title: The effects of honey on the inflammatory response of cells with respect to wound healing

authors: Harcourt, Nichola Robyn

abstract: The primary aim of this thesis was to test the effect of two types of honey, a manuka honey and an Otago pasture honey, on a range of key cell types involved in the inflammatory response of wound healing. A range of in vitro assays were used to test the effect of honey on various cells with a view to the implications of the results for wound healing, focusing on the mechanisms by which honey has been observed to have beneficial effects on both scar formation and inflammation. 
To investigate whether honey could stimulate bovine T cells to proliferate in vitro, both MTT and BrDU assays of proliferation and flow cytometry analysis were used. It was found that low concentrations of honey stimulated resting T cells to proliferate and express the IL-2 receptor in a dose-dependent manner with progressive dilution. This suggests that honey contains lymphomitogenic factors. Manuka honey was stimulatory at higher dilutions than pasture honey. Low concentrations of honey induced cell division profiles similar to those obtained with Con A-stimulated cells. The stimulatory activity of honey was found to be in a high molecular weight fraction. Sugars alone had no effect on T cell proliferation, as demonstrated by use of artificial honey (a syrup of sugars as in honey). 
The ability for honey to induce messenger RNA expression for key cytokines involved in wound healing was investigated. Conventional reverse transcriptase-PCR was used to detect the production of mRNA for honey at 0.25% concentrations for various times (0-24 h). The more sensitive molecular technique, quantitative real-time RT-PCR was then used to quantify the abundance of cytokine mRNA transcripts expressed in bovine blood exposed to 0.25% manuka honey as compared with Con A or control cultures. Transcriptional activity of ten genes, IL-1, IL-5, IL-12, IL-18, IFN-𝛾, HSP70, HSP90, iNOS, TNF-α, and TGF-β were studied at the mRNA level during a 0-24 h exposure of whole blood to honey. To test for any modulatory effects of honey on gene expression in an inflammatory model, whole blood was exposed to honey at the same time as LPS and the mRNA expression for the genes was measured. The results show that honey upregulates a wide range of mediators, including TNF-α, IL-1β, and TGF-β, and this supports the hypothesis that honey induces cytokine release. Honey gave a transient and moderate induction of cytokine mRNA compared with a massive and prolonged induction by the mitogens, Con A and LPS. The inclusion of honey with LPS led to a reduced expression of mRNA for key inflammatory mediators, including iNOS and TGF-β, compared with LPS alone. This supports the hypothesis that honey modulates inflammation. 
To investigate whether honey could induce THP-1 monocytes to release TNF-α, bioassays using WEHI cells were carried out to measure TNF-α production after the monocytes were exposed to honey. Honey was found to stimulate release of TNF-α by the monocytes when at a range of concentrations between 0.000025-0.1%, with no differences between the levels produced at the various concentrations of honey. At concentrations of honey from 0.25-1% the TNF-α production decreased as the concentration of honey increased. This may indicate that an anti-inflammatory action overrides the stimulatory effect at concentrations of honey greater than 0.25%. Sugar content had no effect upon TNF-α release, as demonstrated by the artificial honey control. There were no differences between honey types (manuka honey and pasture honey) in induction of TNF-α release. Time-course analysis confirmed that a 4-6 h incubation period of cells with 0.25% honey gave maximal TNF-α production. A 2 h minimum exposure period of cells to honey was critical for TNF-α production. Incubation of LPS-stimulated monocytes with honey had no effect on their subsequent TNF-α production. A good correlation was found between the TNF measurements detected by ELISA and the WEHI Bioassay. 
To test whether honey could modulate LPS-stimulated NO production by THP-1 monocytes and bovine peripheral blood mononuclear cells, Greiss assays were performed. Both manuka honey and pasture honey at 0.5% and 1% concentrations suppressed LPS-induced nitrite release in a dose-wise manner, indicating modulation of nitric oxide production. Manuka honey had a more potent modulating effect on LPS driven nitrite production than pasture honey, and maintained activity at 0.25%. Sugars alone had no effect. High molecular weight dialysis fractions of either honey contained the activity, but some of the activity was lost by fractionation. An ether extract of manuka honey led to the greatest modulation of nitrite production by LPS-stimulated monocytes.  
To investigate whether honey has an effect on phagocytosis, whole blood was incubated with honey and the ability of neutrophils to take up fluorescent-labelled bacteria was measured using the Phagotest® assay. The artificial honey control provided clear evidence that low concentrations of honey (optimal at 0.25%) induce phagocytosis by neutrophils due to the supply of sugars. Manuka honey had an additional opsonizing effect on bacteria, which enhances the phagocytic response beyond that seen with sugars alone. 
The effects of honey on tight junction (TJ) resistance were assessed for MDCK cell monolayers subjected to an EGTA challenge. It was found that manuka honey and pasture honey have protective effects on TJ following the challenge, and enhance postchallenge recovery of transepithelial resistance. Manuka honey had greater modulatory activity on TJ with increased concentration from 0.1-1%, and 1% concentrations of both honeys gave the greatest protective effects. Manuka honey appeared to have greater protective effects than pasture honey. Application of manuka honey (at 1% concentrations) to both the apical and basolateral sides of the MDCK cell monolayer significantly enhanced TJ tightness beyond the control. Dialysis of the honey confirmed that the high molecular weight fraction contained the active component. Diffusate fractions from either honey type had no effect on TJ. Artificial honey had no effect. 
The effect of various honey concentrations on the proliferation of the 3T3-Ll fibroblast cell-line in vitro was investigated using MTT proliferation assays. Both manuka honey and pasture honey (0.25%) increased fibroblast proliferation. Artificial honey had no effect on fibroblast proliferation, indicating sugars have no role in mitogenic activity. This suggests that honey contains factors which directly promote cell division in fibroblasts. 
An in vitro model was used to test whether honey-induced factors produced by peripheral blood mononuclear cells (PBMC) could activate fibroblast proliferation. These assays were performed to examine whether honey could have an indirect stimulatory effect on fibroblasts. Incubating fibroblasts with supernatants derived from honey-stimulated PBMCs (at 0.025% concentrations of honey) led to significant fibroblast proliferation. Low concentrations of honey (less than 0.1%) do not directly stimulate fibroblast proliferation, therefore factors produced by honey-stimulated PBMCs must promote fibroblast proliferation. A high molecular weight fraction of manuka honey obtained from dialysis contained the active component. The diffusate obtained by dialysis (containing sugars) had no activity. 
To investigate whether honey could modulate the response of fibroblasts to an inflammatory agent, fibroblasts were exposed to honey for various times prior to and at the same time as IL-1β. Honey did not augment fibroblast proliferation when added at the same time as IL-1β. Prior incubation of fibroblasts with honey (0.25-1%) for 2 h before IL-1β-stimulation decreased the cell response to IL-1β, and this anti-inflammatory active component was of a high molecular weight.
It is proposed, on the basis of this in vitro study, that honey provides a neatly controlled therapy for optimising tissue repair, with potential for use in inflammatory disorders. It is the central thesis of this study that the stimulatory agent in honey induces cytokine production necessary for healing to occur, but that the oxidant species produced by these cells are effectively regulated by a second agent, thereby creating a feedback-regulated delivery system. The results presented in the current study suggest that honey can both stimulate and modulate cell activity, and show that honey interferes with a large number of regulatory steps in the inflammatory pathway, e.g. cell division, transcription, tight junction integrity, production of oxidant species. While the stimulatory activity was observed at lower concentrations, the modulatory effects required higher concentrations of honey. The dual ability for honey to stimulate moderate cellular activation in the absence of an immune stimulus, yet not augment mitogenic stimulation, and in some cases to modulate cell response to a mitogen, indicates that it will promote healing without setting up harmful inflammation. 
If further experiments confirm this to be the case, honey will have potential for therapeutic application. The work also identifies some new areas of research, which if completed would further enhance the understanding of the role honey plays in tissue healing.

text: The effects of honey on the inflammatory response of cells with respect to wound healing The primary aim of this thesis was to test the effect of two types of honey, a manuka honey and an Otago pasture honey, on a range of key cell types involved in the inflammatory response of wound healing. A range of in vitro assays were used to test the effect of honey on various cells with a view to the implications of the results for wound healing, focusing on the mechanisms by which honey has been observed to have beneficial effects on both scar formation and inflammation. 
To investigate whether honey could stimulate bovine T cells to proliferate in vitro, both MTT and BrDU assays of proliferation and flow cytometry analysis were used. It was found that low concentrations of honey stimulated resting T cells to proliferate and express the IL-2 receptor in a dose-dependent manner with progressive dilution. This suggests that honey contains lymphomitogenic factors. Manuka honey was stimulatory at higher dilutions than pasture honey. Low concentrations of honey induced cell division profiles similar to those obtained with Con A-stimulated cells. The stimulatory activity of honey was found to be in a high molecular weight fraction. Sugars alone had no effect on T cell proliferation, as demonstrated by use of artificial honey (a syrup of sugars as in honey). 
The ability for honey to induce messenger RNA expression for key cytokines involved in wound healing was investigated. Conventional reverse transcriptase-PCR was used to detect the production of mRNA for honey at 0.25% concentrations for various times (0-24 h). The more sensitive molecular technique, quantitative real-time RT-PCR was then used to quantify the abundance of cytokine mRNA transcripts expressed in bovine blood exposed to 0.25% manuka honey as compared with Con A or control cultures. Transcriptional activity of ten genes, IL-1, IL-5, IL-12, IL-18, IFN-𝛾, HSP70, HSP90, iNOS, TNF-α, and TGF-β were studied at the mRNA level during a 0-24 h exposure of whole blood to honey. To test for any modulatory effects of honey on gene expression in an inflammatory model, whole blood was exposed to honey at the same time as LPS and the mRNA expression for the genes was measured. The results show that honey upregulates a wide range of mediators, including TNF-α, IL-1β, and TGF-β, and this supports the hypothesis that honey induces cytokine release. Honey gave a transient and moderate induction of cytokine mRNA compared with a massive and prolonged induction by the mitogens, Con A and LPS. The inclusion of honey with LPS led to a reduced expression of mRNA for key inflammatory mediators, including iNOS and TGF-β, compared with LPS alone. This supports the hypothesis that honey modulates inflammation. 
To investigate whether honey could induce THP-1 monocytes to release TNF-α, bioassays using WEHI cells were carried out to measure TNF-α production after the monocytes were exposed to honey. Honey was found to stimulate release of TNF-α by the monocytes when at a range of concentrations between 0.000025-0.1%, with no differences between the levels produced at the various concentrations of honey. At concentrations of honey from 0.25-1% the TNF-α production decreased as the concentration of honey increased. This may indicate that an anti-inflammatory action overrides the stimulatory effect at concentrations of honey greater than 0.25%. Sugar content had no effect upon TNF-α release, as demonstrated by the artificial honey control. There were no differences between honey types (manuka honey and pasture honey) in induction of TNF-α release. Time-course analysis confirmed that a 4-6 h incubation period of cells with 0.25% honey gave maximal TNF-α production. A 2 h minimum exposure period of cells to honey was critical for TNF-α production. Incubation of LPS-stimulated monocytes with honey had no effect on their subsequent TNF-α production. A good correlation was found between the TNF measurements detected by ELISA and the WEHI Bioassay. 
To test whether honey could modulate LPS-stimulated NO production by THP-1 monocytes and bovine peripheral blood mononuclear cells, Greiss assays were performed. Both manuka honey and pasture honey at 0.5% and 1% concentrations suppressed LPS-induced nitrite release in a dose-wise manner, indicating modulation of nitric oxide production. Manuka honey had a more potent modulating effect on LPS driven nitrite production than pasture honey, and maintained activity at 0.25%. Sugars alone had no effect. High molecular weight dialysis fractions of either honey contained the activity, but some of the activity was lost by fractionation. An ether extract of manuka honey led to the greatest modulation of nitrite production by LPS-stimulated monocytes.  
To investigate whether honey has an effect on phagocytosis, whole blood was incubated with honey and the ability of neutrophils to take up fluorescent-labelled bacteria was measured using the Phagotest® assay. The artificial honey control provided clear evidence that low concentrations of honey (optimal at 0.25%) induce phagocytosis by neutrophils due to the supply of sugars. Manuka honey had an additional opsonizing effect on bacteria, which enhances the phagocytic response beyond that seen with sugars alone. 
The effects of honey on tight junction (TJ) resistance were assessed for MDCK cell monolayers subjected to an EGTA challenge. It was found that manuka honey and pasture honey have protective effects on TJ following the challenge, and enhance postchallenge recovery of transepithelial resistance. Manuka honey had greater modulatory activity on TJ with increased concentration from 0.1-1%, and 1% concentrations of both honeys gave the greatest protective effects. Manuka honey appeared to have greater protective effects than pasture honey. Application of manuka honey (at 1% concentrations) to both the apical and basolateral sides of the MDCK cell monolayer significantly enhanced TJ tightness beyond the control. Dialysis of the honey confirmed that the high molecular weight fraction contained the active component. Diffusate fractions from either honey type had no effect on TJ. Artificial honey had no effect. 
The effect of various honey concentrations on the proliferation of the 3T3-Ll fibroblast cell-line in vitro was investigated using MTT proliferation assays. Both manuka honey and pasture honey (0.25%) increased fibroblast proliferation. Artificial honey had no effect on fibroblast proliferation, indicating sugars have no role in mitogenic activity. This suggests that honey contains factors which directly promote cell division in fibroblasts. 
An in vitro model was used to test whether honey-induced factors produced by peripheral blood mononuclear cells (PBMC) could activate fibroblast proliferation. These assays were performed to examine whether honey could have an indirect stimulatory effect on fibroblasts. Incubating fibroblasts with supernatants derived from honey-stimulated PBMCs (at 0.025% concentrations of honey) led to significant fibroblast proliferation. Low concentrations of honey (less than 0.1%) do not directly stimulate fibroblast proliferation, therefore factors produced by honey-stimulated PBMCs must promote fibroblast proliferation. A high molecular weight fraction of manuka honey obtained from dialysis contained the active component. The diffusate obtained by dialysis (containing sugars) had no activity. 
To investigate whether honey could modulate the response of fibroblasts to an inflammatory agent, fibroblasts were exposed to honey for various times prior to and at the same time as IL-1β. Honey did not augment fibroblast proliferation when added at the same time as IL-1β. Prior incubation of fibroblasts with honey (0.25-1%) for 2 h before IL-1β-stimulation decreased the cell response to IL-1β, and this anti-inflammatory active component was of a high molecular weight.
It is proposed, on the basis of this in vitro study, that honey provides a neatly controlled therapy for optimising tissue repair, with potential for use in inflammatory disorders. It is the central thesis of this study that the stimulatory agent in honey induces cytokine production necessary for healing to occur, but that the oxidant species produced by these cells are effectively regulated by a second agent, thereby creating a feedback-regulated delivery system. The results presented in the current study suggest that honey can both stimulate and modulate cell activity, and show that honey interferes with a large number of regulatory steps in the inflammatory pathway, e.g. cell division, transcription, tight junction integrity, production of oxidant species. While the stimulatory activity was observed at lower concentrations, the modulatory effects required higher concentrations of honey. The dual ability for honey to stimulate moderate cellular activation in the absence of an immune stimulus, yet not augment mitogenic stimulation, and in some cases to modulate cell response to a mitogen, indicates that it will promote healing without setting up harmful inflammation. 
If further experiments confirm this to be the case, honey will have potential for therapeutic application. The work also identifies some new areas of research, which if completed would further enhance the understanding of the role honey plays in tissue healing.

year: 2005

title: Effects of sample-set size on delayed-matching-to-sample

authors: Adamson, Coreen

abstract: In a series of experiments the discriminative performance of university students was assessed using a Delayed-Matching-to-Sample task. White disks, presented on computer screens, served as stimuli. For the general DMTS task, used here, a sample stimulus was presented to the participant. After a variable delay (0.05, 4, 8 or 16 s) two comparison stimuli were presented on the computer screen. One of these comparison stimuli was the same size as the sample stimulus (matching comparison) and the other was slightly larger or smaller than the sample stimulus (non-matching comparison). The sample stimuli were not intended to appear as comparison stimuli and for each of the sample stimuli there were two possible non-matching comparison sample stimuli, one larger and one smaller than the sample stimulus. The first three experiments showed that it was possible to use a titration procedure to get similar levels of performance across participants, at zero delay. During the titration procedure the size of the non-matching comparison stimuli was changed, for each participant until they were 100% correct at 0.05 s delay. The first two of these experiments also showed that there were generally only small decreases in performance as the delay between the presentation of the sample stimuli and the comparison stimuli was increased. The fourth experiment showed that performance on the DMTS task did not change when feedback was given for correct responses. The fifth and sixth experiments showed that as the number of sample stimuli used in the DMTS task, was increased there was a systematic decrease in a, a measure of discrimination at zero delay, but no systematic change in b, a measure of the rate of decrement in performance with increasing delay. It was not clear whether this decrease in a was a product of the increase in the sample-set size or of the associated decrease in the correspondence of sample stimuli on consecutive trial pairs. Experiment 7 suggested that, when the number of sample stimuli used for the DMTS was held constant and the ratio of corresponding to non-corresponding trial pairs was varied, there was no systematic change in either a or b. However, the results of this experiment were confounded by a ceiling effect and so a different procedure was required. The procedure used for the remaining experiments was a two-sample DMTS task, where the sample stimuli were also used as comparison stimuli. This type of DMTS task is similar to that typically used in discrimination tasks with animal subjects. The eighth and ninth experiments were designed to investigate whether this type of procedure could be successfully used with human participants and found that it could be. Experiment 10 also used this type of two-sample DMTS task to investigate the effect of decreasing the ratio of corresponding to non-corresponding trial pairs. It was found that, when there was no ceiling effect, varying the ratio of corresponding to non-corresponding trial pairs did not result in a systematic change in either a or b as in Experiment 7. These results are discussed in terms of the effects of range, sample-set size and of the ratio of corresponding to non-corresponding trial pairs on human performance on DMTS.

text: Effects of sample-set size on delayed-matching-to-sample In a series of experiments the discriminative performance of university students was assessed using a Delayed-Matching-to-Sample task. White disks, presented on computer screens, served as stimuli. For the general DMTS task, used here, a sample stimulus was presented to the participant. After a variable delay (0.05, 4, 8 or 16 s) two comparison stimuli were presented on the computer screen. One of these comparison stimuli was the same size as the sample stimulus (matching comparison) and the other was slightly larger or smaller than the sample stimulus (non-matching comparison). The sample stimuli were not intended to appear as comparison stimuli and for each of the sample stimuli there were two possible non-matching comparison sample stimuli, one larger and one smaller than the sample stimulus. The first three experiments showed that it was possible to use a titration procedure to get similar levels of performance across participants, at zero delay. During the titration procedure the size of the non-matching comparison stimuli was changed, for each participant until they were 100% correct at 0.05 s delay. The first two of these experiments also showed that there were generally only small decreases in performance as the delay between the presentation of the sample stimuli and the comparison stimuli was increased. The fourth experiment showed that performance on the DMTS task did not change when feedback was given for correct responses. The fifth and sixth experiments showed that as the number of sample stimuli used in the DMTS task, was increased there was a systematic decrease in a, a measure of discrimination at zero delay, but no systematic change in b, a measure of the rate of decrement in performance with increasing delay. It was not clear whether this decrease in a was a product of the increase in the sample-set size or of the associated decrease in the correspondence of sample stimuli on consecutive trial pairs. Experiment 7 suggested that, when the number of sample stimuli used for the DMTS was held constant and the ratio of corresponding to non-corresponding trial pairs was varied, there was no systematic change in either a or b. However, the results of this experiment were confounded by a ceiling effect and so a different procedure was required. The procedure used for the remaining experiments was a two-sample DMTS task, where the sample stimuli were also used as comparison stimuli. This type of DMTS task is similar to that typically used in discrimination tasks with animal subjects. The eighth and ninth experiments were designed to investigate whether this type of procedure could be successfully used with human participants and found that it could be. Experiment 10 also used this type of two-sample DMTS task to investigate the effect of decreasing the ratio of corresponding to non-corresponding trial pairs. It was found that, when there was no ceiling effect, varying the ratio of corresponding to non-corresponding trial pairs did not result in a systematic change in either a or b as in Experiment 7. These results are discussed in terms of the effects of range, sample-set size and of the ratio of corresponding to non-corresponding trial pairs on human performance on DMTS.

year: 2002

title: The centrality of relationships for pedagogy: The Whanaungatanga Thesis

authors: Bishop, Russell

subjects: addressing educational disparities

abstract: Te Kotahitanga is a research and professional development project that seeks to reduce educational disparities between indigenous Māori students and their non-Māori peers in New Zealand secondary schools. While evidence of the impact of the project on teachers’ practice and the associated gains made by Māori students has been published previously, in order for the work of Te Kotahitanga to contribute to the broader educational research community, its pedagogical premises require empirical verification. To do so, we must first establish the validity of the pedagogical data by addressing two questions: (a) To what degree can the data gathered in the collaborative processes of Te Kotahitanga be used as a measure of pedagogical quality? and (b) Do these data support the foundational hypothesis of the project, that “extended family” relationships, as understood by Māori people when using the Māori term, whanaungatanga, are a central necessary component of overall pedagogical quality? This article provides an account of the context of this work then presents an analysis directed to these questions in turn. First, our analysis of the observational data gathered during the Te Kotahitanga professional development process is presented, followed by the measures that were developed for each of the main dimensions of pedagogy addressed in this work. Second, using these measures we present our analysis of the inter-relation among these dimensions of pedagogy to test the Whanaungatanga pedagogical thesis.

text: The centrality of relationships for pedagogy: The Whanaungatanga Thesis Te Kotahitanga is a research and professional development project that seeks to reduce educational disparities between indigenous Māori students and their non-Māori peers in New Zealand secondary schools. While evidence of the impact of the project on teachers’ practice and the associated gains made by Māori students has been published previously, in order for the work of Te Kotahitanga to contribute to the broader educational research community, its pedagogical premises require empirical verification. To do so, we must first establish the validity of the pedagogical data by addressing two questions: (a) To what degree can the data gathered in the collaborative processes of Te Kotahitanga be used as a measure of pedagogical quality? and (b) Do these data support the foundational hypothesis of the project, that “extended family” relationships, as understood by Māori people when using the Māori term, whanaungatanga, are a central necessary component of overall pedagogical quality? This article provides an account of the context of this work then presents an analysis directed to these questions in turn. First, our analysis of the observational data gathered during the Te Kotahitanga professional development process is presented, followed by the measures that were developed for each of the main dimensions of pedagogy addressed in this work. Second, using these measures we present our analysis of the inter-relation among these dimensions of pedagogy to test the Whanaungatanga pedagogical thesis.

year: 2013