Immunohistochemical localisation of caffeine in young tea (Camellia sinensis (L.) O. Kuntze) leaves

The agricultural product using the top young shoots from the Camellia sinensis bush is known as tea. The leaves have many health benefits and contain high contents of antioxidants. The leaves contain caffeine, which is believed to be a phosphodiesterase inhibitor preventing the disposal of cyclic adenosine monophosphate, and thus when consumed, cellular responses are up regulated, and people experience unwanted effects such as being unable to fall asleep. Caffeine is currently believed to be synthesized within chloroplasts and stored within the vacuoles of parenchyma cells. It is hypothesized that the molecule acts as a chemical defence as well as an aid in the sequestration of catechins and polyphenols within the vacuoles of parenchyma cells. Different methods of decaffeination exist using solvents such as methylene chloride, supercritical CO2, and using hot water. Each method has a specific disadvantage. Currently, the Swiss Water Process is a 100 % chemically free method of decaffeination for coffee beans, and a similar process should be envisaged for the tea decaffeination industry as there are many advantages over the other current methods of decaffeination. The anatomical localisation of caffeine within young C. sinensis leaves was investigated using immunohistochemical methods, and confocal scanning laser microscopy. Preliminary fixation experiments were conducted with young C. sinensis leaves to determine which fixation procedure retained caffeine the best as determined by high-performance liquid chromatography analysis. High pressure freezing, freeze substitution, and embedding in Lowicryl K4M resin was deemed the best protocol as it retained most of the caffeine, and allowed for the samples to be sectioned with ease. Immunohistochemical localisation with primary anti-caffeine antibodies, and conjugated secondary antibodies on leaf sections proved at the tissue level that caffeine was localised and accumulated within vascular bundles, mainly the precursor phloem. Immunocytochemical studies using a secondary antibody conjugated to gold were attempted but were inconclusive. With the use of a pressure bomb, xylem sap was analysed by thin-layer chromatography, and the presence of caffeine was determined to be present in a small amount. We hypothesize that caffeine is synthesized in the chloroplasts of photosynthetic cells and transported to vascular bundles where it acts as a chemical defence against various pathogens, and predators. Complex formation of caffeine with chlorogenic acid is also discussed as this may also help explain caffeine’s localisation. Using the knowledge acquired from microscopy analysis, and thin-layer chromatography analysis of xylem sap we investigated three possible methods of decaffeination; vacuum, pressure, and a combination of both. All were based on using the intercellular air within the young leaves to our advantage as this might act as a natural way to ‘squeeze’ caffeine out of its localised areas. Other possible methods of decaffeination were also attempted using hot water, and addition of an external PPO source for black tea production.Dissertation (MSc)--University of Pretoria, 2012.BiochemistryMScUnrestricte

Metabolomics of colistin methanesulfonate treated Mycobacterium tuberculosis

Over the past 5 years, there has been a renewed interest in finding new compounds with anti-TB action. Colistin methanesulfonate or polymyxin E, is a possible anti-TB drug candidate, which may in future be used either alone or in combination to the current 6 month “directly observed treatment short-course” (DOTS) regimen. However its mechanism of action has to date not yet been fully explored, and only described from a histological and genomics perspective. Considering this, we used a GCxGC-TOFMS metabolomics approach and identified those metabolite markers characterising Mycobacterium tuberculosis (Mtb) cultured in the presence of colistin methanesulfonate, in order to better understand or confirm its mechanism of action. The metabolite markers identified indicated a flux in the metabolism of the colistin methanesulfonate treated Mtb towards fatty acid synthesis and cell wall repair, confirming previous reports that colistin acts by disrupting the cell wall of mycobacteria. Accompanying this, is a subsequently elevated glucose uptake, since the latter now serves as the primary energy substrate for the upregulated glyoxylate cycle, and additionally as a precursor for further fatty acid synthesis via the glycerolipid metabolic pathway. Furthermore, the elevated concentrations of those metabolites associated with pentose phosphate, valine, threonine, and pentanediol metabolism, also confirms a shift towards glucose utilization for energy production, in the colistin methanesulfonate treated Mtb.Prof. Anton Stoltz and Prof. Ed Nardell are specifically thanked for their funding towards the cell cultures. The North West University is thanked for financial assistance of the research which forms part of a master's study.http://intl.elsevierhealth.com/journals/tube2019-07-01hj2018Internal Medicin

Feto-Maternal Microchimerism: The Pre-eclampsia Conundrum

Feto-maternal microchimerism (FMM) involves bidirectional cross-placental trafficking during pregnancy, leading to a micro-chimeric state that can persist for decades. In this manner a pregnant woman will harbor cells from her mother, as well as, cells from her child. Historically, eclampsia, a severe disorder of pregnancy provided the basis for FMM following the detection of trophoblast cells in the lungs of deceased women. Bi-directional cell trafficking between mother and fetus is also altered in pre-eclampsia and has been suggested to contribute to the underlying etiology. FMM has been implicated in tolerance promotion, remission of auto-inflammatory disorders during pregnancy, or the development of autoimmune conditions post-partum. The underlying mechanism whereby the host immune system is modulated is unclear but appears to involve HLA class II molecules, in that incompatibility between mother and fetus promotes remission of rheumatoid arthritis, whereas feto-maternal HLA compatibility may assist in the post-partum initiation of scleroderma. Couples having a high degree of HLA class II compatibility have an increased risk for pre-eclampsia, while the occurrence of scleroderma and rheumatoid arthritis is greater in pre-eclamptic cases than in women with normal pregnancies, suggesting a long term autoimmune predisposition. Since pregnant women with pre-eclampsia exhibit significantly lower levels of maternally-derived micro-chimerism, the question arises whether pre-eclampsia and post-partum development of autoimmune conditions occur due to the failure of the grandmothers cells to adequately regulate an inappropriate micro-chimeric constellation

Immunohistochemical localization of caffeine in young Camellia sinensis (L.) O. Kuntze (tea) leaves

The anatomical localization of caffeine within young Camellia sinensis leaves was investigated using immunohistochemical methods and confocal scanning laser microscopy. Preliminary fixation experiments were conducted with young C. sinensis leaves to determine which fixation procedure retained caffeine the best as determined by high-performance liquid chromatography analysis. High pressure freezing, freeze substitution, and embedding in resin was deemed the best protocol as it retained most of the caffeine and allowed for the samples to be sectioned with ease. Immunohistochemical localization with primary anti-caffeine antibodies and conjugated secondary antibodies on leaf sections proved at the tissue level that caffeine was localized and accumulated within vascular bundles, mainly the precursor phloem. With the use of a pressure bomb, xylem sap was collected using a micro syringe. The xylem sap was analyzed by thin-layer chromatography and the presence of caffeine was determined. We hypothesize that caffeine is synthesized in the chloroplasts of photosynthetic cells and transported to vascular bundles where it acts as a chemical defense against various pathogens and predators. Complex formation of caffeine with chlorogenic acid is also discussed as this may also help explain caffeine’s localization.http://link.springer.com/journal/42

Feto-Maternal Microchimerism: The Pre-eclampsia Conundrum

Feto-maternal microchimerism (FMM) involves bidirectional cross-placental trafficking during pregnancy, leading to a micro-chimeric state that can persist for decades. In this manner a pregnant woman will harbor cells from her mother, as well as, cells from her child. Historically, eclampsia, a severe disorder of pregnancy provided the basis for FMM following the detection of trophoblast cells in the lungs of deceased women. Bi-directional cell trafficking between mother and fetus is also altered in pre-eclampsia and has been suggested to contribute to the underlying etiology. FMM has been implicated in tolerance promotion, remission of auto-inflammatory disorders during pregnancy, or the development of autoimmune conditions post-partum. The underlying mechanism whereby the host immune system is modulated is unclear but appears to involve HLA class II molecules, in that incompatibility between mother and fetus promotes remission of rheumatoid arthritis, whereas feto-maternal HLA compatibility may assist in the post-partum initiation of scleroderma. Couples having a high degree of HLA class II compatibility have an increased risk for pre-eclampsia, while the occurrence of scleroderma and rheumatoid arthritis is greater in pre-eclamptic cases than in women with normal pregnancies, suggesting a long term autoimmune predisposition. Since pregnant women with pre-eclampsia exhibit significantly lower levels of maternally-derived micro-chimerism, the question arises whether pre-eclampsia and post-partum development of autoimmune conditions occur due to the failure of the grandmothers cells to adequately regulate an inappropriate micro-chimeric constellation

The antimicrobial effect of colistin methanesulfonate on Mycobacterium tuberculosis in vitro

Polymyxins have previously been described to have activity against M. tuberculosis (MTB), but further research was abandoned due to systemic toxicity concerns to achieve the required MIC. Colistin methanesulfonate (CMS), a polymyxin, is well tolerated when inhaled directly into the lungs, resulting in high local concentrations. We report here for the first time, MIC and MBC data for CMS determined by the microtiter Alamar Blue assay (MABA). We also determined how the MIC would be affected by the presence of pulmonary surfactant (PS) and if any synergy with isoniazid (INH) and rifampicin (RIF) exists. The effect of CMS on the ultrastructure of MTB was also determined. The MIC for CMS was 16 mg/L, while the MBC was 256 mg/L. MIC for CMS in PS was antagonised by eight fold. For synergy, indifference was determined while time-kill assays revealed a greater killing effect when CMS was used together with INH. Ultrastructure analysis suggests that the disruption of the outer polysaccharide layer of MTB by CMS may lead to enhanced uptake of INH. Our findings may provide insight for further investigations of CMS against MTB.http://intl.elsevierhealth.com/journals/tube2016-07-30hb201

New amniotic membrane based biocomposite for future application in reconstructive urology

OBJECTIVE Due to the capacity of the amniotic membrane (Am) to support re-epithelisation and inhibit scar formation, Am has a potential to become a considerable asset for reconstructive urology i.e., reconstruction of ureters and urethrae. The application of Am in reconstructive urology is limited due to a poor mechanical characteristic. Am reinforcement with electrospun nanofibers offers a new strategy to improve Am mechanical resistance, without affecting its unique bioactivity profile. This study evaluated biocomposite material composed of Am and nanofibers as a graft for urinary bladder augmentation in a rat model. MATERIAL AND METHODS Sandwich-structured biocomposite material was constructed from frozen Am and covered on both sides with two-layered membranes prepared from electrospun poly-(L-lactide-co-Ecaprolactone) (PLCL). Wistar rats underwent hemicystectomy and bladder augmentation with the biocomposite material. RESULTS Immunohistohemical analysis (hematoxylin and eosin [H&E], anti-smoothelin and Masson’s trichrome staining [TRI]) revealed effective regeneration of the urothelial and smooth muscle layers. Anti-smoothelin staining confirmed the presence of contractile smooth muscle within a new bladder wall. Sandwich-structured biocomposite graft material was designed to regenerate the urinary bladder wall, fulfilling the requirements for normal bladder tension, contraction, elasticity and compliance. Mechanical evaluation of regenerated bladder wall conducted based on Young’s elastic modulus reflected changes in the histological remodeling of the augmented part of the bladder. The structure of the biocomposite material made it possible to deliver an intact Am to the area for regeneration. An unmodified Am surface supported regeneration of the urinary bladder wall and the PLCL membranes did not disturb the regeneration process. CONCLUSIONS Am reinforcement with electrospun nanofibers offers a new strategy to improve Am mechanical resistance without affecting its unique bioactivity profile.http://www.plosone.orgam2016Internal Medicin

The antimicrobial effect of colistin methanesulfonate (polymyxin E) on Mycobacterium tuberculosis in vitro

Polymyxins have previously been described to have activity against M. tuberculosis (M. tb), but further research was abandoned due to systemic toxicity concerns to achieve the required MIC. Colistin methanesulfonate (CMS), a polymyxin, is well tolerated when inhaled directly into the lungs, resulting in high local concentrations. Reported here for the first time are the MIC and MBC data for CMS, CST and PST determined by the microtiter Alamar Blue® assay (MABA) against H37Ra and multi-drug-resistant (MDR) M. tb. Additionally determined is how the MIC of CMS would be affected by the presence of pulmonary surfactant (PS) and if any synergy with isoniazid (INH) and rifampicin (RIF) exists. The effect of CMS on the ultrastructure of M. tb was also determined. MICs for CMS, CST and PST were determined to be too high for systemic use. CMS can, however, be administered by inhalation allowing for high local concentrations with reduced systemic toxicity. The MIC for CMS was antagonised eight fold in PS. For synergy, indifference was determined for both H37Ra and MDR M. tb. Time-kill assays revealed a bactericidal killing effect when CMS was used together with INH against H37Ra M. tb while no enhanced effect of CMS with INH or RIF was observed against MDR M. tb. The resistant effects caused by rpoB and katG mutations could not be overcome. With regard to H37Ra M. tb, ultrastructure analysis suggests that the disruption of the capsule layer (CL) and cytoplasmic membrane (CM) by CMS may enhance the uptake of INH. These findings may provide insight for further investigations of CMS against M. tb.Thesis (PhD)--University of Pretoria, 2016.Medical MicrobiologyUnrestricte

Elucidating the antimicrobial mechanisms of colistin sulfate on Mycobacterium tuberculosis using metabolomics

Considering the disadvantageous of first line anti-tuberculosis (TB) drugs, including poor patient adherence, drug side effects, the long treatment duration and rapidly increasing microbe resistance, alternative treatment strategies are needed. Colistin sulfate (CS), a polymyxin antibiotic considered a last-resort antibiotics for treating multidrug-resistant Pseudomonas aeruginosa, Klebsiella pneumoniae, and Acinetobacter, has antimicrobial activity towards mycobacteria, and could serve as a possible anti-TB drug. Using GCxGC-TOFMS metabolomics, we compared the metabolic profiles of Mycobacterium tuberculosis (Mtb) cultured in the presence and absence of CS, to elucidate the mechanisms by which this drug may exert its antimicrobial effects. The principal component analysis of the metabolite data indicated significant variation in the underlying metabolite profiles of the groups. Those metabolites best explaining this differentiation, were acetic acid, and cell wall associated methylated and unmethylated fatty acids, and their alcohol and alkane derivatives. The elevated glucose levels, and various glyoxylate and glycerolipid metabolic intermediates, indicates an elevated flux in these metabolic pathways. Since all the metabolites identified in the colistin treated Mtb indicates an increase in fatty acid synthesis and cell wall repair, it can be concluded that CS acts by disrupting the cell wall in Mtb, confirming a similar drug action to other organisms.Prof. Anton Stoltz and Prof. Ed Nardell are specifically thanked for their funding towards the cell cultures. The North West University is thanked for financial assistance of the research which forms part of a master's study.http://intl.elsevierhealth.com/journals/tube2019-07-01hj2018Internal Medicin

Markers of neutrophil extracellular traps predict adverse outcome in community-acquired pneumonia: secondary analysis of a randomised controlled trial.

Neutrophil extracellular traps (NETs) are a hallmark of the immune response in inflammatory diseases. However, the role of NETs in community-acquired pneumonia (CAP) is unknown. This study aims to characterise the impact of NETs on clinical outcomes in pneumonia.This is a secondary analysis of a randomised controlled, multicentre trial. Patients with CAP were randomly assigned to either 50 mg prednisone or placebo for 7 days. The primary end-point was time to clinical stability; main secondary end-points were length of hospital stay and mortality.In total, 310 patients were included in the analysis. Levels of cell-free nucleosomes as surrogate markers of NETosis were significantly increased at admission and declined over 7 days. NETs were significantly associated with reduced hazards of clinical stability and hospital discharge in multivariate adjusted analyses. Moreover, NETs were associated with a 3.8-fold increased adjusted odds ratio of 30-day mortality. Prednisone treatment modified circulatory NET levels and was associated with beneficial outcome.CAP is accompanied by pronounced NET formation. Patients with elevated serum NET markers were at higher risk for clinical instability, prolonged length of hospital stay and 30-day all-cause mortality. NETs represent a novel marker for outcome and a possible target for adjunct treatments of pneumonia