Chrisna Durandt - Academia.edu (original) (raw)
Papers by Chrisna Durandt
Thorax, 2016
from BR patients (n = 115), categorised by bronchiectasis severity index (BSI) scores and sera sa... more from BR patients (n = 115), categorised by bronchiectasis severity index (BSI) scores and sera samples from HV controls (n = 26) Results Endobronchial biopsies from BR airways had a significantly (p < 0.05) higher number of blood vessels per mm of basement membrane than HV samples (18 and 9 blood vessels/ mm basement membrane respectively). Stimulation of HV neutrophils with a variety of molecules (PMA, fMLP, LPS, TNF-a etc.) resulted in a significant increase in VEGF secretion compared to unstimulated (p < 0.05). Although elevated VEGF was found in some patient samples there was no significant correlation between sera/sputa VEGF and individual patient BSI scores. Conclusion The increased presence of vascular tissue seen in BR could indicate a pro-angiogenic airway environment in BR. The in vitro data collected also show that a variety of stimulants can initiate secretion of VEGF by neutrophils. However, our data does not suggest that VEGF levels in sera or sputa can be used to predict disease severity.
Special edition: Recollecting Covid - 365 days in lockdown, 2021
Since the report of the first COVID-19 infected person in South Africa, COVID-19 moved from being... more Since the report of the first COVID-19 infected person in South Africa, COVID-19 moved from being a distant threat to a new reality overnight. Metaphorically, COVID-19 could be described as rain, and in order to be protected one would need to stand under an umbrella. The fundamental question that stems from this is who is holding this protective umbrella? Is the government holding the umbrella or are we holding the umbrella? In this article/commentary/perspective, we briefly discuss the responsibility of the South African government and the individual during this global pandemic, the reasoning behind the implementation of lockdown and the consequences thereof. We conclude that both government and citizens need to cooperatively take responsibility and work together to fight COVID-19. The protective umbrella needs to be held by both government and by ourselves.
Stem Cells International
The potential for human adipose-derived stromal cells (hASCs) to be used as a therapeutic product... more The potential for human adipose-derived stromal cells (hASCs) to be used as a therapeutic product is being assessed in multiple clinical trials. However, much is still to be learned about these cells before they can be used with confidence in the clinical setting. An inherent characteristic of hASCs that is not well understood is their heterogeneity. The aim of this exploratory study was to characterize the heterogeneity of freshly isolated hASCs after two population doublings (P2) using single-cell transcriptome analysis. A minimum of two subpopulations were identified at P2. A major subpopulation was identified as contractile cells which, based on gene expression patterns, are likely to be pericytes and/or vascular smooth muscle cells (vSMCs).
Stem Cells International
There is still much to learn about the cells used for cell- and gene-based therapies in the clini... more There is still much to learn about the cells used for cell- and gene-based therapies in the clinical setting. Stem cells are found in virtually all tissues in the human body. As a result, cells isolated from these tissues are a heterogeneous population consisting of various subpopulations including stem cells. Several strategies have been used to isolate and define the subpopulations that constitute these heterogeneous populations, one of which is the side population (SP) assay. SP cells are identified by their ability to efflux a fluorescent dye at a rate that is greater than the main cell population. This elevated rate of dye efflux has been attributed to the expression of members of the ATP-binding cassette (ABC) transporter protein family. SP cells have been identified in various tissues. In this review, we discuss the research to date on SP cells, focussing on SP cells identified in haematopoietic stem cells, adipose-derived stromal cells, and dental pulp.
Stem cell research, Jan 19, 2016
We have undertaken an in-depth transcriptome analysis of adipogenesis in human adipose-derived st... more We have undertaken an in-depth transcriptome analysis of adipogenesis in human adipose-derived stromal cells (ASCs) induced to differentiate into adipocytes in vitro. Gene expression was assessed on days 1, 7, 14 and 21 post-induction and genes differentially expressed numbered 128, 218, 253 and 240 respectively. Up-regulated genes were associated with blood vessel development, leukocyte migration, as well as tumor growth, invasion and metastasis. They also shared common pathways with certain obesity-related pathophysiological conditions. Down-regulated genes were enriched for immune response processes. KLF15, LMO3, FOXO1 and ZBTB16 transcription factors were up-regulated throughout the differentiation process. CEBPA, PPARG, ZNF117, MLXIPL, MMP3 and RORB were up-regulated only on days 14 and 21, which coincide with the maturation of adipocytes and could possibly serve as candidates for controlling fat accumulation and the size of mature adipocytes. In summary, we have identified gen...
Journal of lipid research, 2016
The ability of mesenchymal stromal cells (MSCs) to differentiate into adipocytes provides a cellu... more The ability of mesenchymal stromal cells (MSCs) to differentiate into adipocytes provides a cellular model of human origin to study adipogenesis in vitro. One of the major challenges in studying adipogenesis is the lack of tools to identify and monitor the differentiation of various subpopulations within the heterogeneous pool of MSCs. CD36 plays an important role in the formation of intracellular lipid droplets, a key characteristic of adipocyte differentiation/maturation. The objective of this study was to develop a reproducible, quantitative method to study adipocyte differentiation by comparing two lipophilic lipid dyes (Nile Red and Bodipy 493/503) in combination with CD36 surface marker staining. We identified a sub-population of adipose-derived mesenchymal stromal cells that express CD36 at intermediate/high levels and show that combining CD36 cell surface staining with neutral lipid-specific staining allows us to monitor differentiation of ASCs that express CD36(intermediate...
Cytotechnology, 2016
Adipose derived mesenchymal stromal/stem cells (ASCs) are a heterogeneous population characterize... more Adipose derived mesenchymal stromal/stem cells (ASCs) are a heterogeneous population characterized by (a) their ability to adhere to plastic; (b) immunophenotypic expression of certain cell surface markers, while lacking others; and (c) the capacity to differentiate into lineages of mesodermal origin including osteocytes, chondrocytes and adipocytes. The long-term goal is to utilize these cells for clinical translation into cell-based therapies. However, preclinical safety and efficacy need to be demonstrated in animal models. ASCs can also be utilized as biological vehicles for vector-based gene delivery systems, since they are believed to home to sites of inflammation and infection in vivo. These factors motivated the development of a labelling system for ASCs using lentiviral vector-based green fluorescent protein (GFP) transduction. Human ASCs were transduced with GFP-expressing lentiviral vectors. A titration study determined the viral titer required to transduce the maximum number of ASCs. The effect of the transduced GFP lentiviral vector on ASC immunophenotypic expression of surface markers as well as their ability to differentiate into osteocytes and adipocytes were assessed in vitro. A transduction efficiency in ASC cultures of approximately 80 % was observed with an MOI of ~118. No significant immunophenotypic differences were observed between transduced and non-transduced cells and both cell types successfully differentiated into adipocytes and osteocytes in vitro. We obtained &amp;amp;amp;amp;amp;gt;80 % transduction of ASCs using GFP lentiviral vectors. Transduced ASCs maintained plastic adherence, demonstrated ASC immunophenotype and the ability to differentiate into cells of the mesodermal lineage. This GFP-ASC transduction technique offers a potential tracking system for future pre-clinical studies.
European Respiratory Journal, 2015
International Journal of Oncology, 2001
The lipophilicity and membrane-destabilizing activities of clofazimine and three tetramethyl-pipe... more The lipophilicity and membrane-destabilizing activities of clofazimine and three tetramethyl-piperidine (TMP)-substituted phenazines were compared with the anti-tumor and multiple drug resistance (MDR) neutralizing potential of these agents using a P-glycoprotein (P-gp)-expressing small cell lung cancer cell line (H69/LX4). Partition coefficients were measured as an index of lipophilicity, while membrane-destabilizing potential was measured using a conventional hemolytic assay. The membrane-destabilizing potential of the TMP-substituted phenazines was found to correlate positively with the degree of lipophilicity, as well as with MDR reversal activity. The presence of a TMP group, as well as chlorine atoms on the phenyl and anilino rings of these agents contributed to the enhancement of anti-tumor activity by potentiating membrane-destabilizing activity. TMP-substituted phenazines may be useful in the design of novel anti-cancer and MDR reversal agents.
Theriogenology, 2015
The aim of this study was to determine whether flow cytometric evaluation of combined merocyanine... more The aim of this study was to determine whether flow cytometric evaluation of combined merocyanine 540 and Yo-Pro 1 (M540-YP) staining would identify viable dog sperm that had undergone membrane stabilization known to be associated with capacitation in other species, and whether such destabilization is detected earlier than when using the tyrosine phosphorylation and ethidium homodimer (TP-EH) stain combination with epifluorescence microscopy. Semen from nine dogs was collected and incubated in parallel in bicarbonate-free modified Tyrode's medium (ÀBIC), medium containing 15 mM bicarbonate (þBIC), dog prostatic fluid, and in PBS. Aliquots for staining were removed at various time points during incubation of up to 6 hours. Staining with M540-YP allowed the classification of dog sperm as viable without destabilized membranes, viable with destabilized membranes, nonviable without destabilized membranes, or nonviable with destabilized membranes. The percentage of viable sperm detected using EH (83.5 AE 1.37%; mean AE SEM) was higher than when using YP (66.7 AE 1.37%: P < 0.05; n ¼ 54 semen samples). On the other hand, M540-YP identified a higher percentage of viable sperm with destabilized membranes than TP-EH (75 AE 1.76% vs. 35 AE 1.70%: P < 0.05; n ¼ 54 semen samples). Staining with M540-YP indicated a rapid increase in the percentage of viable sperm with destabilized membranes, reaching a maximum during the first 30 minutes of incubation in þBIC. For all other treatments (i.e., ÀBIC, prostatic fluid, and PBS), the peak in the percentage of viable sperm with destabilized membranes was reached as much as 90 to 210 minutes later than incubation in þBIC. The lowest percentage of viable sperm showing signs of capacitation was recorded during incubation in PBS. We conclude that YP identifies sperm committed to cell death earlier than EH, and that the M540-YP stain combination identifies membrane destabilization known to be associated with capacitation in other species earlier than the TP-EH stain combination.
International Journal of Oncology, 1993
The riminophenazine agents clofazimine and its analogue B669 displayed anti-tumor activity at 30 ... more The riminophenazine agents clofazimine and its analogue B669 displayed anti-tumor activity at 30 mg/kg/day in benzo[a]pyrene (BP) induced sarcomas of mice as well as dimethylbenz-anthracene (DMBA)-induced rat mammary tumors. No hematological toxicity of these drugs at doses up to 60 mg/kg/day for one month was observed. This is the first study to document in vivo anti-neoplastic activities of clofazimine and B669.
Mediators of Inflammation, 2014
The clinical relevance of the anti-inflammatory properties of beta-2 agonists remains contentious... more The clinical relevance of the anti-inflammatory properties of beta-2 agonists remains contentious possibly due to differences in their molecular structures and agonist activities. The current study has compared the effects of 3 different categories of 2-agonists, namely, salbutamol (short-acting), formoterol (long-acting) and indacaterol (ultra-long-acting), at concentrations of 1-1000 nM, with human blood neutrophils in vitro. Neutrophils were activated with either N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP, 1 M) or platelet-activating factor (PAF, 200 nM) in the absence and presence of the 2-agonists followed by measurement of the generation of reactive oxygen species and leukotriene B4, release of elastase, and expression of the 2-integrin, CR3, using a combination of chemiluminescence, ELISA, colorimetric, and flow cytometric procedures respectively. These were correlated with alterations in the concentrations of intracellular cyclic-AMP and cytosolic Ca 2+ . At the concentrations tested, formoterol and indacaterol caused equivalent, significant ( < 0.05 at 1-10 nM) dose-related inhibition of all of the pro-inflammatory activities tested, while salbutamol was much less effective ( < 0.05 at 100 nM and higher). Suppression of neutrophil reactivity was accompanied by elevations in intracellular cAMP and accelerated clearance of Ca 2+ from the cytosol of activated neutrophils. These findings demonstrate that 2-agonists vary with respect to their suppressive effects on activated neutrophils.
The Journal of Infectious Diseases, 2002
The effects that the Streptococcus pneumoniae-derived, proinflammatory toxin, pneumolysin (8.37 a... more The effects that the Streptococcus pneumoniae-derived, proinflammatory toxin, pneumolysin (8.37 and 41.75 ng/mL), has on the production of interleukin (IL)-8 and tumor-necrosis factor (TNF)-a by human neutrophils have been investigated in vitro. Total and extracellular IL-8 and TNF-a were assayed by enzyme-linked immunosorbent assay, and flow cytometry and colorimetric procedures were used to detect intracellular cytokine and cytokine messenger RNA, respectively. Treatment of neutrophils with pneumolysin either alone or in combination with the chemotactic tripeptide, N-formyl-L-methionyl-L-leucyl-L-phenylalanine (1 mM), resulted in a time-dependent (maximal at 6 h) increase in synthesis and release of IL-8 but not of TNF-a, which was associated with increased expression of IL-8 messenger RNA transcripts and was abrogated by either cycloheximide (10 mg/mL) or depletion of Ca 2+ from the cellsuspending medium. These interactions between the toxin and neutrophils may contribute to the exaggerated pulmonary inflammatory responses caused by pneumolysin-producing strains of the pneumococcus.
BMC Medical Genetics, 2013
The human innate immune system is indispensable for protection against potentially invasive micro... more The human innate immune system is indispensable for protection against potentially invasive microbial and
viral pathogens, either neutralising them or containing their spread until effective mobilisation of the slower,
adaptive (specific), immune response. Until fairly recently, it was believed that the human innate immune
system possessed minimal discriminatory activity in the setting of a rather limited range of microbicidal or
virucidal mechanisms. However, recent discoveries have revealed that the innate immune system possesses
an array of novel pathogen recognition mechanisms, as well as a resourceful and effective alternative
mechanism of phagocyte (predominantly neutrophil)-mediated, anti-infective activity known as NETosis.
The process of NETosis involves an unusual type of programmed, purposeful cell death, resulting in the
extracellular release of a web of chromatin heavily impregnated with antimicrobial proteins. These structures,
known as neutrophil extracellular traps (NETs), immobilise and contribute to the eradication of microbial
pathogens, ensuring that the anti-infective potential of neutrophils is sustained beyond the lifespan of these
cells. The current review is focused on the mechanisms of NETosis and the role of this process in host
defence. Other topics reviewed include the potential threats to human health posed by poorly controlled,
excessive formation of NETs, specifically in relation to development of autoimmune and cardiovascular
diseases, as well as exacerbation of acute and chronic inflammatory disorders of the airways.
The primary objective of the current study was to investigate the potential of the pneumococcal t... more The primary objective of the current study was to investigate the potential
of the pneumococcal toxin, pneumolysin (Ply), to activate neutrophil
extracellular trap (NET) formation in vitro. Isolated human blood
neutrophils were exposed to recombinant Ply (5-20 ng ml21) for 30–90 min
at 378C and NET formation measured using the following procedures to
detect extracellular DNA: (i) flow cytometry using VybrantVR DyeCycleTM
Ruby; (ii) spectrofluorimetry using the fluorophore, SytoxVR Orange (5 lM);
and (iii) NanoDropVR technology. These procedures were complemented
by fluorescence microscopy using 40, 6-diamino-2-phenylindole (DAPI)
(nuclear stain) in combination with anti-citrullinated histone monoclonal
antibodies to visualize nets. Exposure of neutrophils to Ply resulted in
relatively rapid (detected within 30–60 min), statistically significant
(P < 005) dose- and time-related increases in the release of cellular DNA
impregnated with both citrullinated histone and myeloperoxidase.
Microscopy revealed that NETosis appeared to be restricted to a
subpopulation of neutrophils, the numbers of NET-forming cells in the
control and Ply-treated systems (10 and 20 ng ml21) were 43 (42), 14.3
(99) and 165 (75), respectively (n 5 4, P < 00001 for comparison of the
control with both Ply-treated systems). Ply-induced NETosis occurred in the
setting of retention of cell viability, and apparent lack of involvement of
reactive oxygen species and Toll-like receptor 4. In conclusion, Ply induces
vital NETosis in human neutrophils, a process which may either contribute
to host defence or worsen disease severity, depending on the intensity of the
inflammatory response during pneumococcal infection.
Thorax, 2016
from BR patients (n = 115), categorised by bronchiectasis severity index (BSI) scores and sera sa... more from BR patients (n = 115), categorised by bronchiectasis severity index (BSI) scores and sera samples from HV controls (n = 26) Results Endobronchial biopsies from BR airways had a significantly (p < 0.05) higher number of blood vessels per mm of basement membrane than HV samples (18 and 9 blood vessels/ mm basement membrane respectively). Stimulation of HV neutrophils with a variety of molecules (PMA, fMLP, LPS, TNF-a etc.) resulted in a significant increase in VEGF secretion compared to unstimulated (p < 0.05). Although elevated VEGF was found in some patient samples there was no significant correlation between sera/sputa VEGF and individual patient BSI scores. Conclusion The increased presence of vascular tissue seen in BR could indicate a pro-angiogenic airway environment in BR. The in vitro data collected also show that a variety of stimulants can initiate secretion of VEGF by neutrophils. However, our data does not suggest that VEGF levels in sera or sputa can be used to predict disease severity.
Special edition: Recollecting Covid - 365 days in lockdown, 2021
Since the report of the first COVID-19 infected person in South Africa, COVID-19 moved from being... more Since the report of the first COVID-19 infected person in South Africa, COVID-19 moved from being a distant threat to a new reality overnight. Metaphorically, COVID-19 could be described as rain, and in order to be protected one would need to stand under an umbrella. The fundamental question that stems from this is who is holding this protective umbrella? Is the government holding the umbrella or are we holding the umbrella? In this article/commentary/perspective, we briefly discuss the responsibility of the South African government and the individual during this global pandemic, the reasoning behind the implementation of lockdown and the consequences thereof. We conclude that both government and citizens need to cooperatively take responsibility and work together to fight COVID-19. The protective umbrella needs to be held by both government and by ourselves.
Stem Cells International
The potential for human adipose-derived stromal cells (hASCs) to be used as a therapeutic product... more The potential for human adipose-derived stromal cells (hASCs) to be used as a therapeutic product is being assessed in multiple clinical trials. However, much is still to be learned about these cells before they can be used with confidence in the clinical setting. An inherent characteristic of hASCs that is not well understood is their heterogeneity. The aim of this exploratory study was to characterize the heterogeneity of freshly isolated hASCs after two population doublings (P2) using single-cell transcriptome analysis. A minimum of two subpopulations were identified at P2. A major subpopulation was identified as contractile cells which, based on gene expression patterns, are likely to be pericytes and/or vascular smooth muscle cells (vSMCs).
Stem Cells International
There is still much to learn about the cells used for cell- and gene-based therapies in the clini... more There is still much to learn about the cells used for cell- and gene-based therapies in the clinical setting. Stem cells are found in virtually all tissues in the human body. As a result, cells isolated from these tissues are a heterogeneous population consisting of various subpopulations including stem cells. Several strategies have been used to isolate and define the subpopulations that constitute these heterogeneous populations, one of which is the side population (SP) assay. SP cells are identified by their ability to efflux a fluorescent dye at a rate that is greater than the main cell population. This elevated rate of dye efflux has been attributed to the expression of members of the ATP-binding cassette (ABC) transporter protein family. SP cells have been identified in various tissues. In this review, we discuss the research to date on SP cells, focussing on SP cells identified in haematopoietic stem cells, adipose-derived stromal cells, and dental pulp.
Stem cell research, Jan 19, 2016
We have undertaken an in-depth transcriptome analysis of adipogenesis in human adipose-derived st... more We have undertaken an in-depth transcriptome analysis of adipogenesis in human adipose-derived stromal cells (ASCs) induced to differentiate into adipocytes in vitro. Gene expression was assessed on days 1, 7, 14 and 21 post-induction and genes differentially expressed numbered 128, 218, 253 and 240 respectively. Up-regulated genes were associated with blood vessel development, leukocyte migration, as well as tumor growth, invasion and metastasis. They also shared common pathways with certain obesity-related pathophysiological conditions. Down-regulated genes were enriched for immune response processes. KLF15, LMO3, FOXO1 and ZBTB16 transcription factors were up-regulated throughout the differentiation process. CEBPA, PPARG, ZNF117, MLXIPL, MMP3 and RORB were up-regulated only on days 14 and 21, which coincide with the maturation of adipocytes and could possibly serve as candidates for controlling fat accumulation and the size of mature adipocytes. In summary, we have identified gen...
Journal of lipid research, 2016
The ability of mesenchymal stromal cells (MSCs) to differentiate into adipocytes provides a cellu... more The ability of mesenchymal stromal cells (MSCs) to differentiate into adipocytes provides a cellular model of human origin to study adipogenesis in vitro. One of the major challenges in studying adipogenesis is the lack of tools to identify and monitor the differentiation of various subpopulations within the heterogeneous pool of MSCs. CD36 plays an important role in the formation of intracellular lipid droplets, a key characteristic of adipocyte differentiation/maturation. The objective of this study was to develop a reproducible, quantitative method to study adipocyte differentiation by comparing two lipophilic lipid dyes (Nile Red and Bodipy 493/503) in combination with CD36 surface marker staining. We identified a sub-population of adipose-derived mesenchymal stromal cells that express CD36 at intermediate/high levels and show that combining CD36 cell surface staining with neutral lipid-specific staining allows us to monitor differentiation of ASCs that express CD36(intermediate...
Cytotechnology, 2016
Adipose derived mesenchymal stromal/stem cells (ASCs) are a heterogeneous population characterize... more Adipose derived mesenchymal stromal/stem cells (ASCs) are a heterogeneous population characterized by (a) their ability to adhere to plastic; (b) immunophenotypic expression of certain cell surface markers, while lacking others; and (c) the capacity to differentiate into lineages of mesodermal origin including osteocytes, chondrocytes and adipocytes. The long-term goal is to utilize these cells for clinical translation into cell-based therapies. However, preclinical safety and efficacy need to be demonstrated in animal models. ASCs can also be utilized as biological vehicles for vector-based gene delivery systems, since they are believed to home to sites of inflammation and infection in vivo. These factors motivated the development of a labelling system for ASCs using lentiviral vector-based green fluorescent protein (GFP) transduction. Human ASCs were transduced with GFP-expressing lentiviral vectors. A titration study determined the viral titer required to transduce the maximum number of ASCs. The effect of the transduced GFP lentiviral vector on ASC immunophenotypic expression of surface markers as well as their ability to differentiate into osteocytes and adipocytes were assessed in vitro. A transduction efficiency in ASC cultures of approximately 80 % was observed with an MOI of ~118. No significant immunophenotypic differences were observed between transduced and non-transduced cells and both cell types successfully differentiated into adipocytes and osteocytes in vitro. We obtained &amp;amp;amp;amp;amp;gt;80 % transduction of ASCs using GFP lentiviral vectors. Transduced ASCs maintained plastic adherence, demonstrated ASC immunophenotype and the ability to differentiate into cells of the mesodermal lineage. This GFP-ASC transduction technique offers a potential tracking system for future pre-clinical studies.
European Respiratory Journal, 2015
International Journal of Oncology, 2001
The lipophilicity and membrane-destabilizing activities of clofazimine and three tetramethyl-pipe... more The lipophilicity and membrane-destabilizing activities of clofazimine and three tetramethyl-piperidine (TMP)-substituted phenazines were compared with the anti-tumor and multiple drug resistance (MDR) neutralizing potential of these agents using a P-glycoprotein (P-gp)-expressing small cell lung cancer cell line (H69/LX4). Partition coefficients were measured as an index of lipophilicity, while membrane-destabilizing potential was measured using a conventional hemolytic assay. The membrane-destabilizing potential of the TMP-substituted phenazines was found to correlate positively with the degree of lipophilicity, as well as with MDR reversal activity. The presence of a TMP group, as well as chlorine atoms on the phenyl and anilino rings of these agents contributed to the enhancement of anti-tumor activity by potentiating membrane-destabilizing activity. TMP-substituted phenazines may be useful in the design of novel anti-cancer and MDR reversal agents.
Theriogenology, 2015
The aim of this study was to determine whether flow cytometric evaluation of combined merocyanine... more The aim of this study was to determine whether flow cytometric evaluation of combined merocyanine 540 and Yo-Pro 1 (M540-YP) staining would identify viable dog sperm that had undergone membrane stabilization known to be associated with capacitation in other species, and whether such destabilization is detected earlier than when using the tyrosine phosphorylation and ethidium homodimer (TP-EH) stain combination with epifluorescence microscopy. Semen from nine dogs was collected and incubated in parallel in bicarbonate-free modified Tyrode's medium (ÀBIC), medium containing 15 mM bicarbonate (þBIC), dog prostatic fluid, and in PBS. Aliquots for staining were removed at various time points during incubation of up to 6 hours. Staining with M540-YP allowed the classification of dog sperm as viable without destabilized membranes, viable with destabilized membranes, nonviable without destabilized membranes, or nonviable with destabilized membranes. The percentage of viable sperm detected using EH (83.5 AE 1.37%; mean AE SEM) was higher than when using YP (66.7 AE 1.37%: P < 0.05; n ¼ 54 semen samples). On the other hand, M540-YP identified a higher percentage of viable sperm with destabilized membranes than TP-EH (75 AE 1.76% vs. 35 AE 1.70%: P < 0.05; n ¼ 54 semen samples). Staining with M540-YP indicated a rapid increase in the percentage of viable sperm with destabilized membranes, reaching a maximum during the first 30 minutes of incubation in þBIC. For all other treatments (i.e., ÀBIC, prostatic fluid, and PBS), the peak in the percentage of viable sperm with destabilized membranes was reached as much as 90 to 210 minutes later than incubation in þBIC. The lowest percentage of viable sperm showing signs of capacitation was recorded during incubation in PBS. We conclude that YP identifies sperm committed to cell death earlier than EH, and that the M540-YP stain combination identifies membrane destabilization known to be associated with capacitation in other species earlier than the TP-EH stain combination.
International Journal of Oncology, 1993
The riminophenazine agents clofazimine and its analogue B669 displayed anti-tumor activity at 30 ... more The riminophenazine agents clofazimine and its analogue B669 displayed anti-tumor activity at 30 mg/kg/day in benzo[a]pyrene (BP) induced sarcomas of mice as well as dimethylbenz-anthracene (DMBA)-induced rat mammary tumors. No hematological toxicity of these drugs at doses up to 60 mg/kg/day for one month was observed. This is the first study to document in vivo anti-neoplastic activities of clofazimine and B669.
Mediators of Inflammation, 2014
The clinical relevance of the anti-inflammatory properties of beta-2 agonists remains contentious... more The clinical relevance of the anti-inflammatory properties of beta-2 agonists remains contentious possibly due to differences in their molecular structures and agonist activities. The current study has compared the effects of 3 different categories of 2-agonists, namely, salbutamol (short-acting), formoterol (long-acting) and indacaterol (ultra-long-acting), at concentrations of 1-1000 nM, with human blood neutrophils in vitro. Neutrophils were activated with either N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP, 1 M) or platelet-activating factor (PAF, 200 nM) in the absence and presence of the 2-agonists followed by measurement of the generation of reactive oxygen species and leukotriene B4, release of elastase, and expression of the 2-integrin, CR3, using a combination of chemiluminescence, ELISA, colorimetric, and flow cytometric procedures respectively. These were correlated with alterations in the concentrations of intracellular cyclic-AMP and cytosolic Ca 2+ . At the concentrations tested, formoterol and indacaterol caused equivalent, significant ( < 0.05 at 1-10 nM) dose-related inhibition of all of the pro-inflammatory activities tested, while salbutamol was much less effective ( < 0.05 at 100 nM and higher). Suppression of neutrophil reactivity was accompanied by elevations in intracellular cAMP and accelerated clearance of Ca 2+ from the cytosol of activated neutrophils. These findings demonstrate that 2-agonists vary with respect to their suppressive effects on activated neutrophils.
The Journal of Infectious Diseases, 2002
The effects that the Streptococcus pneumoniae-derived, proinflammatory toxin, pneumolysin (8.37 a... more The effects that the Streptococcus pneumoniae-derived, proinflammatory toxin, pneumolysin (8.37 and 41.75 ng/mL), has on the production of interleukin (IL)-8 and tumor-necrosis factor (TNF)-a by human neutrophils have been investigated in vitro. Total and extracellular IL-8 and TNF-a were assayed by enzyme-linked immunosorbent assay, and flow cytometry and colorimetric procedures were used to detect intracellular cytokine and cytokine messenger RNA, respectively. Treatment of neutrophils with pneumolysin either alone or in combination with the chemotactic tripeptide, N-formyl-L-methionyl-L-leucyl-L-phenylalanine (1 mM), resulted in a time-dependent (maximal at 6 h) increase in synthesis and release of IL-8 but not of TNF-a, which was associated with increased expression of IL-8 messenger RNA transcripts and was abrogated by either cycloheximide (10 mg/mL) or depletion of Ca 2+ from the cellsuspending medium. These interactions between the toxin and neutrophils may contribute to the exaggerated pulmonary inflammatory responses caused by pneumolysin-producing strains of the pneumococcus.
BMC Medical Genetics, 2013
The human innate immune system is indispensable for protection against potentially invasive micro... more The human innate immune system is indispensable for protection against potentially invasive microbial and
viral pathogens, either neutralising them or containing their spread until effective mobilisation of the slower,
adaptive (specific), immune response. Until fairly recently, it was believed that the human innate immune
system possessed minimal discriminatory activity in the setting of a rather limited range of microbicidal or
virucidal mechanisms. However, recent discoveries have revealed that the innate immune system possesses
an array of novel pathogen recognition mechanisms, as well as a resourceful and effective alternative
mechanism of phagocyte (predominantly neutrophil)-mediated, anti-infective activity known as NETosis.
The process of NETosis involves an unusual type of programmed, purposeful cell death, resulting in the
extracellular release of a web of chromatin heavily impregnated with antimicrobial proteins. These structures,
known as neutrophil extracellular traps (NETs), immobilise and contribute to the eradication of microbial
pathogens, ensuring that the anti-infective potential of neutrophils is sustained beyond the lifespan of these
cells. The current review is focused on the mechanisms of NETosis and the role of this process in host
defence. Other topics reviewed include the potential threats to human health posed by poorly controlled,
excessive formation of NETs, specifically in relation to development of autoimmune and cardiovascular
diseases, as well as exacerbation of acute and chronic inflammatory disorders of the airways.
The primary objective of the current study was to investigate the potential of the pneumococcal t... more The primary objective of the current study was to investigate the potential
of the pneumococcal toxin, pneumolysin (Ply), to activate neutrophil
extracellular trap (NET) formation in vitro. Isolated human blood
neutrophils were exposed to recombinant Ply (5-20 ng ml21) for 30–90 min
at 378C and NET formation measured using the following procedures to
detect extracellular DNA: (i) flow cytometry using VybrantVR DyeCycleTM
Ruby; (ii) spectrofluorimetry using the fluorophore, SytoxVR Orange (5 lM);
and (iii) NanoDropVR technology. These procedures were complemented
by fluorescence microscopy using 40, 6-diamino-2-phenylindole (DAPI)
(nuclear stain) in combination with anti-citrullinated histone monoclonal
antibodies to visualize nets. Exposure of neutrophils to Ply resulted in
relatively rapid (detected within 30–60 min), statistically significant
(P < 005) dose- and time-related increases in the release of cellular DNA
impregnated with both citrullinated histone and myeloperoxidase.
Microscopy revealed that NETosis appeared to be restricted to a
subpopulation of neutrophils, the numbers of NET-forming cells in the
control and Ply-treated systems (10 and 20 ng ml21) were 43 (42), 14.3
(99) and 165 (75), respectively (n 5 4, P < 00001 for comparison of the
control with both Ply-treated systems). Ply-induced NETosis occurred in the
setting of retention of cell viability, and apparent lack of involvement of
reactive oxygen species and Toll-like receptor 4. In conclusion, Ply induces
vital NETosis in human neutrophils, a process which may either contribute
to host defence or worsen disease severity, depending on the intensity of the
inflammatory response during pneumococcal infection.