title: Investigating the Anti-inflammatory Activity of Honey

authors: Bean, Amanda

subjects: honey

abstract: Honey has various bioactivities with health-benefiting properties.  It has been registered for use in wound dressings but before it can be registered with regulatory authorities for sale with claims for therapeutic actions the components responsible for these bioactivities must be identified to allow standardisation.  This thesis focuses on the anti-inflammatory activity of honey.  Emphasis was put on identifying the mode of action of the anti-inflammatory component, creating an in vitro assay to measure the anti-inflammatory activity of individual honeys and attempting to identify and characterise the component(s) responsible for the activity.  

A cell line (THP-1) was selected for this study based on its macrophage phenotype upon activation and its role as an inflammatory regulator.  Initially this cell line was characterised to better understand the treatments that activate its phagocytosis ability, mobility, phenotype and gene expression profile.  The cell line was then monitored for the change in expression of its pro-inflammatory and anti-inflammatory cytokines and growth factors in response to common cell-activating treatments (LPS, PMA and Vit-D3) and Manuka honey.  It was found that Manuka honey increased the expression of pro-inflammatory cytokines  TNF-α and anti-inflammatory cytokines IL-10, IL-1ra and the growth factors PDGF and TGF-β.  This indicated that Manuka honey may allow inflammation to proceed at a modulated level, allowing healing to occur.    
The cell line THP-1 was further investigated as a model of a phagocyte when activated.  A phagocytosis assay using latex particles was utilised to study the effect of honey on the ability of cells to phagocytose.  It was found that honey reduced phagocytosis, and that Manuka honey had a superior ability to other honey types to do this.  The activity was isolated to the high molecular weight compounds in honey.  As phagocytosis produces large amounts of reactive oxygen species (ROS) and ROS are pro-inflammatory, the ROS produced by phagocytising THP-1 cells was investigated using a dichlorofluorescein assay.  It was found that ROS produced by phagocytising THP-1 cells decrease in the presence of Manuka honey but not artificial honey or Rewarewa honey which has high anti-oxidant activity.  This indicated that the effect of Manuka in decreasing ROS was not solely due to its osmotic effects or anti-oxidant capabilities.  As phagocytosis starts the inflammatory cascade, it was hypothesised that the phagocytosis-inhibiting component resulted in honey having anti-inflammatory activity.
Next, attempts were made to identify the anti-inflammatory component.   Manuka honey was separated using chromatography and the fractions obtained assayed for their inhibitory activity in the phagocytosis assay.  The active fractions were found to contain MRJP-1 and to a small extent MRJP-3, which were identified by MALDI-TOF mass spectrometry.  It was confirmed that these were glycoproteins with a high mannose content by using a ConA lectin column.  
Next a mode of action for the glycoproteins was investigated.  It was demonstrated by using mannan that honey was acting on a mannose-binding phagocytic receptor on the cell surface of macrophages, most likely the mannose receptor.  It was hypothesised that with the glycoproteins bound to the receptor, the cell was unable to have phagocytosis triggered thus ROS production and expression of inflammatory cytokines in response to ROS produced were prevented.  
A wide range of honey varieties was assayed and the anti-inflammatory activity was found in high levels only in Manuka honey and to a lesser extent in Kanuka and Rewarewa.  To identify why this activity was much greater in Manuka honey, as all honey contains the glycoproteins MRJP-1 and MRJP-3, the effect of the recently discovered high levels of MGO in Manuka honey was investigated.  It was found that Manuka honey had a high fluorescence that was not seen in other honey types to the same extent and that this fluorescence was due to MGO modifying the proteins in honey.  It was found that this fluorescence could be used as an indicator of anti-inflammatory activity for Manuka honey and that this fluorescence, and anti-inflammatory activity increased over time upon incubation of honey with high MGO levels.  
The anti-inflammatory activity seen in the phagocytosis assay was compared with that seen in a commonly used in vivo assay of anti-inflammatory activity, the HET-CAM assay.  The HET-CAM assay depends on the experimental production of inflammation and the reduction of this inflammation with an anti-inflammatory agent on the chorionic membrane of a fertilised hen’s egg.  It was concluded that highly fluorescent Manuka honey at a concentration of 5% had and anti-inflammatory effect equivalent to that of 5 mg/ml hydrocortisone, a commonly prescribed anti-inflammatory drug.
The anti-inflammatory activity seen in the honeys assayed in the in vivo HET-CAM assay concurred with the results for the same honeys assayed in the phagocytosis assay which indicated that the phagocytosis assay was a reliable indicator for assaying the anti-inflammatory activity of honey in vitro.

text: Investigating the Anti-inflammatory Activity of Honey Honey has various bioactivities with health-benefiting properties.  It has been registered for use in wound dressings but before it can be registered with regulatory authorities for sale with claims for therapeutic actions the components responsible for these bioactivities must be identified to allow standardisation.  This thesis focuses on the anti-inflammatory activity of honey.  Emphasis was put on identifying the mode of action of the anti-inflammatory component, creating an in vitro assay to measure the anti-inflammatory activity of individual honeys and attempting to identify and characterise the component(s) responsible for the activity.  

A cell line (THP-1) was selected for this study based on its macrophage phenotype upon activation and its role as an inflammatory regulator.  Initially this cell line was characterised to better understand the treatments that activate its phagocytosis ability, mobility, phenotype and gene expression profile.  The cell line was then monitored for the change in expression of its pro-inflammatory and anti-inflammatory cytokines and growth factors in response to common cell-activating treatments (LPS, PMA and Vit-D3) and Manuka honey.  It was found that Manuka honey increased the expression of pro-inflammatory cytokines  TNF-α and anti-inflammatory cytokines IL-10, IL-1ra and the growth factors PDGF and TGF-β.  This indicated that Manuka honey may allow inflammation to proceed at a modulated level, allowing healing to occur.    
The cell line THP-1 was further investigated as a model of a phagocyte when activated.  A phagocytosis assay using latex particles was utilised to study the effect of honey on the ability of cells to phagocytose.  It was found that honey reduced phagocytosis, and that Manuka honey had a superior ability to other honey types to do this.  The activity was isolated to the high molecular weight compounds in honey.  As phagocytosis produces large amounts of reactive oxygen species (ROS) and ROS are pro-inflammatory, the ROS produced by phagocytising THP-1 cells was investigated using a dichlorofluorescein assay.  It was found that ROS produced by phagocytising THP-1 cells decrease in the presence of Manuka honey but not artificial honey or Rewarewa honey which has high anti-oxidant activity.  This indicated that the effect of Manuka in decreasing ROS was not solely due to its osmotic effects or anti-oxidant capabilities.  As phagocytosis starts the inflammatory cascade, it was hypothesised that the phagocytosis-inhibiting component resulted in honey having anti-inflammatory activity.
Next, attempts were made to identify the anti-inflammatory component.   Manuka honey was separated using chromatography and the fractions obtained assayed for their inhibitory activity in the phagocytosis assay.  The active fractions were found to contain MRJP-1 and to a small extent MRJP-3, which were identified by MALDI-TOF mass spectrometry.  It was confirmed that these were glycoproteins with a high mannose content by using a ConA lectin column.  
Next a mode of action for the glycoproteins was investigated.  It was demonstrated by using mannan that honey was acting on a mannose-binding phagocytic receptor on the cell surface of macrophages, most likely the mannose receptor.  It was hypothesised that with the glycoproteins bound to the receptor, the cell was unable to have phagocytosis triggered thus ROS production and expression of inflammatory cytokines in response to ROS produced were prevented.  
A wide range of honey varieties was assayed and the anti-inflammatory activity was found in high levels only in Manuka honey and to a lesser extent in Kanuka and Rewarewa.  To identify why this activity was much greater in Manuka honey, as all honey contains the glycoproteins MRJP-1 and MRJP-3, the effect of the recently discovered high levels of MGO in Manuka honey was investigated.  It was found that Manuka honey had a high fluorescence that was not seen in other honey types to the same extent and that this fluorescence was due to MGO modifying the proteins in honey.  It was found that this fluorescence could be used as an indicator of anti-inflammatory activity for Manuka honey and that this fluorescence, and anti-inflammatory activity increased over time upon incubation of honey with high MGO levels.  
The anti-inflammatory activity seen in the phagocytosis assay was compared with that seen in a commonly used in vivo assay of anti-inflammatory activity, the HET-CAM assay.  The HET-CAM assay depends on the experimental production of inflammation and the reduction of this inflammation with an anti-inflammatory agent on the chorionic membrane of a fertilised hen’s egg.  It was concluded that highly fluorescent Manuka honey at a concentration of 5% had and anti-inflammatory effect equivalent to that of 5 mg/ml hydrocortisone, a commonly prescribed anti-inflammatory drug.
The anti-inflammatory activity seen in the honeys assayed in the in vivo HET-CAM assay concurred with the results for the same honeys assayed in the phagocytosis assay which indicated that the phagocytosis assay was a reliable indicator for assaying the anti-inflammatory activity of honey in vitro.

year: 2012

title: Bayesian network classifiers in Weka

authors: Bouckaert, Remco R.

abstract: Various Bayesian network classifier learning algorithms are implemented in Weka [10].This note provides some user documentation and implementation details.
Summary of main capabilities:
_Structure learning of Bayesian networks using various hill climbing (K2, B, etc) and general purpose (simulated annealing, tabu search) algorithms.
_Local score metrics implemented; Bayes, BDe, MDL, entropy, AIC.
_Global score metrics implemented; leave one out cv, k-fold cv and cumulative cv.
_Conditional independence based causal recovery algorithm available.
_Parameter estimation using direct estimates and Bayesian model averaging.
_GUI for easy inspection of Bayesian networks.
_Part of Weka allowing systematic experiments to compare Bayes net performance with general purpose classi_ers like C4.5, nearest neighbor, support vector, etc.
_Source code available under GPL allows for integration in other systems and makes it easy to extend.

text: Bayesian network classifiers in Weka Various Bayesian network classifier learning algorithms are implemented in Weka [10].This note provides some user documentation and implementation details.
Summary of main capabilities:
_Structure learning of Bayesian networks using various hill climbing (K2, B, etc) and general purpose (simulated annealing, tabu search) algorithms.
_Local score metrics implemented; Bayes, BDe, MDL, entropy, AIC.
_Global score metrics implemented; leave one out cv, k-fold cv and cumulative cv.
_Conditional independence based causal recovery algorithm available.
_Parameter estimation using direct estimates and Bayesian model averaging.
_GUI for easy inspection of Bayesian networks.
_Part of Weka allowing systematic experiments to compare Bayes net performance with general purpose classi_ers like C4.5, nearest neighbor, support vector, etc.
_Source code available under GPL allows for integration in other systems and makes it easy to extend.

year: 2004-09-01

title: The Te Kotahitanga Observation Tool: Development, use, reliability and validity.

authors: Berryman, Mere

subjects: chool reform

abstract: Te Kotahitanga is a New Zealand school reform project aimed at improving the pedagogical contexts in mainstream classrooms in which the indigenous Māori students have traditionally been marginalised. It does this by assisting teachers to implement an Effective Teaching Profile. Part of this process uses an observation tool to monitor the degree to which participating teachers are incorporating the interactions and relationships described in the Effective Teaching Profile into their day-to-day teaching. Given the central importance of these tasks, the Te Kotahitanga team undertook to test the observation tool for measurement reliability and validity. In order to undertake this study, the team conducted synchronous observations amongst trainers (the Professional Development Coordinator and Regional Coordinators) to ascertain their level of consistency when using the tool. The team then conducted synchronous observations between trainers and 38 in-school facilitators in the 12 schools involved in Phase 3 of the project. In total 41 teachers were observed and over 200 Māori students were involved in these observations. This study suggests that the tool can produce consistent and reliable results when observers have been effectively trained.

text: The Te Kotahitanga Observation Tool: Development, use, reliability and validity. Te Kotahitanga is a New Zealand school reform project aimed at improving the pedagogical contexts in mainstream classrooms in which the indigenous Māori students have traditionally been marginalised. It does this by assisting teachers to implement an Effective Teaching Profile. Part of this process uses an observation tool to monitor the degree to which participating teachers are incorporating the interactions and relationships described in the Effective Teaching Profile into their day-to-day teaching. Given the central importance of these tasks, the Te Kotahitanga team undertook to test the observation tool for measurement reliability and validity. In order to undertake this study, the team conducted synchronous observations amongst trainers (the Professional Development Coordinator and Regional Coordinators) to ascertain their level of consistency when using the tool. The team then conducted synchronous observations between trainers and 38 in-school facilitators in the 12 schools involved in Phase 3 of the project. In total 41 teachers were observed and over 200 Māori students were involved in these observations. This study suggests that the tool can produce consistent and reliable results when observers have been effectively trained.

year: 2011