The relevance of apoptosis in the pathogenesis of human immunodeficiency virus-1 disease

Thesis (PhD)--Stellenbosch University, 2004.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.AFRIKAANSE OPSOMMING: ‘n Eenvoudige en vinnige vloei sitometriese toets is ontwikkel om apoptose aan te toon vanuit ‘n gemengde populasie selle. Dit moes geskik wees vir kinders van wie net klein volumes bloed getrek kan word. Die teenwoordigheid van apoptotiese perifere bloed mononuklere selle (PBMS) was vasgestel deur morfologiese beoordeling in kliniese monsters ex vivo en na oornag kultuur. Die ondersoek is gebasseer op die verstrooings patroon van bestraalde PBMS wat apoptose induseer. PBMS is gemerk met a CD4 MAb. CD4+ selle is gesorteer in apoptotiese en lewensvatbare populasies deur verstrooings karakteristieke. Morfologie is beoordeel deur fluoreserende mikroskopie. Die meerderheid van selle met apoptotiese verstrooings karakteristieke (verminderde voorwaartse en verhoogde sywaartse verstrooings patroon) het apoptotiese karakteristieke gehad (80.6%). Twee-en-negentig persent van selle van die lewensvatbare area het normale morfologie gehad. Verstrooings patroon is ook gekorreleer met die TdT meting vir DNA fragmentasie in kliniese monsters van MIV-geinfekteerde kinders. Daarna is Annexin V gekorreleer met verstrooings patroon, apoptotiese morfologie en trypan blou opname in selle wat blootgestel is na verskillende konsentrasies van beauvericin. Apoptose van CD4+ en CD8+ T-selle is bewys in PBMS van MIV-geinfekteerde volwassenes na oornag kultuur. Omdat sel dood ‘n artefak van in vitro kultuur kan wees, en omdat daar min inligting is oor apoptose in paediatriese MIV siekte, het ek onderneem om ‘n deursnee analiese te doen in PBMS wat onmiddelik ex vivo geanaliseer is vanaf MlV-geinfekteerde kinders en volwassenes. Die pasiente is bestudeer in Denver, Colorado, VS A. PBMS van 22 kinders, 4 adolessente en 9 volwassenes en seronegatiewe ouderdoms-gepasde kontroles is gekleur vir CD4+ en CD8+ oppervlaksmerkers. Apoptotiese selle is vloeisitometries aangedui deur verandering in verstrooings patroon. Vir die doeleindes van die verstrooings assay is die PBMS aanvanklik deur ‘n indirekte metode gemerk, wat ‘n intermediere inkubasie in die teenwoordigheid van biogetinileerde MAbs by 37°C vir 30 minute voor dit geinkubeer is met streptavidin- FITC by 4°C vir 20 minute behels. Daarna is die intermediere inkubasie stap verwyder en PBMC is geinkubeer met PE - gekonjugeerde CD4+ and CD8+ MAbs. Beide die CD4+ en CD8+ T-sel apoptose het verhoog voorgekom met die indirekte metode. Die betekenisvolle verskille het verdwyn na data van gelyktydige tyd - gepaarde kontroles afgetrek is. CD4+ en CD8+ T-sel apoptose was betekenisvol hoër in MIV-geinfekteerde studie gevalle as in gelyktydig bestudeerde seronegatiewe kontroles. PBMS assays is gedoen onmiddelik ex vivo en na oornag inkubasie na stimulasie deur ‘n anti-TCR MAb, sowel as spontaan. Daar was ‘n direkte korrelasie tussen CD4+ en CD8+ T sel apoptosis en CD4+ T sel vermindering. ‘n Beduidende korrelasie is ook getoon tussen apoptose onmiddelik ex vivo en na oornag kultuur. Daaropvolgend het ek apoptose in ‘n Suid Afrikaanse populasie van 18 simptomatiese kinders en 4 serologies terukerende gevalle bestudeer. CD4+ en CD8+ T sel apoptose was aansienlik hoër in siptomatiese MIV - 1-geinfekteerde kinders as in die serologies terukerende gevalle en seronegatiewe kontroles. CD4+ T sel apoptose het gekorrelleer met vermindering van CD4+ T sel persentasie. Ek het ook opgemerk dat daar ‘n tendens bestaan het tot verhoogde CD4+ T sel apoptose in pasiente wat besig was om te herstel van bykomende siektes. Ek het CD4+ en CD8+ T sel apoptose in kohorte van Denver, Colorado en Tygerberg, Suid Afrika vergelyk. Suid Afrikaanse pasiente was jonger en meer wangevoed as hul Noord Amerikaanse ewekniee. Suid Afrikaanse kinders het ook meer gevorderde siekte gehad. Wanneer pasiente gepas is vir die graad van ernstigheid van siekte en slegs die minder ernstige (B) en ernstige siekte (C) vergelyk is, was CD8+ T sel apoptose beduidend hoër in Noord Amerikaanse pasiente. Hierdie waarneming ondersteun die hipotese dat CD 8+ T sel aktiwiteit moontlik onderdruk mag wees in simptomatiese Suid Afrikaanse MIV-1-geinfekteerde kinders

Optimizing Research Methods to Understand HIV-Exposed Uninfected Infant and Child Morbidity: Report of the Second HEU Infant and Child Workshop

CITATION: Slogrove, A. L., et al. 2016. Optimizing research methods to understand HIV-exposed uninfected infant and child morbidity : report of the second HEU infant and child workshop. Frontiers in Immunology, 7:576, doi:10.3389/fimmu.2016.00576.The original publication is available at http://journal.frontiersin.orgThe first HIV Exposed Uninfected (HEU) Infant and Child Workshop was held in Vancouver in July 2015, hosted by the Child and Family Research Institute at the British Columbia Children’s Hospital and University of British Columbia. This event brought together 50 clinicians, epidemiologists, and basic scientists to review current knowledge of HEU infants, their clinical course, immunologic differences, and risk for neurodevelopmental and infectious morbidity. This Frontiers in Immunology Research Topic, “Immune mechanisms underlying the increased morbidity and mortality of HIVexposed uninfected (HEU) children,” is a product of the first HEU workshop synthesizing the evidence in the field. It was clear from the first workshop that there is a committed community of researchers who have identified the need to understand the mechanisms of increased morbidity and mortality in HEU infants and children, but evidence to intervene and mitigate these risks is lacking. In high HIV burden countries, all infants and children, irrespective of HIV exposure, are vulnerable to high rates of infant and child mortality (1). In this context, the essential question is whether HEU children are any different than HIV-unexposed uninfected (HUU) children experiencing similar nutritional, environmental, and social constraints to health. To this end, particular research methodological principles require reinforcing in future HEU research. It was these methodological challenges and possible solutions that formed the theme of the second HEU Infant and Child Workshop attended by 75 HEU researchers and hosted by the KwaZulu-Natal Research Institute for Tuberculosis and HIV at the University of KwaZulu-Natal in Durban, South Africa. We report on the specific methodological challenges tackled during the workshop and steps to move forward.http://journal.frontiersin.org/journal/immunology/section/hiv-and-aids#aboutPublisher's versio

Feeding practices and nutritional status of HIV-exposed and HIV-unexposed infants in the Western Cape

Background: Optimal infant- and young child–feeding practices are crucial for nutritional status, growth, development, health and, ultimately, survival. Human breast milk is optimal nutrition for all infants. Complementary food introduced at the correct age is part of optimal feeding practices. In South Africa, widespread access to antiretrovirals and a programme to prevent mother-to-child transmission of HIV have reduced HIV infection in infants and increased the number of HIV-exposed uninfected (HEU) infants. However, little is known about the feeding practices and nutritional status of HEU and HIV-unexposed (HU) infants.Objective: To assess the feeding practices and nutritional status of HIV-exposed and HIV-unexposed (HU) infants in the Western Cape.Design: Prospective substudy on feeding practices nested in a pilot study  investigating the innate immune abnormalities in HEU infants compared to HU infants. The main study commenced at week 2 of life with the nutrition component added from 6 months. Information on children’s dietary intake was obtained at each visit from the caregiver, mainly the mother. Head circumference, weight and length were recorded at each visit. Data were obtained from 6-, 12- and 18-month visits. World Health Organization feeding practice indicators and nutrition indicators were utilised.Setting: Tygerberg Academic Hospital, Western Cape. Mothers were recruited from the postnatal wards.Subjects: Forty-seven mother–infant pairs, 25 HEU and 22 HU infants, participated in this nutritional substudy. Eight (17%) infants, one HU and seven HEU, were lost to follow-up over the next 12 months. The HEU children were mainly Xhosa (76%) and HU were mainly mixed race (77%).Results: The participants were from poor socio-economic backgrounds. In both groups, adherence to breastfeeding recommendations was low with suboptimal dietary diversity. We noted a high rate of sugar- and salt-containing snacks given from a young age. The HU group had poorer anthropometric and nutritional indicators not explained by nutritional factors alone. However, alcohol and tobacco use was much higher amongst the HU mothers.Conclusion: Adherence to breastfeeding recommendations was low. Ethnicity and cultural milieu may have influenced feeding choices and growth. Further research is needed to understand possible reasons for the poorer nutritional and anthropometric indicators in the HU group

Experimental verification of a self-consistent theory of the first-, second-, and third-order (non)linear optical response

We show that a combination of linear absorption spectroscopy, hyper-Rayleigh scattering, and a theoretical analysis using sum rules to reduce the size of the parameter space leads to a prediction of the two-photon absorption cross-section of the dye AF455 that agrees with two-photon absorption spectroscopy. Our procedure, which demands self-consistency between several measurement techniques and does not use adjustable parameters, provides a means for determining transition moments between the dominant excited states based strictly on experimental characterization. This is made possible by our new approach that uses sum rules and molecular symmetry to rigorously reduce the number of required physical quantities.Comment: 10 pages, 9 figure

CLAD: A Complex and Long Activities Dataset with Rich Crowdsourced Annotations

This paper introduces a novel activity dataset which exhibits real-life and diverse scenarios of complex, temporally-extended human activities and actions. The dataset presents a set of videos of actors performing everyday activities in a natural and unscripted manner. The dataset was recorded using a static Kinect 2 sensor which is commonly used on many robotic platforms. The dataset comprises of RGB-D images, point cloud data, automatically generated skeleton tracks in addition to crowdsourced annotations. Furthermore, we also describe the methodology used to acquire annotations through crowdsourcing. Finally some activity recognition benchmarks are presented using current state-of-the-art techniques. We believe that this dataset is particularly suitable as a testbed for activity recognition research but it can also be applicable for other common tasks in robotics/computer vision research such as object detection and human skeleton tracking

HIV sero-conversion during late pregnancy – when to retest

The South African National Prevention of Mother-to-Child Transmission of HIV programme has resulted in significant reductions in vertical transmission, but new infant HIV infections continue to occur. We present two cases of HIV seroconversion during late pregnancy, demonstrating the limitations of the current programme. These could be mitigated by expanding the programme to include maternal testing at delivery and at immunisation clinic visits as we pursue the elimination of mother-to-child transmission

Nevirapine plasma concentrations in premature infants exposed to single-dose nevirapine for prevention of mother-tochild transmission of HIV-1

Background: No pharmacokinetic data exist for premature infants receiving single-dose nevirapine (sd NVP) for prevention of mother-to-child transmission (MTCT) of HIV. Aim: To describe NVP decay pharmacokinetics in two groups of premature infants – those whose mothers either received or did not receive NVP during labour. Methods: Infants less than 37 weeks’ gestation were prospectively enrolled. Mothers received sd NVP during labour if time allowed. Infants received sd NVP and zidovudine. Blood was collected on specified days after birth and NVP concentrations were determined by liquid chromatography-mass spectrometry. Results: Data were obtained from 81 infants, 58 born to mothers who received sd NVP during labour (group I) and 23 to mothers who did not receive NVP (group II). Of the infants 29.6% were small for gestational age (SGA). Median (range) maximum concentration (Cmax), time to reach maximum concentration (Tmax), area under the plasma concentration-time curve (AUC) and halflife (T½) were 1 438 (350 - 3 832) ng/ml, 25h50 (9h40 - 83h45), 174 134 (22 308 - 546 408) ng×h/ml and 59.0 (15.4 - 532.6) hours for group I and 1 535 (635 - 4 218) ng/ml, 17h35 (7h40 - 29h), 168 576 (20 268 - 476 712) ng×h/ml and 69.0 (22.12 - 172.3) hours for group II. For group II, the median (range) volume of distribution (Vd) and body clearance (Cl) were 1 702.6 (623.7 - 6 189.8) ml and 34.9 (6.2 - 163.8) ml/h. The AUC was higher (p=0.006) and Cl lower (p<0.0001) in SGA infants. Plasma concentrations exceeding 100 ng/ml were achieved over 8 days in 78% infants in group I and 70.0% in group II. The MTCT rate was 4.8%. Conclusion: Women in preterm labour often deliver with little advance warning. Our study suggests that NVP dosing of preterm infants as soon as possible after birth without maternal intrapartum dosing may be as effective as combined maternal and infant dosing.Web of Scienc

Nevirapine plasma concentrations in premature infants exposed to single-dose nevirapine for prevention of mother-to-child transmission of HIV-1

The original publication is available at http://www.samj.org.zaBackground. No pharmacokinetic data exist for premature infants receiving single-dose nevirapine (sd NVP) for prevention of mother-to-child transmission (MTCT) of HIV. Aim. To describe NVP decay pharmacokinetics in two groups of premature infants - those whose mothers either received or did not receive NVP during labour. Methods. Infants less than 37 weeks' gestation were prospectively enrolled. Mothers received sd NVP during labour if time allowed. Infants received sd NVP and zidovudine. Blood was collected on specified days after birth and NVP concentrations were determined by liquid chromatography-mass spectrometry. Results. Data were obtained from 81 infants, 58 born to mothers who received sd NVP during labour (group I) and 23 to mothers who did not receive NVP (group II). Of the infants 29.6% were small for gestational age (SGA). Median (range) maximum concentration (Cmax), time to reach maximum concentration (Tmax), area under the plasma concentration-time curve (AUC) and halflife (T) were 1 438 (350-3 832) ng/ml, 25h50 (9h40-83h45), 174 134 (22 308-546 408) ng×h/ml and 59.0 (15.4-532.6) hours for group I and 1 535 (635-4 218) ng/ml, 17h35 (7h40-29h), 168 576 (20 268-476 712) ng×h/ml and 69.0 (22.12-172.3) hours for group II. For group II, the median (range) volume of distribution (Vd) and body clearance (Cl) were 1 702.6 (623.7-6 189.8) ml and 34.9 (6.2-163.8) ml/h. The AUC was higher (p=0.006) and Cl lower (p&lt;0.0001) in SGA infants. Plasma concentrations exceeding 100 ng/ml were achieved over 8 days in 78% infants in group I and 70.0% in group II. The MTCT rate was 4.8%. Conclusion. Women in preterm labour often deliver with little advance warning. Our study suggests that NVP dosing of preterm infants as soon as possible after birth without maternal intrapartum dosing may be as effective as combined maternal and infant dosing

Trends in paediatric bloodstream infections at a South African referral hospital

CITATION: Dramowski, A., Cotton, M.F., Rabie, H. & Whitelaw, A. 2015. Trends in paediatric bloodstream infections at a South African referral hospital. BMC Pediatrics, 15(33), doi:10.1186/s12887-015-0354-3.The original publication is available at http://bmcpediatr.biomedcentral.comPublication of this article was funded by the Stellenbosch University Open Access Fund.Background: The epidemiology of paediatric bloodstream infection (BSI) in Sub-Saharan Africa is poorly documented with limited data on hospital-acquired sepsis, impact of HIV infection, BSI trends and antimicrobial resistance. Methods: We retrospectively reviewed paediatric BSI (0–14 years) at Tygerberg Children’s Hospital between 1 January 2008 and 31 December 2013 (excluding neonatal wards). Laboratory and hospital data were used to determine BSI rates, blood culture contamination, pathogen profile, patient demographics, antimicrobial resistance and factors associated with mortality. Fluconazole resistant Candida species, methicillin-resistant Staphylococcus aureus (MRSA), multi-drug resistant Acinetobacter baumannii and extended-spectrum beta-lactamase (ESBL) producing Enterobacteriaceae were classified as antimicrobial resistant pathogens. Results: Of 17001 blood cultures over 6 years, 935 cultures isolated 979 pathogens (5.5% yield; 95% CI 5.3-5.7%). Contamination rates were high (6.6%, 95% CI 6.4-6.8%), increasing over time (p = 0.003). Discrete BSI episodes were identified (n = 864) with median patient age of 7.5 months, male predominance (57%) and 13% HIV prevalence. BSI rates declined significantly over time (4.6–3.1, overall rate 3.5 per 1000 patient days; 95% CI 3.3–3.7; Chi square for trend p = 0.02). Gram negative pathogens predominated (60% vs 33% Gram positives and 7% fungal); Klebsiella pneumoniae (154; 17%), Staphylococcus aureus (131; 14%) and Escherichia coli (97; 11%) were most prevalent. Crude BSI mortality was 20% (176/864); HIV infection, fungal, Gram negative and hospital-acquired sepsis were significantly associated with mortality on multivariate analysis. Hospital-acquired BSI was common (404/864; 47%). Overall antimicrobial resistance rates were high (70% in hospital vs 25% in community-acquired infections; p < 0.0001); hospital-acquired infection, infancy, HIV-infection and Gram negative sepsis were associated with resistance. S. pneumoniae BSI declined significantly over time (58/465 [12.5%] to 33/399 [8.3%]; p =0.04). Conclusion: Although BSI rates declined over time, children with BSI had high mortality and pathogens exhibited substantial antimicrobial resistance in both community and hospital-acquired infections. Blood culture sampling technique and local options for empiric antimicrobial therapy require re-evaluation.http://www.biomedcentral.com/content/pdf/s12887-015-0354-3.pdfPublisher's versio

Diffusion tensor imaging point to ongoing functional impairment in HIV-infected children at age 5, undetectable using standard neurodevelopmental assessments

Background Perinatal HIV infection negatively impacts cognitive functioning of children, main domains affected are working memory, processing speed and executive function. Early ART, even when interrupted, improves neurodevelopmental outcomes. Diffusion tension imaging (DTI) is a sensitive tool assessing white matter damage. We hypothesised that white matter measures in regions showing HIV-related alterations will be associated with lower neurodevelopmental scores in specific domains related to the functionality of the affected tracts. Methods DTI was performed on children in a neurodevelopmental sub study from the Children with HIV Early Antiretroviral (CHER) trial. Voxel-based group comparisons to determine regions where fractional anisotropy and mean diffusion differed between HIV+ and uninfected children were done. Locations of clusters showing group differences were identified using the Harvard–Oxford cortical and subcortical and John Hopkins University WM tractography atlases provided in FSL. This is a second review of DTI data in this cohort, which was reported in a previous study. Neurodevelopmental assessments including GMDS and Beery-Buktenica tests were performed and correlated with DTI parameters in abnormal white matter. Results 38 HIV+ children (14 male, mean age 64.7 months) and 11 controls (4 male, mean age 67.7 months) were imaged. Two clusters with lower fractional anisotropy and 7 clusters with increased mean diffusion were identified in the HIV+ group. The only neurodevelopmental domain with a trend of difference between the HIV+ children and controls (p = 0.08), was Personal Social Quotient which correlated to improved myelination of the forceps minor in the control group. As a combined group there was a negative correlation between visual perception and radial diffusion in the right superior longitudinal fasciculus and left inferior longitudinal fasciculus, which may be related to the fact that these tracts, forming part of the visual perception pathway, are at a crucial state of development at age 5. Conclusion Even directed neurodevelopmental tests will underestimate the degree of microstructural white matter damage detected by DTI. The visual perception deficit detected in the entire study population should be further examined in a larger study