Blood collection tubes impact expression of activated CD4+ and CD8+ T cells in human whole blood assay

Background: T-lymphocyte subsets CD4 and CD8 play important role in host immune responses. However, little attention has been given to the impact of time lapse and the various anticoagulant blood collection tubes on the expression frequency and activation status of CD4+ and CD8+ T cells. To this end, we explore the impact of time (t<1 h and t=4 h) and collection tubes (EDTA and heparin) on the expression frequency and activation status of CD4+ and CD8+ T cells among healthy Ghanaian individuals. Methods: A cohort of healthy individuals (n=9) is recruited, and blood samples obtained in Ghana for the frequency of CD4+and CD8+ T cells at various time points (<1 h and 4 h). The proportions of activation of these immune markers were profiled using immunophenotyping. Results: Significant statistical differences in the activation frequency of CD69 expressing CD4+T cells (t < 1 h and t=4 h; p=0.02) and CD69 expressing CD8+ T cells from EDTA tubes at times (t < 1 h and t=4 h; p=0.05) was observed. No significant difference were observed with CD69 expressing cells in Heparin tubes. Notably, CD8+ T cell activation frequency was observed to be consistently higher than that of CD4+ T cell at the various study time points and in the collection tubes used. No marked alterations were observed witth the proportion of CD4+ and CD8+ T cells in the samples collected at the time points; <1 h and at 4 h. Conclusion: The study shows that activation of CD4+ and CD8+ T cells in EDTA tubes differed significantly between both time points (t <1 h and t=4 h) but not in the heparin collection tubes. Therefore, it is important to take into account the elapsed time and the type of blood collection tubes when performing phenotypic characterization of activated immune markers

Intracytoplasmic Expression of IL-6 and IL-17A in Circulating CD4+ T Cells Are Strongly Associated with and Predict Disease Activity in Rheumatoid Arthritis: A Case-Control Study in Ghana

Background. T cell cytokines play important roles in the development and progression of rheumatoid arthritis (RA). Loss of Th1/Th2 and Th17/Treg balance has been reported in several inflammatory autoimmune diseases. However, their role in RA within hitherto rare Ghanaian context has not been explored. Here, we evaluated the intracytoplasmic CD4+ T cell cytokine patterns in rheumatoid arthritis patients in Ghana and determined their relationship with disease activity. Methods. This case-control study included 48 newly diagnosed RA patients and 30 apparent healthy controls from two major hospitals in Ghana. Validated structured questionnaires were administered to obtain demographic data; blood samples were collected and processed for flow cytometric analysis. Results. IFN-γ, TNF-α, IL-4, IL-6, IL-10, IL-17A, IL-6/IL-4, and IL-17/IL-10 expressions were significantly higher in RA cases compared to the healthy controls. The expression of IL-6 (0.00 (0.00-0.98) vs. 0.82 (0.34-1.10) vs. 1.56 (1.39-1.68), p<0.0001), IL-17A (0.00 (0.00-0.02) vs. 0.19 (0.09-0.30) vs. 0.99 (0.64-1.25), p<0.0001), and IL-17A/IL-10 (0.00 (0.00-0.39) vs. 0.15 (0.09-0.26) vs. 0.88 (0.41-1.47), p<0.0001) increased significantly from the healthy controls through RA patients with low DAS scores to RA patients with moderate DAS scores. IL-6 (β=0.681, r2=0.527, p<0.0001), IL-17A (β=0.770, r2=0.593, p<0.0001), and IL-17A/IL-10 (β=0.677, r2=0.452, p<0.0001) expressions were significantly directly associated with DAS28 scores. IL-6 (cutoff=1.32, sensitivity=100.0%, specificity=100.0%, accuracy=100.0%, and AUC=1.000) and IL-17A (cutoff=0.58, sensitivity=100.0%, specificity=100.0%, accuracy=100.0%, and AUC=1.000) presented with the best discriminatory power in predicting moderate DAS scores from low DAS scores. Conclusion. Th1- and Th17-related cytokines predominate in the pathophysiology of RA, with IL-6 and IL-17 being principally and differentially expressed based on the severity of the disease. IL-6 and IL-17A could serve as useful prognostic and disease-monitoring markers in RA in the African context

Receptors expressions on peripheral lymphocytes and CD4+CD183+ as a diagnostics biomarker for rheumatoid arthritis: A case–control study in Ghana

Abstract Background T cell receptors play important roles in the development and progression of rheumatoid arthritis (RA). Their involvement has been reported in inflammatory autoimmune diseases. However, their role in predicting RA is still under exploration. This study evaluated the expression of CD183 (CXCR3) receptors on T‐cells and other relevant biomarkers for detecting RA and determine their relationship with disease activity. Methods This unmatched case–control study included 48 newly diagnosed RA patients and 30 apparent healthy controls from the orthopedic units of Komfo Anokye Teaching Hospital (KATH), Kumasi and Korle‐Bu Teaching Hospital (KBTH), Accra, Ghana. Sociodemographic data was obtained, and blood samples were also collected and processed for flow cytometric analysis. Statistical analyses were done using SPSS version 26.0 and R programming language. p < .05 was considered statistically significant. Results This study found a significant difference in age group (p < .0001), marital status (p = .0210), occupation (p = .0140), educational level (p = .0210) and religion (p = .0100) between RA patients and healthy controls. Moreover, hemoglobin level (p = .0010), waist circumference (p < .0001) and hip circumference (p = .0040) were significantly different between RA patients and controls. RA patients had significantly lower levels of CD4+CD183+ compared with the control group (p < .001), and was positively correlated with DAS score (r = .0397, p = .789). In Receiver Operator Characteristics analysis, CD4+CD183+ could significantly detect RA with a high area under the curve (AUC = 0.687, p = .018). At a cut‐off of 0.082, CD4+CD183+ was the best receptor biomarker for detecting RA with a sensitivity of 90.0%, specificity of 25.9%, a positive predictive value of 69.2%, and a negative predictive value of 58.3%. Conclusion CD4+CD183+ best predict RA and is positively correlated with disease activity. CD4+CD183+ could serve as diagnostics and disease‐monitoring biomarker for RA; however, it demonstrates low specificity. Future studies should be directed on CD4+CD183+ and other biomarkers to augment their diagnostics performances and routine management in RA

Cohort profile: Research on Obesity and Diabetes among African Migrants in Europe and Africa Prospective (RODAM-Pros) cohort study

PURPOSE: The Research on Obesity and Diabetes among African Migrants (RODAM) prospective (RODAM-Pros) cohort study was established to identify key changes in environmental exposures and epigenetic modifications driving the high burden of cardiovascular disease (CVD) risk among sub-Saharan African migrants. PARTICIPANTS: All the participants in the RODAM cross-sectional study that completed the baseline assessment (n=5114) were eligible for the follow-up of which 2165 participants (n=638 from rural-Ghana, n=608 from urban-Ghana, and n=919 Ghanaian migrants in Amsterdam, the Netherlands) were included in the RODAM-Pros cohort study. Additionally, we included a subsample of European-Dutch (n=2098) to enable a comparison to be made between Ghanaian migrants living in the Netherlands and the European-Dutch host population. FINDINGS TO DATE: Follow-up data have been collected on demographics, socioeconomic status, medical history, psychosocial environment, lifestyle factors, nutrition, anthropometrics, blood pressure, fasting blood, urine and stool samples. Biochemical analyses included glucose metabolism, lipid profile, electrolytes and renal function, liver metabolism and inflammation. In a subsample, we assessed DNA methylation patterns using Infinium 850K DNA Methylation BeadChip. Baseline results indicated that migrants have higher prevalence of CVD risk factors than non-migrants. Epigenome-wide association studies suggest important differences in DNA methylation between migrants and non-migrants. The follow-up study will shed further light on key-specific environmental exposures and epigenetic modifications contributing to the high burden of CVD risk among sub-Saharan African migrants. FUTURE PLANS: Follow-up is planned at 5-year intervals, baseline completed in 2015 and first follow-up completed in 2021

Analysis of <i>Mycobacterium ulcerans</i>-specific T-cell cytokines for diagnosis of Buruli ulcer disease and as potential indicator for disease progression

<div><p>Background</p><p>Buruli ulcer disease (BUD), caused by <i>Mycobacterium (M</i>.<i>) ulcerans</i>, is the third most common mycobacterial disease after tuberculosis and leprosy. BUD causes necrotic skin lesions and is a significant problem for health care in the affected countries. As for other mycobacterial infections, T cell mediated immune responses are important for protection and recovery during treatment, but detailed studies investigating these immune responses in BUD patients are scarce. In this study, we aimed to characterise <i>M</i>. <i>ulcerans</i>-specific CD4+ T cell responses in BUD patients and to analyse specific cytokine-producing T cells in the context of disease severity and progression.</p><p>Methodology/Principal findings</p><p>For this case-control study, whole blood samples of BUD patients (N = 36, 1.5–17 years of age) and healthy contacts (N = 22, 3–15 years of age) were stimulated with antigen prepared from <i>M</i>. <i>ulcerans</i> and CD4+ T cells were analysed for the expression of TNFα, IFNγ and CD40L by flow cytometry. The proportions and profile of cytokine producing CD4+ T cells was compared between the two study groups and correlated with disease progression and severity. Proportions of cytokine double-positive IFNγ+TNFα+, TNFα+CD40L+, IFNγ+CD40L+ (p = 0.014, p = 0.010, p = 0.002, respectively) and triple positive IFNγ+TNFα+CD40L+ (p = 0.010) producing CD4+ T cell subsets were increased in BUD patients. In addition, TNFα+CD40L-IFNγ- CD4+ T cells differed between patients and controls (p = 0.034). TNFα+CD40L-IFNγ- CD4+ T cells were correlated with lesion size (p = 0.010) and proportion were higher in ‘slow’ healers compared to ‘fast healers’ (p = 0.030).</p><p>Conclusions</p><p>We were able to identify <i>M</i>. <i>ulcerans</i>-specific CD4+ T cell subsets with specific cytokine profiles. In particular a CD4+ T cell subset, producing TNFα but not IFNγ and CD40L, showed association with lesion size and healing progress. Further studies are required to investigate, if the identified CD4+ T cell subset has the potential to be used as biomarker for diagnosis, severity and/or progression of disease.</p></div

TNFα+CD40L-IFNγ- CD4+ T cells differ between fast and slow healers in Buruli ulcer disease.

<p>(<b>A</b>) BUD patients were divided into fast healers (≤ 111 days) and slow healers (> 111 days) based on the time required for complete healing and proportions of cytokine producing CD4+ T cells (<b>B</b>, <b>C</b>) or lesion sizes (<b>D</b>) are compared by non-parametric Mann Whitney <i>U</i> test. The healing rate within the first four weeks (in change if mm/week) was correlated with TNFα+CD40L-IFNγ- CD4+ T cells (<b>E</b>) by Spearman correlation or compared based on time until healing process starts after initiating antibiotic treatment (<b>F</b>).</p

Clinical characteristics of the BUD group.

<p>Clinical characteristics of the BUD group.</p

Description of the study population.

<p>Description of the study population.</p

TNFα+CD40L-IFNγ- CD4+ T cells are correlated with size of lesions in Buruli ulcer disease.

<p>Cytokine producing CD4+ T cells were determined as for <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0005415#pntd.0005415.g001" target="_blank">Fig 1</a> and analysed in regards to type of lesion (<b>A</b>, <b>B</b>) or surface area of lesions <b>(C</b>, <b>D</b>) or widest diameter of the lesions (<b>E</b>). Multiple cytokine producing are shown in (<b>A</b>, <b>C</b>); TNFα+CD40L-IFNγ- and CD40L+IFNγ- are indicated in (<b>B</b>, <b>D</b>). P—values of non-parametric Mann-Whitney <i>U</i> analyses (A, B) and Spearman correlations (C–E) are presented.</p

T_H1 cytokine producing CD4+ T cells in Buruli ulcer disease patients and healthy contacts.

<p>Whole blood was cultured for 17.5 hrs with either <i>M</i>. <i>ulcerans</i> antigen or Staphylococcal enterotoxin b. Cells were analysed by flow cytometry for TNFα, IFNγ and CD40L producing CD4+ T cells. Proportions of double and triple cytokine producing T cells (IFNγ+TNFα+, TNFα+CD40L+, IFNγ+CD40L+ and IFNγ+TNFα+CD40L+) are indicated in (<b>A</b>). Combinations of cytokine producing CD4+ T cells are shown in (<b>B</b>). Medians are indicated in grey and groups are compared by non-parametric Mann-Whitney <i>U</i> test and p values indicated.</p