The HIV-1 glycoprotein gp120 elevates NF-?B levels in human 
cardiomyocytes which may be reversed with the treatment of a 
sesquiterpene lactone isolated from Vernonia staehelinoides 
 
 
 
 
 
 
 
 
 
 
 
Dayaneethie Coopusamy 
 
 
 
 
 
 
 
 
 
A thesis submitted to the Faculty of Science, University of the Witwatersrand, 
Johannesburg, in fulfilment of the requirement for the degree of Master of 
Science. 
 
Johannesburg, January 2009 
 ii 
DECLARATION 
 
I declare that this thesis is my own, unaided work. It is being submitted for the degree 
of Master of Science in the University of the Witwatersrand, Johannesburg. It has not 
been submitted before for any degree or examination in any other University. 
 
 
 
Dayaneethie Coopusamy 
 
 
 
this day of                                         2009 
 iii 
ABSTRACT 
 
Twenty five years of studying HIV-1 structure and replication have improved 
diagnosis and treatment of individuals infected with the virus. In particular, the 
introduction of highly active antiretroviral therapy (HAART) has significantly altered 
the course of HIV-1 infection by increasing life expectancy and reducing 
opportunistic infections. However, chronic cardiovascular complications such as HIV 
associated cardiomyopathy (HIVCM), which manifest later during the course of HIV-
 1 infection, have become increasingly evident. Despite its growing incidence, with 
high cardiovascular morbidity and mortality in young and middle-aged adults, the 
molecular mechanisms of HIVCM remain poorly understood. A number of pathways 
have been implicated in HIVCM, including damage initiated by HIV-1 surface 
glycoprotein, gp120, and dysregulation of NF-?B. NF-?B is a universal transcription 
factor and it regulates a number of genes, many of which are involved in 
inflammation, injury and stress response. The ability of HIV-1 to manipulate host 
signalling pathways, including elevated NF-?B levels, has resulted in efficient viral 
replication and gene expression. The elevation of NF-?B has also been shown to be 
involved in animal models of HIVCM but very little work has been conducted on 
human cells. For this reason, the primary objectives of this thesis were to establish the 
level of NF-?B in a cellular model of HIVCM by challenging human cardiomyocytes 
with HIV-1 or gp120 and to mitigate the effect on NF-?B using natural compounds 
derived from South African indigenous plants. The effect of gp120 and HIV-1 on NF-
 ?B levels in human cardiomyocytes was tested by an ELISA-based assay and 
immunocytochemistry. This was to determine whether the damage induced by HIV-1 
and gp120 is mediated by NF-?B. The results shows that gp120 significantly 
increased NF-?B levels in human cardiomyocytes compared to control unstimulated 
cardiomyocytes (p<0.001). One plant compound, the sesquiterpene lactone 106A, 
significantly reduced the NF-?B response by human cardiomyocytes to gp120 
stimulation (p<0.05). Taken together, this study suggests that the activation of NF-?B 
by gp120 has a role to play in a cellular model of HIVCM and that the sesquiterpene 
lactone 106A could prove valuable in further studies on the modulation of cellular 
responses due to gp120 and HIV-1 induced stress in human cardiomyocytes. 
 iv 
 
ACKNOWLEDGEMENTS 
I would like to thank my supervisors Drs. Makobetsa Khati and Monde Ntwasa for 
their support and guidance. Thanks also to Professor Lynn Morris for the use of her 
laboratory at the NICD, for providing the 293T cell line, HIV-1 strains, and gp120 
plasmids and for her insightful comments. I thank Dr. Vinesh Maharaj for providing 
the plant compounds and the HeLa cell line. I am grateful to Walter Campos for his 
guidance and the rest of the Aptamer Technology Team for the memorable 
experiences. Finally, I thank the CSIR for the MSc studentship and financial 
assistance. 
 v 
TABLE OF CONTENTS 
 
DECLARATION......................................................................................................ii 
ABSTRACT............................................................................................................ iii 
ACKNOWLEDGEMENTS ....................................................................................iv 
LIST OF FIGURES ............................................................................................. viii 
ABBREVIATIONS ..................................................................................................x 
1 INTRODUCTION.................................................................................................1 
1.1 The HIV/AIDS pandemic .................................................................................1 
1.2 HIV-1 structure and infectious cycle.................................................................2 
1.3 HIV pathogenesis .............................................................................................3 
1.4 HIV-associated Cardiomyopathy ......................................................................3 
1.5 Nuclear factor-?B.............................................................................................5 
1.6 The role of NF-?B in HIV infection..................................................................8 
1.7 The role of plant extracts in NF-?B inhibition.................................................10 
2 OBJECTIVES .....................................................................................................11 
3 MATERIALS AND METHODS ........................................................................11 
3.1 Propagation and Maintenance of cell lines ......................................................11 
3.1.1 Cell count ................................................................................................11 
3.1.2 Subculture of cell lines.............................................................................12 
3.1.3 Freezing of cell lines................................................................................12 
3.2 Expression and purification of gp120..............................................................13 
3.3 Detection and quantification of 293T-expressed gp120...................................13 
3.3.1 SDS-PAGE detection of gp120................................................................13 
3.3.2 Western Blot detection of gp120..............................................................14 
3.3.3 Quantification of purified gp120 by the bicinchoninic acid protein assay .14 
3.4 Validation of HIV-1Du151 gp120 biological activity .........................................15 
3.4.1 Immobilisation of HIV-1Du151 gp120 to sensor surface .............................15 
3.4.2 Interaction between immobilised gp120 and IgG1 b12 antibody ..............16 
3.5 Isolation of human peripheral blood mononuclear cells from buffy coats and 
cultivation of human macrophages by monocyte differentiation ...........................16 
3.6 Infection of monocyte-derived macrophages with HIV-1................................17 
3.6.1 Determination of viral replication in MDM..............................................17 
3.7 Immunocytochemistry....................................................................................17 
 vi 
3.8 Cytotoxicity assays .........................................................................................18 
3.9 Quantitative measurement of NF-?B...............................................................19 
3.9.1. Immunofluorescence...............................................................................19 
3.9.2.1 Protein extraction..................................................................................19 
3.9.2.2 ELISA-based NF-?B assay ...................................................................20 
3.10 Statistical Analysis .......................................................................................21 
4 RESULTS............................................................................................................22 
4.1 HIV-1 glycoprotein gp120 expression and validation......................................22 
4.1.1 Expression, purification detection and quantification of gp120.................22 
4.1.2 Validation of HIV-1Du151 gp120 biological activity ..................................23 
4.2 Culture and infection of monocyte-derived macrophages................................24 
4.2.1 Isolation of human peripheral blood mononuclear cells (PBMC) from buffy 
coats and cultivation of human macrophages by monocyte differentiation ........24 
4.2.2 Infection of MDM by Du151 and CM9 HIV-1 strains..............................25 
4.3 Culture and maintenance of cells lines ............................................................25 
4.4 Cytotoxicity of plant compounds ....................................................................27 
4.5 Measurement of NF-?B ..................................................................................29 
4.5.1 Immunocytochemistry .............................................................................29 
4.5.2 Immunofluorescence................................................................................30 
4.5.3 ELISA-based NF-?B assay ......................................................................31 
4.6 Activity of plant compounds against NF-?B activation in HeLa cells..............32 
4.7 Optimisation of NF-?B assay for gp120 stimulation of cardiomyocytes ..........33 
4.8 Stimulation of NF-?B in cardiomyocytes by HIV-1 and gp120.......................34 
4.9 Modulation of NF-?B by plant compounds in gp120-stimulated cardiomyocytes
 .............................................................................................................................38 
5 DISCUSSION ......................................................................................................39 
5.1 HIV-1 glycoprotein gp120 expression and validation......................................39 
5.2 Measurement of NF-?B ..................................................................................40 
5.3 Stimulation of NF-?B in cardiomyocytes by HIV-1 and gp120.......................41 
5.4 Modulation of NF-?B by plant compounds in gp120-stimulated cardiomyocytes
 .............................................................................................................................43 
5.5 Future considerations......................................................................................44 
6 CONCLUSION....................................................................................................45 
7 REFERENCES....................................................................................................46 
 vii 
8 APPENDIX..........................................................................................................55 
Table 1: Panel of natural compounds derived from South African indigenous plant
 .............................................................................................................................55 
SDS PAGE gel and buffer formulations ...............................................................56 
 
 viii 
LIST OF FIGURES 
 
Figure 1: An overview of the HIV-1 proviral genome, genes and proteins together 
with a summary thereof. .............................................................................................2 
Figure 2: Western blot analysis of the lentil-lectin purified gp120. ...........................23 
Figure 3: Sensogram showing the interaction between the anti-gp120 monoclonal 
antibody b12 and HIV-1Du151 gp120. ........................................................................24 
Figure 4: Light microscopy image of Day 16 monocyte-derived macrophages .........25 
Figure 5: Light microscopy image of cultured cell lines ...........................................26 
Figure 6: Phenotyping of cardiomyocytes using anti-cTnI. .......................................27 
Figure 7: Cytotoxicity of plant compounds...............................................................29 
Figure 8: NF-?B induction and nuclear translocation in HeLa cells after PMA 
activation .................................................................................................................30 
Figure 9: Quantification of NF-?B in resting and stimulated HeLa cells by 
immunofluorescence ................................................................................................31 
Figure 10: Quantification of NF-?B in resting and stimulated HeLa cells by ELISA-
 based assay ..............................................................................................................32 
Figure 11: NF-?B DNA-binding levels in stimulated and plant compound treated 
HeLa cells ................................................................................................................33 
Figure 12: NF-?B DNA-binding levels in HIV-1Du151 gp120 treated cardiomyocytes34 
Figure 13: Quantification of NF-?B DNA-binding in cardiomyocytes by treatment 
with gp120 or HIV-1 ................................................................................................35 
Figure 14: Fluorescent images of NF-?B stimulation in cardiomyocytes by HIV-1Du151 
gp120 and HIV-1 .....................................................................................................38 
Figure 15: Mitigation of NF-?B levels in gp120-stimulated cardiomyocytes by 
sesquiterpene lactones ..............................................................................................39 
................................................................................................................................57 
Figure 16: Quantification of NF-?B in resting and stimulated HeLa cells by 
immunofluorescence ................................................................................................57 
Figure 17: NF-?B levels in stimulated and plant compound treated HeLa cells.........57 
Figure 18: NF-?B levels in HIV-1Du151 gp120 treated cardiomyocytes......................58 
Figure 19: Quantification of NF-?B in cardiomyocytes by treatment with gp120 or 
HIV-1 ......................................................................................................................58 
 ix 
Figure 20: Mitigation of NF-?B levels in gp120-stimulated cardiomyocytes by 
sesquiterpene lactones ..............................................................................................59 
 
 x 
ABBREVIATIONS 
 
AIDS Acquired immunodeficiency syndrome 
ATP Adenosine triphosphate 
ARV antiretrovirals 
BCA bicinchoninic acid 
cTnI cardiac troponin I 
CD4/40 cluster of differentiation 4/40 
CCR5 chemokine (C-C motif) receptor 5 
DMSO dimethyl sulphoxide 
DMEM Dulbecco?s Modified Eagle?s medium 
EDTA ethylenediaminetetra-acetic acid 
EDC N-ethyl-N'-(3-diethylaminopropyl)-carbodiimide 
FCS foetal calf serum 
HIV-1 human immunodeficiency virus type-1 
HIVCM HIV associated cardiomyopathy 
HCl hydrochloric acid 
iNOS inducible nitric oxide synthase 
IKK inhibitory kappa kinase 
IL interleukin 
LTR long terminal repeat 
MWCO molecular weight cutoff 
MDM monocyte-derived macrophages 
NF-?B nuclear factor kappa B 
NHS N-hydroxysuccinimide 
NLS nuclear localisation sequence 
PMA phorbol 12-myristate 13-acetate 
PBS  phosphate buffered saline 
PBMC peripheral blood mononuclear cells 
RHD Rel homology domain 
RU response units 
SDS-PAGE sodium dodecyl sulphate ? polyacrylamide gel electrophoresis 
SPR surface plasmon resonance 
TMB 3.3?, 5.5?-tetramethylbenzidine 
TNFR Tumour necrosis factor receptor 
 1 
1 INTRODUCTION 
 
1.1 The HIV/AIDS pandemic  
In 1981 a handful of young homosexual men in the United States were found to have 
a novel type of immunodeficiency (CDC, 1981). It was soon found that this type of 
immunodeficiency was associated with a depletion of CD4+ T helper cells (Gottlieb et 
al., 1981; Masur et al., 1981). The discovery of the disease in intravenous drug users 
and recipients of blood transfusions as well as haemophiliacs receiving pooled 
clotting factors led scientists to believe that the causative agent was viral (CDC, 1982; 
Ragni et al., 1983; Weiss, 2008). Due to the infectious nature of the disease and 
depletion of the immune system cells, it was called Acquired Immunodeficiency 
Syndrome (AIDS). In 1983 a group of scientists at the Pasteur Institute in Paris 
isolated a previously unknown retrovirus from a patient diagnosed with AIDS (Barre-
 Sinoussi et al., 1983). This virus was eventually named Human Immunodeficiency 
Virus type-1 (HIV-1). In 1984, a year after the discovery of HIV-1 and three years 
after the identification of AIDS, there was sufficient data to persuade scientists that 
HIV-1 was the causative agent of AIDS (Weiss, 2008). 
  
Twenty five years of study of this agent?s structure and replication has enabled 
doctors to better diagnose, monitor and treat HIV-1 infection (Weiss, 2008). The 
advances in HIV-1 detection have made it possible to detect the virus early during 
infection, which has helped make blood donation safe. The understanding of the 
action of viral proteins has lead to antiretrovirals that inhibit viral replication, which 
has greatly prolonged the lifespan of HIV-1 positive individuals (Kinloch-De Loes et 
al., 1995; Ruprecht et al., 1990; Weiss, 2008). Knowledge that the virus infects and 
kills CD4+ cells during its replication has allowed the monitoring of disease 
progression by CD4+ cell counts and viral load.  
 
Even with the advances in detection and treatment, HIV-1 prevention programmes 
have not been as effective. The World Health Organisation reports that the worldwide 
estimate for people living with HIV-1 in 2007 was 33.2 million (WHO, 2008). Sub-
 Saharan Africa continues to be the epicentre of the epidemic with a total of 22.5 
 2 
million HIV-1-infected individuals living in this region in 2007, constituting 
approximately 67% of the worldwide HIV-1-infected population (WHO, 2008). 
 
1.2 HIV-1 structure and infectious cycle 
A mature HIV-1 virion contains two copies of viral genomic RNA in its core in 
addition to the protease, reverse transcriptase and integrase viral proteins, which are 
required for early replication events. The outer surface of the virus consists of spikes 
formed by the association of the surface glycoproteins gp120 and gp41, which are 
arranged as trimers. The viral genome is less than 10 kilobases and contains nine 
genes (Figure 1) (Haseltine, 1991). It has, however, managed to hijack host cellular 
pathways while hiding from the immune system.  
 
 
 
Figure 1: An overview of the HIV-1 proviral genome, genes and proteins 
together with a summary thereof. The figure was adapted from Greene and Peterlin 
(Greene and Peterlin, 2002). 
 
The binding of gp120 to the CD4 receptor on macrophages and T cells initiates viral 
entry. This induces conformational changes in gp120 that allow a second interaction 
between the viral protein and either the CCR5 or CXCR4 chemokine co-receptor, 
although other co-receptors have been noted (Eckert and Kim, 2001; Greene and 
Peterlin, 2002). The chemokine receptor binding allows for fusion with the cell and 
the release of the viral core into the cytoplasm of the host cell (Eckert and Kim, 
2001). The viral reverse transcriptase enzyme converts the viral RNA genome to 
DNA once the viral core has disassembled (Haseltine, 1991). The reverse 
transcriptase enzyme has no proofreading ability and is error prone, producing a 
 3 
heterogenic population. Host and viral factors move the proviral DNA to the nucleus 
of the cell where it is inserted into the chromosomal DNA by the viral protein 
integrase. The inserted proviral DNA may be transcriptionally active or latent 
depending on the location that it is integrated into (Greene and Peterlin, 2002). HIV-1 
uses the host cell?s machinery to replicate and express its genome. Viral proteins 
move to the cell membrane where they assemble into immature virus particles, 
acquire a lipid envelope and bud off from the cell (Greene and Peterlin, 2002). The 
viral protease cleaves the Gag-Pol HIV-1 protein to produce mature virions. Without 
this final step, virions remain non-infectious and cannot replicate in other cells 
(Simon et al., 2006). 
 
1.3 HIV pathogenesis 
Active replication can be seen early in infection via the mucosal or parenteral route 
and results in a high initial viral load. This may be accompanied by fever, diarrhoea 
and lymphadenopathy (Barre-Sinoussi et al., 1983; Weiss, 2008), as this replication 
takes place in the regional lymph nodes. The initial state of high viral load then 
resolves to a lower level. This new viral load is predictive of the rate of progression to 
AIDS, with a higher viral load together with a low CD4+ cell count leading to a worse 
prognosis (Mellors et al., 1997). At any given point viral load is predictive of 
transmission risk, with transmission risk being exceptionally high during acute 
infection and co-infection with sexually transmitted diseases (Simon et al., 2006). As 
the infection progresses towards AIDS, the final disease stage, a number of clinical 
syndromes manifest including HIV-associated dementia, nephropathy and 
cardiomyopathy (Moroni and Antinori, 2003). 
 
1.4 HIV-associated Cardiomyopathy 
Preceding the introduction of highly active antiretroviral therapy (HAART), cardiac 
disease was not clinically significant in HIV positive individuals. This was because 
most cases were silent or overshadowed by clinical symptoms in other organs, mainly 
the brain and lungs (Sudano et al., 2006). The arrival of HAART has significantly 
altered the course of HIV disease, resulting in increased life expectancy and the 
reduction of opportunistic infections (Barbaro, 2001; Barbaro et al., 2001). However, 
this has brought with it some side effects in the form of certain chronic conditions 
such as HIV-associated cardiomyopathy (HIVCM). Even though HAART has 
 4 
reduced the incidence of opportunistic infections, the number of cases of coronary 
syndromes is increasing because of the long term effects of HIV infection, HAART 
and opportunistic infections (Barbaro et al., 2001). The Heart of Soweto study 
conducted in 2006 showed 844 new cases of heart failure, 29 of which were attributed 
to HIV-related cardiomyopathy (Stewart et al., 2008). The Data Collection on 
Adverse Events of Anti-HIV Drugs study found that of the 33 347 patients in this 
study, 517 HIV positive patients developed myocardial infarction. Of this group, 509 
individuals were exposed to ARV (Sabin et al., 2008), which if taken together with 
the increasing number of people receiving ARV shows increasing rates of cardiac 
events linked to HIV. Regimens of HAART that include reverse transcriptase and 
protease inhibitors have been shown to reduce the rate of mortality for congestive 
heart failure but increase acute coronary syndromes (Murphy and Barbaro, 2003). 
Unfortunately studies conducted prior to the introduction of HAART are still 
applicable as the vast majority of HIV-1-infected individuals do not have access to 
these drugs (Barbaro et al., 2001; Murphy and Barbaro, 2003).  
 
Many causes of HIVCM have been proposed including both direct and indirect effects 
of HIV-1. Opportunistic infections such as cytomegalovirus, Toxoplasma gondii, 
Staphylococcus aureus and Epstein-Barr virus have also been shown to be 
contributing factors in the advance of cardiac complications (Hajjar et al., 2005; 
Moroni and Antinori, 2003). Certain nutritional deficiencies have been directly or 
indirectly linked to HIVCM that shows left ventricular dysfunction, with selenium 
supplementation in nutritionally depleted patients showing a reversal of 
cardiomyopathy (Barbaro, 2001; Barbaro et al., 2001; Lewis, 2000). In addition to 
these, autoimmune response, in the form of cardiac-specific autoantibodies, as well as 
drug toxicity have been suggested, although it is most likely that a number of these 
causes act in concert (Barbaro et al., 2001). 
 
The molecular mechanisms of the disease are still poorly understood. It has been 
found that this process is inflammatory and mediated by HIV-infected and 
cyclooxygenase 2-expressing CD68+ activated macrophages together with other 
inflammatory cells (Liu et al., 2001). Most of these inflammatory cells were found to 
have CD3 and CD8 receptors. Productive infection, together with expression of the 
viral proteins gp120 and Nef, was found only in macrophages and T cells (Liu et al., 
 5 
2001). This discounts direct infection of cardiomyocytes by HIV as previously 
suggested (Grody et al., 1990), but instead alludes to an indirect mechanism of 
cardiomyocyte damage initiated by inflammatory cells and/or circulating cytokines 
(Fisher et al., 2003). Several different cellular products have been put forth to explain 
this including nitric oxide (Barbaro et al., 1999; Kan et al., 2000) and cytokines, 
particularly tumour necrosis factor-? (TNF-?) and interleukin (IL)-1 (Barbaro et al., 
2001; Lewis, 2000). Although not produced in significant amounts by HIV-1-infected 
cells, TNF-? has been found in high levels in the plasma from 46% of HIV-1 positive 
patients with acute myocarditis (Barbaro et al., 1999; Fisher et al., 2003). It was also 
found to be expressed in 100% of endomyocardial biopsies from HIV-infected 
individuals showing myocarditis, together with IL-1b, IL-6 and IL-8 (Fisher et al., 
2003). This group also showed that the intensity of staining for TNF-? and iNOS in 
these biopsy samples inversely corresponded with CD4 count, with ARV not affecting 
these results (Barbaro et al., 1999). Cardiomyocyte contractility is affected by a 
number of factors in HIVCM including TNF-? (Fisher et al., 2003) and gp120 (Chen 
et al., 2002), suggesting that these factors have a direct effect on cardiomyocyte 
dysfunction. The damage to cardiomyocytes due to HIV-1 infection is possibly due to 
an interaction between viral proteins such as gp120 (Chen et al., 2002; Kan et al., 
2000) and cellular factors leading to the apoptosis of cardiomyocytes, a critical 
mechanism in HIVCM (Twu et al., 2002). A study that found that gp120 stimulates 
the production of nitric oxide in cardiomyocytes through the activation of nuclear 
factor-?B adds weight to this theory (Kan et al., 2000). 
 
1.5 Nuclear factor-?B  
Nuclear factor-?B (NF-?B) was first characterised as a nuclear factor necessary for 
the transcription of immunoglobulin ? light chains in B-cells (Sen and Baltimore, 
1986b). Originally thought to be present in these cells only, NF-?B was found in the 
cytoplasm of most cells from insect to human, sequestered by inhibitory proteins 
(Ghosh et al., 1998). More than twenty years since the discovery of these transcription 
factors, research into the function and regulation of NF-?B continues at a brisk pace 
(Hayden and Ghosh, 2008). 
 
The functional transcription factor consists of homo- or heterodimers made up of 
subunits from the Rel/NF-?B family of proteins (Tak and Firestein, 2001). This 
 6 
family consists of five members; p50/p105, p52/p100, RelA (p65), c-Rel and RelB 
encoded by genes that share an N-terminal Rel homology domain (RHD) (Baldwin, 
1996; Tak and Firestein, 2001). The most studied of the NF-?B proteins is the 
p50:p65 heterodimer. The first two are synthesised as inactive precursor molecules 
that undergo processing to produce transcriptionally active proteins (May and Ghosh, 
1998). The RHD is responsible for the dimerisation of subunits and DNA binding, in 
addition to containing the nuclear localisation sequence (NLS) (Barkett and Gilmore, 
1999; Ghosh et al., 1998; Hayden and Ghosh, 2008). The inhibitory ?B (I?B) proteins 
are a family of structurally and functionally related molecules. They contain 
sequences known as ankyrin repeats, which associate with the RHD of NF-?B dimers 
(May and Ghosh, 1998). NF-?B dimers are inactive in resting cells, either due to this 
association with the I?B proteins or the inactivating precursors (p100 and p105). 
These inactivating agents mask the NLS, maintaining NF-?B predominantly in the 
cytoplasm. The most studied member of I?B, I?B?, only partially masks the NLS of 
the NF-?B dimer but contains a nuclear export sequence, leading to shuttling of 
inactive molecules between the nucleus and cytoplasm (Hayden and Ghosh, 2008; 
Perkins, 2007). Once activated, transcription factors move from the cytoplasm and 
bind to the ?B promoter sites on DNA where they enhance the transcription of target 
genes (Ghosh et al., 1998). The availability of these transcription factors allows for 
rapid response when signalled as it does not require protein synthesis for the signal to 
be transmitted (Ghosh et al., 1998; May and Ghosh, 1998).  
 
There are two major pathways that lead to active NF-?B transcription factors binding 
to their target genes: the canonical (classical) and noncanonical (alternative 
pathways). The canonical pathway involves the degradation of I?B?. The activated 
inhibitory ? kinase (IKK) complex, containing IKK?, IKK? and other proteins, causes 
the phosphorylation of I?B? at Ser32 and Ser36. This leads to the degradation of I?B? 
by the 26S proteasome, which is ubiquitin induced. The degradation of I?B? exposes 
the nuclear localisation sequence of the p50:p65 NF-?B heterodimer. This allows the 
heterodimer to be translocated to the nucleus (Ghosh et al., 1998; May and Ghosh, 
1998; Perkins, 2007). The phosphorylation of the p65 by several kinases is necessary 
for transcriptional activity as this phosphorylation allows the binding of 
transcriptional co-activators to p65 and enhances transcription. In the absence of 
phosphorylation, this dimer binds to DNA but represses transcription (Hayden and 
 7 
Ghosh, 2008). The noncanonical pathway depends on the phosphorylation of p100 by 
active IKK?. This induces ubiquitin-dependent 26S proteasomal processing of the 
p100 precursor molecule to active p52, allowing nuclear translocation of p52-
 containing dimers (Ghosh et al., 1998; May and Ghosh, 1998; Perkins, 2007). The 
transcription of I?B proteins is NF-?B dependent creating a negative feedback loop 
and, together with measures that target DNA-bound dimers, this terminates the NF-?B 
response (Hayden and Ghosh, 2008).  
 
Different pathways regulate distinct subsets of NF-?B, allowing the separate 
pathways to target different genes. A broad range of receptors and stimulants activate 
the canonical pathway (TNF-?, IL-1, LPS) while only those belonging to a subset of 
the TNFR superfamily (CD40, latent membrane protein-1 of Epstein-Barr virus, 
lymphotoxin ? receptor) appear to activate the alternative pathway (Hayden and 
Ghosh, 2008; Pereira and Oakley, 2008). In addition to bacteria and viruses, NF-?B is 
also induced by physiological and physical stress such as haemorrhagic shock and 
irradiation respectively, as well as drugs and environmental stresses. The sequence 
variability of the ?B promoter site together with the varied binding preferences of the 
different NF-?B dimers results in a staggering number of genes that are very precisely 
regulated by NF-?B. Most of these genes are involved in inflammation, injury and 
stress response (Ghosh et al., 1998; May and Ghosh, 1998; Pahl, 1999). These genes 
include receptors essential for immune recognition, proteins required for antigen 
presentation and at least 27 different cytokines (Pahl, 1999). NF-?B therefore relays 
the message of a stress while simultaneously eliciting a response by activating the 
transcription of products to reduce the specific stress. This ensures that the reaction to 
a given stimulus is site and event specific and allows cells to respond to the external 
environment appropriately (Tak and Firestein, 2001). 
 
The dysregulation of NF-?B has huge implications for inflammatory diseases. 
Rheumatoid arthritis shows an overexpression of NF-?B in the inflamed lining of the 
joint. This may increase the production of IL-1, IL-6, IL-8 and TNF-?, which are pro-
 inflammatory cytokines and recruit inflammatory cells to the area. High levels of p50 
and p65 have been found in the synovium and mononuclear cells in the surrounding 
tissue (Han et al., 1998). Gastritis-associated with Helicobacter pylori shows a 
marked increase in NF-?B levels, with the number of NF-?B induced cells 
 8 
determining the severity of the gastritis (Pande and Ramos, 2005; Tak and Firestein, 
2001; van Den Brink et al., 2000). Inflammatory bowel disease shows macrophages 
in the lamina propria of the gastrointestinal tract with activated p50, c-Rel and 
exceptionally high p65. The level of pro-inflammatory cytokine production was 
modulated in these cells by their treatment with antisense p65 oligonucleotides (Tak 
and Firestein, 2001). Bronchial biopsies from asthma patients show NF-?B activation 
and nuclearisation. High levels of pro-inflammatory cytokines and chemokines were 
also found (Hart et al., 1998). NF-?B has also been linked to the inflammation of the 
arteries seen in artherosclerosis. Activated NF-?B has been found in the cells of 
artherosclerotic plaques but not in their healthy counterparts (Pande and Ramos, 
2005). Abnormal levels of NF-?B directly contribute to the pathogenesis of 
inflammation. 
 
NF-?B has been shown to be involved in the cell cycle and cell differentiation. NF-?B 
is also involved in apoptosis but may be pro- or anti-apoptotic, depending on the cell 
type and stimulus (Barkett and Gilmore, 1999; Pahl, 1999). It follows that NF-?B has 
been implicated in oncogenesis, as cancer is a disruption of the healthy cell cycle and 
apoptosis (Barkett and Gilmore, 1999; Pande and Ramos, 2005). The transcription 
factor has been shown to specifically protect cancerous cells by suppressing the 
apoptotic pathways as well as increasing proliferation of these cells (Barkett and 
Gilmore, 1999; Pande and Ramos, 2005). A mutated I?B? gene found in Hodgkin?s 
lymphoma was incapable of regulating NF-?B (Cabannes et al., 1999). 3T3 cells in 
mice were found to have an enhanced tumorigenic potential when there was increased 
expression of p52 (Ciana et al., 1997). The deletion of the ankyrin repeats in mice 
p100 showed increased gastric hyperplasia in these animals (Ishikawa et al., 1997). 
Further evidence of NF-?B participation in oncogenesis has been shown with the 
blockage of tumour formation by NF-?B inhibition through the regulation of several 
proteins upstream of NF-?B (Pande and Ramos, 2005). NF-?B activation has been 
shown in almost all types of cancers in humans (Barkett and Gilmore, 1999; Pande 
and Ramos, 2005).  
 
1.6 The role of NF-?B in HIV infection 
The ability of HIV-1 to manipulate host signalling pathways has resulted in efficient 
viral replication and gene expression. Primary monocytes and myeloid cell lines 
 9 
chronically infected with HIV-1 show elevated levels of NF-?B (Pande and Ramos, 
2005). Ordinarily, this would trigger the expression of pro-inflammatory proteins and 
the immune system recognising and attacking the pathogen. HIV-1 has evolved to 
exploit this activation. Like many other viruses that induce NF-?B activity, HIV-1 
contains NF-?B binding sites in the viral genome (Greene and Peterlin, 2002; Pahl, 
1999). The activation of NF-?B in an infected cell due to viral infection or the host 
immune response results in enhanced HIV-1 replication. HIV-1 mediated NF-?B 
induction is mostly due to IKK activation but the exact mechanism remains unclear 
(Hiscott et al., 2001). Although not essential for viral replication or gene expression, 
the absence of NF-?B slows growth rates of HIV-1 (Hiscott et al., 2001). 
 
The different subtypes of HIV-1 contain varying numbers of NF-?B binding sites in 
the promoter proximal region of the Long Terminal Repeat (LTR) of the viral 
genome. HIV-1 subtype B which is predominant in North America contains two NF-
 ?B binding sites whereas the LTR of HIV-1 subtype C, the predominant subtype in 
Africa, contains three (Barkett and Gilmore, 1999; Hiscott et al., 2001; Jeeninga et 
al., 2000). Subtype C has higher LTR promoter activity than some HIV-1 subtypes 
(Jeeninga et al., 2000), making it tempting to assume that there is a direct correlation 
between the number of NF-?B binding sites and rates of viral replication. However 
the control of viral replication by NF-?B is much more complex when transcription 
factors such as Sp-1 that act synergistically with NF-?B or compete for the same 
binding site (NFAT) are added to the mix (Hiscott et al., 2001; Jeeninga et al., 2000; 
Kinoshita et al., 1997).  
 
Several HIV-1 proteins play a part in the activation of NF-?B. HIV-1 Tat protein 
stimulates cytotoxicity and apoptosis by activating caspases and promoting the 
accumulation of reactive oxygen intermediates through NF-?B induction (Manna and 
Aggarwal, 2000). The HIV-1 accessory protein Vpr activates IL-8 expression in 
macrophages and T cells through an NF-?B dependent mechanism (Hiscott et al., 
2001; Roux et al., 2000). The Nef protein may stimulate T cells through CD3 or 
CD28 to increase the NF-?B dependent secretion of IL-2, a known inducer of NF-?B. 
HIV-1 Nef protein would thereby enhance viral transcription and replication (Wang et 
al., 2000). The HIV-1 envelope glycoprotein gp120 with its interaction with the CD4 
receptor activates NF-?B via two different but closely related pathways, one through 
 10 
the tyrosine kinase p56lck while the other works through phosphatidylinositol-3-kinase 
and IKK (Hiscott et al., 2001). 
 
1.7 The role of plant extracts in NF-?B inhibition 
NF-?B has become an attractive target in the treatment of inflammation due to its 
influence on pro-inflammatory genes (D'Acquisto et al., 2002). Inhibitors of NF-?B 
function in a number of different ways, some scavenge reactive oxygen intermediates, 
others hinder I?B or p100 degradation by affecting the 26S proteasome, while others 
still affect the transcriptional activity of NF-?B bound to DNA (Hehner et al., 1998). 
  
A number of groups have shown inhibition of NF-?B by certain plant extracts, 
especially those rich in sesquiterpene lactones. The first study on extracts from 
medicinal plants as a source of NF-?B inhibition focused on plants native to Mexico 
(Bork et al., 1997). The study found that the extracts negatively interfered with NF-
 ?B activation. The sesquiterpene lactones in the extract were subsequently inhibited 
by the addition of cystein and a loss of inhibitory activity on NF-?B was found, 
suggesting that the inhibition of NF-?B was due to the sesquiterpene lactones found in 
the extracts (Bork et al., 1997). A further study found that one particular compound, 
parthenolide, had a strong inhibitory effect on NF-?B in Jurkat (human T cell 
leukaemia), L929 (mouse fibroblast) and HeLa cell lines (Hehner et al., 1998). This 
sesquiterpene lactone was also shown to have a proapoptotic effect on cancer cells 
(Wen et al., 2002) and reduce inflammation in a mouse model of artherosclerosis 
(Lopez-Franco et al., 2006). Parthenolide has become the model compound for 
studying NF-?B inhibition by sesquiterpene lactones and an attractive possible 
pharmaceutical (Bremner and Heinrich, 2005). The mechanism of action of the 
sesquiterpene lactones is a murky one with one group stating that sesquiterpene 
lactones interfered with the induced degradation of I?B-? and I?B-? (Hehner et al., 
1998), while another finding that a sesquiterpene lactone selectively modifies the p65 
subunit, thereby inhibiting the NF-?B signalling cascade (Lyss et al., 1998).  
 
Flavonoids have also shown an inhibitory effect on NF-?B. Green tea flavonoids as 
well as resveratrol found in red wine inhibit the action of IKK, thereby reducing the 
induction of NF-?B (D'Acquisto et al., 2002; Holmes-McNary and Baldwin, 2000; 
Nomura et al., 2000). Tea flavonoids that have been shown to have an effect on NF-
 11 
?B include (-)-epigallocatechin-3-gallate (EGCG), (-)-epigallocatechin (EGC), (-)-
 epicatechin-3-gallate (ECG) and (-)-epicatechin (EC) (Wiseman et al., 2001). 
Epigallocatechin-3-gallate lowered NF-?B levels in both normal human epidermal 
keratinocytes and A431 cancer cells (Ahmad et al., 2000; Wiseman et al., 2001). 
Decreased binding of NF-?B to DNA was seen in RAW264.7 macrophages treated 
with EGCG, in addition to the inhibition LPS-induced lethality in mice by reducing 
the activation of NF-?B (Wiseman et al., 2001; Yang et al., 1998).  
 
2 OBJECTIVES 
The aims of this thesis were: 
? to measure NF-?B levels in a cellular model of HIV-associated 
cardiomyopathy, established using human stem cell-derived cardiomyocytes 
challenged with purified gp120, cell-free HIV-1 or HIV-1 infected 
macrophages, and  
? to determine whether certain natural compounds derived from South African 
indigenous plants could downregulate or restore NF-?B activation in this 
model of HIVCM. 
 
3 MATERIALS AND METHODS 
 
3.1 Propagation and Maintenance of cell lines 
The human epithelial cell line 293T (American Type Culture Collection, USA) and 
HeLa cells (Sigma, Germany) were propagated and maintained in DMEM (Lonza, 
USA), supplemented with 10% heat inactivated FCS (Sigma, Germany) and 50 mg/L 
gentamicin sulphate (Highveld Biological, RSA). The human cord-blood stem cell-
 derived cardiomyocytes (Celprogen, USA) were cultured in Human Cardiomyocyte 
Expansion Media (Celprogen, USA), a proprietary media containing serum, growth 
factors and antibiotics. All three cell lines grew optimally as monolayers at 37?C in a 
humidified incubator with 5% CO2. 
 
3.1.1 Cell count 
Cells were counted using the Trypan blue exclusion method. Cells were washed with 
PBS pH 7.2 (Lonza, USA), treated with 0.25% trypsin, 0.02% Versene (EDTA) 
 12 
(Lonza, USA) and the clumps removed by gentle pipetting. A 1:10 dilution of the cell 
suspension was made in 0.4% Trypan blue (Sigma, Germany) and loaded into a 
haemocytometer. Dead cells take up the dye and appear blue while live cells do not 
and therefore remain clear. The number of live cells per millilitre of media was 
obtained by multiplying the average number of live cells per haemocytometer square 
by 105. 
 
3.1.2 Subculture of cell lines 
All cell lines were subcultured as per normal tissue culture practises (Phelan, 2003). 
To detach cells from culture flasks, the monolayers were washed with room 
temperature PBS pH 7.2 and treated with 0.25% trypsin, 0.02% Versene (EDTA) for 
2-5 minutes at 37 ?C. Trypsinisation was stopped by adding Human Cardiomyocyte 
expansion media or 10% FCS supplemented DMEM pre-warmed to 37 ?C and cell 
clumps dispersed by careful pipetting. The cells were pelleted at 56 ? g for 5 minutes 
and re-seeded in the supplemented culture medium at the required cell density. Cells 
were not cultured beyond passage 20 as cells lost their phenotype with increasing 
passages. Cells were counted regularly and subcultured to maintain logarithmic 
expansion of cultures. 
 
3.1.3 Freezing of cell lines 
Cells no later than passage 6 were frozen to build a stock of cells. Cultures in the 
logarithmic phase of growth were trypsinised and centrifuged as in 3.1.2 and 
resuspended in 46.25% fresh medium, 46.25% conditioned propagation media, 7.5% 
DMSO (Sigma, Germany) at a density of 1 ? 106-107 cells/ml. These cells were 
quickly aliquoted into 2 ml cryogenic vials (Corning, USA), frozen gradually (-1 ?C 
per minute) and stored at -80 ?C. To thaw the cells, the vial was quickly heated by 
rubbing in hands. The cells were transferred dropwise to 10 ml Human 
Cardiomyocyte expansion media or FCS supplemented DMEM pre-warmed to 37 ?C 
in a sterile 50 ml Falcon tube (Corning, USA) and pelleted at 56 ? g for 5 minutes. 
The cell pellet was resuspended in the propagation medium and seeded as required in 
a tissue culture flask. 
 
 13 
3.2 Expression and purification of gp120 
The mammalian 293T cell line was used to express gp120. These cells were 
transfected with plasmids containing the HIV-1 gp120 inserts from the CAP 45 or 
Du151 HIV-1 strains (National Institute of Communicable Diseases, RSA). The 
transfection was carried out using the ProFection? Mammalian Transfection System-
 Calcium Phosphate Kit (Promega, USA) once the monolayer of cells in a 150cm2 cell 
culture flask (Corning, USA) reached 30-60% confluency. This kit was used to 
maximise the tranfection of cells with the plasmid, and thereby maximise protein 
expression. Three hours prior to transfection the medium in the flasks was replaced 
with fresh 2% FCS supplemented DMEM pre-warmed to 37 ?C and the transfection 
reagents brought to room temperature. Sterile, deionised water was added to 10-20 ?g 
plasmid DNA and 62 ?l 2 M calcium chloride to a final volume of 500 ?l, which was 
added dropwise to 500 ?l 2 ? HBS while gently vortexing. After incubating at room 
temperature for 30 minutes, the solution was added to the 293T cells and incubated at 
37 ?C in a humidified incubator with 5% CO2. 
 
The supernatant was harvested two days post-infection and purified using a Galanthus 
nivalus lectin-agarose column (Sigma, Germany) as previously described (Nkosi et 
al., 2005). This column has affinity for glycosylated proteins and isolates gp120 form 
the supernatant. The supernatant was passed through the column then sequentially 
washed with 5 column volumes of 0.65 M sodium chloride (Sigma, Germany) in PBS 
pH 7.4 (Sigma, Germany), 1 column volume of PBS pH 7.4, 1 column volume of 1 M 
sodium chloride in PBS, and 1 column volume of PBS. Samples were eluted with 3 
ml 1 M methyl-?-D-mannopyranoside (Sigma, Germany), dialyzed against PBS at 4 
?C for 16 hours and an additional 2 hours using the 3.5 kDa MWCO Slide-A-Lyzer? 
dialysis cassette (Thermo Scientific, USA), and concentrated using a 30 kDa MWCO 
Amicon? Ultra-15 centrifugal filter devices (Millipore, USA). 
 
3.3 Detection and quantification of 293T-expressed gp120 
3.3.1 SDS-PAGE detection of gp120 
The column flow through and purified gp120 were separated using denaturing 8% 
acrylamide gels using standard formulations for the gel and all buffers. Samples were 
diluted in sample buffer and heated for 5 minutes at 95 ?C. After loading, samples 
were electrophoresed for 45 minutes at 200 V using a Mini-PROTEAN? 3 Cell 
 14 
(BioRad, USA). The gels were either prepared for Western Blotting or stained with 
Coomassie Brilliant Blue solution [0.25% Coomassie Brilliant blue R250 (Merck, 
Germany), 50% methanol (Sigma, Germany), 10% acetic acid (Sigma, Germany)] for 
30 minutes and destained (20% methanol, 10% acetic acid) until all background was 
removed. 
 
3.3.2 Western Blot detection of gp120 
Resolved bands on the SDS-PAGE gels were transferred electrophoretically to 
Hybond-ECL nitrocellulose membranes (Amersham Biosciences, USA) in transfer 
buffer (25mM Tris base (Merck, Germany), 192 mM glycine (Merck, Germany), 20% 
methanol) at 90 mA, 4 ?C for 16 hours using the Mini Trans-Blot? Electrophoretic 
Transfer Cell (BioRad, USA). The membrane was washed in PBS with Tween? 20 
pH 7.4 (Merck, Germany), blocked for an hour with 5% (w/v) fat free milk powder in 
PBS-Tween 20 and probed with a 1:15000 dilution of human serum from HIV-1 
positive patients in 2.5% (w/v) fat free milk powder in PBS-Tween. The serum from 
HIV-1 positive patients contained high levels of antibodies against HIV-1 gp120. The 
membrane was washed thrice with PBS-Tween 20 for 5 minutes, and reblocked in 
10% (w/v) milk powder in PBS-Tween 20. The anti-human HRP-conjugated 
secondary antibody (Santa Cruz Biotechnology, USA) was diluted 1:20000 in 2.5% 
(w/v) fat free milk powder in PBS-Tween 20. The membrane was probed with this 
diluted antibody for an hour at room temperature. The membrane was washed three 
times as above and detected using ECL Advance? Western Blotting Detection kit 
(GE Healthcare, USA). First, equal parts of solution A and solution B were mixed. 
This was poured over the membrane and allowed to incubate at room temperature for 
a minute. Excess detection reagent was removed and the membrane wrapped in cling 
film. This was exposed to Amersham Hyperfilm ECL X-ray film (GE Healthcare, 
USA). 
 
3.3.3 Quantification of purified gp120 by the bicinchoninic acid protein assay 
Purified gp120 was quantified using the bicinchoninic acid protein assay [Pierce? 
BCA Protein Assay kit (Thermo Scientific, USA)]. This assay allows for the 
colorimetric detection and quantification of total protein content (Smith et al., 1985) 
even in samples that contain detergents, which are included in most protein extraction 
buffers. A standard dilution series (0.025 mg/ml, 0.125 mg/ml, 0.25 mg/ml, 0.5 
 15 
mg/ml, 0.75 mg/ml, 1 mg/ml, 1.5 mg/ml and 2 mg/ml) was set up using the albumin 
standard. The BCA Working Reagent, which contains cupric sulphate and BCA, was 
prepared (50:1 Reagent A: Reagent B) and added to the standard dilutions and 
samples of unknown concentration in a 1:20 ratio. This was heated to 37 ?C for 30 
minutes, cooled to room temperature and the absorbance measured at 562 nm using 
the NanoDrop? ND-100 spectrophotometer (Thermo Scientific, USA). In the alkaline 
medium provided by the BCA Working Reagent, peptide bonds and the amino acids 
cysteine, tryptophan and tyrosine  reduce the Cu2+ of cupric sulphate to Cu1+ 
(Wiechelman et al., 1988). This ion chelates two BCA molecules, producing the 
purple colour measured at 562 nm. A standard curve was prepared from the albumin 
standards and the concentrations for the unknown samples determined from the graph. 
 
3.4 Validation of HIV-1Du151 gp120 biological activity 
The HIV-1Du151 gp120 expressed in 293T cells needed to be assayed for biological 
activity before use in further experiments. The binding of the HIV-1 gp120 
monoclonal antibody IgG1 b12 to the expressed protein was employed to determine 
whether the protein was biologically active, as this antibody has been mapped to the 
CD4 binding site. The following reagent was obtained through the NIH AIDS 
Research and Reference Reagent Program, Division of AIDS, NIAID, NIH: HIV-1 
gp120 Monoclonal Antibody (IgG1 b12) from Dr. Dennis Burton and Carlos Barbas. 
The binding of expressed protein to IgG1 b12 was monitored using surface plasmon 
resonance measured by the BIAcore? 3000 (GE Healthcare, USA). 
 
3.4.1 Immobilisation of HIV-1Du151 gp120 to sensor surface 
The expressed HIV-1Du151 gp120 was bound to a CM5 sensor chip by amine coupling, 
using the BIAcore? amine coupling kit (GE Healthcare, USA) (Brigham-Burke et al., 
1992; Karlsson et al., 1991). Equal amounts of 1 M N-hydroxysuccinimide (NHS) 
(GE Healthcare, USA) and 1 M N-ethyl-N'-(3-diethylaminopropyl)-carbodiimide 
(EDC) (GE Healthcare, USA) were mixed and injected over a flow cell of the 
carboxymethylated dextran surface of the sensor chip (GE Healthcare, USA) to 
activate it. The HIV-1Du151 gp120 (20 ?g/ml in 10 mM sodium acetate (Sigma, 
Germany) pH 4.7) was injected over the activated flow cell. This was followed by 
injection of 1 M ethanolamine-hydrochloride pH 8.5 (GE Healthcare, USA) to block 
remaining amino-ester groups. Finally, 10 mM glycine-HCl (Merck, Germany) pH 
 16 
2.5 was injected over the flow cell to remove noncovalently bound protein. The flow 
rate for all BIAcore? experiments was set at 5 ?l/min. 
 
3.4.2 Interaction between immobilised gp120 and IgG1 b12 antibody 
The IgG1 b12 antibody (50 ?l, 14 ?g/ml in 10 mM sodium acetate pH 4.7) was 
injected over the gp120-coupled flow cell. The flow cell was regenerated by passing 
10 mM glycine-HCl pH 2.5 to remove the antibody from the coupled protein. The 
ligand buffer (10 mM sodium acetate pH 4.7) was injected over the protein-coupled 
flow cell to serve as a negative control. The baseline was set immediately before 
injection of antibody or buffer to determine the number of response units bound after 
injection relative to the baseline. 
 
3.5 Isolation of human peripheral blood mononuclear cells from buffy coats and 
cultivation of human macrophages by monocyte differentiation 
Human buffy coats were obtained from the Gauteng Blood Transfusion Services. 
Buffy coats are the residual material from donated blood after plasma, platelets, and 
red cell separation for patient use. Peripheral blood mononuclear cells (PBMC) were 
isolated form buffy coats as previously described (Khati et al., 2003). Buffy coats 
were layered onto Ficoll-Paque? PLUS (GE Healthcare, USA) and centrifuged at 
900 ? g, 20 ?C for 30 minutes. The serum layer was removed and heat inactivated at 
56 ?C for 30 minutes to provide autologous serum. The PBMC layer was harvested, 
washed once with PBS pH 7.2 and recovered by centrifugation at 56 ? g, 4 ?C for 5 
minutes. The cell pellet was resuspended in 30 ml cold hypotonic ammonium chloride 
solution and incubated at room temperature for 20 minutes to allow for the lysis of 
residual red blood cells. The PBMC were pelleted by centrifugation and incubated in 
gelatine-coated tissue culture flasks containing RPMI medium (Sigma, Germany) 
with 5% autologous serum at 37 ?C, 5% CO2 for 2 hours. Non-adherent cells were 
removed by washing with RPMI pre-warmed to 37 ?C and adherent monocytes 
incubated for an additional 16 hours. Monocytes were harvested by incubating the 
culture at 4 ?C for 30-45 minutes to allow for spontaneous detachment. The 
monocytes were re-seeded in X-VIVO 10 medium (Lonza, USA) containing 5% 
autologous serum. X-VIVO 10 is a conditioned media that allows for the 
differentiation of monocytes into macrophages in 7 to 10 days (Khati et al., 2003). 
 
 17 
3.6 Infection of monocyte-derived macrophages with HIV-1 
Day 7 to 10 monocyte-derived macrophages (MDM) were infected with Du151 or 
CM9 strains of HIV-1 (National Institute of Communicable Diseases, RSA) or mock 
infected with PBS as previously described (Strober, 2004). The cells were exposed to 
1 ? 105 genome equivalents of the HIV-1 or PBS for 3 hours before washing with 
PBS and X-VIVO 10 supplemented with 5% autologous serum. The cells were 
maintained in X-VIVO 10 containing 5% autologous serum for 14 days, with half of 
the media being replaced on Day 7. 
 
3.6.1 Determination of viral replication in MDM 
Day 14 supernatant from the HIV-1 infection was tested for the presence of the HIV-1 
protein p24 using the Vironostika? HIV-1 antigen Microelisa test (bioM?rieux SA, 
France) according to the manufacturer?s recommendations. The use of this kit to 
determine HIV-1 infection and growth rate is a standard protocol of the AIDS unit of 
the National Institute of Communicable Diseases where HIV-1 experiments were 
conducted. The kit contains a plate coated with an anti-HIV-1 p24 monoclonal 
antibody which captures the HIV-1 p24 protein. A standard dilution series and diluted 
culture supernatant were added to the wells and incubated at 37 ?C for 1 hour. The 
wells were washed three times with proprietary 1 ? Wash Buffer and horseradish 
peroxidase-conjugated human anti-HIV-1 p24 antibody added. This was further 
incubated at 37 ?C for an hour and washed as above. Equal parts of the TMB and urea 
peroxidase solutions were premixed before addition to the wells. The absorbance was 
immediately measured at 650 nm as a kinetic reading using a Versa Max Pro 
microplate reader (MDS Analytical Technologies, USA). 
 
3.7 Immunocytochemistry 
HeLa cells or human cord-blood stem cell-derived cardiomyocytes were removed 
from their culture flasks by trypsinisation and incubated in 2-well chamber slides 
(Nunc, USA) overnight to allow cells to adhere and regain their morphology. 
Immunocytochemistry was performed as previously described (Baldwin, 1996; 
Watkins, 1989). After media was removed and cells washed in cold PBS pH 7.2, cells 
were simultaneously fixed and permeabilised by incubating in 2% paraformaldehyde 
(Merck, Germany), 0.1% Triton X-100 (Sigma-Aldrich) for 30 minutes on ice. 
Fixative was removed and cells washed in cold PBS pH 7.2. The fixed cells were 
 18 
incubated first with primary and then fluorochrome-conjugated secondary antibody 
(Invitrogen, USA) for 1 hour at 4 ?C, both of which were diluted in 1% serum to 
prevent nonspecific binding of antibody. Antibody incubations were followed by four 
PBS pH 7.2 washes of 5 minutes each. The upper structure of the chamber slide was 
removed and cells mounted using UltraCruz? Mounting Medium (Santa Cruz 
Biotechnology, USA) containing DAPI for DNA counterstaining. The slides were 
subsequently visualized using the Olympus? BX41 System Fluorescent Microscope, 
together with the analySIS LifeScience? software (Wirsam Scientific and Precision 
equipment, RSA). This technique was used with two very different aims in mind. The 
first was to ensure that the cardiomyocytes retained their phenotype by staining for 
the cardiac specific protein troponin I (cTnI) by a monoclonal antibody (R&D 
Systems, USA). The second was to visualise the activation of NF-?B in HeLa cells 
and cardiomyocytes using either a mouse or rabbit monoclonal antibody raised 
against human p65 (Santa Cruz Biotechnology, USA), a subunit of the NF-?B 
p50/p65 heterodimer. 
 
3.8 Cytotoxicity assays 
Seven natural compounds derived from South African indigenous plants 
(Bioprospecting Group, CSIR Biosciences, RSA) were screened for cytotoxicity 
against HeLa cells, MDM and cardiomyocytes. These compounds were from three 
different classes of compounds; flavonoids, sesquiterpene lactones and a 
sesquiterpene (Table 1). Cells were seeded at a density of 2 ? 104 cells per well in 96 
microwell white optical bottom plates (Nunc, USA). HeLa cells and cardiomyocytes 
were allowed to adhere overnight, while monocytes were seeded and allowed to 
differentiate into macrophages in X-VIVO 10 containing 5% autologous serum over 
seven to ten days. The cells were incubated with 10-fold serial diluted (100 ?M, 10 
?M, 1 ?M or 100 nM) plant compounds for 3 hours at 37 ?C, 5% CO2 with humidity. 
The experiments were conducted in triplicate, including cell controls, which were not 
treated. After the 3 hour incubation with the compounds, the cells were lysed and 
ATP levels measured as a measure of cell viability using the CellTiter-Glo? 
Luminescent cell viability assay (Promega, USA). This assay detects the number of 
viable cells in the culture as a function of ATP levels, as ATP levels correlate to cell 
viability. The CellTiter-Glo? Reagent lyses cells but prevents the degradation of ATP 
by ATPases. It then uses the bioluminescent luciferin-luciferase reaction to detect the 
 19 
amount of ATP in the cell culture. The reagents were brought to room temperature 
before mixing the CellTiter-Glo? buffer with the CellTiter-Glo? substrate by gentle 
inversion to prevent the formation of bubbles. A volume of CellTiter-Glo? reagent 
equal to the media in the wells was added to the wells. This was incubated at room 
temperature to allow all cells to be lysed and the luminescent signal to stabilise. The 
luminescence measurement was acquired using the Biotek? FLx800? Fluorescence 
microplate reader and the Gen 5? Data Analysis software (Analytical and Diagnostic 
Products, RSA). 
 
3.9 Quantitative measurement of NF-?B 
 
3.9.1. Immunofluorescence 
HeLa cells were seeded in sterile 96 microwell black optical bottom plates (Nunc, 
USA) at 1 ? 104 cells per well and allowed to adhere overnight. Some cells were left 
unstimulated while others were simulated to release NF-?B by incubating with 50 
ng/ml phorbol 12-myristate 13-acetate (PMA) for 20 and 60 minutes at 37 ?C, 5% 
CO2 with humidity (Bork et al., 1997). After medium was removed and cells washed 
in cold PBS, cells were simultaneously fixed and permeabilised by incubating in 2% 
paraformaldehyde (Merck, Germany), 0.1% Triton X-100 (Sigma-Aldrich) for 30 
minutes on ice. Fixative was removed and cells washed in cold PBS pH 7.2. The fixed 
cells were incubated first with the p65 antibody (Santa Cruz Biotechnology, USA) 
and then fluorescein isothiocyanate-conjugated secondary antibody (Invitrogen, USA) 
for 1 hour at 4 ?C, both of which were diluted in 1% serum. Each incubation of 
antibody was followed by four PBS washes of 5 minutes each. Secondary antibody 
staining only was used as a blank for background fluorescence. All conditions were 
carried out in triplicate. The plate was read at 520 nm on the Biotek? FLx800? 
Fluorescence microplate reader and the Gen 5? Data Analysis software (Analytical 
and Diagnostic Products, RSA). 
 
3.9.2.1 Protein extraction 
The whole cell lysate was obtained using the 5 ? Reporter Lysis Buffer (Promega, 
USA). Lysis buffers which were freshly prepared were attempted but these yielded 
protein levels that were too low to be used in further experiments. Cells in 24-well 
plates (Corning, USA) were washed with room temperature PBS pH 7.2. The 
 20 
Reporter Lysis Buffer was diluted five-fold and 125 ?l 1 ? Reporter Lysis Buffer was 
added to each well. The plate was incubated at room temperature with gentle rocking 
for 20 minutes. The plate was then frozen at -20 ?C overnight. This was thawed to 
room temperature with gentle rocking for 30 minutes. The cells were scraped and 
centrifuged at 12 000 ? g for 2 minutes at 4 ?C. The supernatant, or whole cell lysate, 
was used undiluted in the ELISA-based NF-?B assay. 
 
3.9.2.2 ELISA-based NF-?B assay 
Previously electromobility shift assays have been used to show increases in NF?B. 
However, this used radioactivity and resulted in streaky gels that required 
densitometry to quantify these increases. In this thesis the TransAM? NF?B Family 
Transcription Factor Assay kit (Active Motif, USA) was utilized to detect and 
quantify NF-?B. This assay is a modification on traditional enzyme-linked 
immunosorbent assays because it uses the NF-?B consensus sequence to immobilize 
NF-?B instead of the conventional capture antibody. This assay could have been 
prepared from scratch but would have been more expensive to purchase the individual 
components. Complete Binding Buffer (30 ?l), which contained dithiothreitol and 
herring sperm DNA, was added to each DNA-coated well and 20 ?l of the whole cell 
lysate added to this. The plate was sealed and incubated at room temperature with 
mild shaking for 1 hour. Each well was washed three times with 1 ? Wash Buffer. 
The p65 antibody was diluted 1:1000 in 1 ? Antibody Binding Buffer, 100 ?l added to 
each well and the plate incubated at room temperature for 1 hour. After washing each 
well thrice with 1 ? Wash Buffer, horseradish peroxidase-conjugated secondary 
antibody was diluted and incubated as above. The wells were washed thrice and 100 
?l of the developing solution was added to each well at room temperature. This was 
incubated for 2-10 minutes protected from light until the solution turned a medium to 
dark blue. The reaction was stopped by adding 100 ?l of the stop solution to each 
well. The absorbance was read within 5 minutes at 450 nm using the Biotek? 
ELx800? Absorbance microplate reader and KC? junior software (Analytical and 
Diagnostic Products, RSA). 
 
 21 
3.10 Statistical Analysis 
Statistical analyses (Normality and t-tests) were performed using the GraphPad 
Prism? software Version 3.02. 
 
 22 
4 RESULTS 
 
4.1 HIV-1 glycoprotein gp120 expression and validation  
 
4.1.1 Expression, purification detection and quantification of gp120 
Large scale expression (? 2 litres) of gp120 yielded approximately 0.4 mg/L and 0.2 
mg/L of HIV-1Du151 and HIV-1CAP45 gp120 respectively as determined by the BCA 
assay. This yield was much lower than expected. After five months of poor yield and 
trouble shooting, a luminescence-based detection for mycoplasma enzymes found that 
the cells were contaminated with mycoplasma. This contamination caused a marked 
decrease in cell metabolism, thereby reducing transfection and expression 
efficiencies. New mycoplasma-free 293T cells were obtained, propagated and 
tranfected as described in section 3.2. The protein expressed using a new batch of 
293T cells resulted in improved yield of about 2 mg per 200ml (equivalent to 10 
mg/L) of HIV-1Du151 gp120 and 0.5 mg per 60 ml (approximately 8 mg/L) HIV-1CAP45 
gp120 as measured by the BCA assay and confirmed by western blot (Figure 2). Lane 
2 of the western blot contains the positive control HIV-1Ba-L gp120 which runs at 
approximately 97 kDa on the blot and is therefore smaller than the 120 kDa protein 
expressed. This is because the positive control was expressed in insect cells and is 
only partially glycosylated, resulting in this protein being smaller than the fully 
glycosylated gp120 expressed in mammalian cells (Figure 2). Due to the amount of 
protein expressed, only HIV-1Du151 gp120 at a final concentration of 2 mg/ml was 
validated and used in subsequent experiments. 
 
 23 
 
 
Figure 2: Western blot analysis of the lentil-lectin purified gp120. M1 and M2 
represent two different molecular weight markers. Lane 2 contains 7 ?g HIV-1Ba-L 
gp120, Lane 3 is flow-through from the lentil-lectin column of the HIV-1CAP45 gp120 
transfection, Lane 4 contains flow-through from the HIV-1Du151 gp120 transfection, 
Lane 5 and 6 contain purified 4 ?g HIV-1CAP45 gp120 and 4 ?g HIV-1Du151 gp120 
proteins respectively. The membrane was probed with serum from HIV-1 positive 
patients and anti-human HRP-conjugated secondary antibody, before detection of 
gp120-bound antibodies by chemiluminescence. 
 
4.1.2 Validation of HIV-1Du151 gp120 biological activity 
Following expression and purification, the functionality of HIV-1Du151 gp120 was 
validated by binding to IgG1 b12 using surface plasmon resonance technology. A 
total of 17500 RU of HIV-1Du151 gp120 was coupled to one flow cell of a CM5 sensor 
chip. The ligand buffer (10 mM sodium acetate pH 4.7) was used to dilute the 
antibody before passing it over the gp120-coupled flow cell. This buffer served as an 
excellent negative control as it showed how the buffer reacted with the protein. A 
small change of 120 RU was seen after the injection of the ligand buffer compared to 
the 3000 RU of IgG1 b12 bound (Figure 3). The sensogram curve of IgG1 b12 was 
typical of protein binding. 
 
 24 
-250 0 250 500 750 1000
 -10000
 -5000
 0
 5000
 10000
 15000
 20000 b12 antibody
 Ligand buffer
 Time (s)
 Re
 lat
 ive
  R
 es
 po
 ns
 e u
 nit
 s
 (R
 U)
  
Figure 3: Sensogram showing the interaction between the anti-gp120 monoclonal 
antibody b12 and HIV-1Du151 gp120. The pink graph represents the binding of the 
b12 antibody to HIV-1Du151 gp120, the blue shows the ligand buffer passed over the 
gp120-coupled flow cell and corresponds to non-specific binding. 
 
4.2 Culture and infection of monocyte-derived macrophages  
 
4.2.1 Isolation of human peripheral blood mononuclear cells (PBMC) from buffy 
coats and cultivation of human macrophages by monocyte differentiation 
Day 16 MDM were cultured and thereafter seeded in a chamber slide and bright field 
images obtained using Olympus? BX41 System Fluorescent Microscope, together 
with the analySIS LifeScience? software. The macrophages exhibited a heterogeneous 
population (Figure 4) and had strong adherence properties. 
 
 25 
 
 
 
 
Figure 4: Light microscopy image of Day 16 monocyte-derived macrophages 
(Magnification, 100?). Different morphologies can be seen because MDM exhibit a 
diverse and heterogeneous population. The arrows indicate the fried egg-like shape 
typical of circulating macrophages and the asterisk shows a spherical macrophage. 
 
4.2.2 Infection of MDM by Du151 and CM9 HIV-1 strains 
Infection of MDM by HIV-1 was measured by the Vironostika? HIV-1 antigen kit. 
An increase of the viral protein p24 was used as a measure of viral replication. A 
standard curve was plotted after measuring the absorbance of the dilution series of the 
kit standard. Unfortunately the measurements for all infections on Day 14 were 
approximately 0 mg/ml, indicating that the macrophages were not infected with the 
two strains of HIV-1 used. It was also found that the viral stocks of HIV-1 DU151 were 
no longer viable and needed to be re-established. Due to the amount of time this takes, 
a different viral strain HIV-1 CM9 was used in subsequent experiments. 
 
4.3 Culture and maintenance of cells lines 
The HeLa cells or human cord-blood stem cell-cardiomyocytes were incubated in a 
chamber slide overnight and bright field images obtained using Olympus? BX41 
System Fluorescent Microscope, together with the analySIS LifeScience? software. 
The HeLa cells rapidly proliferated with a high density (Figure 5A). The 
cardiomyocytes were spindle-shaped, as is typical of muscle cells (Figure 5B).  
 
 26 
 
 
Figure 5: Light microscopy image of cultured cell lines; HeLa cells (A) or human 
cord-blood stem cell-derived cardiomyocytes (B). (Magnification, 100?) 
 
The human cord-blood stem cell-derived cardiomyocytes were routinely tested to 
determine whether they maintained their cardiomyocyte phenotype. This was 
accomplished by immunocytochemistry, with staining for cTnI. Two negative 
controls were conducted, the first using HeLa cells to show specificity of the antibody 
to cardiomyocytes and the second using human cord-blood stem cell-derived 
cardiomyocytes stained with only rhodamine-conjugated secondary antibody 
(Invitrogen, USA) to determine whether the fluorochrome or secondary antibody 
showed any nonspecific binding (Figure 6 A and B respectively). The human cord-
 27 
blood stem cell-derived cardiomyocytes stained positive for cTnI (Figure 6C), 
confirming that these cells were cardiomyocytes. 
 
 
 
Figure 6: Phenotyping of cardiomyocytes using anti-cTnI. Fluorescence 
microscopy images of HeLa cells stained with anti-cTnI (A) as a negative control, 
human cord-blood stem cell-derived cardiomyocytes stained with secondary antibody 
only (B) as a second negative control, and with anti-cTnI (C). The red dye indicates 
the presence of cTnI and the blue the DAPI-stained nuclei. (Magnification, 500?) 
 
4.4 Cytotoxicity of plant compounds 
The plant compounds were diluted to a stock concentration of 2 mg/ml in DMSO and 
stored at -20 ?C until use. The compounds were serially diluted in PBS pH 7.2 just 
before use. The serially diluted compounds were incubated with the respective cell 
lines to test their cytotoxicity. The compounds significantly reduced cell viability 
 28 
(p<0.05) in very few cases; in HeLa cells with compounds 8B and 8C; and in MDM 
with compounds 8A and 10B. This significant reduction of cell viability was only 
seen with treatment at the highest concentration of 100 ?M (Figure 7A and B 
respectively). However, a very significant decrease in cell viability was observed after 
incubation of the cardiomyocytes with compounds 106A and 38B at the highest 
concentration (p<0.001) (Figure 7C). 
 
0 5 10
 0
 20
 40
 60
 80
 100
 120
 140 8A
 8B
 8C
 106A
 38B
 124D
 10B
 100 110
 Concentration (mM)
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Ce
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 0 5 10
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 140 8A
 8B
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 106A
 38B
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 100 110
 Concentration (mM)
 % 
Ce
 ll S
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 viv
 al
 0 5 10
 0
 20
 40
 60
 80
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 120
 140 8A
 8B
 8C
 106A
 38B
 124D
 10B
 100 110
 Concentration (mM)
 % 
Ce
 ll s
 ur
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 % 
Ce
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Ce
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Ce
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Ce
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Ce
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Ce
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Ce
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Ce
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Ce
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Ce
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Ce
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Ce
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 29 
 
Figure 7: Cytotoxicity of plant compounds. Graphs showing the percentage cell 
viability after 3 hours of plant compound treatment of HeLa cells (A), MDM (B), and 
human cord-blood stem cell-derived cardiomyocytes (C). 
 
4.5 Measurement of NF-?B 
 
4.5.1 Immunocytochemistry 
HeLa cells were used to illustrate the induction of NF-?B and the nuclear localisation 
thereof. This was to show the difference between resting cells and cells exhibiting 
constitutively activated NF-?B. Cells were stimulated with 50 ng/ml PMA for the 
stated time before staining with an anti-p65 mouse monoclonal antibody. This was 
visualised using a fluorescein isothiocyanate-conjugated secondary antibody 
(Invitrogen, USA) which emits a green fluorescence. Unstimulated HeLa cells 
exhibited low levels of NF-?B in their cytoplasm (Figure 8A). The incubation with 
PMA showed an increase in cytoplasmic NF-?B after 20 minutes (Figure 8B) and 
visible nuclear translocation after 60 minutes (Figure 8C). 
 
 30 
 
 
Figure 8: NF-?B induction and nuclear translocation in HeLa cells after PMA 
activation. Fluorescence microscopy images with green indicating NF-?B and blue 
the DAPI-stained nuclei. These images show untreated HeLa cells with low levels of 
NF-?B in the cytoplasm (A), cells stimulated with 50 ng/ml PMA for 20 minutes (B) 
clearly showing an increase in cytoplasmic NF-?B, and for 60 minutes (C), 
illustrating the movement of NF-?B to the nucleus. (Magnification, 500?) 
 
4.5.2 Immunofluorescence 
HeLa cells were cultured on a 96 well black optical bottom plate and stimulated to 
release NF-?B by incubating with 50 ng/ml PMA for 20 or 60 minutes or left 
untreated to show the difference between resting cells and those exhibiting activated 
NF-?B. Cells were stained for NF-?B with the p65 primary antibody and fluorescein 
isothiocyanate-conjugated secondary antibody before detection by the Fluorescence 
microplate reader (Relative fluorescence values can be found in the Appendix Figure 
16). No significant difference between the resting and stimulated cells (p>0.05) could 
 31 
be seen after measurement of fluorescence emitted (Figure 9). This may be due to the 
test not being sensitive enough to determine the difference between resting and 
stimulated levels of NF-?B or there being no significant difference between NF-?B 
levels in resting and stimulated cells. This could only be determined by using a 
different method to quantify NF-?B levels. 
 
0.8
 0.85
 0.9
 0.95
 1
 1.05
 Unstimulated 20 60
 PMA stimulation time (m in)
 n-
 fo
 ld
  di
 ffe
 ren
 ce
  o
 f U
 ns
 tim
 ul
 ate
 d
  
Figure 9: Quantification of NF-?B in resting and stimulated HeLa cells by 
immunofluorescence. NF-?B levels relative to unstimulated cells show no significant 
difference between unstimulated cells and those stimulated with PMA 20 and 60 
minutes respectively (p>0.05). 
 
4.5.3 ELISA-based NF-?B assay 
HeLa cells were cultured on 24 well plates at the specified cell densities. Some of 
these cells were stimulated to release NF-?B by incubating with 50 ng/ml PMA for 60 
minutes at 37 ?C (Bork et al., 1997). Cellular proteins were extracted (3.9.2.1) and 
this whole cell lysate was used in the TransAM? NF?B Family Transcription Factor 
Assay kit. The Raji nuclear extract, a protein extract high in NF-?B, was used as a 
positive control to ensure that the kit was working optimally. A clear increase in the 
levels of NF-?B after PMA stimulation and a concentration dependent increase in NF-
 ?B were observed (Figure 10). This demonstrates that the protocols for protein 
extraction and NF-?B quantification are compatible and effective for the measurement 
of the shifting NF-?B levels. 
 
 32 
0.0000
 0.2000
 0.4000
 0.6000
 0.8000
 1.0000
 100000 200000 300000 Positive
 Number of cells
 Ab
 so
 rb
 an
 ce
  (4
 50
 nm
 )
 Unstimulated
 PMA stimulated
 Raji Nuclear extract
  
Figure 10: Quantification of NF-?B in resting and stimulated HeLa cells by 
ELISA-based assay. NF?B Family Transcription Factor Assay of HeLa cells 
showing an increase in the transcription factor after PMA stimulation as well as a cell 
concentration-dependant increase in NF-?B. All PMA stimulated were significantly 
different from their unstimulated counterparts (p<0.05). 
 
4.6 Activity of plant compounds against NF-?B activation in HeLa cells 
HeLa cells were seeded at 2 ? 105 cells per well on a 24 well plate and allowed to 
adhere overnight. These were treated with 50 ?M of each plant compound for 1 hour 
before stimulation with PMA for 60 minutes as previously described (Bork et al., 
1997). Controls for PMA stimulation only and unstimulated cells were also 
conducted. Cellular proteins were extracted and NF-?B measured using the 
TransAM? NF?B Family Transcription Factor Assay as described above. The 
significant reduction in NF-?B that was clear with 106A and 38B (p<0.05 vs. 
unstimulated), both sesquiterpene lactones, was not evident with the flavonoids (8A, 
8B and 8C) and the sesquiterpene (10B) (Figure 11). The sesquiterpene lactone 124D 
reduced NF-?B levels but this reduction was not significant. Absorbance values can 
be found in the Appendix (Figure 17). 
 
 33 
0.000
 1.000
 2.000
 3.000
 4.000
 5.000
 6.000
 PMA only 8A 8B 8C 106A 38B 124D 10B Raji NE
 Treatment
 DN
 A-
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 nd
 in
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ac
 tiv
 ity
  (r
 el.
  va
 lu
 e)
  
Figure 11: NF-?B DNA-binding levels in stimulated and plant compound treated 
HeLa cells. TransAM? NF?B Family Transcription Factor Assay of HeLa cells 
showing NF-?B levels after treatment with plant compounds and stimulation with 
PMA. As in Figure 9 the Raji Nuclear extract was used as a positive control. 
(*significantly lowered, p<0.05) 
 
4.7 Optimisation of NF-?B assay for gp120 stimulation of cardiomyocytes 
Human cord-blood stem cell-derived cardiomyocytes were seeded at 3 ? 105 cells per 
well on a 24 well plate and allowed to adhere overnight. These cells were treated with 
4 ?g/ml HIV-1Du151 gp120 for 2, 3 and 4 hours to induce NF-?B levels or were left 
untreated. Thereafter, cellular proteins were extracted and NF-?B measured using the 
TransAM? NF?B Family Transcription Factor Assay as above. Absorbance values 
can be found in the Appendix (Figure 18). Stimulation with gp120 for two hours gave 
the best stimulation of 2.6-fold, although longer stimulation continued to show an 
elevation in NF-?B levels of at least two-fold (Figure 12). Only stimulation of 
cardiomyocytes with gp120 for two hours significantly raised NF-?B levels (p<0.05). 
This was used in future experiments with gp120 and HIV-1. 
 
 *  * 
 34 
0.000
 0.500
 1.000
 1.500
 2.000
 2.500
 3.000
 Unstimulated 2 3 4
 gp120 stimulation (h)
 DN
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ac
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  (r
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  va
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 e)
  
Figure 12: NF-?B DNA-binding levels in HIV-1Du151 gp120 treated 
cardiomyocytes. NF?B Family Transcription Factor Assay of human cord-blood 
stem cell-derived cardiomyocytes showing a stimulation of NF-?B levels after 
treatment with HIV-1Du151 gp120. Treatment with gp120 for two hours significantly 
elevated NF-?B levels as compared to unstimulated cells (* p<0.05). 
 
4.8 Stimulation of NF-?B in cardiomyocytes by HIV-1 and gp120 
Human cord-blood stem cell-derived cardiomyocytes were seeded at 3 ? 105 cells per 
well on a 24 well plate and allowed to adhere overnight as above. These cells were 
treated with 4 ?g/ml HIV-1Du151 gp120, HIV-1 CM9 (8 ng/ml of p24) or left untreated 
for two hours. Several controls were conducted: treatment with 10 ng/ml TNF-? for 
30 minutes as a positive control for NF-?B stimulation (Osborn et al., 1989); heat 
inactivation of gp120 to determine if denaturation affects NF-?B stimulation; and 
media from uninfected PBMC as a control for HIV-1 stimulation. Cellular proteins 
were extracted and NF-?B measured using the TransAM? NF?B Family 
Transcription Factor Assay as before. All treatments elevated NF-?B levels compared 
to unstimulated cells (Figure 13). Treatments with TNF-?, HIV-1 and HIV-1 negative 
were significantly different from unstimulated cells (p<0.05), with gp120 resulted in 
higher NF-?B levels that were highly significant as compared to HIV-1 stimulation 
(p<0.001). Absorbance values can be found in the Appendix (Figure 19). 
 
 
 
* 
 35 
 
0.00
 0.50
 1.00
 1.50
 2.00
 2.50
 3.00
 Unstimulated TNF-? HI gp120 gp120 HIV-1
 negative
 HIV-1
 Treatment
 DN
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ac
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  (r
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Figure 13: Quantification of NF-?B DNA-binding in cardiomyocytes by 
treatment with gp120 or HIV-1. NF?B Family Transcription Factor Assay of human 
cord-blood stem cell-derived cardiomyocytes showing a stimulation of NF-?B levels 
after treatment with HIV-1Du151 gp120 and HIV-1. All treatments including the 
controls for gp120 and HIV-1, as well as TNF-?, were significantly higher than 
unstimulated cells (* p<0.05; **p<0.001). 
 
These results were confirmed by immunocytochemistry. Human cord-blood stem cell-
 derived cardiomyocytes were seeded at 1.5 ? 105 cells per well on a chamber slide 
and allowed to adhere overnight. These cardiomyocytes were treated with 4 ?g/ml 
HIV-1Du151 gp120, HIV-1 CM9 (8 ng/ml of p24) or left untreated for two hours as 
above. Cells were fixed and stained as described earlier (3.7). A rabbit anti-p65 
antibody was used as the primary antibody. This was visualised using an Alexa Fluor? 
532-conjugated secondary antibody (Invitrogen, USA). Unstimulated cardiomyocytes 
exhibited low levels of NF-?B in their cytoplasm (Figure 14A). The incubation with 
TNF-? resulted in an increase in cytoplasmic and nuclear NF-?B after 30 minutes 
(Figure 14B). Both heat inactivated and functional gp120 show an increase in nuclear 
NF-?B (Figure 14C and D respectively), although functional gp120 shows higher 
cytoplasmic levels of the transcription factor than its heat inactivated counterpart. 
Treatment with HIV-1 negative media from PBMC and HIV-1 (Figure 14E and F 
* ** ** 
* * 
 36 
respectively) showed a marginal increase in nuclear NF-?B, with HIV-1 stimulation 
appearing brighter than HIV-1 negative PBMC media. 
 37 
 
 38 
 
Figure 14: Fluorescent images of NF-?B stimulation in cardiomyocytes by HIV-
 1Du151 gp120 and HIV-1. Fluorescence microscopy images of human cord-blood stem 
cell-derived cardiomyocytes stained with anti-p65. These images show untreated 
cardiomyocytes with low levels of NF-?B in the cytoplasm (A), cells stimulated with 
10 ng/ml TNF-? for 30 minutes (B), cardiomyocytes treated with 4 ?g/ml heat 
inactivated HIV-1Du151 gp120 (C), 4 ?g/ml HIV-1Du151 gp120 (D), HIV-1 negative 
media (E), and HIV-1 (8 ng/ml of p24) (F). The red dye indicates the presence of the 
p65 subunit of NF-?B and the blue the DAPI-stained nuclei. Other than the TNF-? 
positive control, only stimulation with heat inactivated and functional gp120 clearly 
induced nuclear and cytoplasmic NF-?B. (Magnification, 500?). 
 
4.9 Modulation of NF-?B by plant compounds in gp120-stimulated 
cardiomyocytes 
Human cord-blood stem cell-derived cardiomyocytes were seeded at 3 ? 105 cells per 
well on a 24 well plate and allowed to adhere overnight. Cardiomyocytes were pre-
 incubated with compounds 106A or 38B at the stated concentration in cell culture 
medium for one hour before washing these cells with PBS and treating with 4 ?g/ml 
HIV-1Du151 gp120 for two hours (Hehner et al., 1998). Controls were carried out by 
stimulation using gp120 without treatment by either sesquiterpene lactone, together 
with a control for resting cells. Cellular proteins were extracted and NF-?B measured 
using the TransAM? NF?B Family Transcription Factor Assay. Treatment with 
106A or 38B at the two stated concentrations in gp120 stimulated cells did not bring 
NF-?B levels back to those seen in unstimulated cells (Figure 15). However, 
treatment with 5 ?M 106A caused a significant decrease in NF-?B levels as compared 
to gp120 only (p<0.05). Absorbance values can be found in the Appendix (Figure 20). 
 39 
0.00
 0.50
 1.00
 1.50
 2.00
 2.50
 3.00
 Unstimulated gp120 only 1?M 106A +
 gp120
 5?M 106A  +
 gp120
 1?M 38B  +
 gp120
 5?M 38B +
 gp120
 Treatment
 DN
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 ct
 ivi
 ty
  (r
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 )
  
Figure 15: Mitigation of NF-?B levels in gp120-stimulated cardiomyocytes by 
sesquiterpene lactones. Downregulation of NF-?B DNA binding activity can be seen 
in both treatments at 5 ?M but only treatment with 5 ?M 106A significantly lowered 
NF-?B levels after gp120 stimulation (* p<0.05 vs. gp120 stimulation only). 
 
5 DISCUSSION 
 
5.1 HIV-1 glycoprotein gp120 expression and validation  
The HIV-1 surface protein gp120 is a heavily glycosylated protein. This post-
 translational modification cannot be accomplished in the traditional Escherichia coli 
expression system (Gerstein, 2001). Even the baculovirus expression system, which 
uses the insect cell line Sf9, can only partially glycosylate the protein it produces 
(Fraser, 1992). Only mammalian cells appear to be able to process this protein as is 
seen in the infection of human CD4+ cells (Gerstein, 2001). For this reason, as well as 
the permissibility of the cell line to transfection, the mammalian 293T cell line was 
used to express gp120. Once expressed and determined by western blotting that the 
correct protein was recovered (Figure 2), the glycoprotein needed to be validated. 
Surface plasmon resonance was the method chosen to do this as it shows the 
interaction of native proteins, with the advantage of this interaction being in real-time. 
The IgG1 b12 antibody was used in this experiment because it binds to the same site 
on the gp120 that CD4 does (Burton et al., 1994). The CD4-gp120 interaction allows 
the virus to dock onto CD4+ cells and is essential for viral entry into these cells. The 
IgG1 b12 antibody as it is a potent neutraliser of a broad range of HIV-1 isolates 
(Burton et al., 1994) and recognises only functional gp120. The binding of this 
 * 
 40 
antibody to the expressed HIV-1Du151 gp120 confirms that the gp120 is biologically 
active (Figure 3). 
 
5.2 Measurement of NF-?B 
Within the first year of the discovery of NF-?B as a novel transcription factor, it was 
found that this protein is inducible (Sen and Baltimore, 1986a). One of the first 
compounds found to induce the transcription factor was PMA (Sen and Baltimore, 
1986a), which is still used as an NF-?B inducer today at the concentration first 
published by Sen and Baltimore. Tumour necrosis factor-? is also routinely employed 
to activate NF-?B levels (Pahl, 1999). Both of these inducers of NF-?B were used in 
this study to elevate NF-?B levels in the various experiments. The phorbol ester was 
used in the initial experiments to establish the methods. After comparison in HeLa 
cells (results not shown), TNF-? was found to elevate NF-?B to higher levels than 
PMA in a shorter space of time than the phorbol ester and was therefore used in the 
final experiments. Stimulation with PMA helped illustrate the activation of NF-?B in 
HeLa cells by immunocytochemistry, which initially resulted in a higher level of the 
transcription factor in the cytoplasm and appeared as brighter cytoplasmic 
fluorescence compared to unstimulated HeLa cells (Figure 8A and B). A longer 
incubation of HeLa cells with PMA showed nuclear translocation with fluorescence 
mostly in nuclei (Figure 8C). The immunofluorescence experiment was conducted 
using the same protocol as immunocytochemistry in a 96-well format and measured 
using a fluorometer. Unlike the clear results seen in the immunocytochemistry, the 
immunofluorescence test was not sensitive enough to determine the difference 
between resting and stimulated levels of NF-?B (Figure 9). This is possibly due to the 
additional washing steps which may affect the number of cells left on the plate, but 
the fixation and permeabilisation of the cells may also affect the readout. The 
TransAM? NF?B Family Transcription Factor assay proved to be a better measure 
of NF-?B when it could be seen that PMA stimulation significantly raised NF-?B 
levels (Figure 10). The phorbol ester was also used to stimulate HeLa cells after 
treatment with the various plant compounds to determine their effect on NF-?B levels 
(Bork et al., 1997). Only the plant compounds that fall into the sesquiterpene lactone 
category showed reduced NF-?B stimulation after incubation with PMA (Figure 11). 
The two sesquiterpene lactones that showed a significant decrease in NF-?B levels 
 41 
after PMA stimulation (106A and 38B) were used in further experiments. In the final 
experiments TNF-? was used to show that the cardiomyocytes can be stimulated to 
release NF-?B (Figures 13, 14 and 15). 
 
5.3 Stimulation of NF-?B in cardiomyocytes by HIV-1 and gp120 
A number of groups have conducted studies on the effects of HIV-1 proteins on 
cardiomyocytes to determine the molecular mechanism of HIVCM (Fiala et al., 2004; 
Kan et al., 2000; Twu et al., 2002). Most of these groups have had to rely on data 
from rat or rabbit cells due to a lack of a human cardiomyocyte cell line. Recently 
cord-blood stem cells were used to produce a human cardiomyocyte cell line, 
allowing for the testing of live human cardiomyocytes against HIV-1 and its proteins. 
Before conducting the experiments with HIV-1, the exposure time of the 
cardiomyocytes to gp120 needed to be optimised. This was to ensure that the 
exposure was long enough to possibly stimulate NF-?B but not cause cell death due to 
apoptosis (Fiala et al., 2004; Twu et al., 2002). Human cord-blood stem cell-derived 
cardiomyocytes were exposed to the expressed gp120 for two to four hours and NF-
 ?B levels tested by the ELISA-based NF-?B assay. All time-points raised NF-?B 
levels to at least double that of unstimulated cardiomyocytes (Figure 12). The 
exposure time of two hours was chosen for gp120 and HIV-1 stimulation in future 
experiments as it was the only treatment that showed a significant elevation of NF-?B 
levels and correlates to gp120 studies on cardiomyocytes conducted by other groups 
(Kan et al., 2000). The exposure of human cardiomyocytes to gp120 significantly 
increased NF-?B levels (Figures 12 and 13) as was shown previously in rat myocytes 
(Kan et al., 2000). Heat inactivation of the protein showed almost identical results as 
native gp120 i.e. it did not lower the NF-?B response as compared to gp120 (Figure 
13). However, immunocytochemistry of cardiomyocytes stimulated with the heat 
inactivated control and gp120 (Figure 14C and D respectively) showed that although 
the nuclear levels of the transcription factor may appear similar, there is a higher 
cytoplasmic component to the gp120 stimulation. This may simply mean that gp120 
treatment of cardiomyocytes elicits a prolonged NF-?B response, one that may be 
shortened by heat inactivation of the viral protein. It is also possible that the protein 
remains in its native conformation due to inefficient heating of the protein as various 
temperatures and heating periods have been previously described (Barak et al., 2002; 
Lee et al., 2005; Zauli et al., 1996). A common contaminant of protein expression is 
 42 
the heat-stable Escherichia coli LPS (Cardoso et al., 2007). It is unlikely that the 
gp120 stock was contaminated with LPS as a mammalian expression system was used 
and sterile techniques and endotoxin-free reagents employed. Incubation of HIV-1 
isolate CM9 resulted in significantly elevated levels of NF-?B but this elevation was 
mirrored by its control of PBMC media (Figure 13). This makes it impossible to know 
whether any part of the elevation may be attributed to the virus. These conditions 
were repeated and tested using immunocytochemistry where the intensity of staining 
after HIV-1 stimulation was slightly higher than that seen after treatment with the 
PBMC medium (Figure 14E and F). Unfortunately, the software for the fluorescent 
microscope cannot distinguish whether there is a significant difference in the 
fluorescence of any two pictures. The elevation seen in the control of medium from 
uninfected PMBC was expected due to the presence of IL-2 (Hazan et al., 1990) but it 
was hoped that the stimulation with HIV-1 would be higher than this. The stimulation 
with gp120 elicited a higher NF-?B response than stimulation with HIV-1. This is 
probably due to a higher concentration of gp120 in the incubation with the protein 
only as compared to the virus-associated protein in the HIV-1 stimulation which was 
quantified by p24 assay. Another consideration is that there are many more exposed 
epitopes on free gp120 than on the viral envelope and these epitopes may elicit a 
higher NF-?B response. 
 
The choice of HIV-1 isolates used in this study was intentional. Both Du151 and CM9 
strains are HIV-1 subtype C viruses, the predominant subtype found in Sub-Saharan 
Africa. These isolates are also both R5 viruses i.e. they use the CCR5 chemokine 
receptor to facilitate viral entry (Cilliers et al., 2003; Williamson et al., 2003). This 
allows for infection and replication of these viral strains in macrophages. However, it 
is always a challenge to replicate HIV-1 in macrophages in vitro, as was evident in 
this study. Had the infection and replication in macrophages worked, it could have 
provided data on the possible role of cytokines produced by HIV-1 infected 
macrophages on human cardiomyocytes. It may have also been possible to see 
whether IL-2 was solely responsible for the increase in NF-?B levels seen in the HIV-
 1 treatment of cardiomyocytes, as macrophages do not require IL-2 to aid infection. 
 
 43 
5.4 Modulation of NF-?B by plant compounds in gp120-stimulated 
cardiomyocytes 
A number of groups have shown the damaging effects of gp120 on cardiomyocytes. A 
study on the effects of gp120 on rat cardiac myocytes found that co-stimulation of 
these cells with gp120 and IL-1? raised nitric oxide levels significantly as compared 
to IL-1? stimulation alone (Kan et al., 2000). The study also connected this nitric 
oxide elevation to p38-mediated stimulation of NF-?B. This group realised for the 
first time that gp120 has a direct effect on cytokine production in cardiomyocytes and 
proposed that the interaction between viral proteins and cytokines contribute to 
HIVCM. Another study found that gp120 inhibits the contraction of rabbit 
cardiomyocytes and their L-type Ca2+ current (Chen et al., 2002). This group took 
note of the fact that nitric oxide has been shown to modulate Ca2+ currents in 
ventricular myocytes. This alludes to elevated nitric oxide levels causing the 
inhibition of contraction in gp120-stimulated cardiomyocytes. NF-?B may be the 
common element in all of these pathways and an NF-?B inhibitor that has shown 
efficacy in human cardiomyocytes against HIV-1 or gp120-induced stress could prove 
to be a valuable asset. Sesquiterpene lactones have been shown to be inhibitors of NF-
 ?B activation by many studies (Bork et al., 1997; Hehner et al., 1998; Lopez-Franco 
et al., 2006; Lyss et al., 1998). These compounds have also been shown to have a 
high unspecific toxicity and to be particularly cardiotoxic in the 10-4 to 10-3 M range 
(Schmidt, 1999). The cytotoxicity assays conducted in this thesis is consistent with 
these observations, where the only cardiotoxic compounds were 106A and 38B, both 
sesquiterpene lactones (Figure 7C). These compounds were used at non-cardiotoxic 
concentrations to modulate the effect that gp120 and HIV-1 had on NF-?B levels in 
cardiomyocytes. These concentrations showed at least 80% cell viability in the 
cytotoxicity assays after a three hour incubation with the compound at the low 
micromolar range, a range that most sesquiterpene lactones show their bioactivity 
(Schmidt, 1999). An interesting note is that low concentrations of sesquiterpene 
lactones like those used in the present study may enhance cardiomyocyte contractility 
by increasing the amount of intracellular Ca2+ released on stimulation (Schmidt, 
1999), thereby possibly negating the effect of gp120 on cardiomyocyte contractility. 
Of all the treatments conducted on gp120-stimulated cardiomyocytes in this thesis, 
only 5 ?M 106A significantly lowered NF-?B levels as compared to gp120 
stimulation alone (Figure 15). This did not bring NF-?B levels to within the range of 
 44 
unstimulated cardiomyocytes but does show that the compound is effective against 
gp120 stimulation. The 106A compound was isolated from the Vernonia 
staehelinoides plant and was shown to have antiplasmodial activity in vitro but at a 
concentration that was toxic to mammalian cells (Pillay et al., 2007). This plant has 
been reportedly used in traditional medicine but no particulars of its use have been 
detailed (Watt and Breyer-Brandwijk, 1932). A methanol extract from a different 
South African species of the same genus, Vernonia stipulacea, was found to stimulate 
the activity of the HIV-1 reverse transcriptase enzyme at a concentration of 100 ?g/ml 
in an assay that measures activity of the expressed enzyme only and does not test the 
whole virus and infection (Bessong et al., 2005). It is important to note that the crude 
extract of the plant was used in the study above while a pure compound was used in 
this thesis. 
 
5.5 Future considerations 
There needs to be a fine balance when it comes to the administration of sesquiterpene 
lactones in the clinical setting. A general administration, especially one at too high a 
dosage, could cause immunosuppression and lowered host defence (Ghosh et al., 
1998). This is further complicated by liver apoptosis if there is an elevation in TNF-? 
levels (Tak and Firestein, 2001), as is seen in patients with HIVCM (Barbaro et al., 
1999; Twu et al., 2002). Oral absorption of sesquiterpene lactones has been shown to 
be incomplete (Schmidt, 1999), making it next to impossible to estimate the correct 
dosage to recommend. More advanced drug delivery systems will be needed to ensure 
only targeted cells receive the compound and at the correct dosage. 
 
There are a number of issues related to this study that still need to be investigated. 
The effects of HIV-1 without the presence of IL-2 as well as HIV-infected 
macrophages on NF-?B levels in cardiomyocytes merits further study. These results 
could provide information on the possible role of cell-free HIV-1 and cytokines 
produced by HIV-1 infected macrophages on human cardiomyocytes. The elucidation 
of the membrane receptor that relays the presence of gp120 may also provide valuable 
insights into HIVCM and the signalling pathways involved. The 106A compound 
appears to be a candidate for further study on the modulation of cellular responses due 
to gp120 stimulation in human cardiomyocytes. A higher dosage or longer incubation 
time with this sesquiterpene lactone may lower NF-?B levels to within the range of 
 45 
resting cells. Further study on the effect of gp120 and HIV-1 on nitric oxide levels 
and contractility of human cord-blood stem cell-derived cardiomyocytes could 
provide answers to the molecular mechanism of HIVCM. The effect of the 106A 
compound on these parameters may also provide a much needed reduction in the 
damage of cardiomyocytes seen in HIVCM.  
 
6 CONCLUSION 
The HIV-1 surface glycoprotein gp120 elevated NF-?B levels in human cord-blood 
stem cell-derived cardiomyocytes by more than 2-fold compared to unstimulated 
control cells. This suggests that the damage to cardiomyocytes caused by gp120 in 
HIVCM is mediated by upregulation of the NF-?B transcription factor. 
 
The sesquiterpene lactone 106A, at a concentration of 5 ?M, significantly lowered the 
NF-?B response due to gp120 stimulation in human cord-blood stem cell-derived 
cardiomyocytes. The 106A compound could prove valuable in further studies on the 
modulation of cellular responses due to HIV-1 or gp120 induced stress in human 
cardiomyocytes. 
 
 46 
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 55 
8 APPENDIX 
 
Table 1: Panel of natural compounds derived from South African indigenous 
plant 
Sample number Structure Class of compound Origin 
8A 
 
Flavonoid Helichrysum 
aureonitens 
8B 
 
Flavonoid Helichrysum 
aureonitens 
8C  
 
Flavonoid Helichrysum 
aureonitens 
106A  
 
Sesquiterpene lactone Vernonia 
staehelinoides 
38B  
 
Sesquiterpene lactone Brachylaena 
transvaalensis 
124D  
 
Sesquiterpene lactone Oneosiphon 
piluliferum 
10B  
 
Sesquiterpene Siphonochilus 
aethiopicus 
 
O
 O
 O
 O
 O
 OAc
 O
 OAc
 10
 11
 14
 13
 12
 4'
 1'
 1
 4
 6
 7
 8
 15
 5'
 OH
 O
 O
 OH
  
O
 OH
 HO
 O
 OH
  
O
 OH
 HO
 O
  
O
 OH
 O
 O
 O
 O
 1
 2
 33a4
 H axHeqMe Me
 4a56
 7
 8
 Me
 9 9a
 8a
 56 
SDS PAGE gel and buffer formulations 
8% Resolving gel 
 40% Acrylamide / Bis-acrylamide (19:1)  2 ml 
 1.5 M Tris-HCl, pH 8.8    2.5 ml 
20% (w/v) SDS     0.05 ml 
 10% (w/v) Ammonium persulphate   0.1 ml 
 Tetramethylethylenediamine    0.01 ml 
 H20       5.34 ml 
 
4% Stacking gel 
 40% Acrylamide / Bis-acrylamide (19:1)  0.5 ml 
 0.5 M Tris-HCl, pH 6.8    1.25 ml 
20% (w/v) SDS     0.025 ml 
 10% (w/v) Ammonium persulphate   0.05 ml 
 Tetramethylethylenediamine    0.005 ml 
 H20       3.17 ml 
 
1? SDS Running buffer 
 25 mM Tris-HCl 
 200 mM Glycine 
 0.1% (w/v) SDS 
 
2? SDS Sample buffer 
 4% (w/v) SDS 
 4 mM ?-Mecaptoethanol 
 8% (w/v) Glycerol 
 80 mM Tris-HCl, pH 6.8 
 0.02% (w/v) Bromophenol Blue 
 57 
0
 200
 400
 600
 800
 1000
 1200
 1400
 Unstimulated 20 60 Positive
 PMA stim ulation tim e (min)
 Re
 lat
 ive
  Fl
 uo
 res
 ce
 nc
 e u
 nit
 s (
 RF
 U)
  
Figure 16: Quantification of NF-?B in resting and stimulated HeLa cells by 
immunofluorescence. NF-?B levels relative to unstimulated cells show no significant 
difference between unstimulated cells and those stimulated with PMA 20 and 60 
minutes respectively (p>0.05). The Raji Nuclear extract was used as a positive 
control. 
 
0.000
 0.100
 0.200
 0.300
 0.400
 0.500
 0.600
 0.700
 0.800
 0.900
 Un
 stim
 ula
 ted
 PM
 A o
 nly 8A 8B 8C 106
 A 38B 124
 D 10B
 Ra
 ji N
 E
 Treatment
 Ab
 so
 rb
 an
 ce
  (4
 50
 nm
 )
  
Figure 17: NF-?B levels in stimulated and plant compound treated HeLa cells. 
TransAM? NF?B Family Transcription Factor Assay of HeLa cells showing 
absorbance values after treatment with plant compounds and stimulation with PMA. 
(*significantly lowered, p<0.05) 
 
* * 
 58 
0.000
 0.500
 1.000
 1.500
 2.000
 2.500
 Unstimulated 2 3 4 Positive control
 gp120 stim ulation (h)
 Ab
 so
 rb
 an
 ce
  (4
 50
 nm
 )
  
Figure 18: NF-?B levels in HIV-1Du151 gp120 treated cardiomyocytes. NF?B 
Family Transcription Factor Assay of human cord-blood stem cell-derived 
cardiomyocytes showing a stimulation of NF-?B levels after treatment with HIV-
 1Du151 gp120. Treatment with gp120 for two hours significantly elevated NF-?B levels 
as compared to unstimulated cells (* p<0.05). 
 
0.000
 0.500
 1.000
 1.500
 2.000
 2.500
 Unstimulated TNF-? HI gp120 gp120 HIV-1
 negative
 HIV-1 Positive
 Contro l
 Treatment
 Ab
 so
 rb
 an
 ce
  (4
 50
 nm
 )
  
Figure 19: Quantification of NF-?B in cardiomyocytes by treatment with gp120 
or HIV-1. NF?B Family Transcription Factor Assay of human cord-blood stem cell-
 derived cardiomyocytes showing a stimulation of NF-?B levels after treatment with 
HIV-1Du151 gp120 and HIV-1. All treatments including the controls for gp120 and 
HIV-1, as well as TNF-?, were significantly higher than unstimulated cells (* p<0.05; 
**p<0.001). 
* 
* ** ** * * 
 59 
 
0.000
 0.500
 1.000
 1.500
 2.000
 2.500
 Unstimulated gp120 only 1?M 106A +
 gp120
 5?M 106A  +
 gp120
 1?M 38B  +
 gp120
 5?M 38B +
 gp120
 Positive
 Control
 Treatment
 Ab
 so
 rb
 an
 ce
  (4
 50
 nm
 )
  
Figure 20: Mitigation of NF-?B levels in gp120-stimulated cardiomyocytes by 
sesquiterpene lactones. Downregulation of NF-?B levels can be seen in both 
treatments at 5 ?M but only treatment with 5 ?M 106A significantly lowered NF-?B 
levels after gp120 stimulation (* p<0.05 vs. gp120 stimulation only). 
 
*