Medical Microbiology

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Determination of the permeability of biological membranes to various chemical markers, including anti-HIV drugs
(Stellenbosch : University of Stellenbosch, 2009-12) Pretorius, Erina; Bouic, Patrick J. D.; University of Stellenbosch. Faculty of Health Sciences. Dept. of Pathology. Medical Microbiology.
ENGLISH ABSTRACT: Due to modern high-throughput technologies, large numbers of compounds are produced by parallel synthesis and combinatorial chemistry. The pharmaceutical industry therefore requires rapid and accurate methods to screen new drugs leads for membrane permeability potential in the early stages of drug discovery. Around 50 % of all investigational new drugs fail in pre-clinical and clinical phases of development due to inadequate absorption/permeation, distribution, metabolism, excretion and/or unacceptable toxicity. This may be decreased by applying in vitro screening methods early in the discovery process. Reliable in vitro models can be applied to determine permeation of the test compounds, which will help avoid the wasting of valuable resources for the development of drugs that are destined to fail in preclinical and clinical phases due to insufficient permeability properties. It is important to decide as early as possible on the most promising compound and physical formulation for the intended route of administration. With awareness of the increasing importance of in vitro models in the investigations of the permeability properties of drug compounds, this research project was specifically devoted to determine the suitability of our in vitro model to evaluate and predict drug permeability. A continuous flow-through diffusion system was employed to evaluate the permeability of nine different compounds/drugs with different chemical properties, across three biological membranes. The biological membranes chosen for the present study were human vaginal mucosa, human skin tissue and human small intestine mucosa. The continuous flow-through diffusion system was furthermore utilised to investigate the effects of de-epithelialisation of mucosal surfaces, chemical enhancers, temperature, permeant concentration and formulation on the permeability of the test compounds/drugs. The in vitro permeability information and data from the flow-through diffusion model were compared to in vitro and in vivo literature studies and drug profile. An in vitro model that is able to reliably predict in vivo data will shorten the drug development period, economise resources and may potentially lead to improved product quality. In this thesis research results are reported on the permeability of the mentioned biological membranes to the various chemical markers, including anti-HIV (human immunodeficiency virus) drugs. The permeability studies will be discussed in three sections: vaginal mucosa, skin tissue, small intestine mucosa. The results of the vaginal permeability studies showed that the three peptides (MEA- 5, MDY-19 and PCI) readily penetrated the vaginal mucosa. MDY-19 had a higher flux rate than MEA-5, commensurate with its smaller molecular size (weight). The surfactant enhanced the flux rate of MDY-19 approximately 1.3 times and decreased the lag time of the peptide. Removal of the vaginal epithelium increased the flux rates of the peptides across the mucosa and may have implications for a more rapid uptake of these and other microbicides in vivo. The permeability of 1 mM MDY-19 and PCI at 37 °C were significantly (p<0.05) higher than at 20 °C. At 37 °C the AUCs of the overall mean flux values of MDY-19 and PCI increased with concentration according to well-established diffusion theory. The experiments on the permeability of different terbinafine hydrochloride formulations through human skin demonstrated that the terbinafine hydrochloride formulations used in this study, readily diffused into the skin tissue. However, no flux values for any of the terbinafine hydrochloride formulations through the skin into the acceptor fluid were found. The mean terbinafine concentrations in the skin after 24 h exposure to the three commercial, terbinafine hydrochloride formulations were 3.589, 1.590 and 4.219 μg/ml respectively. The mean terbinafine concentration in the skin exposed to the 10 mg/ml PBS/Methanol solution was higher than those from the three commercial formulations. The results of the temperature study demonstrated that an increase of 5 ºC caused a significant increase in flux values of tritiated water across skin. The flux values for tritiated water across skin at 37 ºC were on average double those at a temperature of 32 ºC. The permeability of excised human small intestine mucosa to different oral dosage drugs was investigated over a 24 h period. The four drugs selected were zidovudine, propranolol hydrochloride, didanosine and enalapril maleate. They were selected as representative model compounds of drug classes 1 (high solubility, high permeability) and 3 (high solubility, low permeability) according to the Biopharmaceutics Classification System. The flux rates of the four chosen test drugs were influenced by the length of the experiment. Between the time periods 2-4 h and 4-6 h, zidovudine’s mean flux values across small intestine tissue were respectively 1.8 and 2.0 times higher than didanosine and 2.3 and 2.2 times higher than enalapril. Propranolol’s mean flux values were respectively 1.2 and 1.4 times higher than didanosine and 1.6 higher than enalapril during both the 2-4 and 4-6 h time periods. Between both the time periods 2-4 and 4-6 h AZT’s mean flux values were 1.4 times higher than propranolol and didanosine’s mean flux values were respectively 1.3 and 1.1 times higher than enalapril during the mentioned time periods. Class 1 drugs showed a significantly higher flux rate across the jejunal mucosa compared to the class 3 drugs and these results are in line with their Biopharmaceutics Classification System classification. The in vitro model has proved to be reliable to predict permeability of class 1 and 3 drugs and also showed correlation with human in vivo data. It seems that the in vitro flow-through diffusion model used in the present study have the potential to overcome some of the problems and limitations demonstrated by other in vitro techniques and may potentially serve as a future tool for pharmaceutical companies to predict the diffusion characteristics of new drugs and different formulations, across different biological membranes. Furthermore, it may serve as a prospective method for assessing the bioequivalence of alternative (generic) vehicles or formulations containing the same drug/compound.
Development and validation of an in vitro model of dendritic cell identification and activation
(Stellenbosch : Stellenbosch University, 2008-03) Clark, Anel; Bouic, Patrick J. D.; Stellenbosch University. Faculty of Health Sciences. Dept. of Pathology. Medical Microbiology.
ENGLISH ABSTRACT: The aim of this study was to investigate the effect of MBV and Coley’s Toxin on dendritic cells in vitro. The dendritic cell system of antigen presenting cells is the initiator and modulator of the immune response. The principle function of the dendritic cells is to present antigens to resting naïve T lymphocytes: these cells are the only APCs that prime naïve T cells and only mature DCs can carry out this function.Previous studies done on dendritic cells showed that bacterial peptides can induce the maturation of dendritic cells. With the results of these studies in mind we hypothesized that these two vaccines will also induce the maturation of dendritic cells. Chapter 1 is a literature review on the immune system explaining the organs and cells of the immune system. Chapter 2 includes a full description of DCs, the MBV and Coley’s toxin. Also included in this chapter is a short explanation of the principle of the technique being used for the identification and maturation of both mDCs and pDCs, namely the technique of flow cytometry. Chapter 3 describes the method for the phenotypic identification of DCs: the subsets are distinguished by their absence of expression of several lineage markers for lymphocytes, monocytes and NK cells and the expression of CD11c (in the case of myeloid DCs) and CD123 (in the case of plasmacytoid DCs). The inclusion of HLA-DR in addition to the previous described markers allows the discrimination of CD123+ DCs from basophils. The assay requires three tubes per sample which enables quick analysis of these rare subsets with a small sample volume. This assay was applied to peripheral blood samples obtained from healthy individuals and individuals with cancer, HIV and HIV and TB co-infected patients. Our results showed that the maturation status of DCs in HIV and lymphoma were low but those measured in the case of HIV + TB patients were even higher than in the control group. Chapter 4 and 5 describe the in vitro activation and maturation status of DCs following their incubation with bacterial-derived products. Interactions between DCs and microbial pathogens are fundamental to the generation of innate and adaptive immune responses and upon contact with bacteria or bacterial components such as lipopolysaccharide (LPS), immature DCs undergo a maturation process that involves expression of costimulatory molecules, HLA molecules, and cytokines and chemokines, thus providing critical signals for lymphocyte development and differentiation. In this study, we investigated the response of human DCs to MBV and Coley’s Toxin. Previous studies showed DCs can be activated with killed Streptococcus pyogenes. With this study in mind it was hypothesized that the MBV and Coley’s Toxin used in this study might modulate DC maturation. The results of this study showed that the MBV and Coley’s toxin did induce the maturation of both pDCs and mDCs as measured by increased surface expression of costimulatory molecules such as CD80 and CD83. Chapter 6 presents the measurement of cytokines released after the PMBCs had been were incubated with Coley’s Toxin and Mixed Killed bacteria. The BD™ Cytometric Bead Array (CBA) flex set was used for the simultaneous detection of multiple soluble analytes. The results indicated that both Coley’s Toxin and the MBV activated the DCs and subsequently induced TH1 as well as a TH2 responses in the T cells present in the cell cultures. Finally, a general conclusion discussing the significance and implications of our results as well as possible future research required is discussed in Chapter 7. DCs are potent antigen presenting cells (APCs) which play a critical role in the regulation of the immune response. There is great interest in exploiting DCs to develop immunotherapies for cancer, chronic infections, immunodeficiency diseases and autoimmune diseases.
Development and validation of stabilized whole blood samples expressing T-cell activation markers as quality control reference material
(Stellenbosch : Stellenbosch University, 2008-03) Louw, Anne-Rika; Bouic, Patrick J. D.; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Medical Microbiology.
ENGLISH ABSTRACT: Introduction: Flow cytometry has progressively replaced many traditional laboratory tests due to its greater accuracy, sensitivity and rapidity in the routine clinical settings especially clinical trails. It is a powerful tool for the measuring of chemical (the fluorochrome we add) and physical (size and complexity) characteristics of individual cells. As these instruments became major diagnostic and prognostic tools, the need for more advanced quality control, standardized procedures and proficiency testing programs increased as these instrumentations and their methodology evolve. Minor instrument settings can affect the reliability, reproducibility and sensitivity of the cytometer and should be monitored and documented in order to ensure identical conditions of measurement on a daily basis. This can be accomplished by following an Internal Quality Assurance (IQA) and/ or External Quality Assurance (EQA) program. Currently there are no such programs available in South Africa and poorer Africa countries. HIV is a global concern and the laboratories and clinics in these places are in need of such IQA programs to ensure quality of their instrumentation and accurate patient results. Quality assurance programs such as CD Chex® and UK Nequas are available but due to bad sample transport, leave the receiving laboratories with nightmares. It would be best if there was a laboratory in South Africa that could provide the surrounding laboratories with stabilized whole blood samples that can be utilized as IQA. The transport of these samples can be more efficient due to shorter distance and thus the temperature variations limited. Aims and Objectives: The aim of Chapter one is to familiarize the reader with general terminology and concepts of immunology. Chapter two describes in detail the impact stabilized whole blood had on clinical immunology concerning Quality Control and Quality Assurance. The objective of this study is to stabilize whole blood with a shelf life of greater than 30 days to serve as reference control material for South African Immunophenotyping. It is further an objective to use these in-house stabilized control samples for poorer African countries as Internal Quality Assurance reference material. It is a still further objective to stimulate various lymphocyte subsets to express activation antigens and then stabilize these cells for more specialized immunological test and can serve as a QC for those required samples. Study design: In Chapter three, the method currently used to stabilize whole blood was modified. The stability of different concentrations of a first stabilizing agent (Chromium Chloride hexahydrate) was investigated. Incubation periods and concentrations of paraformaldehyde as second stabilizing agent were investigated. Blood samples from healthy individuals (n=10) were stabilized and monitored for the routine HIV phenotypic surface antigens over a period of 40 days. These samples (n=10) were compared on the Becton Dickinson Biosciences (BD) FACSCalibur™ versus BD FACSCount™ instrumentation. Blood samples (n=3) were stabilized and monitored to identify phenotypic cell surface molecules for as long as possible. They were quantified on both flow cytrometric instruments. In addition, these stabilized samples (n=3) were investigated as control blood for calibration purposes on the BD FACSCount™ instrument. In Chapter four, lymphocytes were isolated and activated with various stimuli to express sufficient activation antigens such as CD25, CD69, HLA-DR and CD40 Ligand on the T helper cell surfaces. These activated antigens were analyzed on the BD FACSCalibur™ and further stabilized to serve as possible IQA samples in future. Results: In Chapter three, the ten individual stabilized samples had non-significant P values (P > 0.05) for CD3, CD4 and CD8 percentages and absolute values comparing day 3 until day 40. Comparing the BD FACSCalibur™ versus BD FACSCount™, resulted in a R2 = 0.9848 for CD4 absolute values and a R2 = 0.9636 for CD8 absolute values. Stabilized blood samples (n=3) were monitored for routine HIV phenotypic markers until day 84. The cells populations were easily identifiable and could be quantified on both BD FACSCalibur™ and BD FACSCount™ instruments. In Chapter four; for the activation study purposes, activated T helper lymphocytes expressed approximately 25 to 35% CD40 Ligand cell surface molecules. The stimulant of choice was Ionomycin at a 4μM concentration. Cells were incubated for four hours at 37 degree Celsius in a 5% CO2 environment. For CD69 surface expression, 6 hour incubation was optimum. The stimulus of choice in this case was 4μM Ionomycin which induced 84.21% CD69 expression in the test samples. For CD25 expression; 6 hour incubation with PHA resulted in approximately 43% of CD25 expression. For HLA-DR surface expression; 6 hour incubation with PHA resulted in approximately 43.32% of HLA-DR expression. Activated lymphocytes expressing CD40 Ligand showed stability until day 23. Activated Lymphocytes expressing CD69, CD25 and HLA-DR were stabilized in the same manner and stability could be achieved until day 16. Conclusion: This thesis was related to the preparation of control samples (IQA) designed to simulate whole blood having defined properties in clinical laboratory situations. In future kits can be developed with a low, medium and high control sample for the various immunological phenotypic determinants. Another kit can be compiled where various activation markers can be identified, quantified with a “zero”, low and high control. These whole blood IQA kits and “activation IQA kits” can be implemented for training of newly qualified staff, competency testing of staff, method development, software testing, panel settings and instrument setting testing. Control samples ideally must have a number of properties in order to be effective. For instance stability during storage times, preferably lasting more than a few weeks, reproducibility and ease of handling. These will provide the information on day-to-day variation of the technique or equipment which will enhance accuracy and improve patient care.
Die effek van musiek op die immuunsisteem, emosies en longfunksie tydens die standaard fisioterapeutiese behandeling van spesifieke longpatologie
(Stellenbosch : University of Stellenbosch, 2005-03) Le Roux, Frances Hendriehetta; Bouic, Patrick J. D.; University of Stellenbosch. Faculty of Health Sciences. Dept. of Pathology. Medical Microbiology.
There has recently been a significant transformation in the medical world, in particular regarding the relation between the mind/health and mind/illnesses. The changes are briefly a revolution whereby the new approach sees the development of an illness as an interaction between the psychological, biochemical and physiological factors. Music, which is used as a clinical intervention, is perceived first through the brain, affirms this interaction between the body systems, as well as having the capacity to modify the mind and thus the biochemistry of the body. The aim of this study was essentially to supply empirical data by measuring selective parameters while the patients were receiving music intervention during the physiotherapeutic treatment for pneumonia and bronchitis. Forty adult patients who were divided into an experimental and control group, according to a random scale, participated in the research. The dependant variables that had shown significant changes amongst the experimental group after three days of physiotherapeutic treatment were as follows: the cortisol, the cortisol: DHEA ratio plasma levels, the POMS scale (that measures different moods), the peak flow measurements of the lung functions and the immune parameters, namely, CD4+ : CD8+ ratio and B-cells. The results showed that the experimental group that was exposed to the acoustic stimuli of the Magnificat in D, BWV 243 of JS Bach, experienced a more positive mood and lower cortisol levels, while the immune markers as well as the peak flow of the lungs had improved. The results of the control group showed significant implications, in that its cortisol levels increased and the POMS subscale of anger and depression showed no significant change, while the tension decreased significantly. This research provided sufficient scientific evidence to confirm the concept of a bidirectional communication between the brain and the immune system. It also showed clearly that music had the capacity to modify emotional conditions, which again influenced the endocrine and autonomic nervous system and modulated the immune systems.
Generation of a database of mass spectra patterns of selected Mycobacterium species using MALDI-ToF mass spectrometry
(Stellenbosch : Stellenbosch University, 2008-12) Oduwole, Elizabeth O.; Bouic, Patrick J. D.; Wasserman, E. W.; Stellenbosch University. Faculty of Health Sciences. Dept. of Pathology. Medical Microbiology.
The genus Mycobacterium is a group of acid–fast, aerobic, slow- growing organisms which include more than 90 different species. A member of this genus, Mycobacterium tuberculosis, belonging to the Mycobacterium tuberculosis complex (MTB), is the causative agent of tuberculosis (TB). This disease is currently considered a global emergency, with more than 2 million deaths and over 8 million new cases annually. TB is the world’s second most common cause of death after HIV/AIDS. About one-third of the world’s population is estimated to be infected with TB. This catastrophic situation is further compounded by the emergence of Multi Drug Resistant tuberculosis (MDR-TB) and in more recent times, Extensive Drug Resistant tuberculosis (XDR-TB). Early diagnosis is critical to the successful management of patients as it allows informed use of chemotherapy. Also, early diagnosis is also of great importance if the menace of MDR-TB and XDR-TB is to be curbed and controlled. As MTB is highly infectious for humans, it is of paramount importance that TB be diagnosed as early as possible to stop the spread of the disease. Traditional conventional laboratory procedures involving microscopy, culture and sensitivity tests may require turnaround times of 3-4 weeks or longer. Tremendous technological advancement over the years such as the advent of automated liquid culture systems like the BACTEC® 960 and the MGITTM Tube system, and the development of a myriad of molecular techniques most of which involves nucleic acid amplification (NAA) for the rapid identification of mycobacterial isolates from cultures or even directly from clinical specimens have contributed immensely to the early diagnosis of tuberculosis. Most of these NAA tests are nevertheless fraught with various limitations, thus the search for a rapid, sensitive and specific way of diagnosing tuberculosis is still an active area of research. The search has expanded to areas that would otherwise not have been considered ‘conventional’ in diagnostic mycobacteriology. One of such areas is mass spectrometry. This study joins the relatively few studies of its kind encountered in available literature to establish the ground work for the application of mass spectrometry, specifically Matrix Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (MALDI-ToF MS) in the field of diagnostic mycobacteriology. This is an area which is in need of the speed, sensitivity and specificity that MALDI-ToF technique promises to offer. Since this technology is still in its infancy, the use of utmost care in the preparation of reagents, and the handling and storage of the organisms used to generate reference mass spectra for the database cannot be overemphasized. Similarly, the optimization of certain crucial experimental factors such as inactivating method and choice of matrix is of paramount importance. The main aim of this thesis was to generate a database of reference mass spectra fingerprints of selected (repository) Mycobacterium species. This necessitated the standardization of an experimental protocol which ensured that experimental factors and the various instrument parameters were optimized for maximum spectra generation and reproducibility. A standard operating procedure (SOP) for generating the database of reference mass spectra finger print of selected Mycobacterium species was developed and used to investigate the ability of the database to differentiate between species belonging to the same clinical disease complex as well as the nontuberculosis complex. The findings of this study imply that if the defined protocol is followed, the database generated has the potential to routinely identify and differentiate (under experimental conditions) more species of Mycobacterium than is currently practical using PCR and its related techniques. It is therefore a realistic expectation that when the database is clinically validated and tested in the next phase of the study, it will contribute immensely to the diagnosis of tuberculosis and other mycobacterioses. It will also aid in the identification of emerging pathogens particularly amongst the non-tuberculous mycobacteria.
An in vitro investigation of the anti-inflammatory and immunosuppressive effects of the synthetic contraceptives medroxyprogesterone acetate (MPA) and norethisterone acetate (NET-A)
(Stellenbosch : University of Stellenbosch, 2005-03) Kriek, W. J.; Bouic, Patrick J. D.; Hapgood, J. P.; University of Stellenbosch. Faculty of Health Sciences. Dept. of Pathology. Medical Microbiology.
The aim of this study was to investigate the anti-inflammatory and immunosuppressive effects of the synthetic progestins, MPA and NET-A on human cells in vitro. These injectable contraceptives are used extensively throughout the world, including Africa. The potential of these two synthetic hormones to have certain immunosuppressive and GC properties have previously been shown. Therefore, it was of concern to us to investigate whether these two hormones could possibly demonstrate any of these GC-like properties at contraceptive doses. This was achieved by determining the effects of these two synthetic hormones in vitro on certain immunologic parameters. Chapter 1 is a literature review on MPA, NET and GCs. This chapter starts with a short introduction that sets the scene. The mode of action, effectiveness, sideeffects as well as previously reported relevant data on both MPA and NET-A is portrayed in this review. Research on the known GC, Dex, is also included in the section dealing with GCs, because this synthetic hormone was used as a comparative GC in all our experiments. This chapter soon makes the reader realize how much evidence exists that indicate the possible immunosuppressive effects these two contraceptive hormones, in particular MPA, could have. The possible anti-inflammatory or pro-inflammatory effects of MPA and NET-A are investigated in Chapter 2. This was done in vitro by measuring the effects of these two synthetic hormones on the inflammatory markers, IL-6 and TNFα, by means of ELISA. In this chapter we demonstrate that MPA, even at contraceptive doses, exhibits significant anti-inflammatory properties on both cytokines tested, while NETA displayed considerably less anti-inflammatory tendencies. In its true antiinflammatory manner, we found that Dex significantly inhibited the release of both inflammatory markers from human monocytes. In Chapter 3, we investigated the effects of MPA and NET-A on the activation of human lymphocytes. This was achieved by flow cytometric measurement of the expression of the activation membrane marker CD69 by CD4 and CD8 T cells. Here we discovered that MPA had a very significant inhibitory effect on the activation of both CD4+ and CD8+ T cells, while NET-A only significantly inhibited the activation of CD8+ T cells. In addition, we found that the inhibition of CD4+ and CD8+ T cell activation by MPA was more or less the same as the known GC, Dex, and in some cases even more potent. Chapter 4 consists of an investigation of the effects of MPA and NET-A on the cytokines belonging to TH1 and TH2 subsets of CD4 T cells. This was achieved by determining whether MPA and/or NET-A targeted specific subsets of T helper cells by measuring the distinct regulatory cytokines, IFNγ and IL-4. The mechanism and role of the T helper subsets are discussed in the introduction of this chapter. Our results were portrayed as a ratio of TH2: TH1 on which the statistical analysis was done. In addition to the analysis done on the ratio, we analyzed the helper subsets separately in order to determine which subset(s) were influenced. The results of this chapter showed that neither MPA nor NET-A significantly affected either one of the helper subsets, while Dex significantly decreased this ratio. After our observed effects of MPA and NET-A on CD8 T cells, it became of interest in Chapter 5 to investigate the effects of these two synthetic hormones on the CD8 T cell-specific chemokine, RANTES. This was achieved by measuring the effects MPA and NET-A had on RANTES production in vitro by means of ELISA. Surprisingly, we discovered in this chapter that MPA and NET-A enhanced RANTES production before and after activation of CD8 T cells. We also found that Dex had the same effect on RANTES production, but to a lesser degree. Finally, a general conclusion depicting the significance and implications of our results as well as possible future research that is required is presented in Chapter 6. It was of great importance to discuss and interpret the magnitude of data generated out of all our experiments to the utmost of our capabilities. We found that MPA, even at contraceptive doses, displayed significant immunosuppressive as well as anti-inflammatory properties. NET-A, on the other hand, demonstrated weaker immunosuppressive properties in our research and no significant anti-inflammatory properties. These findings could have clinical implications in females being treated with these synthetic contraceptives. We also demonstrated significant variation found amongst genders in response to MPA, NET-A and Dex.
The measurement of apoptosis in HIV-1 infection
(Stellenbosch : University of Stellenbosch, 2006-03) Yu, J.; Bouic, Patrick J. D.; Cotton, Mark F.; University of Stellenbosch. Faculty of Health Sciences. Dept. of Pathology. Medical Microbiology.
Acquired immunodeficiency syndrome (AIDS) was first reported in 5 homosexual men in Unite States of America in 1981 as a series of opportunistic infections which occasionally occurred in adults. Subsequently, it has been achieved that human immunodeficiency virus type 1 (HIV-1) is the cause of AIDS and this aetiological agent has spread all over the world. The virus primarily attacks CD4+ T cells and gradually leads to progressive depletion of CD4 T lymphocytes from peripheral blood and lymphoid organs. Since CD4+ T cells are vital immune cells in induction and regulation of both cell-mediated and humoral immune responses, depletion of these cells ultimately results in a profound immunodeficiency characterized by susceptibility to variety of opportunistic infection. Apoptosis have been commonly proposed as the mechanism of CD4 depletion because elevated levels of apoptosis were observed in HIV-1 infected individuals (Ameisen et al., 1991; Groux et al., 1992 & Oyaizu et al., 1993). Nevertheless, there was evidence showing that HIV-1 infected cells died not from apoptosis (Bolton et al., 2002) and another study reported that inhibition of apoptosis resulted in high viral production (Antoni et al., 1995). These controversial views indicated that the mechanism of CD4 depletion and the immuno-pathogenesis of apoptosis should be considered. As a pilot sub-study, eight HIV-1 infected subjects were enrolled to determine the methods in measuring apoptosis. Three different cell separations: (1) whole blood cells, (2) buffy coat cells and (3) isolated PBMCs were prepared to determine whether different cell preparations result in different measurements of apoptosis. In addition, FITC-labelled Annexin V, an early marker of apoptosis, and flow-cytometer based scatter methods based on characteristics of apoptotic cells were used to investigate the difference in analytical methods in determining the levels of apoptosis. Firstly, it was found that whole blood samples yielded more precise measurements in measuring apoptosis, followed by Buffy coat and then PBMC samples. Secondly, this sub-study also indicated that the scatter method as well as fluorenscent labelled Annexin V could be useful markers for apoptosis. Secondly, different surface markers of apoptosis were used to investigate apoptosis in HIV-1 infected adults. Fifty-eight HIV-1 infected adults were involved in this sub-study. They were classified into three categories based on CDC CD4 category classification (CDC, 1993). According to the data, the level of apoptotic CD4+ T cells measured by the scatter method was high in CD4 category 1, decreased in category 2 and finally increased again in category 3. This tendency was in parallel with CD95 (Fas) expression on CD4+ T cells. The curve formed a “V” shape according to the three CD4 categories. Together with the gradually increased plasma viral load, these data reflect an activated immune response at early stage of infection and under controlled viraemia. This possibly represents the immune response trying to eliminate infected cells as a means of survival. The high level of apoptosis in category 3 could indicate a disordered immune system accounting for the rapid loss of CD4+ T cells and progression to AIDS. A novel finding of this study was the presence of two CD4+ populations in 10 HIV-1 infected subjects, which were CD4dim and CD4bright. These 10 subjects had relatively high CD4 count and low viral replication. Statistical analysis showed they had significantly higher levels of apoptosis in CD4 and CD8 T lymphocytes, measured by the scatter method, than those subjects presenting single CD4 population. In addition, when comparing the two CD4 subpopulations, it was found that CD4dim cells had significant higher level of apoptosis and CD95 expression than the CD4bright cells. Finally, the virological and immunological effects of antiretroviral therapy (ART) were investigated in two cohorts of HIV-1 infected children. Fourteen HIV-1 infected children were involved in investigation of 12-month long-term effect, while another five children were involved in a short-term 1-month follow-up study. In addition, a different assay of detecting apoptosis: terminal deoxynucleotidyltransferase deoxyuridine triphosphates nick end labeling (TUNEL) was conducted to measure the level of apoptotic PBMCs. According to the findings from 12-month and 1-month sub-studies, it appeared that ART could be effective in suppression of viral replication at an early stage. However, the immunological effect, such as CD4 reconstitution, could only be seen as a long-term effect, since immune recovery would take a long time. In addition, different regimens containing protease inhibitors (PIs) might be more effective in inhibiting apoptosis than non-nucleoside reverse transcriptase inhibitors (NNRTIs).
Molecular diagnostic approach to determine the degree of photoaging of the skin
(Stellenbosch : Stellenbosch University, 2015-04) Wilcox, Stephany Vanessa; Bouic, Patrick J. D.; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Pathology. Medical Microbiology.
ENGLISH ABSTRACT: Context: Excessive exposure to ultraviolet radiation (UV) results in the risk of acquiring long-term harmful effects such as photoaging, which is characterised by deep wrinkles, roughness, dyspigmentation and an increased loss in elasticity. As a result, the detection of photoaging at an early stage is crucial to improving morbidity, whilst preventing the advancement of skin cancer. Aim: The aim of the study was to develop and to validate a diagnostic real-time PCR method in order to establish the gene expression profiles of potential biomarkers in the skin so as to quantify the degree of photoaging: this was conducted by retrieving total RNA from cells adherent to tape strips from sun exposed and non-exposed skin areas. Materials and methods: Twenty healthy volunteers consisting of seven males and thirteen females aged 25 to 67 years were included in this study. Tape stripping was performed using pre-cut D-Squame® 22 mm adhesive discs. Samples were collected on the right medial thigh area 20 cm above the patella and 2 cm below the lateral canthus of the right eye. Total RNA was extracted and relative standard curve method of gene expression was performed. TGF-β, MMP 9, TNF-α and IL-6 mRNA transcripts were selected as representative cytokines to determine the relative fold-change in sun exposed and non-exposed areas of the skin so as to determine extent of photoaging. Results: Repeatability and reproducibility was determined by the coefficient of variation (CV) was within an acceptable range. Thirty five percent (n=7) samples displayed down-regulatory effects for TGF-β. Down regulation of MMP 9 was observed within 30% (n=6) of samples, while 15% (n=3) showed marked up regulation. Only two samples showed measurable levels of TNF-α in the assay, of which one showed significant up regulation. Furthermore, we were unable to detect any IL-6 expression in any of the samples prepared. Conclusion: we have shown that epidermal cytokines can be retrieved from tape stripped samples and can be quantified via real-time PCR. However, the choices of cytokine biomarkers reveal that they are as important as the concentration of starting material. In this study cytokines such as IL-6 is not as informative in determining the extent of photoaging without high doses of ultraviolet radiation before sample collection as opposed to the other explored cytokines.
The relevance of apoptosis in the pathogenesis of human immunodeficiency virus-1 disease
(Stellenbosch : Stellenbosch University, 2004-12) Cotton, Mark F.; Bouic, Patrick J. D.; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Pathology. Medical Microbiology.
ENGLISH ABSTRACT: A simple and rapid scatter-based flow cytometric assay was developed to detect apoptosis in CD4+ and CD8+ T cells from a mixed population of cells. The assay was suitable for children. Apoptotic PBMCs were confirmed by morphologic assessment in clinical samples ex vivo and after overnight culture. The scatter-based assay was validated in a number of ways. Firstly, PBMCs were irradiated with 500 rads and cultured overnight to induce apoptosis. Thereafter, PBMCs were labeled with a CD4 MAb. CD4+ cells were sorted into apoptotic and viable populations by scatter characteristics (diminished forward and increased side scatter). Morphology was assessed by fluorescence microscopy. The majority of cells with apoptotic scatter characteristics had apoptotic morphology (chromatin condensation) (80.6%). Ninety-two percent of cells from the viable region had normal morphology. CD4+ T cell apoptosis measured by scatter was then correlated with the TdT assay for DNA fragmentation. Lastly, CD4+ T cell apoptosis by scatter and annexin V uptake were also shown to correlate. In the latter experiments, PBMC morphology and cell death by trypan blue uptake were studied simultaneously and confirmed the two flow cytometric assays. Apoptosis of CD4+ and CD8+ T cells has been shown in PBMCs from HIV infected adults analyzed after overnight culture. Since cell death may be an artifact of in vitro culture, and because there is little information on apoptosis in paediatric HIV disease, I undertook a cross-sectional analysis in PBMCs analyzed immediately ex vivo from HIV infected children and adults. Patients were studied in Denver, CO, USA. PBMCs from 21 children, 4 adolescents and 9 adults and seronegative age-matched controls were stained for CD4 and CD8 surface markers. Apoptotic cells were detected in a newly characterized flow cytometric assay by diminished forward and increased side scatter. For the scatter assay, PBMCs had been labeled initially by an indirect method involving an intermediary incubation in the presence of biotinylated MAbs at 37°C for 30 minutes prior to incubating with streptavidin-FITC at 4°C for 20 minutes. Thereafter, the intermediary incubation step was removed and PBMCs were incubated with PE-conjugated CD4+ and CD8+ MAbs. Both CD4+ and CD8+ T cell apoptosis appeared enhanced in the indirect method. The significant differences were abolished after subtraction of data from simultaneously studied time-matched controls. CD4+ and CD8+ T cell apoptosis were significantly higher in HIV-infected study subjects than in simultaneously studied seronegative controls. PBMCs were assayed immediately ex vivo and after overnight culture after stimulation by an anti-TCR MAb as well as spontaneously. There was a direct correlation between CD4+ and CD8+ T cell apoptosis and CD4+ T cell depletion. A significant correlation was also shown between apoptosis immediately ex vivo and after overnight culture. I then studied apoptosis in a South African population comprising 18 symptomatic children and 4 seroreverters. CD4+ and CD8+ T cell apoptosis were significantly higher in symptomatic HIV-1-infected children than in seroreverters and seronegative controls. CD4+ T cell apoptosis correlated with depletion of CD4+ T cell percentage in symptomatic HIV-1-infected children. I also noted elevated CD4+ T cell apoptosis in patients recovering from intercurrent disease in comparison to those who were either acutely ill or relatively asymptomatic outpatient attendees. Lastly, I compared CD4+ and CD8+ T cell apoptosis in cohorts from Denver, CO and Tygerberg Children’s Hospital, South Africa. I selected only patients with moderate or severe HIV infection from both centers. South African patients were significantly younger, more malnourished, had higher gamma globulin levels and were less likely to receive ART. CD8+ T cell apoptosis was higher in North American patients suggesting a possible impairment in CD8+ activity in the South African study subjects.
Sequence-based molecular diagnosis of X-linked agammaglobulinemia in South African individuals
(Stellenbosch : University of Stellenbosch, 2011-03-04) Leo, Melanie Joy; Esser, Monika; Scholtz, Charlotte; Bouic, Patrick J. D.; University of Stellenbosch. Faculty of Health Sciences. Dept. of Pathology. Medical Microbiology.
ENGLISH SUMMARY: Background: Primary immunodeficiency disorders (PID) disrupt the proper functioning of the immune system. The prototypic PID is X-linked Agammaglobulinemia (XLA). This disorder is caused by mutations in the Bruton tyrosine kinase (Btk) gene and results in an arrest in B cell development which leads to a profound reduction of all classes of serum immunoglobulins (i.e antibodies). Patients with a lack of antibodies experience recurring bacterial infections during early childhood that can be fatal if not treated. Intravenous gammaglobulin replacement therapy (IVIg) is the standard treatment for XLA. It provides passive immunity thereby reducing the number and severity of infections as well as limiting many of the infectious complications. Early detection and treatment of XLA allows affected individuals to live a relatively normal life. Objective: The purpose of this study was to determine the molecular basis of XLA in South Africa using a direct sequence-based method to detect abnormalities in the Btk gene to aid clinical diagnosis of the disease. Methods : Male patients with a clinical diagnosis of XLA were included in this study. Genetic analysis was used to explore the exonic region of the Btk gene of 5 unrelated male patients and compared to 10 healthy controls. Family members were followed up to determine carrier status, where possible. Results: Mutational analysis revealed Btk abnormalities in 4 of the 5 patients leading to a definitive diagnosis of XLA. Two of the three mutations found in this study have been previously described while one mutation appears to be novel. The novel mutation is a one base pair deletion in exon 16 which leads to the truncation of the Btk protein. Despite the clinical findings suggestive of XLA, no mutation was identified in the exonic region of the Btk gene of the remaining patient, indicating that this patient might have a different form of PID. Maternal follow-up confirmed the maternal inheritance pattern as all mothers screened were carriers of the Btk mutation present in the affected individual. Discussion : Using a direct sequence-based method abnormalities were identified in the Btk gene of three patients. Molecular diagnosis coupled to clinical history of the patient provides a definitive XLA diagnosis. This study supports the use of molecular techniques in the diagnosis of PID and underlines the synergy that could be possible in a clinical setting.

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