The trapping of methylglyoxal by phenolic acids : effect on antioxidant and antibacterial activity

Methylglyoxal (MGO) is a reactive carbonyl species found in Manuka honey reported to cause advanced glycation end products (AGE) formation. AGE’s increase the risk for hyperglycaemia resulting in neuropathy, arteriosclerosis, retinopathy and Alzheimer’s disease. Phenolic acids such as pyrogallol (PY) are known to trap MGO, lessening the harmful effects of MGO as an AGE precursor. However, MGO is also a very effective antibacterial agent therefore; its trapping could have negative side effects. Manuka honey contains both phenolic acids such as gallic acid (GA), caffeic acid (CA) as well as MGO and it is unknown whether trapping of MGO by phenolic acids reduces the antioxidant activity of phenolic acids or the antibacterial activity of MGO. Phenolic acids PY, GA and CA were combined with MGO in a 1:1 and 1:2 ratio. The trapping of MGO with polyphenolic acids was determined with Liquid chromatography-mass spectrometry (LCMS). Total polyphenolic acids (TPC) was determined with the TPC assay. Antioxidant activity was determined with 2,2-diphenyl-2-picrylhydrazyl (DPPH), Trolox equivalent antioxidant capacity (TEAC) and Oxygen Radical Absorbance Capacity (ORAC) assays. The effect on cell number and viability was determined with crystal violet and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays on Caco-2 and SC-1 cells. Cellular antioxidant activity was determined with Dichlorodihydrofluorescein diacetate assay. Lastly, antibacterial activity was determined with the turbidity assay on Gram positive B. subtilis and Gram negative E. coli and the ultrastructural morphology of B. subtilis was further investigated with scanning electron microscopy. PY was the only phenolic acid used with trapping ability, forming mono- and di- adducts with MGO reported with the LCMS results, resulting in a decrease in TPC and antioxidant activity measured with the DPPH assay. GA did not show any alteration when combined with MGO at 1:1 and 1:2 ratio in all antioxidant content and activity assays. The antioxidant content of CA in combination with MGO was decreased, although its antioxidant activity (DPPH) was increased at 1:2 ratio. The antioxidant activity measured with the ORAC assay was increased with PY and CA combined with MGO. TEAC assay did not show any changes when phenolic acids were combined with MGO a 1:1 and 1:2 ratio. The cytotoxicity of phenolic acids combined with MGO did not cause a change in cell number or viability of SC-1 and Caco-2 cells. MGO and phenolic acids alone and in combination did no cause oxidative damage (without 2,2'-Azobis(2- amidinopropane) dihydrochloride (AAPH). All phenolic acids in combination with MGO retained the ability to reduce AAPH induced oxidative damage. The polyphenolic acids showed minor inhibition of the growth of B. subtilis and E. coli. PY only reduced the antibacterial activity of MGO at a 1:1 combination of B. subtilis. GA and CA did not alter the antibacterial activity of MGO when combined at 1:1 or 1:2 ratio. This study showed that phenolic acids with the ability to trap MGO can be altered by the mono- and di-MGO adduct formation, altering its antioxidant activity and can further alter the antibacterial activity of MGO.Dissertation (MSc)--University of Pretoria, 2017.AnatomyMScUnrestricte

Unbiased Profiling Reveals Compartmentalization of Unconventional T-Cells Within the Intestinal Mucosa Irrespective of HIV Infection

The intestinal mucosa is enriched for unconventional T-cells, including mucosal associated invariant T-cells (MAIT), invariant natural killer T-cells (iNKT) and gamma delta T-cells. These cells are activated by bacterial metabolites, lipid antigens and cytokines, and are important for intestinal barrier integrity. The loss of gut homeostasis observed in HIV infection is central to disease pathogenesis, and studies have highlighted impairment of particular unconventional T-cell subsets within a specific gut compartment. However, although the small and large intestine are distinct niches, the overall impact of HIV on unconventional T-cells across the gut mucosal has not been well-studied. We hypothesized that compartment specific differences in the unconventional T-cell repertoire would exist between the small and large intestine, due to increasing bacterial loads and microbial diversity;and that the impact of HIV infection might differ depending on the compartment examined. We used mass cytometry, flow cytometry and unbiased T-cell receptor profiling to quantify unconventional T-cells in blood and tissue from the small (duodenum) and large (colon) intestine in HIV infected and uninfected participants undergoing examination for a range of intestinal conditions. Overall, we find distinct compartmentalisation of T-cells between blood, duodenum and colon, with iNKT cells significantly enriched in the duodenum and delta-1 expressing gamma delta T-cells in the colon. In addition, we observe greater clonal expansion of conventional TCRs in the duodenum, suggestive of stronger adaptive immunity in this compartment. Conversely, we find evidence of an expanded unconventional TCR repertoire in the colon, which contained far more overlapping "donor unrestricted" sequences than the duodenum. Twelve of these TCRs were highly "MAIT-like" and 3 were unique to the colon, suggesting an enrichment of donor unrestricted T-cells (DURTs) in this compartment. Unexpectedly, however, no significant impact of HIV infection on any of the unconventional T-cell subsets measured was observed in either mucosal site in terms of frequency or TCR repertoire. Further studies are required to investigate the importance of these unconventional T-cell subsets to intestinal homeostasis within the different gut compartments and determine if they are functionally impaired during HIV infection

B cell heterogeneity in human tuberculosis highlights compartment-specific phenotype and functional roles

B cells are important in tuberculosis (TB) immunity, but their role in the human lung is understudied. Here, we characterize B cells from lung tissue and matched blood of TB patients and found they are decreased in the blood and increased in the lungs, consistent with recruitment to infected tissue, where they are located in granuloma associated lymphoid tissue (GrALT). Flow cytometry and transcriptomics identified multiple B cell populations in the lung, including those associated with tissue resident memory, germinal centers, antibody secretion, proinflammatory atypical B cells, and regulatory B cells, some of which are expanded in TB disease. Additionally, TB lungs contained high levels of Mtb-reactive antibodies, specifically IgM, which promoted Mtb phagocytosis. Overall, these data reveal the presence of functionally diverse B cell subsets in TB diseased lung and suggest several potential localized roles that may represent a target for interventions to promote immunity or mitigate immunopathology