunomodulator and if used as an inhaled medication, it could improve mucociliary clearance, decelerate rate of bacterial growth and infection, alleviate inflammation and hinder airway remodeling. As it is hypothesized, CT could unsettle the vicious cycle of airway obstruction–infection–inflammation in all of its three steps. Thereby cholera toxin (CT) could shed a new light on future treatments and approaches to CF (see Fig. 1).
Discussion and conclusion There is a vicious cycle between airway obstruction, infection and inflammation which leads to progressive respiratory disease in CF patients [14]. Thereby therapeutic strategies should focus on unsettling the vicious cycle by [15]: (1) Diluting the thickened mucus to facilitate airway clearance. (2) Diminishing bacterial growth and infection. (3) Modifying inflammation to slow down airway remodeling.
CT as a mucodiluter CT impedes inactivation of Adenylate Cyclase, resulting in constitutive action of this enzyme. Thereby CT increases concentration
As it is hypothesized, CT, if inhaled by CF patients, could unsettle the vicious cycle of airway obstruction–infection–inflammation, because:
A. Azimi / Medical Hypotheses 85 (2015) 715–717
(1) As a mucodiluter, CT could increase excretion of Cl and HCO3 in the thickened mucus which dilutes it and facilitates its clearance. (2) As an immune-booster, CT improves function of APCs and enhances presentation of antigens to the immune system which can strengthen immunity of airway mucosa and attenuate bacterial growth and development of infections [9–12]. (3) As an immunomodulator, CT modulates cellular immune response and airway inflammation as it suppresses production of TNFa which may slow down remodeling of airways and respiratory deterioration [13]. All in all cholera may be the explanation for high prevalence of CF genes in general population, but now it is hypothesized that cholera, itself can also offer a remission for CF. Conflict of interest Arsalan Azimi reports no conflict of interest. References [1] Rodman DM, Zamudio S. The cystic fibrosis heterozygote — advantage in surviving cholera? Med Hypotheses 1991;36(3):253–8. PubMed PMID: 1724059. [2] Goodman BE, Percy WH. CFTR in cystic fibrosis and cholera: from membrane transport to clinical practice. Adv Physiol Educ 2005;29(2):75–82. PubMed PMID: 15905150. [3] Juzych NS, Resnick B, Streeter R, Herbstman J, Zablotsky J, Fox M, et al. Adequacy of state capacity to address noncommunicable disease clusters in the era of environmental public health tracking. Am J Public Health 2007;97 (Suppl. 1):S163–9. http://dx.doi.org/10.2105/AJPH.2006.096453. PMCID: PMC1854996.
717
[4] Glenn GM, Rao M, Matyas GR, Alving CR. Skin immunization made possible by cholera toxin. Nature 1998;391(6670):851. [5] Lycke NY. Cholera toxin promotes B cell isotype switching by two different mechanisms. cAMP induction augments germ-line Ig H-chain RNA transcripts whereas membrane ganglioside GM1-receptor binding enhances later events in differentiation. J Immunol 1993;150(11):4810–21. [6] Gagliardi MC, De Magistris MT. Maturation of human dendritic cells induced by the adjuvant cholera toxin: role of cAMP on chemokine receptor expression. Vaccine 2003;21(9–10):856–61. [7] Eriksson K, Fredriksson M, Nordström I, Holmgren J. Cholera toxin and its B subunit promote dendritic cell vaccination with different influences on Th1 and Th2 development. Infect Immun 2003;71(4):1740–7. PubMed PMID: 12654787; PubMed Central PMCID: PMC152034. [8] Bromander A, Holmgren J, Lycke N. Cholera toxin stimulates IL-1 production and enhances antigen presentation by macrophages in vitro. J Immunol 1991;146(9):2908–14. [9] Chen KS, Strober W. Cholera holotoxin and its B subunit enhance Peyer’s patch B cell responses induced by orally administered influenza virus: disproportionate cholera toxin enhancement of the IgA B cell response. Eur J Immunol 1990;20(2):433–6. [10] Abraham E, Robinson A. Oral immunization with bacterial polysaccharide and adjuvant enhances antigen-specific pulmonary secretory antibody response and resistance to pneumonia. Vaccine 1991;9(10):757–64. [11] Walsh EE. Mucosal immunization with a subunit respiratory syncytial virus vaccine in mice. Vaccine 1993;11(11):1135–8. [12] Yamamoto M, McDaniel LS, Kawabata K, Briles DE, Jackson RJ, McGhee JR, et al. Oral immunization with PspA elicits protective humoral immunity against Streptococcus pneumoniae infection. Infect Immun 1997;65(2):640–4. PubMed Central PMCID: PMC176108. [13] Cong Y, Oliver AO, Elson CO. Effects of cholera toxin on macrophage production of co-stimulatory cytokines. Eur J Immunol 2001;31(1):64–71. PubMed PMID: 11169439. [14] Hoffman LR, Ramsey BW. Cystic fibrosis therapeutics: the road ahead. Chest 2013;143(1):207–13. http://dx.doi.org/10.1378/chest.12-1639. PubMed PMID: 23276843; PubMed Central PMCID: PMC3610617. [15] Ramsey BW, Davies J, McElvaney NG, Tullis E, Bell SC, Drˇevínek P, et al. A CFTR potentiator in patients with cystic fibrosis and the G551D mutation. N Engl J Med 2011;365(18):1663–72. http://dx.doi.org/10.1056/NEJMoa1105185. PubMed PMID: 22047557; PubMed Central PMCID: PMC3230303.