Human exposure to diesel exhaust induces CYP1A1 expression and AhR activation without a coordinated antioxidant response.

BACKGROUND: Diesel exhaust (DE) induces neutrophilia and lymphocytosis in experimentally exposed humans. These responses occur in parallel to nuclear migration of NF-κB and c-Jun, activation of mitogen activated protein kinases and increased production of inflammatory mediators. There remains uncertainty regarding the impact of DE on endogenous antioxidant and xenobiotic defences, mediated by nuclear factor erythroid 2-related factor 2 (Nrf2) and the aryl hydrocarbon receptor (AhR) respectively, and the extent to which cellular antioxidant adaptations protect against the adverse effects of DE. METHODS: Using immunohistochemistry we investigated the nuclear localization of Nrf2 and AhR in the epithelium of endobronchial mucosal biopsies from healthy subjects six-hours post exposure to DE (PM10, 300 µg/m3) versus post-filtered air in a randomized double blind study, as a marker of activation. Cytoplasmic expression of cytochrome P450s, family 1, subfamily A, polypeptide 1 (CYP1A1) and subfamily B, Polypeptide 1 (CYP1B1) were examined to confirm AhR activation; with the expression of aldo-keto reductases (AKR1A1, AKR1C1 and AKR1C3), epoxide hydrolase and NAD(P)H dehydrogenase quinone 1 (NQO1) also quantified. Inflammatory and oxidative stress markers were examined to contextualize the responses observed. RESULTS: DE exposure caused an influx of neutrophils to the bronchial airway surface (p = 0.013), as well as increased bronchial submucosal neutrophil (p < 0.001), lymphocyte (p = 0.007) and mast cell (p = 0.002) numbers. In addition, DE exposure enhanced the nuclear translocation of the AhR and increased the CYP1A1 expression in the bronchial epithelium (p = 0.001 and p = 0.028, respectively). Nuclear translocation of AhR was also increased in the submucosal leukocytes (p < 0.001). Epithelial nuclear AhR expression was negatively associated with bronchial submucosal CD3 numbers post DE (r = -0.706, p = 0.002). In contrast, DE did not increase nuclear translocation of Nrf2 and was associated with decreased NQO1 in bronchial epithelial cells (p = 0.02), without affecting CYP1B1, aldo-keto reductases, or epoxide hydrolase protein expression. CONCLUSION: These in vivo human data confirm earlier cell and animal-based observations of the induction of the AhR and CYP1A1 by diesel exhaust. The induction of phase I xenobiotic response occurred in the absence of the induction of antioxidant or phase II xenobiotic defences at the investigated time point 6 h post-exposures. This suggests DE-associated compounds, such as polycyclic aromatic hydrocarbons (PAHs), may induce acute inflammation and alter detoxification enzymes without concomitant protective cellular adaptations in human airways.

Documented higher burden of advanced and very advanced HIV disease among patients, especially men, accessing healthcare in a rapidly growing economic and industrial hub in South Africa: A call to action.

BACKGROUND: Lephalale Municipality in Limpopo Province, South Africa, has seen significant economic and industrial development owing to expansion of the coal mining and power generation sectors. This development has coincided with substantial population growth of 65% between 2001 and 2016, attributable to largely (migrant) males living in the area who, overall, outnumbered females by ~121:100. The local HIV prevalence is reported to be higher than national rates. OBJECTIVES: Anonymised National Health Laboratory Service CD4+ data were used to document increasing laboratory services workload and to establish the burden of advanced (CD4+ count <200 cells/µL) and very advanced (<100 cells/µL) HIV disease among adult patients accessing public healthcare in Lephalale between 2006 and 2015. METHODS: A cross-sectional design was used to analyse CD4+ laboratory data. CD4+ outcomes were categorised by volumes of tests, year, health facility type, age categories (15 - 19, 20 - 24, 25 - 29, 30 - 34, 35 - 39, 40 - 44, 45 - 49 and >49 years), CD4+ test range (≤50, 51 - 100, 101 - 200, 201 - 350, 351 - 500 and ≥501 cells/µL) and gender. Median CD4+ counts were calculated. RESULTS: Extracted Lephalale data comprised 57 490 CD4+ results, with a mean patient age of 34 years. Considerably fewer male than female patients had CD4+ counts reported (male/female ratio 0.45:1). CD4+ test volumes showed a five-fold escalation over the study period, increasing from 1 458 tests in 2006 to 8 239 in 2015. A considerable burden of advanced and very advanced HIV disease (exceeding 50% of all cases) was noted in 2006/2007; by 2015 the proportion had fallen, but was still high at 27%. The overall median CD4+ count in 2006 (192 cells/µL) confirmed a high burden of advanced disease, with modest improvement to 289 cells/µL by 2015. Between 2006 and 2015, the median CD4+ count for females increased from 204 to 405 cells/µL, while that for males increased from 126 to 285 cells/µL. Age analysis further revealed that men aged <20 years or >25 years, and specifically those aged 30 - 45 years, had up to 44% more advanced HIV disease. CONCLUSIONS: Lower median CD4+ counts and a dramatic increase in volumes of CD4+ tests performed from 2007 onwards revealed a high burden of advanced and very advanced HIV disease in patients accessing care in Lephalale. Viewed together with Statistics South Africa census documentation of a disproportionately high number of males compared with females living in the area, these figures suggest that improved systems are urgently needed to encourage and accommodate access to HIV care for male (migrant worker) patients living and working in emerging industrial centres.

Prostate cancer age-standardised incidence increase between 2006 and 2016 in Gauteng Province, South Africa: A laboratory data-based analysis.

BACKGROUND: Prostate cancer (PCa) is the leading male neoplasm in South Africa (SA) and is the second most frequently diagnosed cancer among men globally. Age-specific incidence rates (ASIRs) vary by up to 189-fold globally, with an ASIR of 68.0 per 100 000 in 2018 in SA. OBJECTIVES: To describe PCa among men undergoing prostate biopsy in Gauteng Province, SA. METHODS: We undertook a retrospective descriptive study using prostate biopsy data collected from the National Health Laboratory Service (NHLS) database between 2006 and 2016. We extracted the Systematized Nomenclature of Medicine (SNOMED) clinical terms morphology and topography codes to assign histological findings using the International Classification of Diseases for Oncology. PCa was defined as adenocarcinoma with a reported Gleason Score (GS). The new grade group (GG) based on the GS is defined as follows; (i) GG1 for a GS ≤6; (ii) GG2 for a GS of 3 + 4 = 7 ; (iii) GG3 for a GS of 4 + 3 = 7; (iv) GG4 for a GS of 8; and (v) GG5 for a GS ≥9. Higher-grade disease was defined as GG4 and GG5 (GS ≥8), in line with local guidelines. We reported associations of PCa with a GS ≥7 with age and race and used provincial and world standard population data to determine annual ASIRs. RESULTS: We identified 22 937 biopsies referred to the NHLS between 2006 and 2016. Of the 6 448 biopsies (39%) with a PCa finding for black Africans, 46% were diagnosed with high-risk PCa compared with 36 - 40% for other race groups (p<0.0001). Black Africans were more likely than whites to have GG4 or GG5 PCa (odds ratio 1.45; 95% confidence interval 1.27 - 1.67). The ASIR increased from 44.9 per 100 000 in 2006 to 57.3 per 100 000 in 2016. CONCLUSIONS: Black African men were significantly more likely to present with PCa with a GS ≥8 (GG4 and GG5) compared with the other racial groups in Gauteng. The ASIR increased dramatically during the study period, perhaps as a result of increased screening and awareness. There is a need for additional research to better understand why black African men present with higher-grade disease.

Using Big Data Techniques to Improve Prostate Cancer Reporting in the Gauteng Province, South Africa.

Prostate cancer (PCa) data is of public health importance in South Africa. Biopsy data is recorded as semi-structured narrative text that is not easily analysed. Our study reports a pilot study that applied predictive analytics and text mining techniques to extract prognostic information that guides patient management. In particular, the Gleason score (GS) reported in a number of formats were extracted successfully. Our study reports that predominantly older men were diagnosed with PCa reporting a high-risk GS (8-10). Where cell differentiation was reported, 64% of biopsies reported poor differentiation. The approaches demonstrated in our study should be extended to a larger dataset to assess whether it has the potential to scale up to the national level.

Piloting a national laboratory electronic programme status reporting system in Ekurhuleni health district, South Africa.

BACKGROUND: The National Health Laboratory Service (NHLS) performs ~4 million CD4 tests per annum for the public health sector at 61 CD4 testing laboratories across South Africa. Currently, CD4 laboratory data captured do not differentiate between antiretroviral treatment (ART) and pre-ART care. METHODS: A cross-sectional study was undertaken to evaluate a redesigned Comprehensive Care, Management and Treatment of HIV and AIDS (CCMT) request form, incorporating a two-tick collection procedure linking the CD4 test request to patient CCMT programme status. Field testing was undertaken at three health facilities, where healthcare personnel were required to capture whether the CD4 count requested was a 'first-ever CD4', 'CD4 taken previously, not yet in ART care' or 'in ART care'. All data were extracted from the NHLS Corporate Data Warehouse and analysed using Microsoft Excel and Stata-12. RESULTS: A substantial increase in the number of request forms with a CCMT programme status (28.1% v. 84.4%) was reported pre- and post-implementation. Post-implementation data (N=1 004) revealed that 30.8% patients were ART naive ('first-ever CD4'), with 7.4% 'not yet on ART' (median CD4 counts of 150 and 328 cells/µL, respectively). Patients on ART comprised 61.9% of the study group (median CD4 count ~346 cells/µL). Sixty percent of patients were aged between 30 and 44 years, and females predominated (male/female ratio 0.7:1). CONCLUSIONS: A simple modification to the CCMT request form can successfully facilitate collection of programme status. For national implementation, it would be advantageous to have a unique patient identifier to further enhance laboratory-based programmatic monitoring and evaluation.

Implementation of a new 'community' laboratory CD4 service in a rural health district in South Africa extends laboratory services and substantially improves local reporting turnaround time.

BACKGROUND: The CD4 integrated service delivery model (ITSDM) provides for reasonable access to pathology services across South Africa (SA) by offering three new service tiers that extend services into remote, under-serviced areas. ITSDM identified Pixley ka Seme as such an under-serviced district. OBJECTIVE: To address the poor service delivery in this area, a new ITSDM community (tier 3) laboratory was established in De Aar, SA. Laboratory performance and turnaround time (TAT) were monitored post implementation to assess the impact on local service delivery. METHODS: Using the National Health Laboratory Service Corporate Data Warehouse, CD4 data were extracted for the period April 2012-July 2013 (n=11,964). Total mean TAT (in hours) was calculated and pre-analytical and analytical components assessed. Ongoing testing volumes, as well as external quality assessment performance across ten trials, were used to indicate post-implementation success. Data were analysed using Stata 12. RESULTS: Prior to the implementation of CD4 testing at De Aar, the total mean TAT was 20.5 hours. This fell to 8.2 hours post implementation, predominantly as a result of a lower pre-analytical mean TAT reducing from a mean of 18.9 to 1.8 hours. The analytical testing TAT remained unchanged after implementation and monthly test volumes increased by up to 20%. External quality assessment indicated adequate performance. Although subjective, questionnaires sent to facilities reported improved service delivery. CONCLUSION: Establishing CD4 testing in a remote community laboratory substantially reduces overall TAT. Additional community CD4 laboratories should be established in under-serviced areas, especially where laboratory infrastructure is already in place.

Tree-Based Methods for Discovery of Association between Flow Cytometry Data and Clinical Endpoints.

We demonstrate the application and comparative interpretations of three tree-based algorithms for the analysis of data arising from flow cytometry: classification and regression trees (CARTs), random forests (RFs), and logic regression (LR). Specifically, we consider the question of what best predicts CD4 T-cell recovery in HIV-1 infected persons starting antiretroviral therapy with CD4 count between 200 and 350 cell/muL. A comparison to a more standard contingency table analysis is provided. While contingency table analysis and RFs provide information on the importance of each potential predictor variable, CART and LR offer additional insight into the combinations of variables that together are predictive of the outcome. In all cases considered, baseline CD3-DR-CD56+CD16+ emerges as an important predictor variable, while the tree-based approaches identify additional variables as potentially informative. Application of tree-based methods to our data suggests that a combination of baseline immune activation states, with emphasis on CD8 T-cell activation, may be a better predictor than any single T-cell/innate cell subset analyzed. Taken together, we show that tree-based methods can be successfully applied to flow cytometry data to better inform and discover associations that may not emerge in the context of a univariate analysis.

Affordable CD4+ T cell counts by flow cytometry. II. The use of fixed whole blood in resource-poor settings.

We tested the feasibility and precision of affordable CD4+ T cell counting in resource-poor settings using a recently standardised fixative, TransFix in whole blood (WB) by flow cytometry (FCM). The precision of the assays was established under optimal conditions for single-platform FCM such as the volumetric CytoronAbsolute and the bead-based FACSCan. Fresh WB samples from HIV-seropositive and seronegative patients were tested in Tanzania and South Africa, fixed and sent to the UK for reanalysis 7 days later. Correlation, bias and limits of agreements were analysed by linear regression and the Bland-Altman test. Absolute CD4+ T cell counts remained stable for at least 10 days when TransFix was added to WB in 1:10 dilution at 20-25 degrees C, and for 7 days when added in 1:10 or 1:5 dilution to samples stored to mimic 'tropical' conditions at 37 degrees C. Higher temperatures such as 42 degrees C were tolerated for only short periods since the recovery had decreased to 63% by day 3. The reproducibility of lymphocyte subset analysis remained unchanged by TransFix with coefficient of variations <6% for all T cell subsets. Absolute CD4+ T cell counts and CD4+ T cell % values on fixed samples in the UK showed a high correlation with the results using fresh samples in Tanzania (r=0.993 and 0.969, respectively) and with the samples handled in Johannesburg (r=0.991 and 0.981) with minimal bias. Primary CD4 gating using only a single CD4 antibody also remained accurate in TransFixed samples (r=0.999). Thus, TransFix permits optimal fixation and transport of WB samples in the developing world for FCM to local regional laboratories and for quality assurance in international centres. When used together with inexpensive primary CD4 gating, TransFix will allow reliable and affordable CD4+ T cell counting by FCM in resource-poor settings.

CD4 cell monitoring for HIV/AIDS: old options; new insights.

Cost effective solutions are needed for laboratory monitoring that do not compromise on quality but address costs. Current recommended methods of CD4+ T cell enumeration are complex and costly. Monitoring typically utilises viral load assessment (PCR based) and CD4+ T cell counting to assess disease progression and response to therapy in HIV/AIDS. This paper reviews CD4 testing with the focus on different methods of CD4+ T cell enumeration including state-of-the-art flow cytometric testing, the advantages and disadvantages of these systems and quality control. Lastly, recent new work undertaken at the University of the Witwatersrand is discussed that addresses the problems of cost and precision and accuracy of CD4 T cell testing.

CD4+ T cell enumeration in HIV infection with limited resources.

The incidence of human immunodeficiency virus (HIV) infection continues to increase in South Africa. Limited resources are available for diagnosis and management of the disease and the development of affordable strategies is required. Absolute CD4 counts are used locally predominantly to monitor disease progression and institute prophylaxis against opportunistic infections. A dramatic increase in demand for CD4 counts prompted an investigation for a more cost-effective flow cytometry method than those currently recommended by the Centers for Disease Control (CDC). CD4 counts generated by two different single tube methods using CD3/CD4/CD8 [1(3)] and CD4 [1(1)] antibodies, respectively, were compared to the CDC recommended 6 tube 2 colour panel [6(2)]. Whole blood analysis using the Coulter Multi-Q-Prep system and an Epics XL Flow Cytometer (Coulter, Hialeah, FL) was performed for each of the three methods. Random samples from HIV positive adult patients were compared. A mean difference in the absolute CD4 counts of less than 10x10(6)/l was generated by both of the alternative panels when compared with the 6(2) panel. The precision of the three methods is comparable. In reagents alone, the 1(3) and 1(1) methods represent a cost saving of 76% and 93%, respectively, over the 6(2) method. The 1(3) and 1(1) panels would permit more affordable CD4 counts to be determined by the gold standard methodology of flow cytometry with no clinically significant sacrifices in accuracy or precision.

Microvolume fluorimetry for the determination of absolute CD4 and CD8 lymphocyte counts in patients with HIV: a comparative evaluation.

This study compared CD4 and CD8 lymphocyte counts obtained by microvolume fluorimetry (MVF) with those derived by flow cytometry (FC). Samples from 192 patients with known or suspected HIV were analysed, and the distribution of CD4 counts for these samples ranged from 0 and 1,279/microl, with 142/192 (74%) of the samples having CD4 values of less than 400/microl. Good agreement between FC and MVF CD4 counts was found (MVF = 0.98 x FC + 7.30) although there was a minor constant inter-method bias of approximately +7 cells/microl for the MVF data. For CD8 counts there was a constant bias between the two methods of approximately +23 cells/microl for FC. Most outliers were associated with higher FC CD8 counts. Supplementary analyses showed a high level of agreement between FC and MVF methods for the CD4:CD8 ratios (MVF = 0.98 x FC). This suggests that observed discrepancies between FC and MVF methods were almost certainly a result of the influence of the absolute lymphocyte counts obtained from the haematology analyser. The results confirm that the IMAGN 2000 microvolume fluorimeter system can be used as an alternative to conventional flow cytometry for the enumeration of CD4 and CD8 counts.

Melatonin has no effect on the growth, morphology or cell cycle of human breast cancer (MCF-7), cervical cancer (HeLa), osteosarcoma (MG-63) or lymphoblastoid (TK6) cells.

Melatonin was previously shown to inhibit proliferation of MCF-7 human breast cancer cells. In this study the effect of melatonin on MCF-7 cells was further examined, while human cervical carcinoma (HeLa), osteosarcoma (MG-63) and lymphoblastoid (TK6) cells were tested for the first time. Haemocytometer counts, DNA content, flow cytometry and indirect immunofluorescence for nucleolar proteins, actin and beta-tubulin showed no differences in the growth, cell cycle or morphology between melatonin-exposed and control cells. The direct antiproliferative effect of melatonin thus seems to be confined to a melatonin-responsive subclone of MCF-7 cells and not applicable to the majority of cancer cells.